0107
A functional interaction between plant MEINOX-HD and BLH genes is substantiated by the synergism observed when pennywise a null-allele of BLH9 (18), allelic to bellringer, replumless, vaamana (19,22,23) is combined with the brevipedicellus1 (bp1) mutant (24).
0030
This dominant-negative function upon expression of the MEINOXSTM domain is strengthened in combination with the Drosophila engrailed repressor domain and best explained by squelching: the abundant MEINOXSTM domain depleting the native STM protein from essential interaction partners.
0018
The meristem-enriched cDNA library in the vector pACT2 (Clonetech) was prepared from immature inflorescence shoots (Lumbrineris erecta, 4–5 mm size) containing inflorescence and multiple floral meristems (FMs). cDNA synthesis exactly followed the manual for the cDNA synthesis kit (Stratagene). Directional cloning into pACT2 was achieved via an XhoI site addition to the oligo-dT primer (3′) and an EcoRI adapter at the cDNA 5′-terminus. The cDNA library (1.25 × 106 clones) was colony amplified in Escherichia coli, plasmid DNA purified by CsCl2-gradient centrifugation and used for yeast transformation (strain AH109). Screening of the library followed the Matchmaker manual applying quadruple selection (HIS3, ADE2, LacZ and MEL1).

0018
Positive clones (223) were isolated and cDNA inserts in pACT2 were subjected to N-terminal sequence analysis. The specificity of each protein interaction was confirmed by retransformation of sequence-verified cDNA clones (ATH1, BHL3 or BHL9) into yeast strains providing the MEINOXSTM or MEINOXBP1 bait constructs in pGDKT7.
0809
Verified cDNA inserts were either directly transferred into appropriate secondary vectors for bimolecular fluorescence complementation (BiFC), co-immunoprecipitation or transgenic experiments and served as templates in second-round PCR-amplification to remove the stop codon or to adjust open reading frames (ORFs) for translational fusions.

0019
Verified cDNA inserts were either directly transferred into appropriate secondary vectors for bimolecular fluorescence complementation (BiFC), co-immunoprecipitation or transgenic experiments and served as templates in second-round PCR-amplification to remove the stop codon or to adjust open reading frames (ORFs) for translational fusions.
0030
Non-radioactive in situ hybridization experiments were performed essentially as described in Bradley et al.

0889
Non-radioactive in situ hybridization experiments were performed essentially as described in Bradley et al.
0007
HA-tagged (1/3 aliquot) partner proteins were identified using an anti-HA horseradish peroxidase (HRP)-conjugated antibody (clone 3F10; Roche 2012819).

0004
Epidermal proteins after bombardement of leek epidermal cells were isolated in 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton and size-fractionated by SDS–PAGE.
0107
The method of choice to prove a physical interaction between candidate partner proteins in plant cells is BiFC (31,36).

0663
As a potential transcription factor, STM was expected to direct GFP fluorescence into the plant cell nucleus.
0663
Additional support for nuclear exclusion was obtained by several STM deletion constructs, none of which targeted GFP fluorescence to the plant cell nucleus (data not shown).
0018
Besides obvious controls in yeast such as retransformation of bait or prey plasmids and the combination with the empty bait vector, we included the MEINOXBp1 domain of the BP1 gene as bait as it encodes the closest relative to the MEINOXSTM domain in the Arabidopsis genome.

0019
In addition to the interactions in yeast, we performed co-immunoprecipitation experiments with epitope-tagged full-length proteins.

0019
The co-immunoprecipitation experiments therefore confirm that the full-length STM protein interacts with full-length ATH1, BLH3 and BLH9 proteins in vitro and substantiate the affinity of BLH/STM interactions.
0096
To verify STM interaction partners we constructed translational fusions between the N- or the C-terminal YFP sub-domains and the full-length STM protein or individual full-length BLH partner proteins.

0107
To verify STM interaction partners we constructed translational fusions between the N- or the C-terminal YFP sub-domains and the full-length STM protein or individual full-length BLH partner proteins. Optimal combinations of chimeric gene constructs are indicated in Figure 4 above each corresponding experiment.

0055
Any detectable fluorescence was dependent on the combination of the C-terminal with the N-terminal YFP sub-domain fused to STM or BLH partner proteins. In contrast, the fusion of the C- or the N-terminal YFP sub-domain to the STM protein and their co-bombardment in plant cells gave no fluorescence (data not shown).
0030
However, without analysing the cellular localization of the STM protein, Bhatt et al.

0889
However, without analysing the cellular localization of the STM protein, Bhatt et al.

0107
This may indicate a weak interaction between STM and BLH9 as suggested by the yeast two-hybrid results, that chimeric BLH9-NYFP protein levels remain low in leak epidermal cells, or that steric constraints interfere with a reconstitution of YFP fluorescence.
0809
According to our BiFC results, however, STM is targeted into the nucleus as a heterodimer with ATH1, BLH3 and BLH9.
0030
In Drosophila leg imaginal discs, the EXD protein is nuclear localized in proximal regions, which correspond to the expression domain of the HTH protein.

0107
The accepted scheme of nuclear import control of MEINOX TALE-HD proteins through interaction with the second class of TALE-HD proteins, BLH or PBC in plants and animals, respectively, suggests an ancient and conserved mechanism. In animals, the interaction between PBC and MEINOX proteins requires integrity of the bipartite PBC-A and B domains in the PBC and the MEINOX domain in MEIS proteins (13).
0096
Based on protein–protein interaction data, the animal PBC-A/B domain finds its counterpart in the BELL domain of plant BLH proteins although conservation at the amino acid level is weak.
0081
The PBC-B domain contains several conserved phosphorylation sites for Ser/Thr kinases and PBX1 sub-cellular localization correlates with the phosphorylation state of these residues whose dephosphorylation induces nuclear export (49).
0107
The evolutionary conservation of a functional interaction between members of two families of TALE-HD gene products, MEIS/PBX and KNOX/BLH proteins in animals or plants, respectively, suggests an ancient and possibly extant function.
0107
The positive controls upper panel to the left shows the interaction between p53 and the SV40 T-antigen (pGBKT7-53 and pADT7-T, respectively, provided in the MATCHMAKER Biosensor kit).

0007
(B) In vitro translated full-length BLH proteins tagged with the hemaglutine (HA) epitope which were used in co-precipitation experiments with His-tagged STM protein and visualized by an anti-HA HRP-conjugated antibody.

0096
(B) In vitro translated full-length BLH proteins tagged with the hemaglutine (HA) epitope which were used in co-precipitation experiments with His-tagged STM protein and visualized by an anti-HA HRP-conjugated antibody.
0809
(A–C) BiFC staining of the nucleus obtained after coexpression of STM/BLH constructs as indicated above each photograph in leek epidermal cells.

0107
(H) Interaction between BLH3ΔC-NYFP and STM-CYFP.
0107
Functional interaction between STM-GR and partner proteins in transgenic Arabidopsis plants.
0416
Herpes simplex virus type-1 expresses a heterodimeric Fc receptor, gE-gI, on the surfaces of virions and infected cells that binds the Fc region of host immunoglobulin G and is implicated in the cell-to-cell spread of virus.

0114
Here we identify the C-terminal domain of the gE ectodomain (CgE) as the minimal Fc-binding domain and present a 1.78-Å CgE structure. A 5-Å gE-gI/Fc crystal structure, which was independently verified by a theoretical prediction method, reveals that CgE binds Fc at the CH2-CH3 interface, the binding site for several mammalian and bacterial Fc-binding proteins. The structure identifies interface histidines that may confer pH-dependent binding and regions of CgE implicated in cell-to-cell spread of virus.
0411
Herpes simplex virus type-1 (HSV-1) has evolved several strategies to escape detection by the host's immune system, including the expression of an Fc receptor (FcR) called gE-gI that is found on the surface of virions and infected cells [1–3].

0416
Herpes simplex virus type-1 (HSV-1) has evolved several strategies to escape detection by the host's immune system, including the expression of an Fc receptor (FcR) called gE-gI that is found on the surface of virions and infected cells [1–3].

0416
Antibody bipolar bridging has been shown to protect the virus and infected cells from IgG-mediated immune responses, such as antibody- and complement-dependent neutralization [6], antibody-dependent cell-mediated cytotoxicity [5], and granulocyte attachment [8]. Experiments in HSV-1–infected mice comparing the effectiveness of human anti-HSV IgG versus nonimmune IgG or murine anti-HSV IgG (which does not bind gE-gI) have provided support for the importance of antibody bipolar bridging mediated by gE-gI [7].
0107
Residues critical to the interaction were identified in binding studies comparing the affinities of human IgG subtypes for gE-gI, which found that gE-gI binds to IgG1, IgG2, and IgG4 with similar affinities (equilibrium dissociation constant [KD] ˜40–400 nM), but does not bind several IgG3 allotypes or an IgG4 mutant in which His435 was changed to an arginine [21,22].
0107
In order to study the interaction between gE-gI and Fc in more detail, we initiated structural studies of gE, gE-gI, and a gE-gI/Fc complex.
0096
To determine whether isolated CgE (gE residues 213–390, where residue 1 is the first residue of the hydrophobic leader peptide in the immature gE and residue 420 is the first residue of the predicted transmembrane region) binds Fc and to measure the affinity of the interaction, we performed a surface plasmon resonance assay using two forms of recombinant Fc derived from human IgG1: wild-type Fc (wtFc) and nbFc, which contains six point mutations in the CH2-CH3 linker that abrogate binding to gE-gI (Met252 to Gly, Ile253 to Gly, His310 to Glu, His433 to Glu, His435 to Glu, and Tyr436 to Ala) [12]. The Fc proteins were immobilized on the surface of a biosensor chip, and the binding of CgE was assayed at pH 8 and pH 6.

0676
To determine whether isolated CgE (gE residues 213–390, where residue 1 is the first residue of the hydrophobic leader peptide in the immature gE and residue 420 is the first residue of the predicted transmembrane region) binds Fc and to measure the affinity of the interaction, we performed a surface plasmon resonance assay using two forms of recombinant Fc derived from human IgG1: wild-type Fc (wtFc) and nbFc, which contains six point mutations in the CH2-CH3 linker that abrogate binding to gE-gI (Met252 to Gly, Ile253 to Gly, His310 to Glu, His433 to Glu, His435 to Glu, and Tyr436 to Ala) [12].

0104
Significant binding of CgE to nbFc was not observed under any conditions (unpublished data), but CgE binds wtFc with a KD of ˜7 μM at pH 8 (Figure 1A and1C).

0019
The relatively low affinity of the CgE/Fc interaction likely explains why a CgE/Fc binding interaction was not detectable by coimmunoprecipitation analyses [16].

0104
The affinity of CgE for Fc is similar to that determined for the interaction of gE with Fc at pH 8 (KD ˜30 μM), but ˜50-fold weaker than the affinity of the gE-gI/Fc interaction, which has KD values of 340 nM and 930 nM for the first and second binding sites on wtFc, respectively [12].

0107
The affinity of CgE for Fc is similar to that determined for the interaction of gE with Fc at pH 8 (KD ˜30 μM), but ˜50-fold weaker than the affinity of the gE-gI/Fc interaction, which has KD values of 340 nM and 930 nM for the first and second binding sites on wtFc, respectively [12]. As previously observed for the interaction of gE-gI with Fc [12], the binding of CgE to Fc is much weaker at pH 6 than at pH 8 (Figure 1A and1B).
0114
A 3-D search of the protein database using the DALI server [29] identified the coxsackie virus and adenovirus receptor domain 1 (pdb entry 1eaj) and a mouse Fv (pdb entry 1mfa) as two of its closest structural neighbors, with root mean square deviations of 2.6 Å over 108 Cα atoms and 2.8 Å over 106 Cα atoms, respectively (Figure S1). The structural similarity between CgE and its structural homologs does not include the third β-sheet found in CgE.

0114
Further structural differences include the extension of strands C, C′, and C′′ as well as the A′–B and C′–C′′ loops, and the shortening of strands D and E as compared to a representative Ig V domain (Figure S1A), which leads to a more open organization of the first two β-sheets (sheets DEBB′A and A′GFCC′C′′).
0107
The magnitude of the energy gap between the correct models and other models is correlated with the binding affinity of the interaction [33]. Perhaps because of the relatively low binding affinity of the CgE/Fc interaction (Figure 1C), we did not observe a significant energy gap in this case, and hence could not confidently identify a single model as correct based on the global docking calculations alone.
0114
The complex structure shows that CgE interacts with Fc using regions of the first and second β-sheets, which correspond to the two β-sheets found in Ig V domains: strands A and B′ and the B′–C and D–E loops in the DEBB′A sheet and strands C, C′, and C′′ and the C′–C′′ loop in the A′GFCC′C′′ sheet (Figures 2C and4).
0416
Previous studies demonstrated a sharply pH-dependent interaction for HSV-1–infected cells binding to rabbit IgG [37] and for soluble gE-gI or gE monomers binding to human Fc [12], such that binding occurs at neutral or slightly basic pH but not at acidic pH.

0676
An analysis of the change in the affinity of the gE-gI/Fc complex versus pH suggested that two ionizable residues, likely histidines because they have a pKa near neutral pH, participate in the pH-dependent affinity transition [12].
0419
Although we cannot rule out local conformational variability that occurs as a function of pH, it seems unlikely that CgE undergoes large changes in conformation due to its relative compactness as a single domain, and arguments against pH-induced conformational changes in Fc have been made based on the similarity of Fc crystal structures at pH values between 4.1 and 8.0 [27].
0416
Insertion mutants in gE were also tested for their effects on viral spread in cultured epithelial cells and HSV-1 infected mice [7,17,18,38,39]. Although the mechanism of viral spread is unknown, it has been hypothesized that the extracellular domains of gE-gI bind to receptors at cell junctions, allowing entry into adjacent cells [17,40].
0114
The position of the C-terminus of CgE in the gE-gI/Fc complex structure, which is ˜30 amino acids before the predicted transmembrane domain, suggests that IgG bound by gE-gI is in an upright orientation with respect to the membrane (Figure 5), similar to FcαRI-bound IgA [41], but opposite to the orientation predicted for antibodies bound by other cellular Fc receptors such as FcγRIII and FcɛRI [42–44].
0114
Because we were able to solve the crystal structure of the CgE subunit of the gE-gI ectodomain at high resolution, the identification of CgE as a minimal Fc-binding domain (Figure 1) made its structure useful for interpreting the low-resolution gE-gI/Fc complex structure. An independent prediction of the complex structure using the coordinates of the unbound CgE and Fc molecules revealed a close agreement with the experimentally determined gE-gI/Fc structure (Figure 3C), demonstrating the power of new protein docking methods [33,34], and their potential for facilitating difficult crystallographic problems.
0114
As revealed in the low resolution crystal structure of a gE-gI/Fc complex, the CgE portion of gE-gI binds to Fc at the CH2-CH3 interdomain junction (Figure 3A), consistent with previous studies of the gE-gI/Fc interaction [12,21,23] and allowing a comparison of the Fc-binding properties of CgE with other proteins that bind to the same site on Fc [19,20,27].

0081
An analysis of the CgE/Fc interface predicts that the six residues that are the hallmark of the consensus binding site on Fc [19,27] are also central to Fc binding by CgE (Figure 4A).
0081
The crystallographic data can be used to deduce a model for how IgG binds gE-gI on the surface of virions or infected cells, whereby CgE binds the consensus Fc-binding site on each half of the Fc dimer, and gI and the NgE are located underneath the CgE domain between the CH3 domains of Fc and the membrane (Figure 5).

0416
The crystallographic data can be used to deduce a model for how IgG binds gE-gI on the surface of virions or infected cells, whereby CgE binds the consensus Fc-binding site on each half of the Fc dimer, and gI and the NgE are located underneath the CgE domain between the CH3 domains of Fc and the membrane (Figure 5).

0426
The crystallographic data can be used to deduce a model for how IgG binds gE-gI on the surface of virions or infected cells, whereby CgE binds the consensus Fc-binding site on each half of the Fc dimer, and gI and the NgE are located underneath the CgE domain between the CH3 domains of Fc and the membrane (Figure 5).
0081
The gE-gI binding site on Fc does not directly overlap with the binding sites for the FcγRs or the C1q component of complement, which bind on or near the CH2 domain [43,44,51], thus the structure of the gE-gI/Fc complex does not directly suggest how gE-gI binding to the Fc region of IgG leads to evasion of FcγR- and complement-mediated immune responses.

0114
The gE-gI binding site on Fc does not directly overlap with the binding sites for the FcγRs or the C1q component of complement, which bind on or near the CH2 domain [43,44,51], thus the structure of the gE-gI/Fc complex does not directly suggest how gE-gI binding to the Fc region of IgG leads to evasion of FcγR- and complement-mediated immune responses.

0416
The demonstration that the gE-gI/Fc structure is compatible with antibody bipolar bridging (Figure 5) raises the possibility that anti-HSV IgG/HSV antigen complexes interacting with gE-gI on the surface of infected cells are endocytosed by gE-gI and degraded in the lysosomes after dissociation at acidic pH, resulting in destruction of antiviral antibodies and removal of viral antigens from the cell surface.
0081
The expression vector also encoded the gE hydrophobic leader peptide N-terminal to the inserted fragment and a C-terminal Factor Xa cleavage site and 6x-His tag.

0411
Recombinant baculovirus stocks were generated by cationic liposome cotransfection of the expression plasmid with linear wild-type baculovirus DNA in High 5 insect cells (Invitrogen, Carlsbad, California, United States) as described by the manufacturer.

0004
Insect cell supernatants containing secreted CgE were buffer-exchanged into nickel-binding buffer (40 mM Tris [pH 8], 300 mM NaCl, 10 mM imidazole) and passed over a Ni-NTA agarose column (Qiagen, Valencia, California, United States). Bound protein was eluted in buffer containing 40 mM Tris (pH 8), 300 mM NaCl, and 250 mM imidazole, and further purified by size-exclusion chromatography on a Superdex 75 HiLoad 16/60 column (GE Healthcare, Piscataway, New Jersey, United States) that was equilibrated in 10 mM HEPES (pH 7.6) and 150 mM NaCl. Peak fractions were concentrated and buffer exchanged into 10 mM HEPES (pH 7.5).

0071
Insect cell supernatants containing secreted CgE were buffer-exchanged into nickel-binding buffer (40 mM Tris [pH 8], 300 mM NaCl, 10 mM imidazole) and passed over a Ni-NTA agarose column (Qiagen, Valencia, California, United States). Bound protein was eluted in buffer containing 40 mM Tris (pH 8), 300 mM NaCl, and 250 mM imidazole, and further purified by size-exclusion chromatography on a Superdex 75 HiLoad 16/60 column (GE Healthcare, Piscataway, New Jersey, United States) that was equilibrated in 10 mM HEPES (pH 7.6) and 150 mM NaCl. Peak fractions were concentrated and buffer exchanged into 10 mM HEPES (pH 7.5).

0416
Briefly, insect cells that were infected with the CgE-recombinant virus were grown in Ex-Cell 400 media (JRH Biosciences, Lenexa, Kansas, United States) for 16 hours, pelleted, and then resuspended in Sf-900 II SFM media (Invitrogen) supplemented with 30 mg/l of L-cysteine (Sigma, St.
0004
Equilibrium binding data for CgE binding to wtFc (coupling density, 440 resonance units), nbFc (coupling density, 405 resonance units), and a mock-coupled flow cell were collected for a CgE concentration series (three-fold dilutions of CgE from 30 μM to 5 nM) at pH 8 (50 mM HEPES [pH 8.0], 150 mM NaCl, 3 mM EDTA, 0.005% [vol/vol] P-20 surfactant) and pH 6 (50 mM sodium phosphate [pH 6.0], 150 mM NaCl, 3 mM EDTA, 0.005% [vol/vol] P-20 surfactant). After each injection of CgE, a 30-s injection of 250 mM di-ammonium hydrogen citrate (pH 5.0) was used to disrupt the interaction and restore the surface.
0096
In the next stage, a second constraint was imposed requiring that CgE make contact with both the CH2 and CH3 domains of the Fc protein, as observed for other proteins binding to the Fc “hot spot,” [19] which resulted in pruning some of the top-ranking clusters.
0114
A half complex (one CgE and one chain of the Fc dimer) is shown with Fc in green, the CgE as positioned in the crystal structure in blue, and the CgE as positioned by the docking prediction in pink.
0107
Hence, understanding the interaction between the Munc13–1 C2A domain and the α-RIM ZF domain at atomic detail can provide key insights into the functional diversification of C2 domains and ZF domains in general.
0114
NMR spectroscopy and X-ray crystallography provide complementary tools to study protein structure. X-ray crystallography is better suited to elucidate the structures of large proteins and accurately define interfaces of protein complexes, but requires crystallization, which can yield artifacts due to crystal packing.

0077
NMR spectroscopy and X-ray crystallography provide complementary tools to study protein structure.

0077
NMR spectroscopy can be performed in solution, and low-resolution information on the conformational and aggregation states of proteins can be quickly obtained even for large species using heteronuclear NMR experiments such as1H-15N heteronuclear single quantum coherence (HSQC).

0114
In this study, we took advantage of the strengths of both techniques, using NMR spectroscopy to optimize protein complexes for crystallization and to obtain structural information in solution that was later employed to interpret the high-resolution structures of these complexes elucidated by X-ray crystallography.

0077
In this study, we took advantage of the strengths of both techniques, using NMR spectroscopy to optimize protein complexes for crystallization and to obtain structural information in solution that was later employed to interpret the high-resolution structures of these complexes elucidated by X-ray crystallography.
0104
Interestingly, the apparent molecular weights observed for isolated Munc13–13–132, Munc13–13–150, and Munc13–13–209 in gel filtration were significantly higher than their monomeric molecular weights (Figure 1B and unpublished data), suggesting that they form stable dimers.
0104
We next used analytical ultracentrifugation to determine the oligomerization state of Munc13–13–209, Munc13–13–150, and Munc13–13–132, and of a shorter fragment that we prepared during optimization for our crystallographic studies (Munc13–13–128; see below). Fitting the data to single ideal species yielded molecular weights of 49,100 Da, 35,200 Da, 30,100 Da, and 26,700 Da, respectively, which are approximately twice the predicted molecular weights of these fragments (24683.4 Da, 17821.3 Da, 16168.4 Da, and 14856.9 Da, respectively).
0114
The Munc13–1 C2A domain monomer exhibits a β-sandwich structure formed by two four-stranded β-sheets that is characteristic of C2 domains (Figure 2B and2C), but the aforementioned β-hairpin formed by a long loop in monomer C (strands 8 and 9) has not been previously observed in other C2 domains. A structural comparison using DALI [26] showed that, among C2 domains deposited in the Protein Data Bank (http://www.rcsb.org/pdb, the PLC-δ1 C2-domain shares the highest structural similarity with the Munc13–1 C2A domain (1.65 Å rms deviation for 106 equivalent Cα carbons).
0114
However, this β-hairpin is well packed against strand 6 of monomer C itself and appears to be an intrinsic feature of the Munc13–1 C2A domain, because the two Munc13–1 monomers present in the crystal structure of the Munc13–1/RIM2 heterodimer also contain this β-hairpin (see below).
0114
Hence, we designed two charge-reversal mutations, K32E and E63K, to disrupt Munc13–1 homodimerization based on the crystal structure of the C2A-domain homodimer.
0104
Gel filtration showed that both point mutations disrupt dimerization of Munc13–13–128, Munc13–13–150, and Munc13–13–209, but preserve heterodimerization with RIM2α82–142 (Figure 4A and unpublished data).

0676
We attribute this increased apparent affinity compared to the wild-type complex to the lack of competition with homodimerization.
0104
Thus, the binding mode between monomer A and RIM2α82–142 observed in the crystals is consistent with all available data obtained in solution, whereas that involving monomer B is not.
0114
The structure of the RIM2α ZF domain in the heterodimer with Munc13–13–150(K32E) (Figure 5E, blue) is similar to the solution structure of the isolated RIM2α ZF domain (Figure 5E, red) [20], exhibiting two zinc-binding sites and a central β-hairpin that is flanked on one side by N-terminal loops and on the other side by another β-hairpin and a short C-terminal α-helix (helix a2). Superposition of the two structures yielded an rms deviation of 1.9 Å for 54 Cα carbons, revealing that heterodimer formation induces significant conformational changes in the ZF domain that suggest a substantial malleability and that are consistent with the widespread changes in its1H-15N HSQC spectrum caused by Munc13–1 binding [20].
0107
As shown inFigure 6D, the interaction of the C-terminal α-helix of Munc13–13–150(K32E) with the crevice between the C-terminal β-hairpin and helix a2 of the ZF domain involves a combination of hydrophobic and polar interactions.
0114
Our Munc13–13–150(K32E)/RIM2α82–142 structure, together with the crystal structure of a rabphilin/Rab3A complex [27], and the sequence homology between rabphilin and RIM2α, also provide an explanation for our previous observations that Munc13–1, RIM2α, and Rab3A form a tripartite complex and that Munc13–1 alters the RIM2α/Rab3A binding mode [20].
0107
Disrupting the interaction between the N-terminal region of Munc13–1 and α-RIMs causes a severe impairment in priming [10,20] that is comparable to that observed in RIM1α knockout mice [16]. The marked decrease in Munc13–1 levels observed in these mice further supports the physiological importance of the Munc13–1/α-RIM interaction [14].
0114
The structure of the Munc13–1/RIM2α ZF domain complex described here shows that Munc13–1 binding to the α-RIM/Rab3A complex should lead to a partial steric clash with Rab3A (Figure 7B), explaining our previous observation that the interaction of the α-RIM SGAWFY motif with Rab3A is released upon Munc13–1 binding [20]. The Munc13–1/α-RIM interaction still allows formation of the tripartite complex because helix a1 of α-RIMs is sufficient for Rab3A binding, but is expected to induce a significant reorientation of the N-terminal structural elements of α-RIMs (Figure 7C).
0081
It is worth noting that phosphorylation of RIM1α by PKA at an N-terminal site between the ZF and PDZ domains is key for mossy-fiber LTP [17], and that the surfaces of Munc13–1 involved in homodimerization and heterodimerization (Figures 2G,6D, and6E) contain several predicted phosphorylation sites (Y23, T25, and Y140). Hence, phosphorylation may be crucial for regulation of inter- and intramolecular interactions of Munc13–1 and α-RIMs during plasticity processes.
0114
In the past, NMR spectroscopy and X-ray crystallography were largely viewed as alternative methods for structure determination of biomolecules, but the usefulness of combining the strengths of both techniques is increasingly being recognized [32,33]. On the other hand, two structural genomics studies recently indicated that there is no overt correlation between1H-15N HSQC spectral quality and successful crystallization of protein targets [34,35]. However, it is unclear to what extent the fragment length of each target was optimized in these studies, and a separate structural genomics effort suggested that NMR spectra can be used to identify promising targets for structure determination by X-ray crystallography [36]. The data presented here, together with our previous NMR analysis of Munc13–1/α-RIM/Rab3 interactions [20], provide a particularly compelling illustration of how NMR spectroscopy can assist in X-ray diffraction studies of protein complexes that present particularly challenging problems for crystallization, and at the same time can provide complementary information.

0077
In the past, NMR spectroscopy and X-ray crystallography were largely viewed as alternative methods for structure determination of biomolecules, but the usefulness of combining the strengths of both techniques is increasingly being recognized [32,33].

0077
However, it is unclear to what extent the fragment length of each target was optimized in these studies, and a separate structural genomics effort suggested that NMR spectra can be used to identify promising targets for structure determination by X-ray crystallography [36]. The data presented here, together with our previous NMR analysis of Munc13–1/α-RIM/Rab3 interactions [20], provide a particularly compelling illustration of how NMR spectroscopy can assist in X-ray diffraction studies of protein complexes that present particularly challenging problems for crystallization, and at the same time can provide complementary information.

0114
Altogether, these observations suggest that combining the strengths of NMR spectroscopy in fragment optimization and analysis of protein–protein interactions in solution with the high accuracy of structure determination by X-ray crystallography for biomolecules of any size will be particularly useful to study complex protein networks.

0077
Altogether, these observations suggest that combining the strengths of NMR spectroscopy in fragment optimization and analysis of protein–protein interactions in solution with the high accuracy of structure determination by X-ray crystallography for biomolecules of any size will be particularly useful to study complex protein networks.
0096
Unlabeled and isotopically labeled proteins were expressed in bacteria as GST fusions as described [20]. The fusion proteins were isolated on glutathione-Sepharose beads (Amersham Biosciences, Little Chalfont, United Kingdom), cleaved from the GST moiety, and further purified by size-exclusion or ion-exchange chromatography.

0004
Gel-filtration binding experiments were performed on Superdex S75 or S200 columns (Amersham) in 30 mM Tris-HCl buffer containing 150 mM NaCl, and 1 mM Tris(2-carboxyethyl)-phosphine (TCEP) at pH 7.4.

0071
Gel-filtration binding experiments were performed on Superdex S75 or S200 columns (Amersham) in 30 mM Tris-HCl buffer containing 150 mM NaCl, and 1 mM Tris(2-carboxyethyl)-phosphine (TCEP) at pH 7.4.
0004
ITC experiments were performed using a VP-ITC system (MicroCal, Northampton, Massachusetts, United States) at 20 °C in a buffer composed of 30 mM Tris (pH 7.4), 150 mM NaCl, and 1 mM TCEP.

0065
ITC experiments were performed using a VP-ITC system (MicroCal, Northampton, Massachusetts, United States) at 20 °C in a buffer composed of 30 mM Tris (pH 7.4), 150 mM NaCl, and 1 mM TCEP.

0065
Data were fit with a non-linear least-squares routine using a single-site binding model with Origin for ITC v.5.0 (Microcal), varying the stoichiometry(n), the enthalpy of the reaction (ΔH) and the association constant (Ka).
0004
The purified Munc13–13–128 fragment and the Munc13–13–150(K32E)/Rim2α82–142 complex were concentrated to 12 and 10 mg/ml, respectively, in buffer containing 30 mM Tris (pH 7.4), 150 mM NaCl, and 1 mM TCEP. Munc13–13–128 was crystallized in 0.4 M magnesium formate, 0.1 M sodium acetate (pH 4.5) at 20 °C using the hanging-drop vapor-diffusion method.
0004
The loading concentration of Munc13–13–128, Munc13–13–132, Munc13–13–150, and Munc13–13–209 were 13.52 μM, 13.65 μM, 12.46 μM, and 12.55 μM in 30 mM Tris (pH 8.0), 150 mM NaCl, 1 mM TCEP.
0065
(B) ITC analysis of the binding of Munc13–13–150(K32E) to RIM2α82–142.
0107
The model represents how formation of the tripartite Munc13–1/α-RIM/Rab3 complex involves dissociation of the Munc13–1 homodimer and release of the interaction between the SGAWFY motif and Rab3A.
0081
It requires the coordination of adhesion receptor–matrix interactions on the cell surface, trafficking of the receptors to and from the sites of adhesion (adhesion site turnover), and cytoskeletal reorganization inside the cell.
0030
Integrins, like several other proteins lacking the AP-2 localization signal are internalized from the cell membrane via nonclathrin-derived structures, and some integrins have been shown to subsequently fuse with compartments containing clathrin-derived cargo proteins (Ng et al., 1999; Naslavsky et al., 2003; Upla et al., 2004; Weigert et al., 2004).

0889
Integrins, like several other proteins lacking the AP-2 localization signal are internalized from the cell membrane via nonclathrin-derived structures, and some integrins have been shown to subsequently fuse with compartments containing clathrin-derived cargo proteins (Ng et al., 1999; Naslavsky et al., 2003; Upla et al., 2004; Weigert et al., 2004).

0663
Depending on the cell type and the stimulus, integrins have been shown to be transported through caveolin-1–positive structures or early endosomes, either directly or via the perinuclear recycling compartment, back to the plasma membrane (Caswell and Norman, 2006).
0030
Integrin traffic is regulated by several kinases (Ng et al., 1999; Roberts et al., 2001, 2003, 2004; Ivaska et al., 2002; Woods et al., 2004) and motor proteins (Zhang et al., 2004).

0889
Integrin traffic is regulated by several kinases (Ng et al., 1999; Roberts et al., 2001, 2003, 2004; Ivaska et al., 2002; Woods et al., 2004) and motor proteins (Zhang et al., 2004).

0107
Although Rab proteins are known to bind a multiplicity of diverse effectors (Zerial and McBride, 2001), only a few examples demonstrate an interaction between a Rab GTPase and a cargo molecule (Seachrist et al., 2002; van IJzendoorn et al., 2002).

0030
Although Rab proteins are known to bind a multiplicity of diverse effectors (Zerial and McBride, 2001), only a few examples demonstrate an interaction between a Rab GTPase and a cargo molecule (Seachrist et al., 2002; van IJzendoorn et al., 2002).

0889
Although Rab proteins are known to bind a multiplicity of diverse effectors (Zerial and McBride, 2001), only a few examples demonstrate an interaction between a Rab GTPase and a cargo molecule (Seachrist et al., 2002; van IJzendoorn et al., 2002).

0096
We identify Rab21 and Rab5 as integrin-associated proteins and positive regulators of integrin traffic.
0030
Rab21 is a ubiquitously expressed and poorly characterized member of the Rab family that has recently been shown to function on the endocytic pathway (Simpson et al., 2004).

0889
Rab21 is a ubiquitously expressed and poorly characterized member of the Rab family that has recently been shown to function on the endocytic pathway (Simpson et al., 2004).
0096
Finally, endogenous β1-integrins readily associated with endogenous Rab21 and to some extent Rab5 proteins in vivo. These associations were specific, as Rab7 and Rab11 failed to coprecipitate β1-integrin from these cells, even though separately all proteins were efficiently immunoprecipitated (Fig.
0107
Interaction between p53 and large T antigen was used as a positive control.

0416
(G) Nontransfected MDA-MB-231 cells were analyzed with three-color immunofluorescence staining (Rab5 or Rab21, green; EEA1, red; and β1-integrin, blue).
0030
A Rab21 mutant (Simpson et al., 2004) that has defects in GTP hydrolysis (Rab21 Q76L, designated Rab21GTP) showed a 10–73% increase in its ability to associate with integrins in different cell lines when compared with Rab21 wild type (WT).

0889
A Rab21 mutant (Simpson et al., 2004) that has defects in GTP hydrolysis (Rab21 Q76L, designated Rab21GTP) showed a 10–73% increase in its ability to associate with integrins in different cell lines when compared with Rab21 wild type (WT).
0030
α/β1- Integrin heterodimers can be expressed in active (extracellular domain detected by a monoclonal antibody HUTS21; active-β1 antibody) and inactive forms on the cell surface (extracellular domain of both forms detected by P5D2; pan-β1 antibody; Lenter et al., 1993; Luque et al., 1996).

0889
α/β1- Integrin heterodimers can be expressed in active (extracellular domain detected by a monoclonal antibody HUTS21; active-β1 antibody) and inactive forms on the cell surface (extracellular domain of both forms detected by P5D2; pan-β1 antibody; Lenter et al., 1993; Luque et al., 1996).

0416
Expression of Rab21 in MDA-MB-231 breast cancer cells altered the subcellular localization of the total pool of β1-integrins. Cells expressing GFP-Rab21 contained numerous β1-integrin−positive vesicles (compare the nontransfected cell [Fig.

0411
Expression of Rab21 in MDA-MB-231 breast cancer cells altered the subcellular localization of the total pool of β1-integrins. Cells expressing GFP-Rab21 contained numerous β1-integrin−positive vesicles (compare the nontransfected cell [Fig.

0030
The recruitment of integrins to Rab21-positive intracellular structures was confirmed using two additional antibodies recognizing active-β1-integrin (12G10 [Mould et al., 1995] and P4G11 [Wayner et al., 1993]; Fig.

0889
The recruitment of integrins to Rab21-positive intracellular structures was confirmed using two additional antibodies recognizing active-β1-integrin (12G10 [Mould et al., 1995] and P4G11 [Wayner et al., 1993]; Fig.

0030
Similar to previous observations on breast cancer cells (Ng et al., 1999), the ECM proteins collagen and fibronectin were also detected in the endocytic structures (Fig.

0411
Similar to previous observations on breast cancer cells (Ng et al., 1999), the ECM proteins collagen and fibronectin were also detected in the endocytic structures (Fig.

0889
Similar to previous observations on breast cancer cells (Ng et al., 1999), the ECM proteins collagen and fibronectin were also detected in the endocytic structures (Fig.
0030
Mutagenesis of the putative COOH-terminal prenylation motif (CCXXX; Opdam et al., 2000) in Rab21 resulted in a complete loss of vesicular localization of Rab21 (GFP-Rab21CCSS; Fig.

0889
Mutagenesis of the putative COOH-terminal prenylation motif (CCXXX; Opdam et al., 2000) in Rab21 resulted in a complete loss of vesicular localization of Rab21 (GFP-Rab21CCSS; Fig.
0096
Interestingly, Rab5 and Rab21 antibodies very weakly coimmunoprecipitated biotinylated ∼160-kD proteins located on the plasma membrane (Fig.
0030
We are aware that we have not excluded the effects of Rab21 expression for all types of unspecific internalization (e.g., macropinocytosis), but at least its closest homologues (Rab5 and Rab22) have not been shown to affect macropinocytosis (Rosenfeld et al., 2001; Kauppi et al., 2002).

0889
We are aware that we have not excluded the effects of Rab21 expression for all types of unspecific internalization (e.g., macropinocytosis), but at least its closest homologues (Rab5 and Rab22) have not been shown to affect macropinocytosis (Rosenfeld et al., 2001; Kauppi et al., 2002).

0411
Interestingly, the internalized receptor recycled rapidly back to the cell surface in Rab5- and Rab21-expressing cells with >50% of the labeled pool being recycled during a 15-min chase (detected by the reduction in the amount of biotinylated receptor in these cells where cell surface biotin is cleaved). Integrin internalization was reduced by expression of GFP-Rab21GDP, whereas GFP-Rab21GTP induced a steady accumulation of the internalized receptor when compared with GFP-, Rab5-, Rab11-, and Rab21GDP-expressing cells (Fig.
0030
In density gradient fractionations, expression of GFP-Rab21 shifted the integrins toward the denser Rab-positive fractions (Hughes et al., 2002), and GFP-Rab21 cofractionated with α2-integrin in fractions 3–9 (Fig.

0889
In density gradient fractionations, expression of GFP-Rab21 shifted the integrins toward the denser Rab-positive fractions (Hughes et al., 2002), and GFP-Rab21 cofractionated with α2-integrin in fractions 3–9 (Fig.

0027
In the lighter fractions (3–5), GFP-Rab21 and integrin were found to cosediment with the Golgi-marker GM130, whereas in the denser fractions, cosedimentation was observed with the ER marker P115 (fractions 6–8) and EEA1 (fractions 7–9; Fig.
0040
Further characterization of the large intracellular structures induced by GFP-Rab21 overexpression was performed by electron microscopy and immunogold labeling of GFP (Fig.
0416
Combined total internal reflection fluorescence microscopy (TIRFM; pseudocolored green) and conventional widefield epifluorescence analysis (pseudocolored red) showed GFP-Rab21 vesicles emanating from the membrane into the cell (changing from green to red/yellow) and back or vice versa (although formally this technique does not exclude the possibility that some vesicles will by chance come closer and further away from the plasma membrane).
0411
We also addressed whether Rab21 expression alters the motility of integrins in live cells.
0416
(B and C) MDA-MB-231 cells transfected with GFP–α2-integrin and Rluc-Rab21 (B) or with GFP–α2-integrin alone (C) were plated on collagen for 1 h, and time-lapse series were acquired with widefield epifluorescence microscopy. Cotransfected cells (B; see Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200509019/DC1) show green fluorescent vesicles moving in the cytosol.
0411
To investigate the importance of Rab21-mediated integrin traffic in β1-integrin–dependent cell adhesion, we studied MDA-MB-231 breast and PC3 prostate cancer cells having relatively high and low endogenous Rab21 expression, respectively (unpublished data). Of all the Rabs tested, overexpressed Rab21 was most efficient in increasing cell adhesion to collagen (86 ± 8% in PC3 cells and 22% in MDA-MB-231 cells; Fig.

0411
Rab21 expression did not enhance the initial adhesion step of cells plated on fibronectin or vitronectin (Fig.

0411
Expression of Rab5 increased adhesion by 48 ± 13% in PC3 cells.

0411
6 C) and PC3 cells (not depicted), suggesting that efficient transport of endocytosed integrins to newly formed sites of adhesion, rather than a change in the steady-state expression of integrins on the cell surface, is the basis of Rab21-induced cell adhesion. Interestingly, GFP-Rab21GDP caused a modest increase in β1-integrin surface expression in MDA-MB-231 cells (Fig.

0411
CHO cells lack endogenous collagen receptors. Transient expression of α2WT, α2AA (deficient Rab21 association), or α2A (unaltered Rab21 association), together with Rluc, enabled equivalent adhesion of these cells to collagen (Fig.
0054
(A and B) Adhesion of transiently transfected PC3 or MDA-MB-231 cells was studied on collagen (0.25 μg/ml CI, 30 min; means ± SD; n = 12; **, P < 0.02; ***, P < 0.003). (C) Cell surface expression of β1-integrin was analyzed with dual-color FACS from transiently transfected MDA-MB-231 cells. Mean fluorescence intensities (633-nm laser; β1-integrins) were detected from GFP-positive and -negative cells and are presented as a ratio (relative means ± SEM; n = 4).

0411
(A and B) Adhesion of transiently transfected PC3 or MDA-MB-231 cells was studied on collagen (0.25 μg/ml CI, 30 min; means ± SD; n = 12; **, P < 0.02; ***, P < 0.003). (C) Cell surface expression of β1-integrin was analyzed with dual-color FACS from transiently transfected MDA-MB-231 cells. Mean fluorescence intensities (633-nm laser; β1-integrins) were detected from GFP-positive and -negative cells and are presented as a ratio (relative means ± SEM; n = 4).

0416
(F) Subcellular localization of GFP-integrin in the transfected collagen adhering cells was studied by widefield fluorescence microscopy.
0411
Conversely, stable Rab21-shRNA (short hairpin RNA)–transfected cells with reduced Rab21 expression (Fig. 3 C) migrated poorly, covering only sim;59% of the wound area, whereas Scr-shRNA–transfected control cells closed the wound almost completely (Fig.

0411
During this time, there were no obvious differences in the proliferation of the stable transfected cells (not depicted). These data suggest that expression of Rab21 regulates migration in these cells.
0411
Furthermore, Rab21 expression is shown to regulate integrin-containing focal adhesions and adhesion and migration of breast and prostate cancer cells. Live-cell imaging revealed that Rab21-positive vesicles move between the plasma membrane and the cell body and that expression of Rab21 modulates vesicular motility of integrins in vivo.
0107
The identification of integrin association with the putative early endosomal Rab21 led us to identify a functional relationship between integrin association with Rab21 and Rab5 and the regulation of cell adhesion.

0030
Upon growth factor stimulus, internalized integrins either recycle very rapidly back to the plasma membrane from the perinuclear compartment (β1-integrins; Powelka et al., 2004) or are completely rerouted to a short-loop trafficking pathway directly back to the membrane (αvβ3-integrin; Roberts et al., 2004).

0889
Upon growth factor stimulus, internalized integrins either recycle very rapidly back to the plasma membrane from the perinuclear compartment (β1-integrins; Powelka et al., 2004) or are completely rerouted to a short-loop trafficking pathway directly back to the membrane (αvβ3-integrin; Roberts et al., 2004).

0030
This is in agreement with the recent finding that integrins in newly forming protrusions travel on actin cables associated to motor protein Myosin X and that normal cell adhesion and spreading during the initial stages of adhesion seem to require the efficient motility of integrins close to the plasma membrane (Zhang et al., 2004).

0889
This is in agreement with the recent finding that integrins in newly forming protrusions travel on actin cables associated to motor protein Myosin X and that normal cell adhesion and spreading during the initial stages of adhesion seem to require the efficient motility of integrins close to the plasma membrane (Zhang et al., 2004).
0030
One study demonstrates the internalization of GFP-tagged integrin in live cells (Laukaitis et al., 2001).

0889
One study demonstrates the internalization of GFP-tagged integrin in live cells (Laukaitis et al., 2001).

0030
Although the role of Rab proteins was not addressed in the study of Laukaitis et al.

0889
Although the role of Rab proteins was not addressed in the study of Laukaitis et al.

0030
Evidence from detailed biochemical and nuclear magnetic resonance studies (Stefansson et al., 2004; Vinogradova et al., 2004) indicates that upon integrin activation the membrane-proximal regions of the integrin cytoplasmic tails move out of the membrane into the cytoplasm, revealing the highly conserved residues to the cytoplasmic face and involving substantial structural changes in the cytoplasmic tails.

0077
Evidence from detailed biochemical and nuclear magnetic resonance studies (Stefansson et al., 2004; Vinogradova et al., 2004) indicates that upon integrin activation the membrane-proximal regions of the integrin cytoplasmic tails move out of the membrane into the cytoplasm, revealing the highly conserved residues to the cytoplasmic face and involving substantial structural changes in the cytoplasmic tails.

0889
Evidence from detailed biochemical and nuclear magnetic resonance studies (Stefansson et al., 2004; Vinogradova et al., 2004) indicates that upon integrin activation the membrane-proximal regions of the integrin cytoplasmic tails move out of the membrane into the cytoplasm, revealing the highly conserved residues to the cytoplasmic face and involving substantial structural changes in the cytoplasmic tails.
0030
Prenylation-dependent membrane targeting of Rabs is crucial for Rab function as regulators of vesicle fusion in intracellular protein trafficking (Desnoyers et al., 1996).

0889
Prenylation-dependent membrane targeting of Rabs is crucial for Rab function as regulators of vesicle fusion in intracellular protein trafficking (Desnoyers et al., 1996).
0030
Integrins have been shown to internalize in a dynamin and PKCα-dependent manner after ligand binding (Ng et al., 1999). Internalized β1-integrins are then targeted to caveosomes (Upla et al., 2004) or transferrin-positive endosomes (Laukaitis et al., 2001) depending on the heterodimer, the cell type, and the stimulus used. Recent data showing that the caveolar and endosomal pathways intersect (Pelkmans et al., 2004) and our data showing caveolin-1–positive domains on Rab5- and Rab21-positive integrin–containing vesicles provide a possible explanation for these variable integrin trafficking routes.

0889
Integrins have been shown to internalize in a dynamin and PKCα-dependent manner after ligand binding (Ng et al., 1999). Internalized β1-integrins are then targeted to caveosomes (Upla et al., 2004) or transferrin-positive endosomes (Laukaitis et al., 2001) depending on the heterodimer, the cell type, and the stimulus used. Recent data showing that the caveolar and endosomal pathways intersect (Pelkmans et al., 2004) and our data showing caveolin-1–positive domains on Rab5- and Rab21-positive integrin–containing vesicles provide a possible explanation for these variable integrin trafficking routes.
0889
This correlates well with the previous work showing that α5-integrin engagement initiates the newly forming adhesions and serves to organize proteins like paxillin in these sites (Laukaitis et al., 2001).

0030
This correlates well with the previous work showing that α5-integrin engagement initiates the newly forming adhesions and serves to organize proteins like paxillin in these sites (Laukaitis et al., 2001).
0030
Interestingly, emerging evidence implicates alterations in the Rab small GTPases and their associated regulatory proteins and effectors in multiple human diseases, including cancer (Cheng et al., 2004).

0889
Interestingly, emerging evidence implicates alterations in the Rab small GTPases and their associated regulatory proteins and effectors in multiple human diseases, including cancer (Cheng et al., 2004).
0030
Antibodies against the following antigens were used: EEA1, Rab5A, Rab7, Rab11, caveolin-1 (all from Santa Cruz Biotechnology, Inc.), Rab21 (Opdam et al., 2000), β1-integrin (P5D2, P4G11, and AIIB2), α5-integrin (BIIG2), EGFR (151-IgG; all from the Drosophila Studies Hybridoma Bank), α2 (mAb MCA2025; Serotec), pAb AB1934 (Chemicon), α1 (MAB1973; Chemicon), α6 (MAB699; Chemicon), β1 (HUTS-21 [BD Biosciences] and MAB2252 [Chemicon]), collagen type 1 (RAHC11; Imtek), GFP polyclonal antibody, fluorescently conjugated secondary antibodies, Cell Tracker dyes, and labeled transferrin (all from Invitrogen).

0889
Antibodies against the following antigens were used: EEA1, Rab5A, Rab7, Rab11, caveolin-1 (all from Santa Cruz Biotechnology, Inc.), Rab21 (Opdam et al., 2000), β1-integrin (P5D2, P4G11, and AIIB2), α5-integrin (BIIG2), EGFR (151-IgG; all from the Drosophila Studies Hybridoma Bank), α2 (mAb MCA2025; Serotec), pAb AB1934 (Chemicon), α1 (MAB1973; Chemicon), α6 (MAB699; Chemicon), β1 (HUTS-21 [BD Biosciences] and MAB2252 [Chemicon]), collagen type 1 (RAHC11; Imtek), GFP polyclonal antibody, fluorescently conjugated secondary antibodies, Cell Tracker dyes, and labeled transferrin (all from Invitrogen).
0030
Plasmids encoding GFP-Rab5a, GFP-Rab7, YFP-Rab9, and GFP-Rab11 have been described (Wilcke et al., 2000; Barbero et al., 2002; Lebrand et al., 2002; Gomes et al., 2003), and YFP-mouse talin was provided by D.

0889
Plasmids encoding GFP-Rab5a, GFP-Rab7, YFP-Rab9, and GFP-Rab11 have been described (Wilcke et al., 2000; Barbero et al., 2002; Lebrand et al., 2002; Gomes et al., 2003), and YFP-mouse talin was provided by D.

0030
α2-Integrin was subcloned from the α2 cDNA in pawneo2 vector (Ivaska et al., 1999) into pEGFP-C2 vector.

0889
α2-Integrin was subcloned from the α2 cDNA in pawneo2 vector (Ivaska et al., 1999) into pEGFP-C2 vector.
0413
Rluc-tagged Rab21 constructs alone or with GFP-α2 variants (CHO cells that lack endogenous collagen binding integrins) were transfected into 95% confluent cells using Lipofectamine 2000 and incubated for 18 h. For immunoprecipitations with endogenous proteins, confluent MDA-MB-231 cells (20 × 106) were collected from plastic plates with cold PBS.

0030
Reimmunoprecipitations were performed as described earlier (Mattila et al., 2005).

0889
Reimmunoprecipitations were performed as described earlier (Mattila et al., 2005).
0030
These were performed as described previously (Roberts et al., 2001; Ivaska et al., 2002) with some modifications.

0889
These were performed as described previously (Roberts et al., 2001; Ivaska et al., 2002) with some modifications.

0411
The cells were lysed (200 mM NaCl, 75 mM Tris, 15 mM NaF, 1.5 mM Na3VO4, 7.5 mM EDTA, 7.5 mM EGTA, 1.5% Triton-X-100, and Complete), and the amount of biotinylated integrin was assayed using the anti–β1-integrin antibody AIIB2 to capture the integrins and HRP anti-biotin antibody for ELISA detection. As control, the cells were lysed after the labeling to determine the amount of total biotinylated integrin.
0030
48 h after transfection, the cells were harvested and fractionated on a sucrose density gradient and analyzed by Western blotting as described previously (Hughes et al., 2002).

0889
48 h after transfection, the cells were harvested and fractionated on a sucrose density gradient and analyzed by Western blotting as described previously (Hughes et al., 2002).
0018
The α2-integrin COOH-terminal tail (28 residues) Gal4 DNA binding domain fusion (pGBKT7 vector) was used to screen a mouse E17 Matchmaker cDNA library (CLONTECH Laboratories, Inc.) as described previously (Mattila et al., 2005). In yeast mating tests, pGADT7-Rab21 (95–222) prey was transformed in Y187 host strain and cytoplasmic tails of α2- and α11-integrin (pGBKT7-α2 and -α11) and their variants in AH109 host strain.

0030
The α2-integrin COOH-terminal tail (28 residues) Gal4 DNA binding domain fusion (pGBKT7 vector) was used to screen a mouse E17 Matchmaker cDNA library (CLONTECH Laboratories, Inc.) as described previously (Mattila et al., 2005).

0889
The α2-integrin COOH-terminal tail (28 residues) Gal4 DNA binding domain fusion (pGBKT7 vector) was used to screen a mouse E17 Matchmaker cDNA library (CLONTECH Laboratories, Inc.) as described previously (Mattila et al., 2005).
0030
Saos-2 cells express no endogenous α2 (Ivaska et al., 1999). Stable Saos-2 cells expressing equal levels of chimeric integrins (extracellular domain of α2 fused with α1 or α5 cytoplasmic tails; Ivaska et al., 1999) have been described (Mattila et al., 2005).

0889
Saos-2 cells express no endogenous α2 (Ivaska et al., 1999). Stable Saos-2 cells expressing equal levels of chimeric integrins (extracellular domain of α2 fused with α1 or α5 cytoplasmic tails; Ivaska et al., 1999) have been described (Mattila et al., 2005).
0663
Slides were examined using an inverted fluorescence microscope (Carl Zeiss MicroImaging, Inc.) or a confocal laser-scanning microscope (Axioplan 2 with LSM 510; Carl Zeiss MicroImaging, Inc.) equipped with 100×/1.4 Plan-Apochromat oil-immersion objectives. Confocal images represent a single z section of ∼1.0 μm.

0030
β1-Integrin and transferrin internalization were studied as described previously (Powelka et al., 2004).

0889
β1-Integrin and transferrin internalization were studied as described previously (Powelka et al., 2004).
0416
TIRFM was combined with conventional widefield epifluorescence microscopy and time-lapse series (frame rate ∼2/s) Widefield images were pseudocolored red and TIRFM images green. Transiently transfected GFP-Rab21 cells were plated on acid-washed glass-bottomed dishes (MatTek Corporation) coated with 10 μg/ml collagen type I and allowed to adhere for 1 h before microscopy.
0411
To confirm whether these structures also contain Ago2 as recently reported [28, 31], we transfected HeLa cells with expression vectors containing YFP-Ago1, CFP (cyan fluorescent protein)-Ago1, YFP-Ago2, and CFP-Ago2.

0663
To examine the contents of these cytoplasmic foci, HeLa cells were transfected with expression vectors for YFP-Lsm1 and CFP-Ago2, or YFP-RCK/p54 and CFP-Ago2, and visualized 24 h later by confocal microscopy.

0411
To examine the contents of these cytoplasmic foci, HeLa cells were transfected with expression vectors for YFP-Lsm1 and CFP-Ago2, or YFP-RCK/p54 and CFP-Ago2, and visualized 24 h later by confocal microscopy.

0416
To examine the contents of these cytoplasmic foci, HeLa cells were transfected with expression vectors for YFP-Lsm1 and CFP-Ago2, or YFP-RCK/p54 and CFP-Ago2, and visualized 24 h later by confocal microscopy.
0019
In cells expressing CFP-Ago2 and YFP-Lsm1, FRET efficiency was not significant (1.62% ± 1.11%), corroborating our immunoprecipitation results (Figure 1A).

0107
Similar to the YFP-RCK/p54 and CFP-Ago2 pair, YFP-Ago1 and CFP-Ago2 showed an efficient FRET (19.61% ± 4.51%), indicating a direct interaction between Ago1 and Ago2 in vivo [28].
0676
To determine the functional interactions of RCK/p54 with miRISC, we employed affinity purification of RISC and target mRNA cleavage capabilities of miRISC when the target has perfectly complementary sequences to the miRNAs.
0411
HeLa cells were transfected with siRNA against Lsm1, harvested at 24, 48, and 72 h post-transfection, and TCEs were analyzed by immunoblot.
0411
P-bodies were disrupted in HeLa cells by transfecting them with siRNA against Lsm1, and cytoplasmic extracts were prepared 48 h post-transfection.

0019
After immunoprecipitation, RISC activities were analyzed by incubating the supernatant (S) or bead (B) phases with 182-nt 32P-cap-labeled let-7 substrate mRNAs having a perfectly complementary or mismatched sequence to the let-7 miRNA.
0663
To examine whether the P-body protein RCK/p54, also a component of RISC, is involved in siRNA-mediated gene silencing, the siRNA dose dependence of RNAi-mediated gene silencing was quantified in RCK/p54-depleted HeLa cells using a dual fluorescence reporter assay. Briefly, GFP and red fluorescent protein (RFP) were constitutively expressed in cells transfected with reporter plasmids for enhanced GFP (EGFP) and RFP, respectively.

0411
GFP expression was silenced by treating cells with a 21-nt siRNA targeting nt 238–258 of the EGFP mRNA.

0663
The fluorescence intensity ratio of target (GFP) to control (RFP) fluorophore was determined in the presence of siRNA duplexes and normalized to that observed in control-treated cells [52].

0416
The fluorescence intensity ratio of target (GFP) to control (RFP) fluorophore was determined in the presence of siRNA duplexes and normalized to that observed in control-treated cells [52].
0411
When HeLa cells were transfected with siRNA mismatched for CDK9 (control), the second transfection with GFP siRNA silenced GFP expression in a dose-dependent manner, i.e., GFP/RFP ratios decreased with increasing concentrations of siRNA (Figure 6A, gray bars).
0411
To test this hypothesis, we first determined whether RCK/p54 was a general translation repressor in human cells. To this end, RCK/p54 expression was silenced in HeLa cells and general translational activity was analyzed by [35S]methionine incorporation (Figure 7A). The translational rates measured by [35S]-labeling were significantly greater in RCK/p54-depleted cells than in control siRNA-treated cells (Figure 7A), indicating that RCK/p54 was a translational repressor in human cells, consistent with results in yeast and reticulocyte extracts [45].
0007
HeLa cells were co-transfected with siRNAs directed against P-body proteins (RCK/p54, GW182, Lsm1, and Ago2) and with siRNA or miRNA reporters in the absence or presence of 25 nM CXCR4 siRNA.

0411
At 24 h post-transfection, cells were harvested and RL activities were analyzed. RL signals were normalized to Photinus pyralis luciferase (FL) signals from cells co-transfected with pGL3 plasmid as control.
0411
We next hypothesized that the expression of a specific cellular protein, known to be controlled by miRNAs, might be up-regulated in RCK/p54-depleted cells.

0411
When cells were treated with let-7 inhibitor, RAS protein levels increased, consistent with previous findings [13]. Strikingly, RAS protein expression in RCK/p54-depleted cells also showed a robust increase (Figure 7C) while RAS levels were not significantly affected when cells were treated with control siRNA or 2′-O-Me oligonucleotides. RAS mRNA levels did not differ drastically in HeLa cells after treatment with let-7 inhibitor, RCK/p54 silencing, or mock treatment (Figure S3).
0411
For control experiments, we treated cells with a CDK9 mm siRNA and a 2′ -O-Me oligonucleotide complementary to HIV-1 TAR RNA sequence [53]. Cells transfected with a control siRNA and let-7 inhibitor induced more firefly luciferase expression when reporter plasmids contained 3′ UTR sequences for NRAS and KRAS than for the control siRNA and a 2′-O-Me oligo control (Figure 7D), consistent with a recent report [13].
0107
Where in the cell does this physical interaction between RISC and RCK/p54 take place and does it require P-body structures? To address these questions, we disrupted P-bodies in cells by depleting Lsm1 and immunopurified endogenous miRISC, and analyzed its ability to cleave a target mRNA with perfect complementarity to let-7 miRNA (Figure 5).
0411
We chose RAS because it is an endogenous target of let-7, and 3′ UTRs of human RAS genes contain multiple complementary sites for let-7 to bind and regulate RAS expression levels [13]. Furthermore, let-7 inhibitors are known to enhance RAS protein expression in HeLa cells [13]. Depleting RCK/p54 in HeLa cells up-regulated RAS protein, and this increase in RAS levels was higher than that in general translation of control actin (Figure 7C), suggesting that multiple sites of let-7 miRISC binding to target 3′ UTR dictate the potency and specificity of translation suppression.
0030
Recently, Petersen et al.

0889
Recently, Petersen et al.
0004
TCEs were prepared by incubating cells in lysis buffer (20 mM HEPES [pH 7.9], 10 mM NaCl, 1 mM MgCl2, 0.2 mM EDTA, 0.35% [v/v] Triton X-100, 1/100 [v/v] dilution in protease inhibitor cocktail) and centrifuging at 14,000 rpm for 10 min at 4 °C. Protein concentration was determined by Dc protein assay (Bio-Rad, Hercules, California, United States).

0019
To examine the RNA dependence of protein–protein interactions, TCEs (250 μg) were treated before immunoprecipitation with 0.2 μg/ul of RNase A for 20 min at room temperature.
0411
HeLa cells were cultured in 35 mm dishes with glass coverslip bottoms (MatTek Corporation, Ashland, Massachusetts, United States). Expression vectors for CFP- or YFP-tagged proteins were transfected into cells using Lipofectamine as described above.

0055
24 h later, the live cells were monitored for CFP and YFP signals of the transiently expressed proteins.

0663
The signals were detected by a Leica (Wetzlar, Germany) confocal imaging spectrophotometer system (TCS-SP2) attached to a Leica DMIRE inverted fluorescence microscope equipped with an argon laser, two HeNe lasers, an acousto-optic tunable filter (AOTF) to attenuate individual visible laser lines, and a tunable acousto-optical beam splitter (AOBS).

0055
For FRET studies, HeLa cells co-expressing CFP- and YFP-tagged proteins were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min and washed three times with PBS.

0055
FRET experiments were performed by an acceptor photobleaching method as described [48,49,68]. FRET efficiencies were measured and images were analyzed using Leica confocal software.

0416
For immunofluorescence studies, cells transfected with Myc-Ago2 were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min and permeabilized with 0.25% (v/v) Triton X-100 for 5 min.

0040
Samples were washed three times with PBST (0.1% [v/v] Triton X-100 in PBS) and blocked for 30 min in PBST containing 2% (w/v) BSA. Primary and secondary antibodies were diluted in blocking solution during incubation.
0663
A dual fluorescence assay was used to quantify the RNAi activity of siRNAs against GFP.

0411
To quantify RNAi effects, cell lysates were prepared from siRNA-treated cells 24 h post-transfection.

0663
GFP fluorescence was detected in cell lysates by exciting at 488 nm and recording emissions from 504–514 nm. The spectrum peak at 509 nm represents the fluorescence intensity of GFP. RFP fluorescence was detected in the same cell lysates by exciting at 558 nm and recording emissions from 578–588 nm. The spectrum peak at 583 nm represents the fluorescence intensity of RFP. The fluorescence intensity ratio of target (GFP) to control (RFP) fluorophores was determined in the presence of siRNA duplexes and normalized to the emissions measured in mock-treated cells.

0416
The fluorescence intensity ratio of target (GFP) to control (RFP) fluorophores was determined in the presence of siRNA duplexes and normalized to the emissions measured in mock-treated cells.
0007
HeLa cells transfected with siRNAs directed against P-body proteins (RCK/p54, GW182, Lsm1, Ago2) were co-transfected again with siRNA or miRNA reporters in the absence or presence of 25 nM CXCR4 siRNA.

0411
At 24 h post-transfection, cells were harvested and RL activities were analyzed. RL signals were normalized to the FL signals from cells co-transfected with pGL3 plasmid as control.
0411
HeLa cells expressing YFP-RCK/p54 and CFP-Ago2 were fixed at 24 h post-transfection.
0019
After immunoprecipitation, RISC activities were analyzed by incubating the supernatant (S) or bead (B) phases with 182-nt 32P-cap-labeled let-7 substrate mRNAs having a perfectly complementary or mismatched sequence to the let-7 miRNA.
0411
HeLa cells were transfected with siRNA against Lsm1. At 48 h post-transfection, cells were analyzed by immunofluorescence using antibodies against Lsm1 and Myc tag for Ago2. Cells were stained with Hoechst33258 to visualize nuclei, and images were digitally merged.

0416
HeLa cells were transfected with siRNA against Lsm1. At 48 h post-transfection, cells were analyzed by immunofluorescence using antibodies against Lsm1 and Myc tag for Ago2. Cells were stained with Hoechst33258 to visualize nuclei, and images were digitally merged.
0019
After immunoprecipitation, RISC activities were analyzed by incubating the supernatant (S) or bead (B) phases with 182-nt 32P-cap-labeled let-7 substrate mRNAs having a perfectly matched or a mismatched sequence to the let-7 miRNA.
0663
24 h later cells were transfected again with EGFP and RFP reporter plasmids and varying amounts (2, 10, and 50 nM) of siRNA against EGFP.

0663
To quantify the effect of depleting RCK/p54 and Ago2 on RNAi, the ratio of GFP/RFP signals was normalized to that observed in the absence of GFP siRNA (0 nM).
0426
Incorporation of [35S]methionine into HeLa cells was used to measure general translational activity. Cells were transfected with 50 nM siRNAs targeting mismatched CDK9 (control) or RCK/p54.

0411
Mock control cells were treated with the transfection reagent only. At 24 h post-transfection, cells were incubated for 1 h in medium lacking Met and Cys, and metabolically labeled with [35S]methionine (see Materials and Methods). As a control for passive uptake of [35S], mock cells were treated with 40 μg/ml of the translation inhibitor, cycloheximide.
0081
24 h later, cells were co-transfected with siRNA (1 × perfectly matched [PM] site) or miRNA (4 × bulged sites) luciferase reporters in the presence of CXCR4 siRNA.
0411
Sorting of ephrin-B1–positive and –negative cells following X-inactivation has been observed in ephrin-B1+/− mice; however, the mechanisms by which mosaic ephrin-B1 expression leads to cell sorting and phenotypic defects remain unknown. Here we show that ephrin-B1+/− mice exhibit calvarial defects, a phenotype autonomous to neural crest cells that correlates with cell sorting.
0411
As a result of random X-inactivation, X-linked ephrin-B1 expression is mosaic in ephrin-B1+/− mice and ephrin-B1–positive and ephrin-B1–negative cells segregate from one another.
0411
Indeed, studies in zebrafish have shown that expression of Eph receptors and ephrins in animal cap cells was sufficient to block GJC at the boundary between both cell populations.
0411
It has been reported previously that patchy expression of ephrin-B1 in ephrin-B1+/− limb buds reflects sorting between ephrin-B1–positive and –negative cell populations that are generated in the ephrin-B1 heterozygous females via random X-inactivation and that this abnormal expression of ephrin-B1 in the limb bud correlates with a polydactyly phenotype that is observed in ephrin-B1+/− females [7,8]. Our data demonstrate that the calvarial phenotypes observed in ephrin-B1 heterozygous females correlate with an abnormal expression of ephrin-B1 and EphB2 in the presumptive frontal bone, likely due to cell sorting between ephrin-B1–positive and ephrin-B1–negative cells in the craniofacial mesenchyme.
0411
Using AP activity as a marker for osteogenic differentiation, we observed that cells isolated from ephrin-B1+/− embryos were consistently less prone to differentiate in vitro (Figure 3C), indicating that these defects are autonomous to the osteoprogenitor cells. In these primary cultures, expression of ephrin-B1 was detected in a punctate pattern in both AP-positive as well as AP-negative cells (Figure S3A), indicating that expression of ephrin-B1 and AP do not strictly correlate, and suggesting that ephrin-B1 does not regulate AP activity directly.
0030
Compagni et al.

0889
Compagni et al.

0107
We found that interaction between ephrin-B1 and Eph-B2 resulted in inhibition of GJC in vitro (Figure 5).

0107
These results demonstrate that interaction between EphB2 and ephrin-B1 impairs establishment of GJC.
0411
Expression of ephrin-B1 and Cx43 was partially overlapping in untreated NIH 3T3 cells, especially at interfaces between cells expressing ephrin-B1, which exhibited strong Cx43 staining (Figure 6Ba–c).
0081
Western-blot analysis of whole cell lysates indicated that both proteins were expressed, albeit at different levels, and that expression of ephrin-B1 did not influence the phosphorylation status of Cx43 (detected by differences in mobility on a SDS-PAGE), which is known to regulate GJC (Figure 7A).

0412
Cx43 could be detected in the pull downs from cells expressing either ephrin-B1 wild type or ephrin-B1ΔPDZ, however, the relative abundance of phosphorylated versus unphosphorylated band was changed.
0107
Because the lack of cell sorting in the experiments described above precluded the establishment of a functional link between the calvarial phenotype and the regulation of GJC in ephrin-B1 heterozygote embryos, we performed a genetic rescue experiment.
0030
Moreover, heterozygosity for the transcription factor Twist can lead to craniosynostosis, as well as to foramina, and both transcription factors regulate differentiation of frontal bone osteoprogenitors (Ishii et al., 2003).

0889
Moreover, heterozygosity for the transcription factor Twist can lead to craniosynostosis, as well as to foramina, and both transcription factors regulate differentiation of frontal bone osteoprogenitors (Ishii et al., 2003).

0107
The genetic interaction suggests, however, that the discrepancy in coronal suture phenotype between ephrin-B1 heterozygous mice and humans could be due to genetic modifiers.
0107
In our pull-down assay, wild-type ephrin-B1 interacted preferentially with phosphorylated Cx43 whereas ephrin-B1ΔPDZ interacted preferentially with unphosphorylated Cx43, suggesting that the interaction between ephrin-B1 and Cx43 might not be direct, and that these proteins might interact differently when at the cell surface or in the cytoplasm.
0052
Trypsin was inactivated by addition of complete medium (DMEM containing 15% FCS) and cells were dissociated by trituration with a glass Pasteur pipette.
0004
Cells were scraped in 1% NP40 lysis buffer (50 mM Hepes [pH 7.5], 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1mM EGTA, 100 mM NaF), except for the experiment presented in Figure 7A (100 mM Tris [pH 7.4], 150 mM NaCl, 1mM EDTA). Protein lysates were incubated with either 5-μg EphB2-Fc or 4-μg Cx43 monoclonal antibody (generous gift from P.
0411
NIH 3T3 cells were plated on glass coverslips and transiently transfected with an expression vector for ephrin-B1. Forty hours after transfection, cells were either fixed in 2% PFA or incubated with 4-μg/ml EphB2-Fc for 30 min at 37 °C and then fixed in PFA.

0411
For the sorting experiments, HEK293T cells were transiently transfected with either DsRed or a Cx43 expression construct. Twenty-four hours after transfection, cells were trypsinized into a single-cell suspension and replated onto glass coverslips.
0040
For immunofluorescence on sections, paraffin sections were rehydrated and subjected to a citrate boil to reveal antigens. Tissue sections were blocked in Blocking solution (PBS/5% horse serum) 1 h at room temperature and incubated overnight at 4 °C in Cx43 antibody (1/100 in Blocking solution [Sigma]). Incubation with the Cy3-conjugated secondary antibody was for 1 h at room temperature (1/250 in Blocking solution).
0416
(A) Primary mesenchymal cells isolated from presumptive calvaria of wild-type (a) and (b) or ephrin-B1+/− embryos (c) and (d) were stained for AP activity (a) and (c) and subsequently processed for Cx43 immunofluorescence (b) and (d).
0416
(B) Detection of Cx43 by immunofluorescence in limb bud sections of an X-gal–stained chimeric embryo obtained by injecting ephrin-B1 null cells into a ROSA26-βgal blastocyst.
0411
(B) Primary NCCs were loaded with Calcein-AM and dropped onto NIH 3T3 cells that were transfected either with a control plasmid (pcDNA3 [a]), or an expression construct for ephrin-B1 (b) or Eph-B2 (c). In the control situation, the majority of cells transferred the dye (arrowheads).
0676
(C) Ephrin-B1 was affinity precipitated from NIH 3T3 cells using EphB2-Fc. The presence of Cx43 in the affinity complex was assessed by Western-blot (a).

0006
Protein lysates from NIH 3T3 cells expressing ephrin-B1 were incubated with ephrinB1-Fc, and the presence of Cx43 in the affinity complexes was assessed by Western blot (b) Cx43 was immunoprecipitated from NIH 3T3 cells expressing ephrin-B1 and the presence of ephrin-B1 in the immunocomplexes was detected by Western blot (right panel).
0416
(B) NIH 3T3 cells were transiently transfected with ephrin-B1ΔPDZ and treated with EphB2-Fc. Subcellular localization of Cx43 (a) and ephrin-B1ΔPDZ (b) was analyzed by immunofluorescence.
0107
At a developmental boundary and at ectopic ephrin boundaries in ephrin-B1+/− embryos, Eph/ephrin interaction leads to inhibition of GJC, possibly through endocytosis of Cx43, concomitant with a loss of stable cell–cell interactions between the two cell types. Sorting between these cells and inhibition of GJC concur to establish distinct developmental compartments.
0423
Also, biochemical analysis of purified Cdc23 (Mcm10) protein demonstrates that it is required for efficient phosphorylation of the Mcm2-7 complex by Dfp1-Hsk1 (Dbf4-Cdc7) kinase in vitro, and that Cdc23 (Mcm10) can directly interact with Dfp1-Hsk1 (Dbf4-Cdc7) [37].
0018
Ars binding protein 1 (Abp1) was first identified in a search for fission yeast proteins that could retard an ARS (Autonomously Replicating Sequence)-containing DNA fragment in a gel-shift mobility assay [38].

0096
Ars binding protein 1 (Abp1) was first identified in a search for fission yeast proteins that could retard an ARS (Autonomously Replicating Sequence)-containing DNA fragment in a gel-shift mobility assay [38].
0107
The two-hybrid interaction between Snf1 and Snf4 is shown as a positive control (Figure 1A, row 4).
0107
Therefore the genetic interaction observed between cdc23-M36 and Δabp1 appears to be Abp1-specific.
0054
Cell cycle progression profiles of Δabp1, cdc23-M36 and cdc23-M36 Δabp1 at 30°C (from 1–6 hrs) and 25°C at time zero, by flow cytometry anaylsis.
0054
We analyzed DNA content by flow cytometry to determine the precise arrest points for the different strains (Figure 3B). As expected, wildtype or mutant cells grown at the permissive temperature of 25°C display a 2C DNA content, indicating that most cells when growing exponentially are in the G2 phase of the cell cycle.
0018
Yeast genetics has provided a powerful tool to identify some of the key proteins that are required for assembly of these complexes. One of these proteins, called Cdc23 (Mcm10), is conserved from yeast to man, and is required for the early events of DNA replication initiation.
0018
First, Abp1 not only interacts with Cdc23 (Mcm10) protein in the two-hybrid analysis, but deletion of Abp1 lowers the restrictive temperature for cdc23-M36, consistent with its proposed role in DNA replication.
0054
Our flow cytometry analysis suggests that cdc23+ (MCM10) is required for DNA replication initiation. Previous studies also indicate that following a shift to the restrictive temperature of 36°C, cdc23 temperature-sensitive mutant cells arrest in early-S phase [32].
0426
In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage–induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-xL deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-xL deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo.
0411
Surprisingly, DNA damage–triggered deamidation in primary wild-type cells is mediated not enzymatically, but by intracellular alkalinisation caused by increased expression of the NHE-1 Na+/H+ exchanger (antiport), events blocked by expression of the oncogenic tyrosine kinase (OTK). In the case of either murine or human cancer cells, enforced alkalinisation triggers Bcl-xL deamidation, crippling its ability to provide protection from the pro-apoptotic consequences of DNA damage, thereby indicating possible novel approaches to cancer therapy.
0426
Because the BH3-only protein Puma, not Bim, plays a major role in DNA-damage triggered apoptosis [19,20], we also showed that both Puma and Bim are found in Bcl-xL immunoprecipitates from etoposide treated CD45−/−LckF505 thymocytes, whereas sequestration is ablated in wild-type cells, correlating with Bcl-xL deamidation (Figure 2B).
0004
Until now, the in vivo mechanism for the deamidation of internal protein Asn residues has not been described for any protein. Because protein Asn deamidation is accelerated by increased pH in vitro, we investigated intracellular pH change (pHi) as a possible regulatory mechanism in thymocytes.

0426
To investigate Bcl-xL deamidation, pHi, and apoptosis in parallel, we manipulated pHi values artificially by incubating cells at varying pHe values in the absence of monensin.
0426
Interestingly, in the cells expressing these mutant forms of Bcl-xL, the apoptosis induced by enforced alkalinisation was reduced 4-fold compared to cells transduced with empty vector, or more than 2-fold in comparison with the wild-type protein (Figure 3G, right panel), which of course undergoes deamidation in response to alkali treatment.

0426
Nevertheless, protection was not absolute, suggesting that Bcl-xL may not be the only mechanism protecting cells from apoptosis triggered by alkalinisation.
0426
Taken overall, these results demonstrate that intracellular alkalinisation following DNA damage is both necessary and sufficient for nonenzymatic Bcl-xL deamidation, that the oncogenic suppression of Bcl-xL deamidation in pretumourigenic thymocytes is caused by inhibition of alkalinisation, and that versions of Bcl-xL competent for BH3-only protein sequestration are sufficient per se to protect cells from apoptosis at alkaline pHi.
0411
Because the NHE-1 Na/H antiport is a well-established regulator of pHi [22] and has previously been implicated in the regulation of thymic apoptosis [23], we measured its expression in wild-type thymocytes after DNA damage and found that the NHE-1 level increased 2.5-fold within 5 h, whereas this increase was completely suppressed in pretumourigenic thymocytes (Figure 4B). No inhibition of increased NHE-1 expression in wild-type thymocytes was observed following addition of the Z-VAD-fmk caspase inhibitor (Figure S4A) nor following depletion of Bax and Bak from the cells (Figure S4B). We therefore carried out a further series of experiments to demonstrate that there was a direct causal linkage between the regulation of NHE-1 expression, pHi, Bcl-xL deamidation, and apoptosis. Given that the OTK blocks DNA-damage induced NHE-1 expression in pretumourigenic thymocytes, this provides a powerful system for examining the consequences of experimentally enforcing NHE-1 expression in these cells by retroviral transduction. As Figure 4C illustrates (upper panel), an enforced 2-fold–3-fold increase in NHE-1 expression in pretumourigenic thymocytes, without DNA damage, restored Bcl-xL deamidation to a level comparable to that observed in a retrovirally transduced wild-type control in five separate experiments, thereby bypassing the OTK-mediated inhibition in deamidation.
0426
We measured apoptosis by two different methods to ensure that DNA damage–induced cell death following retroviral transduction was by apoptosis and not by necrosis. Figure 5C and Figure S6B illustrate that double staining for Annexin V and propidium iodide (PI) followed by FACS analysis revealed a major increase in Annexin V+ PI− (apoptotic) cells following transduction with the negative control shRNA followed by either γ irradiation or treatment with etoposide, whereas there was no increase in apoptotic cells above baseline in the cells depleted of NHE-1: DNA damage–induced apoptosis was blocked 100%.

0054
Figure 5C and Figure S6B illustrate that double staining for Annexin V and propidium iodide (PI) followed by FACS analysis revealed a major increase in Annexin V+ PI− (apoptotic) cells following transduction with the negative control shRNA followed by either γ irradiation or treatment with etoposide, whereas there was no increase in apoptotic cells above baseline in the cells depleted of NHE-1: DNA damage–induced apoptosis was blocked 100%. Comparable results were obtained by measuring the sub-G1 peak by FACS (unpublished data) and NHE-1 depletion also correlated with increased survival (Figure S5B).
0081
For example, a number of serine kinases have been shown to regulate NHE-1 phosphorylation and activity [24,25], so we investigated the pSer and pThr levels in NHE-1 immunoprecipitates from irradiated wild-type and pretumourigenic thymocytes, but the basal level of phosphorylation did not change after DNA damage and was comparable between the two cell types (Figure S6C). Nevertheless, we cannot formally exclude the possibility that not all pSer/pThr sites were recognised by the cocktail of monocolonal antibodies (mAbs) used.
0426
Figure S7 shows that this was indeed the case: murine tumour cells resistant to genotoxic insult at physiological pHi values can be sensitised to die by enforced alkalinisation leading to Bcl-xL deamidation. Furthermore, a modest rise in pHi following incubation in a mildly alkaline buffer produces levels of Bcl-xL deamidation and apoptosis in murine tumour cells comparable to those observed by adding a DNA damaging reagent to wild-type thymocytes incubated at physiological pH.
0426
We therefore determined whether genotoxic treatment in vitro of primary human B lineage CLL (B-CLL) cells might cause increased NHE-1, alkalinisation, Bcl-xL deamidation, and apoptosis, as in primary murine thymocytes (Figures 3–5), or whether this might be inhibited, as with the murine cancer cells (Figure S7). In addition, we examined the consequences for these parameters of incubating cancer cells in alkaline pH buffers. To perform these investigations, we divided each sample of patient cancer cells into nine aliquots that were either untreated, subjected to γ irradiation, or exposed to etoposide, followed by incubation at pH 7.2, pH 8.0, or pH 8.5 for 24 h. Each aliquot was then further subdivided into three samples to measure pHi, Bcl-xL deamidation, and apoptosis. As expected, exposure of cells to mildly alkaline buffers generated pHi values that displayed some variation between samples from different patients within a narrow range.

0426
Interestingly, unlike the murine tumour cells expressing an OTK, the B-CLL cells behaved somewhat more like wild-type thymocytes in that DNA damage at physiological pHe caused a mean increase of pHi of 0.22 units, an 8% increase in Bcl-xL deamidation, and an 18% increase in the number of cells undergoing apoptosis (Figure 6A and Figure S8A), compared to the higher thymocyte values of 0.45 pHi units, 40% increase, and 37% increase, respectively (Figure 3). The human cancer cell values for these parameters were greatly increased at alkaline pHe, generating tight correlations between increasing pHi, Bcl-xL deamidation, and apoptosis (r values shown in Figure 6A).

0426
This point is further illustrated by the gray shaded area shown in Figure 6A, which encompasses the overlap in sub-G1 (apoptosis) values that were obtained either by DNA damage at physiological pH or by enforced alkalinisation without DNA damage. Conversely, incubation of B-CLL cells at lower pH inhibited DNA damage–induced Bcl-xL deamidation and apoptosis (Figure 6B). Therefore with respect to enforced changes in pHi, the B-CLL cells behaved in a comparable way to both murine thymocytes and tumour cells.
0426
These increases correlate with the observed increases in Bcl-xL deamidation and apoptosis in patients' cells (Figure 6A) and at the 2.6-fold level, at least, are comparable with the increases observed in wild-type thymocytes (Figure 4B). Furthermore, DNA damage–induced Bcl-xL deamidation in B-CLL cells was prevented by addition of either cycloheximide (CHX) (Figure S8C) or DMA (Figure S8D), establishing a possible linkage between DNA damage, NHE-1 function, and Bcl-xL deamidation in human cancer cells.
0114
The structural importance of protein iso-Asp residues is likewise underlined by the expression of the putative repair enzyme L-isoaspartate O-methyltransferase which converts iso-Asp to Asp residues: its deletion has striking effects on protein functions [28–30]. Furthermore, comparison of the crystal structures of native rat Bcl-xL with its deamidated version has revealed significant differences [10]; the structural implications of introducing iso-Asp residues into the disordered loop environment of Asn52/Asn66 merits further work.
0411
The resistance to genotoxic attack by CD45−/−lckF505 murine tumour cells correlates, as in their pretumourigenic counterparts, with the inhibition of DNA damage–induced NHE-1 antiport expression, alkalinisation, Bcl-xL deamidation, and apoptosis (Figure S7), which is an apparent example of ”oncogene addiction”, whereby oncogene expression continues to be important for survival [36]. By contrast, DNA damage of human B-CLL cells, which should not express OTKs, triggered increased NHE-1 expression and apoptosis, achieving levels comparable with wild-type thymocytes (Figure 6C).

0426
The resistance to genotoxic attack by CD45−/−lckF505 murine tumour cells correlates, as in their pretumourigenic counterparts, with the inhibition of DNA damage–induced NHE-1 antiport expression, alkalinisation, Bcl-xL deamidation, and apoptosis (Figure S7), which is an apparent example of ”oncogene addiction”, whereby oncogene expression continues to be important for survival [36]. By contrast, DNA damage of human B-CLL cells, which should not express OTKs, triggered increased NHE-1 expression and apoptosis, achieving levels comparable with wild-type thymocytes (Figure 6C). However, enforced alkalinisation of either the murine (Figure S7) or human (Figure 6) cancer cells triggered significant increases in Bcl-xL deamidation and apoptosis, even in the absence of genotoxic attack (Figure 7C). In the case of the B-CLL cells, we cannot yet exclude the possibility that the tight correlation observed between these events does not reflect causal efficacy, and further work will be necessary to elucidate this point.

0426
For example, the down-regulation of Bcl-xL promoted the apoptosis of KARPAS-299 cells derived from a patient with anaplastic large cell lymphoma [38], and down-regulation of Bcl-xL suppresses the tumourigenic potential of the causative NPM-ALK oncogenic fusion protein in vivo [39]. Knockdown of Bcl-xL also significantly reduces the viability of pancreatic cancer cells to tumour necrosis factor α (TNF-α)– and TNF-α - related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by antitumour drugs [40].

0412
Knockdown of Bcl-xL also significantly reduces the viability of pancreatic cancer cells to tumour necrosis factor α (TNF-α)– and TNF-α - related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by antitumour drugs [40].
0081
These digestion conditions were chosen after careful optimisation to give good and consistent yields of the peptides SDVEENRTEAPEGTESEMETPSAINGNPSW (peptide 1) and HLADSPAVNGATGHSSSL (peptide 2), containing the putative deamidation sites N52 and N66, respectively, but without inducing further deamidation.

0004
Peptides were eluted from the column (0.075 mm × 100 mm, Vydac C18) with a gradient of 5%–35% acetonitrile (containing 10 mM ammonium acetate pH 5.3) over 30 min at a flow rate of 250 nl/min.

0081
During the development phase of the methodology, the mass spectrometer was operated in MS/MS mode to conclusively identify the peptide digestion products and to confirm the sites of deamidation as N52 and N66. Once the identities of the peptides had been established, the mass spectrometer was operated in MS mode for subsequent analyses.
0004
Cells were lysed in 50 mM HEPES (pH 7.2), 150 mM NaCl, 1mM EDTA, 0.2% NP-40, and complete protease inhibitors. Cell lysates were resolved by standard Laemmli's SDS-PAGE (pH 8.8) unless otherwise stated.
0054
Intracellular pH was measured using a standard ratiometric method with a pH-sensitive fluorophore SNARF-1 by flow cytometry [44]. Briefly, cells in phosphate-buffered saline (PBS) were loaded with 10 μM SNARF-1 for 40 min at 37 °C, followed by washing and incubation in PBS at room temperature for 30 min prior to measurement of pHi.

0054
FACS data were analysed using Flowjo software to obtain the ratio based on the Fl3/Fl2 channels.
0055
(C) Plasmids of shRNA Bax (GFP) and shRNA Bak (DsRed) were cotransfected into purified DN thymocytes using an Amaxa nucleofactor kit. 48 h later, GFP+ DsRed+ cells were purified by flow cytometry and treated with etoposide (Etop, 25 μM) for 30 h or exposed to irradiation (IR, 5 Gy) followed by 30 h in culture.

0663
(C) Plasmids of shRNA Bax (GFP) and shRNA Bak (DsRed) were cotransfected into purified DN thymocytes using an Amaxa nucleofactor kit. 48 h later, GFP+ DsRed+ cells were purified by flow cytometry and treated with etoposide (Etop, 25 μM) for 30 h or exposed to irradiation (IR, 5 Gy) followed by 30 h in culture.

0054
48 h later, GFP+ DsRed+ cells were purified by flow cytometry and treated with etoposide (Etop, 25 μM) for 30 h or exposed to irradiation (IR, 5 Gy) followed by 30 h in culture.
0006
Bcl-xL was immunoprecipitated from lysates derived from 1.5 × 106 sorted GFP-positive cells per lane, followed by immunoblotting for Bim or Puma.

0426
Note that in the vector lane, at this exposure endogenous Bcl-xL is not visible because of the small number of cells used.
0081
(F) Peptides SDVEENRTEAPEGTESEMETPSAINGNPSW (peptide 1) and HLADSPAVNGATGHSSSL (peptide 2), and the corresponding deamidated forms, containing the putative deamidation sites N52 and N66, respectively, were generated by digestion of rBcl-xL with chymotrypsin. The chromatographic conditions used for the separation of the peptides in the LC-MS analyses were optimised so as to resolve the Asn, Asp, and iso-Asp forms of peptides 1 and 2.
0004
Wild-type thymocytes were maintained in RPMI-1640/10% bovine fetal calf serum buffered at the indicated pH with Tris-HCl for 20 h in the presence of 20 μM monensin prior to lysis and immunoblotting for Bcl-xL. To minimize any deamidation produced during the gel-running process, the resolving gel buffer was adjusted to pH 8.0 in this experiment.
0054
The lower left FACS histogram shows the infection efficiency for nontransfected (non), empty-vector transfected (vector), or NHE-1 transfected (NHE-1) cells as percentage GFP-positive cells.

0426
The lower right histograms show the mean pHi and apoptosis (sub-G1) values ± SD (n = 5) analysed on GFP-negative and positive cells.
0054
pHi was measured by FACS on live CD4−CD8− cells, and the sub-G1 peak was analysed by FACS on CD4−CD8− cells to assess apoptosis.

0426
pHi was measured by FACS on live CD4−CD8− cells, and the sub-G1 peak was analysed by FACS on CD4−CD8− cells to assess apoptosis.
0054
Patients' cells (PBMC, in the range 85%–95% CD19+B220+) were incubated at pHe values of 7.2, 8.0, or 8.5, and the pHi values were monitored by SNARF-1 staining using flow cytometry. Apoptosis was evaluated by measurement of sub-G1 peaks using flow cytometry.
0052
The same cell aliquots cultured in RPMI/10% FCS for 24 h or 48 h were analysed for apoptosis by sub-G1 staining (right panel).
0426
(C) Enforced alkalinisation of murine tumour cells, or human B-CLL cells, causes Bcl-xL deamidation and subsequent apoptosis, even in the absence of external genotoxic attack.
0030
Even though these highly homologous receptors both activate the G protein stimulatory for adenylyl cyclase (Gs), signaling through β1AR and β2AR produces clearly distinguishable biological effects (Xiang and Kobilka, 2003; Xiao et al, 2004).
0006
To probe for a possible signaling complex including the β1AR and a PDE, mouse neonatal cardiomyocytes were infected with an adenovirus encoding a Flag-tagged β1AR, and the receptor was subsequently immunoprecipitated using an antibody against the tag.

0019
A significant amount of endogenous PDE activity was recovered in the β1AR immunoprecipitation (IP) pellet (Figure 1A).

0019
Whereas ablation of PDE4A or PDE4B had no effect, inactivation of the PDE4D gene prevented co-IP of PDE activity with the β1AR (Figure 1B).
0081
All PDE4 long forms are activated by phosphorylation at a conserved PKA consensus site in UCR1 (see Figure 2D); this mechanism provides a ubiquitous negative-feedback loop critical for cAMP signaling (Conti et al, 2003).
0676
Conversely, PDE4D5 is the variant tethered to the β2AR/β-arrestin complex (Baillie et al, 2003) concurring with the preferential activation of PDE4D5 upon β2AR signaling (Figure 5B). In pull-down experiments using purified proteins (Figure 3D and E), β1AR efficiently interacts with PDE4D, whereas β2AR has negligible affinity for PDE4D, underscoring the direct mode of PDE4D–β1AR interaction versus the indirect, β-arrestin-dependent mode of PDE4D–β2AR interaction.
0030
The latter may include the competition of PDE4D with other proteins for binding to the receptor or, in an opposite fashion, PDE4D may act as a scaffold by tethering additional proteins to the receptor complex such as the exchange protein activated by cAMP or PKA-anchoring proteins (Dodge-Kafka et al, 2005).

0096
The latter may include the competition of PDE4D with other proteins for binding to the receptor or, in an opposite fashion, PDE4D may act as a scaffold by tethering additional proteins to the receptor complex such as the exchange protein activated by cAMP or PKA-anchoring proteins (Dodge-Kafka et al, 2005).
0426
PAN-selective antibodies against PDE4A (AC55), PDE4B (K118), and PDE4D (M3S1), as well as splice variant-selective antibodies against PDE4D3, 4, 5, 8, and 9 (Richter et al, 2005), were used in IPs to determine the expression of the respective PDE4 subtype and splice variant in cardiac myocytes (Figure 2), as well as their PKA-dependent activation after β-adrenergic stimulation (Figure 5; Supplementary Figure 2). A PKA-site-specific antibody from Cell Signaling (Danvers, MA) was used to measure PKA phosphorylation of the β1AR (Figure 6).
0411
All cells were cultured at 37°C and under a 5% CO2 atmosphere. For expression of exogenous βARs and/or PDE4D constructs, cells were infected with adenoviruses 40 h before experimentation at an MOI of 2–6 (HEK293), 20–40 (cardiac myocytes), and 100 (MEFs), respectively.

0416
All cells were cultured at 37°C and under a 5% CO2 atmosphere. For expression of exogenous βARs and/or PDE4D constructs, cells were infected with adenoviruses 40 h before experimentation at an MOI of 2–6 (HEK293), 20–40 (cardiac myocytes), and 100 (MEFs), respectively. As ‘mock' controls, cells were infected with comparable titers of an adenovirus encoding green fluorescent protein (GFP).
0004
After the respective cell treatment, cells were rinsed once with ice-cold PBS and then lysed in 500 μl of 20 mM HEPES, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% N-dodecyl-β-D-maltopyranoside (DDM, Anatrace), 1 μM microcystin-LR (Calbiochem), and Complete protease inhibitor cocktail (Roche).

0006
Flag-tagged receptors were then immunoprecipitated using M1-affinity resin (α-Flag antibody resin; Sigma Aldrich).
0676
Rat PDE4D3, expressed in Sf9 insect cells using a recombinant baculovirus, was purified to >90% purity using an anti-PDE4D antibody (M3S1) covalently coupled to ProteinG Sepharose as described previously (Salanova et al, 1998). Flag-tagged β1AR and β2AR were also expressed in Sf9 cells and subsequently purified in a two-step procedure consisting of an initial affinity chromatography using M1-resin (immobilized anti-Flag antibody; Sigma Aldrich), followed by an alprenolol-sepharose affinity column.

0007
Flag-tagged β1AR and β2AR were also expressed in Sf9 cells and subsequently purified in a two-step procedure consisting of an initial affinity chromatography using M1-resin (immobilized anti-Flag antibody; Sigma Aldrich), followed by an alprenolol-sepharose affinity column.

0004
For βAR/PDE4D IP, equal amounts of purified β1AR and β2AR (1 μg) were coupled to M1 resin and then incubated in 500 μl of 20 mM HEPES (pH 7.5), 100 mM NaCl, 0.1% DDM, 4 mM CaCl2, and 0.01% cholesterol hemisuccinate with 0.5 μg of purified PDE4D under continuous rotation for 4 h at 4°C.
0004
After 4 days of culture and the respective cell treatment, neonatal cardiac myocytes were harvested in buffer containing 50 mM Tris–HCl (pH 7.4), 1 mM EDTA, 0.2 mM EGTA, 150 mM NaCl, 5 mM β-mercaptoethanol, 10% glycerol, 1 μM microcystin-LR, Complete protease inhibitor cocktail (Roche Diagnostics), and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF; Roche Diagnostics).
0019
(B) Co-IP of β1AR and PDE activity from cardiomyocytes deficient in PDE4A, PDE4B, or PDE4D, and wild-type controls.
0081
The PKA phosphorylation site conserved among long splice variants is indicated with red circles.
0019
Interaction of exogenous β1AR and PDE4D. (A, B) Co-IP of exogenous β1AR and Myc-tagged PDE4D splice variants expressed in HEK293 cells.

0019
(C) Shown is the co-IP of exogenous β1AR and PDE4D8-Myc from extracts of MEFs derived from mice deficient in β-arrestin 1 and 2 (βarr1/2KO) or from wild-type controls (WT-MEF).
0411
AIRE is expressed in thymic medullary epithelial cells, where it promotes the expression of tissue-restricted antigens.
0411
AIRE promotes the thymic expression of many tissue-restricted antigens, enabling the negative selection of developing T cells and thus precluding self-reactivity (Anderson et al, 2002; Liston et al, 2003); however, the mechanisms are so far unknown.
0019
Western blot analysis of H3/AIRE–PHD1 complex formation by using antibodies for H3K4me1, H3K4me3 and H3K9me3 indicated that H3K4 trimethylation hinders interaction (Fig 1F), whereas H3K9 trimethylation does not.
0081
The mapping of the H3/AIRE interaction site uniquely to AIRE–PHD1 was further confirmed by NMR titrations of histone H3 peptides into AIRE–PHD2, which bound neither methylated nor H3K4me0 peptides (data not shown).
0114
We generated a model of AIRE–PHD1 complexed with the H3K4me0 peptide on the basis of the crystal structure of the BPTF–PHD finger bound to H3K4me3 and performed molecular dynamics calculations for 10 ns.
0077
Remarkably, residues G305, G306 and G313 showed strong shifts when bound to H3K4me2 and disappeared completely from the NMR spectrum owing to line-shape broadening on binding to H3K4me3, indicating an involvement of this region in peptide binding.
0107
Although there are many similarities between these two structures and the AIRE–PHD1/H3K4me0 complex presented here, the AIRE–PHD1 finger differs in the additional recognition of the H3R2 side chain, which makes an important contribution to the high affinity of this interaction, as shown by our peptide mutagenesis experiments.

0676
Although there are many similarities between these two structures and the AIRE–PHD1/H3K4me0 complex presented here, the AIRE–PHD1 finger differs in the additional recognition of the H3R2 side chain, which makes an important contribution to the high affinity of this interaction, as shown by our peptide mutagenesis experiments.
0411
We have shown previously that transiently transfected AIRE enhances target gene expression in human embryonic kidney (HEK)293 cells (Pitkanen et al, 2005). So far, no cell line has been described with endogenous AIRE expression; therefore, we transfected HEK293 cells with an AIRE-encoding or control plasmid and generated stable cell lines called HEK-AIRE and HEK-control. We first tested HEK-AIRE compared with HEK-control cell lines for expression levels of tissue-restricted antigens that are downregulated in AIRE-deficient mouse thymic medullary epithelial cells (Derbinski et al, 2005). Indeed, the HEK-AIRE cell line showed enhanced expression of such antigens, including insulin, the principal autoantigen in type I diabetes (Babaya et al, 2005), involucrin and S100A8 (Fig 5A).

0019
Next, we studied in vivo histone binding by protein chromatin immunoprecipitation (ChIP) assays and observed that AIRE is found in complexes with a small fraction of histone H3 but not with H3K4me3.

0030
Although AIRE specificity towards chromatin might be influenced by other protein and DNA interactions (Ruan et al, 2007), the data presented here indicate that AIRE preferentially binds to and activates the promoters containing low levels of H3K4me3.

0411
On the basis of these results, we propose a speculative model for the regulation of tissue-restricted antigen expression in thymic epithelial cells (supplementary Fig S8 online). Normally, tissue-restricted antigens are silenced in immature thymic epithelial cells as they lack the active chromatin mark H3K4me3 on their promoters. During differentiation into mature thymic medullary epithelial cells, activation of AIRE expression (Kyewski & Klein, 2006) enables the read-out of non-methylated H3K4 as a signal to activate tissue-restricted antigen genes.
0065
NMR binding, fluorescence titration assays and isothermal titration calorimetry thermodynamic analysis.

0077
NMR binding, fluorescence titration assays and isothermal titration calorimetry thermodynamic analysis. Details on NMR titrations, fluorescence spectroscopy and thermodynamic measurements are described in the supplementary information online.
0096
(E) GST-PHD1, but not GST alone, interacts with native mononucleosomes detected by western blot against anti-H3 (middle) and anti-H2B (bottom). Equal input of GST proteins is shown with Ponceau red staining (top).

0107
(F) Interaction between GST-AIRE fusion proteins and whole histones, detected by anti-H3K4me1, anti-H3K4me3 and anti-H3K9me3. (G) Interaction between GST-AIRE–PHD1 fusion proteins and amino-terminal histone H3 peptides (H3K4me0, H3K4me1, H3K4me3 and H3K9me3), all detected by anti-GST.
0107
(B) Interaction between AIRE–PHD1 (PHD1), AIRE–PHD1-D297A (D297A) mutant proteins and H3K4me0 peptide detected by anti-GST.
0019
AIRE, autoimmune regulator; ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human embryonic kidney; H3K4me3, histone H3 trimethylated at lysine 4.
0019
AIRE, autoimmune regulator; ChIP, chromatin immunoprecipitation; HEK, human embryonic kidney; PHD, plant homeodomain.
0889
Compared to chemiluminescence, the detection with infrared dye is linear over a very wide range and amounts from 1 pg up to 10 ng can be detected more reliably (Chen et al, 2005).
0413
To test whether the increased Plx1 binding to chromatin was required for promoting DNA replication in the presence of stalled replication forks, we measured DNA replication in Plx1-depleted extracts treated with low amounts of aphidicolin.
0030
One of the truncated proteins contained the protein interaction domain known as the Polo box, which has been shown to interact with phosphorylated proteins (Elia et al, 2003).
0676
The fact that Plx1 binding to the Mcm complex is enhanced by checkpoint activation suggests that post-translational modifications of the Mcm proteins induced by the activation of the DNA damage response could increase Plx1 affinity for the Mcm complex.

0081
Mcm2 is strongly phosphorylated on serine 92 by ATM/ATR (Yoo et al, 2004b), and its phosphorylation can be induced by pA/pT, aphidicolin or etoposide (Supplementary Figure 9A). To test whether phosphorylation of serine 92 of Mcm2 has a role in Plx1 recruitment to chromatin and in Plx1-mediated suppression of the checkpoint, we produced recombinant wild-type Mcm2 (Mcm2-WT) and Mcm2 in which serine 92 had been substituted with alanine (Mcm2-S92A) rendering it resistant to ATR-mediated phosphorylation (Supplementary Figure 9B).
0107
As the interaction between Plx1 and the Mcm complex is mediated by the Polo box, which is a phospho-binding domain (Elia et al, 2003), it is plausible that direct phosphorylation of Mcm7 and/or Mcm2 by ATM/ATR could favour Plx1 interaction with the Mcm complex on chromatin. Indeed, we found that the interaction of Plx1 with Mcm proteins is increased by the induction of the checkpoint.
0030
Claspin inactivation by protein degradation restrains Chk1 activity and promotes recovery from the DNA replication checkpoint in mammalian organisms (Mailand et al, 2006; Peschiaroli et al, 2006). We have recently shown that Tipin, a factor critical for replication fork stabilization (Chou and Elledge, 2006), is required for Claspin binding to chromatin and for efficient Chk1 activation (Errico et al, 2007).
0411
Downregulation of Plk1 expression leads to cell death in transformed cells (Liu and Erikson, 2003a, 2003b), suggesting that Plk1 is required for the cell cycle of tumour cells. The genome of tumour cells is highly unstable and has many DNA lesions that would normally interfere with replication progression.
0004
The eggs were collected in 90 mM NaCl. To prepare interphase extracts, the eggs were dejelled in a buffer (20 mM Tris, pH 8.5, 110 mM NaCl, 5 mM DTT) for 5 min, washed with ¼ Marc's modified Ringer (MMR) (5 × MMR: 100 mM HEPES, pH 7.5, 2 M NaCl, 10 mM KCl, 5 mM MgSO4, 10 mM CaCl2, 0.5 mM EDTA) and activated with 1 μg/ml calcium ionophore A23187 in MMR for 5 min. The activated eggs were washed with ¼ MMR and then washed three times with ice-cold S-buffer (0.25 M sucrose, 50 mM KCl, 2.5 mM MgCl2, 2 mM β-mercaptoethanol, 15 μg/ml leupeptin and 50 mM HEPES–KOH at pH 7.5).
0411
6xHistidine-tagged geminin (a gift from M Micheal) was expressed in BL21(DES) cells from a pET28 plasmid and purified on a nickel-NTA column according to standard protocols.

0426
Baculoviruses encoding 6xHistidine-tagged Plx1 (a gift from J Gautier), Mcm2-WT and Mcm2-S92A (a gift from W Dunphy) were used to infect Sf9 cells, which were expanded in large flasks using complete GRACE medium according to standard protocols (Invitrogen).
0004
Nuclei were lysed for 1 h at 4°C in a lysis solution at pH 9.5 (2.5 M NaCl, 100 mM EDTA, 10 mM Tris base, 1% Triton X-100 and 10% dimethyl sulphoxide). The slides were washed three times in the electrophoresis buffer (100 mM Tris, 300 mM sodium acetate, adjusted to pH 8.3) and then equilibrated for 60 min in the same buffer.

0426
Images of 100 randomly selected ethidium bromide-stained cells were analysed from each coded slide at × 400 magnification. To measure the extent of DNA damage, image analysis software (Comet IV, Perceptive Instruments) was used. DNA damage was assessed by monitoring comet head length analysing 100 cells for each slide.
0858
(A) Immunodepletion of Plx1.
0104
After pull-down, proteins were consecutively eluted using 3 mg/ml of phosphopeptide solution (first elution=E1, second elution=E2, see Supplementary data for the sequence of the peptide and the Polo box) and subjected to MALDI-TOF analysis.

0004
The GST fusion protein containing the Polo box from Plk1 is shown at the bottom.
0426
The percentage increase in the head length of the comets subtracted from the average nuclear diameter of undepleted samples with high Mcm was used as a parameter to measure DSBs.
0030
In relation to transcriptional machinery, so far the structures of TBP (TATA box-binding protein) subunit (Nikolov et al, 1992) from TFIID, TBP–DNA (Kim et al, 1993a, 1993b), TBP–DNA–TFIIB (Nikolov et al, 1995), Pol II (Cramer et al, 2000, 2001) and Pol II–TFIIB (Bushnell et al, 2004) have been determined.
0107
With regard to the interaction between hTFIIE and hTFIIH, it has been shown that the C-terminal acidic region of hTFIIEα is necessary for native hTFIIH binding (Ohkuma et al, 1995) and hTFIIEα strongly binds to the p62 subunit of hTFIIH (Yamamoto et al, 2001; Okuda et al, 2004).

0030
hTFIIEα AC-D is found to share its binding surface on p62 PH-D with the acidic transactivation domains (TADs) of tumour supressor protein p53 (Di Lello et al, 2006) and herpes simplex virus protein VP16 (Di Lello et al, 2005).
0004
After purification by glutathione-Sepharose column chromatography, all samples containing the C-terminal region, namely full-length GST–p621−548, GST–p62109−548, GST–p62238−548 and GST–p62333−548, were considerably degraded or incompletely translated (data not shown). Though such instability of the C-terminal half of p62 has previously been reported (Jawhari et al, 2004), we found that full-length GST–p621−548, GST–p621−108, GST–p621−238 and GST–p621−333 bound to hTFIIEα AC-D, whereas no binding was observed with GST–p62109−548, GST–p62238−548, GST–p62333−548 and GST alone (Figure 2B).
0077
To obtain the p62 PH-D-bound structure of hTFIIEα AC-D using NMR, we performed NMR titration experiments in buffers both with and without 100 mM NaCl for both domains (Supplementary Figures 1–3). Although in the 100 mM NaCl buffer the dissociation constant (Kd) between AC-D and p62 PH-D was estimated from the titration plots as 376±81 nM (Supplementary Figure 1C) or 237±82 nM (Supplementary Figure 2C), the NaCl-free buffer NMR titration experiment showed much stronger binding affinity between AC-D and p62 PH-D because of the slow exchange timescale with a Kd below about 150 nM (Supplementary Figure 3).

0107
Although in the 100 mM NaCl buffer the dissociation constant (Kd) between AC-D and p62 PH-D was estimated from the titration plots as 376±81 nM (Supplementary Figure 1C) or 237±82 nM (Supplementary Figure 2C), the NaCl-free buffer NMR titration experiment showed much stronger binding affinity between AC-D and p62 PH-D because of the slow exchange timescale with a Kd below about 150 nM (Supplementary Figure 3).

0104
Although in the 100 mM NaCl buffer the dissociation constant (Kd) between AC-D and p62 PH-D was estimated from the titration plots as 376±81 nM (Supplementary Figure 1C) or 237±82 nM (Supplementary Figure 2C), the NaCl-free buffer NMR titration experiment showed much stronger binding affinity between AC-D and p62 PH-D because of the slow exchange timescale with a Kd below about 150 nM (Supplementary Figure 3).
0030
Furthermore, the herpes simplex virus protein 16 (VP16) TAD also interacts with virtually identical sites of Tfb1 and p62 PH-D (Di Lello et al, 2005).

0081
Interestingly, the binding sites of p62 for p53 TAD2 and VP16 TAD significantly overlap with a part of the binding site for hTFIIEα AC-D.

0081
The binding site of p53 TAD2 peptide is disordered in an unbound state, but it forms a nine-residue amphipathic α-helix upon binding to Tfb1 PH-D and p62 PH-D.
0107
In the present study, the specific interaction between hTFIIEα AC-D and hTFIIH p62 PH-D was explored, and structures of both the free and PH-D-bound forms of hTFIIEα AC-D were determined. This is the first report of the structural determination of the complex describing the interaction between TFIIE and TFIIH at the molecular level.
0107
For yeast TFIIEα (Tfa1), functional significance of the C-terminal region and specific interaction between Tfa1 and Tfb1 has been reported (Bushnell et al, 1996; Kuldell and Buratowski, 1997). We aligned sequences of TFIIEα AC-D from other species to ascertain whether the interaction observed in human is evolutionally conserved (Figure 1A).
0030
In many cases, acidic TADs are disordered in an unbound form, but form an amphipathic helix upon binding to target proteins, for example, p53 TAD2–RPA70 (replication protein A 70) (Bochkareva et al, 2005), p53 TAD1–MDM2 (ubiquitin ligase) (Kussie et al, 1996), VP16 TAD–hTAFII31 (human TBP-associated factor) (Uesugi et al, 1997) complexes as well as the recently determined Tfb1 PH-D–p53 TAD2 complex (Di Lello et al, 2006).

0096
In many cases, acidic TADs are disordered in an unbound form, but form an amphipathic helix upon binding to target proteins, for example, p53 TAD2–RPA70 (replication protein A 70) (Bochkareva et al, 2005), p53 TAD1–MDM2 (ubiquitin ligase) (Kussie et al, 1996), VP16 TAD–hTAFII31 (human TBP-associated factor) (Uesugi et al, 1997) complexes as well as the recently determined Tfb1 PH-D–p53 TAD2 complex (Di Lello et al, 2006).

0077
In the binding of VP16 TAD to Tfb1 PH-D, the Kd value estimated by NMR titration experiment was ∼4000–7000 nM (Di Lello et al, 2005).

0107
In the binding of VP16 TAD to Tfb1 PH-D, the Kd value estimated by NMR titration experiment was ∼4000–7000 nM (Di Lello et al, 2005). Compared with these Kd values, the binding of hTFIIEα AC-D to p62 PH-D is rather strong.

0889
p62 has been shown to interact with the TADs of not only VP16 and p53 (Xiao et al, 1994) but also E2F-1 (Pearson and Greenblatt, 1997), and the oestrogen receptor α (ERα) (Chen et al, 2000).

0402
It can be imagined that if TFIIH is recruited by an activator near the promoter through its TAD then the recruited TFIIH could be captured by TFIIE instead of the activator to form the PIC. TFIIE regulates the enzymatic activities of TFIIH, which are necessary for the next stage after the PIC formation, that is, promoter melting or promoter clearance.
0411
The NdeI–BamHI fragment of mutated hTFIIEα cDNA was subcloned into the pET11d vector (Novagen) making the N-terminal hexa histidine-tagged hTFIIEα (6H–hTFIIEα) expression plasmid. The NdeI–BamHI fragment of mutated hTFIIH p62 cDNA was subcloned into the pET vector making the N-terminal FLAG-tagged hTFIIH p62 PH-D (FLAG–p62 PH) expression plasmid.
0071
After digestion with thrombin to remove the 6H tag, sample was again loaded onto the Ni-NTA agarose column. Fractions passing through the column were concentrated and applied onto Superdex30 (GE Healthcare).
0096
GST fusion proteins were used for protein interaction assays (Okamoto et al, 1998).

0004
GST fusion proteins were used for protein interaction assays (Okamoto et al, 1998).

0030
GST fusion proteins were used for protein interaction assays (Okamoto et al, 1998).

0007
The bound proteins were released by boiling in SDS–PAGE loading buffer, separated by SDS–PAGE and detected by western blotting with anti-hTFIIEα rabbit antiserum (1:3000 dilution), anti-FLAG M2 monoclonal antibody (Sigma) and anti-p53 (DO-1) (Santa Cruz) using the enhanced chemiluminescence detection system (GE Healthcare).
0077
Left, superposition of the backbone heavy atoms of the 20 lowest energy NMR structures.
0077
(A) Superposition of the backbone heavy atoms of the 20 lowest energy NMR structures.
0019
Interacting residues are indicated with the side chains and interaction areas are roughly marked in yellow.

0007
The top panel shows 5 ng of FLAG-tagged p62 PH-D mutant proteins (p62 PH-D mutants).

0007
Bound proteins were subjected to SDS–PAGE and detected by western blotting with anti-FLAG, anti-p53 and anti-hTFIIEα antisera.
0419
Vav proteins share a common structure, including a N-terminal calponin homology (CH) domain involved in Ca+2 mobilization and transforming activity, an acidic domain (AD) containing three regulatory tyrosines, a Dbl homology (DH) domain with a conserved region that promotes the exchange of GDP for GTP on Rac/Rho GTPases, a pleckstrin homology (PH) domain binding to PIP3 that enables its movement to the inner face of the plasma membrane, two Src-homology 3 (SH3) domains interacting with proteins containing proline-rich sequences, and a Src-homology 2 (SH2) domain interacting with proteins containing phosphorylated tyrosines [4,5].

0114
Vav proteins share a common structure, including a N-terminal calponin homology (CH) domain involved in Ca+2 mobilization and transforming activity, an acidic domain (AD) containing three regulatory tyrosines, a Dbl homology (DH) domain with a conserved region that promotes the exchange of GDP for GTP on Rac/Rho GTPases, a pleckstrin homology (PH) domain binding to PIP3 that enables its movement to the inner face of the plasma membrane, two Src-homology 3 (SH3) domains interacting with proteins containing proline-rich sequences, and a Src-homology 2 (SH2) domain interacting with proteins containing phosphorylated tyrosines [4,5].

0419
Upon phosphorylation of the tyrosines in the AD domain, the folding is opened and the DH domain is exposed. Thus, Vav protein is activated and interacts with substrate proteins, and the PH domain is exposed for PIP3 binding [6].

0004
Thus, Vav protein is activated and interacts with substrate proteins, and the PH domain is exposed for PIP3 binding [6].

0096
Thus, Vav protein is activated and interacts with substrate proteins, and the PH domain is exposed for PIP3 binding [6].
0004
Briefly, aliquots of samples with the same amount of protein, determined using the Bradford assay (BioRad, Hercules, CA), were mixed with loading buffer (final concentrations of 62.5 mM Tris-HCl, pH 6.8, 2.3% SDS, 100 mM dithiothreitol, and 0.005% bromophenol blue), boiled, fractionated in a SDS-PAGE, and transferred onto a 0.45-um nitrocellulose membrane (BioRad).
0004
GST-Vav3-DH+PH and control GST vectors were transformed into BL21 bacteria, respectively (Protein Express, Inc.

0004
The transformed bacteria were cultured in L-Broth with addition of 100 uM of IPTG to induce GST-fusion protein expression. Then, the bacteria were harvested and subjected to GST fusion protein purification by Sonication and using Glutathione Sepharose 4B (Amersham Bioscience).
0004
For pull down reaction, 5~10 ug of GST or GST-Vav3-DH+PH was incubated with 1 mg of cell extracts from MCF7 cells in a binding buffer [20 mM of Tris.CL, PH. 7.9; 300 mM of KCL; 0.05% of NP-40; 0.2 mM of EDTA; 20% of Glycerol; 1 mM of Dithiothritol; 1 mM of phenylmethylsulfonyl fluoride (PMSF), 1× of protease inhibitor cocktail (Roche Diagnostics)] for overnight [28,29].

0413
For pull down reaction, 5~10 ug of GST or GST-Vav3-DH+PH was incubated with 1 mg of cell extracts from MCF7 cells in a binding buffer [20 mM of Tris.CL, PH.
0411
Vav3 is involved in growth of breast cancer cells. (A) Expression analysis of Vav3 and ERα in breast cancer MCF7 and T47D cells and nontumoral breast epithelial MCF-10A cells. The cell extracts were prepared from T47D, MCF7, and MCF-10A cells and subjected to western blot analysis for Vav3 and ERα.

0411
(B) MCF7 cells were transiently transfected with Vav3 expression vector or control empty vector and then cultured in stripped medium in the absence or presence of EGF for 5 days, followed by MTT assay.

0411
(C) MCF7 cells were transiently transfected with Vav3 expression vector or empty pHEF vector for 3 days, followed by cell extracts preparation and western blot analysis for Vav3.
0411
We then determined whether knockdown expression of Vav3 inhibits growth of these breast cancer cells using Vav3 siRNA that has been characterized previously [25].

0426
Lipofectamine 2000 used for transfection of siRNA has been shown to knock down more than 80% activity of the endogenous gene in a panel of cells tested (Invitrogen).

0411
A growth stimulatory effect in response to estrogen stimulation was observed in T47D and MCF7 cells (Figure 2Eand 2F). We found that knockdown expression of Vav3 significantly inhibited both estrogen-dependent and -independent growth in these breast cancer cells (Figure 2Eand 2F). At 1.2 pM of siRNA, the growth of T47D and MCF7 cells was inhibited by 64% and 68% in the absence of estrogen and 46% and 77% in the presence of estrogen relative to their respective controls.
0411
Hela cells were transiently cotransfected with a luciferase reporter driven by the estrogen response element (ERE), ERα expression vector, and either Vav3 or Vav3* expression vector.

0411
Upregulation of the endogenous ERα activity by Vav3 relative to the empty vector pHEF was also confirmed in MCF7 cells by stimulation of luciferase reporter expression driven by ERE (Figure 3B) and natural promoter of ERα target gene pS2 (Figure 3C).
0411
(A) Hela cells (105 cells/well in 12-well plate) were cotransfected with ERE-Luc (0.5 ug), expression vectors for Vav3, Vav3*, or empty vector pHEF (200 ng) and ERα (50 ng), respectively. Then, the cells were treated without or with E2 (10-9 M) and without or with Tamoxifen. (B and C) MCF7 cells (105 cells/well in 12-well plate) were cotransfected with ERE-Luc (0.5 ug) (B) or pS2-Luc (0.5 ug) (C), and expression vector (0.25 ug) for Vav3*, or empty vector pHEF, respectively. Then, the cells were treated with E2.
0004
The truncated Vav3 protein with deletion of both PH and SH domains failed to activate ERα.
0411
(B) Hela cells (105 cells/well in 12-well plate) were cotransfected with ERE-Luc (0.5 ug) and expression vectors (200 ng) for Vav3, Vav3*, Vav3*-ΔDH, Vav3*-ΔSH, or empty vector pHEF, as well as expression vector (50 ng) for ERα, respectively.
0411
(A) Hela cells were cotransfected with ERE-Luc (0.25 ug), expression vectors ERα (25 ng), Vav3* or empty vector pHEF (50 ng), p85+p110 or empty vector pCR3.1 (50 ng). (B) Hela cells were cotransfected with ERE-Luc reporter (0.25 ug/well in 12-well plate), expression vectors ERα (25 ng), dominant-negative Akt expression vector or empty vector pCR3.1 (0.1 ug), and Vav3* expression vector or empty vector pHEF (0.1 ug), respectively. (C) T47D cells were cotransfected with ERE-Luc (0.5 ug) and expression vector for Vav3 or empty vector pHEF (0.25 ug). Then, the cells were treated with Wortmannin (0.5 um). (D) T47D cells were cotransfected with pS2-Luc (0.5 ug) and expression vector for Vav3 or empty vector pHEF (0.25 ug). Then, the cells were treated with EGF (20 ng/ml).
0411
Hela cells were transiently cotransfected with ERE-Luc reporter, and expression vectors ERα, Vav3*, and p85 (the regulatory subunit of PI3K) and p110 (the catalytic subunit of PI3K).
0004
A nuclear localization signal (NLS) in the PH domain was shown to be solely responsible for the nucleus localization of Vav1 protein, indicating a role of Vav family protein as a transcription coregulator.

0419
A nuclear localization signal (NLS) in the PH domain was shown to be solely responsible for the nucleus localization of Vav1 protein, indicating a role of Vav family protein as a transcription coregulator. We have demonstrated that the DH domain of Vav3 is essential for AR and ERα activation (Figure 4) [25]. We performed sequence analysis of Vav proteins and found that the DH domain of Vav3 contains three consensus sequences of the LXXLL motifs or NR boxes, which have been well characterized and involved in interaction with nuclear receptors (Figure 6A). In addition, homologous analysis of Vav3 and Vav1 genes identified a conserved NLS in the PH domain of Vav3 (Figure 6B).
0419
(B) A consensus sequence of the nucleus localization signal (NLS) in Vav3 was localized in the PH domain.
0107
We performed GST pull down experiment to confirm the interaction between Vav3 and ERα.

0004
A GST fusion protein including the DH and PH domain of Vav3 (GST-Vav3-DH+PH) was generated (Figure 7A). Cell extract derived from MCF7 cells was incubated with immobilized GST-Vav3-DH+PH fusion protein or GST protein.

0419
A GST fusion protein including the DH and PH domain of Vav3 (GST-Vav3-DH+PH) was generated (Figure 7A).

0004
We found that GST-Vav3-DH+PH fusion protein, but GST protein, interacted with ERα (Figure 7C).

0419
In summary, we found that Vav3 contains NLS in the PH domain and three LXXLL motifs in the DH domain. The deletion mutation and functional analysis by luciferase reporter assay showed that the DH domain of Vav3 is essential for enhancing ERα activity and is involved in complex with ERα.
0004
(A) GST-Vav3-DH+PH fusion protein. (B) GST-Vav3-DH+PH fusion protein (lane 3 and 4) and control GST protein (lane 1 and 2) were subjected to pull down reaction in the absence (lane 1 and 3) and presence (land 2 and 4) of cell extract derived from MCF7 cells.
0411
The knockdown expression of Vav3 compromised both estrogen-stimulated and -independent growth of breast cancer cells.
0419
Vav3 is activated upon ligand stimulation of EGF, insulin, Ros, and IGF receptors and physically associates with a variety of signaling molecules, including Rac1, Cdc42, PI3K, Grb2, and PLC-γ, leading to alteration in cell morphology and cell transformation [33].
0004
A nuclear localization signal (NLS) in the PH domain is solely responsible for nucleus localization of Vav1 protein, indicating a role of Vav family proteins as a transcription coregulator.

0419
A nuclear localization signal (NLS) in the PH domain is solely responsible for nucleus localization of Vav1 protein, indicating a role of Vav family proteins as a transcription coregulator. We found that Vav3 contains the LXXLL motifs in the DH domain and NLS in the PH domain.
0004
Our findings support the notion that Vav3 overexpression may play a role in breast cancer, based on the following reasons: 1) Vav3 is overexpressed and correlated with poorly differentiated tumors in human breast cancer; 2) Vav3 contains the LXXLL motifs and complexes with ERα; 3) Vav3 enhances ERα activity partially via the PI3K-Akt pathway; 4) Vav3 is a protein with multiple domains and functions, including the SH2 domain interacting with receptor protein tyrosine kinase, the PH domain binding PIP3 involved in association with the cell membrane, and the DH domain involved in interaction with ERα; 5) Vav3 potentiates EGF for cell growth and ERα activation.

0419
Our findings support the notion that Vav3 overexpression may play a role in breast cancer, based on the following reasons: 1) Vav3 is overexpressed and correlated with poorly differentiated tumors in human breast cancer; 2) Vav3 contains the LXXLL motifs and complexes with ERα; 3) Vav3 enhances ERα activity partially via the PI3K-Akt pathway; 4) Vav3 is a protein with multiple domains and functions, including the SH2 domain interacting with receptor protein tyrosine kinase, the PH domain binding PIP3 involved in association with the cell membrane, and the DH domain involved in interaction with ERα; 5) Vav3 potentiates EGF for cell growth and ERα activation.
0096
KL generated GST fusion protein for pull down analysis.

0004
KL generated GST fusion protein for pull down analysis.
0426
Although Bcl-XL and Bax are structurally similar, activated Bax forms large oligomers that permeabilize the outer mitochondrial membrane, thereby committing cells to apoptosis, whereas Bcl-XL inhibits this process.
0426
During development and under stress, cells can become committed to die via programmed cell death (apoptosis).
0096
However, there is also considerable overlap between these two competing models: both models recognize that Bcl-XL directly binds to and inhibits a proapoptotic Bcl-2 family protein that is directly involved in membrane permeabilization, while other proapoptotic Bcl-2 family proteins indirectly induce apoptosis by binding to Bcl-XL and preventing this function.

0096
On the basis of these data and evidence from the literature, we have proposed a model termed “embedded together”, in which interaction of these proteins with each other changes after binding to the membrane as this causes conformational changes that alter and/or expose new binding surfaces [10,11].
0426
The large increase in fluorescence that accompanies the release of the fluorophore/quencher pair 8-aminonaphthalene 1,3,6-trisulfonic acid (ANTS)/p-xylene-bis-pyridinium (DPX) from liposomes was used to measure Bax-dependent membrane permeabilization and its inhibition by Bcl-XL [7].

0663
As seen previously, single addition of recombinant Bax (100 nM) or tBid (20 nM) to liposomes had little effect, but in combination the two proteins caused an increase in fluorescence due to membrane permeabilization (Figure 1A).
0071
Membrane-bound proteins were separated from soluble proteins by Sepharose CL-2B gel filtration chromatography.

0019
Individual fractions were analyzed by immunoblotting (IB) using Bid, Bax, or Bcl-XL antibodies, as indicated.
0071
In incubations of 20 nM tBid and liposomes, tBid bound effectively to liposomes as assayed by gel filtration chromatography (Figure 1B).
0107
In the absence of membranes, interaction between Bcl-XL and tBid could not be detected by co-immunoprecipitation (unpublished data).
0006
Samples were immunoprecipitated (IP) in either 2% CHAPS or 0.2% NP-40, as indicated, using an antibody with the indicated specificity and immunoblotted (IB) for the indicated protein.
0019
Interaction of this peripheral membrane (indicated by the shadow) Bax with membrane-bound tBid causes a further conformational change such that Bax integrates in the membrane in an oligomerization competent form (step 2).

0096
Conversely, cytoplasmic Bax may interact with other activator proteins to integrate into membranes, or spontaneously active Bax molecules may integrate into the membrane without binding an activator protein.
0107
When tested in the absence of tBid, a stable interaction between Bax and Bcl-XL was not detected (Figure 2A, left panel, lane 3), suggesting that Bcl-XL does not sequester Bax in solution.

0426
However as expected, co-immunoprecipitation of Bcl-XL and Bax was observed in control experiments where membranes were solubilized with the nonionic detergent NP-40, known to induce a conformational change in Bax required for heterodimerization with Bcl-XL that is also seen in cells when apoptosis is induced [20,26].
0107
When tested in the presence of tBid (20 nM) and membranes, we did not detect an interaction between Bcl-XL (100 nM) and Bax (100 nM) (Figure 2A, left panel, lane 2).
0107
The absence of a stable interaction between tBid-mt1 and Bcl-XL significantly decreased Bcl-XL-mediated inhibition of Bax binding to membranes at all tBid concentrations assayed.
0426
At the onset of apoptosis in cells, Bcl-XL binds to membranes [23].
0019
Unlike the conformational change that accompanies tBid-induced insertion of Bax into membranes, the liposome-induced conformational change also disappears if liposomes are solubilized in CHAPS prior to immunoprecipitation (Figure 5A, compare lanes 1 and 3).
0006
Conformation-altered Bax was immunoprecipitated using the 6A7 antibody with or without the addition of 2% CHAPS to solubilize the liposomes prior to immunoprecipitation and analyzed by immunoblotting using an α-Bax antibody. The asterisk denotes the light chain of the 6A7 antibody.
0096
On the basis of the effects previously published for mutations in BH3 domains of proapoptotic proteins and peptides, we selected two peptides that both caused membrane binding by Bcl-XL (Figure S5A) but differed in their functional effects (Figure S5B).
0426
Pioneering experiments to identify relevant binding partners for Bcl-2 and Bcl-XL using immunoprecipitation in transfected cells suggested a lack of correlation between Bax binding and inhibition of apoptosis, as only certain Bcl-XL point mutants that could no longer bind to Bax lost function [40]. Further analysis of two of these Bcl-XL mutants that did not bind to Bax (the F131V/D133A mutant that remained functional and the inactive G138E/R139L/I140N mutant) suggested that prevention of apoptosis required binding to the BH3-only proteins tBid, Bim, and Bad, a function that was specifically lost in the latter mutant [19]. However, as these experiments were conducted on whole cells where Bax was also present, it is possible that the lack of function of this mutant is caused by the loss of binding to both BH3-only proteins (e.g., tBid) and Bax , a result entirely consistent with our observations. Conversely, early work with the M97A/D98A Bid mutant that does not bind to Bcl-XL [27,30] that we have used in our study (tBid mt1) indicated that Bcl-XL inhibited the apoptosis caused by this Bid mutant, in cells and in purified mitochondria, implying that the interaction with activated Bax rather than Bid was critical to the antiapoptotic function of Bcl-XL in this context.

0019
Conversely, early work with the M97A/D98A Bid mutant that does not bind to Bcl-XL [27,30] that we have used in our study (tBid mt1) indicated that Bcl-XL inhibited the apoptosis caused by this Bid mutant, in cells and in purified mitochondria, implying that the interaction with activated Bax rather than Bid was critical to the antiapoptotic function of Bcl-XL in this context.
0081
We therefore determined whether peptides from the BH3 regions of Bid, Bim, Bad, Bax, and Bak caused insertion of Bcl-XL into liposomes, assayed by flotation on a sucrose gradient (Figure S6). All five peptides caused Bcl-XL to insert into membranes, while a mutant Bid peptide that fails to bind Bcl-XL [2] did not.
0019
Previous reports using transfected cells have indicated that the removal of the C-terminal insertion sequence of Bcl-XL severely impairs membrane insertion but has varying effects on Bcl-XL function, from moderate [29] to severe [50] reduction in function.

0426
Consistent with this variability, we have shown previously [37] that the function of ΔTM Bcl-XL compared to that of wild type is heavily dependent on the stimulus used to initiate apoptosis (and therefore possibly the BH3 protein(s) involved).

0426
However, in certain circumstances in cells ΔTM Bcl-XL may retain at least part of its antiapoptotic function in the cytoplasm depending on the location of relevant binding partners at the onset of apoptosis.
0096
The liposomes that we used to model physiologic membranes have a high intrinsic curvature and lipid composition that facilitate membrane binding of the recombinant proteins and induction of the 6A7 conformational change in Bax.

0426
In cells, these feature are likely represented by complex and dynamic physiologic processes such as mitochondrial membrane fission and fusion shown to be important in apoptosis [52–54] and the interaction of mitochondria with other membrane systems [39,52,55]. The major observations reported here were confirmed for cytochrome c release from mitochondria (Figure 3B–D), suggesting that they will be relevant in live cells. Moreover, the simplicity and power of our in vitro system using recombinant proteins and membranes has allowed us to identify and measure functionally important and potentially “druggable” interactions important for the regulation of apoptosis (Figure 6).
0676
Recombinant full-length human Bcl-XL (or Bcl-XL Y101K) with no additional amino acids was expressed in Escherichia coli as a C-terminal intein/chitin-binding domain fusion and purified by affinity chromatography on a chitin column followed by further purification on a phenyl-Sepharose column, similar to a method described previously [7] but with a final dialysis step to remove detergents.
0081
The signal peptide is shaded black, and putative O-linked glycosylation sites are indicated by vertical lines.
0004
However, OSM-11 and DOS-3 also have a consensus proprotein convertase protease cleavage site and a C-terminal transmembrane domain, suggesting that they may be translated as transmembrane preproproteins prior to proteolytic processing and release of a soluble DOS protein.
0030
C901 is a predicted Drosophila protein of unknown function containing a DSL domain and multiple EGF repeats [56]; it is unclear whether C901 is a transmembrane DSL domain protein.
0411
In animals harboring this transgene, GFP expression was observed in numerous unidentified cells during embryonic development from the comma stage onward (unpublished data). GFP expression was observed in the VPCs during larval development, as well as various hypodermal cells during larval stages (Figure 4).

0411
OSM-11 immunoreactivity was also observed in the seam cells of L1 larvae and adult animals (Figure 4A and 4D). In adult animals, osm-11p::gfp was expressed only in hypodermal seam cells in adult animals; hypodermal seam cell expression in adult animals was also confirmed with staining with OSM-11 antisera (Figure 4D). The larval hypodermal expression pattern of osm-11p::gfp is reminiscent of the osm-7p::gfp expression pattern described previously, but osm-7p::gfp expression in seam cells was not reported [52]. OSM-11 protein was also expressed in the developing uterus of L4 larvae (Figure 4B) and in the spermatheca (Figure 4D); the LIN-12 Notch receptor plays a developmental role in these tissues as well [64], but only OSM-11 expression in VPCs was characterized further.
0411
(D) OSM-11 expression in seam cells and spermatheca in adult animals. An osm-11p::gfp reporter gene containing unc-54 3′ UTR sequences is expressed in adult seam cells (left); α-OSM-11 antisera was used to confirm seam cell and spermatheca expression (right). No OSM-11 was detected in neurons of larvae or adult animals (unpublished data); embryonic expression was not characterized.

0426
An osm-11p::gfp reporter gene containing unc-54 3′ UTR sequences is expressed in adult seam cells (left); α-OSM-11 antisera was used to confirm seam cell and spermatheca expression (right).
0411
OSM-11 expression in Pn.p cells is consistent with a role for OSM-11 in initial cell fate specification.
0030
Because the predicted peptide sequence of OSM-11 contains a signal peptide, we tested whether OSM-11 is a secreted protein. When an osm-11 cDNA was expressed in Drosophila S2 tissue culture cells, OSM-11 protein accumulates in the media and not in cells (Figure 5A), consistent with OSM-11 acting in vivo as a soluble protein in the extracellular milieu.
0411
(A) Western blot of conditioned media from Drosophila S2 cells containing an OSM-11 cDNA expression construct or empty vector.
0423
We found that OSM-11 also interacted with LIN-12 extracellular EGF repeats 1 through 6 (Figure 8); OSM-11 did not interact with DSL-1 or LAG-2 ligands. We also confirmed previous studies [50] in which murine DLK1 EGF repeats 1 and 2 containing the DOS motif interacted specifically with murine Notch1 EGF repeats 12 and 13 in the same two-hybrid assay format (unpublished data).
0030
Dionne, et al., unpublished data), we also found that OSM-11 activates both LIN-12 and germline proliferation defective-1 (GLP-1) in the adult nervous system to regulate behavior.

0889
Dionne, et al., unpublished data), we also found that OSM-11 activates both LIN-12 and germline proliferation defective-1 (GLP-1) in the adult nervous system to regulate behavior.
0411
OSM-11 mRNA localization by in situ hybridization is consistent with expression in VPCs and hypoderm in young larvae and in seam cells in adult animals (see NEXTDB, http://nematode.lab.nig.ac.jp/db2/ShowCloneInfo.php?clone=59g10; Y.
0018
The osm-11 cDNA clone was obtained by PCR from the Vidal laboratory ORFeome cDNA library [94] and agrees exactly with the predicted sequence in WormBase and at NCBI. osm-11 cDNA constructs used herein for rescue contained the unc-54 3′ UTR, whereas genomic rescue clones contained the osm-11 3′ UTR.
0030
Cell adhesion and migration are coordinated by dynamic membrane-associated protein assemblies called focal adhesions (FAs). With over 150 components, FAs play a crucial role in transmitting signals bidirectionally across the cell membrane and provide a physical link between integrin receptors and the actin cytoskeleton (Zaidel-Bar et al., 2007).

0889
With over 150 components, FAs play a crucial role in transmitting signals bidirectionally across the cell membrane and provide a physical link between integrin receptors and the actin cytoskeleton (Zaidel-Bar et al., 2007).

0030
LD-binding proteins include the kinases FAK (Turner et al., 1999), ILK (Nikolopoulos and Turner, 2001), and PAK3 (Hashimoto et al., 2001), the Arf-GAP PKL (Turner et al., 1999), the antiapoptotic protein Bcl-2 (Sheibani et al., 2008), the papillomavirus oncoprotein E6 (Tong et al., 1997), and the cytoskeletal proteins vinculin (Turner et al., 1999) and α-parvin (Nikolopoulos and Turner, 2000). While several of these proteins, such as FAK, PKL, and vinculin, bind LD motifs through parallel α-helical bundles (Gao et al., 2004; Hoellerer et al., 2003; Liu et al., 2002; Scheswohl et al., 2008; Schmalzigaug et al., 2007; Zhang et al., 2008), others employ topologically distinct modules, such as the kinase domain of ILK (Tu et al., 2001), the BH4 domain of Bcl-2 (Sorenson, 2004), and the calponin homology (CH) domain of α-parvin (Nikolopoulos and Turner, 2000).

0889
LD-binding proteins include the kinases FAK (Turner et al., 1999), ILK (Nikolopoulos and Turner, 2001), and PAK3 (Hashimoto et al., 2001), the Arf-GAP PKL (Turner et al., 1999), the antiapoptotic protein Bcl-2 (Sheibani et al., 2008), the papillomavirus oncoprotein E6 (Tong et al., 1997), and the cytoskeletal proteins vinculin (Turner et al., 1999) and α-parvin (Nikolopoulos and Turner, 2000). While several of these proteins, such as FAK, PKL, and vinculin, bind LD motifs through parallel α-helical bundles (Gao et al., 2004; Hoellerer et al., 2003; Liu et al., 2002; Scheswohl et al., 2008; Schmalzigaug et al., 2007; Zhang et al., 2008), others employ topologically distinct modules, such as the kinase domain of ILK (Tu et al., 2001), the BH4 domain of Bcl-2 (Sorenson, 2004), and the calponin homology (CH) domain of α-parvin (Nikolopoulos and Turner, 2000).
0030
α-parvin (Olski et al., 2001), also known as actopaxin (Nikolopoulos and Turner, 2000) or CH-ILKBP (Tu et al., 2001), is part of the ILK signaling complex (Tu et al., 2001), plays an essential role in adhesion-dependent PKB/Act activation (Fukuda et al., 2003) and in the regulation of actin organization (LaLonde et al., 2005) and Rac activation (LaLonde et al., 2005; Zhang et al., 2004). It belongs to the highly conserved parvin family that shares a common architecture composed of a variable N-terminal region followed by two CH domains (Olski et al., 2001).

0889
α-parvin (Olski et al., 2001), also known as actopaxin (Nikolopoulos and Turner, 2000) or CH-ILKBP (Tu et al., 2001), is part of the ILK signaling complex (Tu et al., 2001), plays an essential role in adhesion-dependent PKB/Act activation (Fukuda et al., 2003) and in the regulation of actin organization (LaLonde et al., 2005) and Rac activation (LaLonde et al., 2005; Zhang et al., 2004). It belongs to the highly conserved parvin family that shares a common architecture composed of a variable N-terminal region followed by two CH domains (Olski et al., 2001).

0030
However, the primary sequences of both CH domains of α-parvin are highly diverged from the typical type-1 and type-2 CH domains found in ABDs; they have therefore been classified as type-4 and type-5 CH domains (Gimona et al., 2002). An ability to recognize paxillin LD motifs has only been reported for the type-5 CH domains (Nikolopoulos and Turner, 2000; Yoshimi et al., 2006).

0889
However, the primary sequences of both CH domains of α-parvin are highly diverged from the typical type-1 and type-2 CH domains found in ABDs; they have therefore been classified as type-4 and type-5 CH domains (Gimona et al., 2002). An ability to recognize paxillin LD motifs has only been reported for the type-5 CH domains (Nikolopoulos and Turner, 2000; Yoshimi et al., 2006).
0030
While this manuscript was in preparation, an independent study reported the NMR structure of the C-terminal CH domain of α-parvin in complex with a 10-residue peptide derived from the paxillin LD1 motif (Wang et al., 2008).

0114
While this manuscript was in preparation, an independent study reported the NMR structure of the C-terminal CH domain of α-parvin in complex with a 10-residue peptide derived from the paxillin LD1 motif (Wang et al., 2008).

0889
While this manuscript was in preparation, an independent study reported the NMR structure of the C-terminal CH domain of α-parvin in complex with a 10-residue peptide derived from the paxillin LD1 motif (Wang et al., 2008).
0081
Three cocrystal structures of α-parvin-CHC with 20-residue peptides representing LD1, LD2, and LD4 allow us to characterize the interaction at atomic resolution and to highlight binding-induced conformational changes in α-parvin-CHC. This analysis together with NMR studies of a spin-labeled LD1 peptide supports the surprising finding that LD motifs can associate with a single binding site bidirectionally.
0114
We initially attempted to solve the crystal structure of full-length human α-parvin, but no crystals could be obtained.

0030
This fragment includes residues 242–372 and thereby spans the entire C-terminal CH domain as defined by SMART (Schultz et al., 1998) and a portion of the inter-CH domain linker (Figure 1A).

0889
This fragment includes residues 242–372 and thereby spans the entire C-terminal CH domain as defined by SMART (Schultz et al., 1998) and a portion of the inter-CH domain linker (Figure 1A).

0114
The crystal structure of α-parvin-CHC at 1.05 Å resolution was determined by molecular replacement using an ensemble of homologous type-1 CH domain structures as search model.

0114
The refined structural model includes α-parvin residues 246–372 (molecule A) or 247–372 (molecule B) and represents the first high-resolution crystal structure of a type-5 CH domain (Figure 1B).

0030
In spite of very low levels of sequence conservation (≤26% identity) compared to canonical type-1 CH domains (see Figure S1 available online), α-parvin-CHC exhibits a typical CH domain core composed of four main α-helices (αA, αC, αE, and αG; nomenclature from Djinovic Carugo et al., 1997), which are connected by loops and shorter helical elements (αD and αF).

0889
In spite of very low levels of sequence conservation (≤26% identity) compared to canonical type-1 CH domains (see Figure S1 available online), α-parvin-CHC exhibits a typical CH domain core composed of four main α-helices (αA, αC, αE, and αG; nomenclature from Djinovic Carugo et al., 1997), which are connected by loops and shorter helical elements (αD and αF).

0114
However, conformational differences in this region may not be significant, since the C/E-loop structure varies between α-parvin-CHC molecules both in the same and different crystal forms and is involved in crystal packing (data not shown).
0030
However, we previously demonstrated that LD-binding to the FAT domain of FAK does not reside in a local peptide sequence such as the PBS (Hoellerer et al., 2003) and thus investigated the interaction of α-parvin-CHC with LD motifs using solution NMR.

0889
However, we previously demonstrated that LD-binding to the FAT domain of FAK does not reside in a local peptide sequence such as the PBS (Hoellerer et al., 2003) and thus investigated the interaction of α-parvin-CHC with LD motifs using solution NMR.
0004
As measured by NMR, none of these substitutions substantially altered the binding affinity for α-parvin-CHC, indicating that the predicted electrostatic contacts are energetically neutral under the assay conditions (50 mM sodium phosphate and 100 mM NaCl [pH 6.9]) used.

0081
Likewise, comparison of the chemical shift perturbations imposed by oppositely aligned peptides on backbone amide resonances of α-parvin-CHC (Figure 2) is inconclusive. We note that the peptide side-chains contacting the LD-binding site of α-parvin-CHC are similar in either orientation (Figure S4), as is the conformational rearrangement induced by peptide binding, so that the perturbation patterns of either unidirectional binding mode (or a bidirectional mixture) may be indistinguishable.
0077
This phenomenon is also illustrated in Figure 5, using two diagnostic NMR signals as examples: the resonance originating from residue 257 is predicted to experience strong PRE in the forward mode (calculated distance from spin-label 10.7 Å) but should only be weakly affected in the backward mode (calculated distance 20.2 Å).
0081
Our results demonstrate that full-length α-parvin contains a single LD binding site, formed by the N-linker helix, helix A, and helix G of the C-terminal type-5 CH domain. This binding site is consistent with the one described in a recent solution NMR structure of α-parvin-CHC in complex with an LD1 peptide (Wang et al., 2008).

0889
This binding site is consistent with the one described in a recent solution NMR structure of α-parvin-CHC in complex with an LD1 peptide (Wang et al., 2008).

0030
This binding site is consistent with the one described in a recent solution NMR structure of α-parvin-CHC in complex with an LD1 peptide (Wang et al., 2008).

0030
The significantly lower value of ∼1.2 mM recently measured (Wang et al., 2008) might be due to the restricted length of the peptide (10 versus 20 residues) used in their study.

0889
The significantly lower value of ∼1.2 mM recently measured (Wang et al., 2008) might be due to the restricted length of the peptide (10 versus 20 residues) used in their study.
0030
The FAT domain of FAK contains two independent LD binding sites, which bind equally well to LD2 and LD4 in vitro (Hoellerer et al., 2003; Gao et al., 2004), but it also interacts to some degree with the other LD motifs (M.K.H., unpublished results). Recent NMR studies (Zhang et al., 2008) have also demonstrated that the single LD binding site of PKL/GIT1 binds to both LD4 (Turner et al., 1999) and LD2. Taken together, these observations suggest that paxillin LD motifs are promiscuous protein interaction modules.

0889
The FAT domain of FAK contains two independent LD binding sites, which bind equally well to LD2 and LD4 in vitro (Hoellerer et al., 2003; Gao et al., 2004), but it also interacts to some degree with the other LD motifs (M.K.H., unpublished results). Recent NMR studies (Zhang et al., 2008) have also demonstrated that the single LD binding site of PKL/GIT1 binds to both LD4 (Turner et al., 1999) and LD2.
0030
On the basis of our results for α-parvin, we can make two general predictions with respect to LD recognition by CH domains: First, as noted previously (Wang et al., 2008), we propose that the presence of an N-linker helix is a prerequisite for the interaction with LD motifs.

0889
On the basis of our results for α-parvin, we can make two general predictions with respect to LD recognition by CH domains: First, as noted previously (Wang et al., 2008), we propose that the presence of an N-linker helix is a prerequisite for the interaction with LD motifs.

0030
Interestingly, γ-parvin was shown to associate with paxillin in vivo (Yoshimi et al., 2006). Available experiments with β-parvin, however, have proved negative to date (Yamaji et al., 2004), although this protein is more similar in sequence to α-parvin than is γ-parvin (Figure 1C).

0889
Interestingly, γ-parvin was shown to associate with paxillin in vivo (Yoshimi et al., 2006). Available experiments with β-parvin, however, have proved negative to date (Yamaji et al., 2004), although this protein is more similar in sequence to α-parvin than is γ-parvin (Figure 1C).
0030
The structure of α-parvin-CHC is topologically distinct from other LD-binding domains, such as the FAT domain of FAK; it associates with a single LD motif across three oblique helices, whereas FAT accommodates two LD motifs in parallel fashion on opposite sites of its 4-helical bundle (Hoellerer et al., 2003).

0114
The structure of α-parvin-CHC is topologically distinct from other LD-binding domains, such as the FAT domain of FAK; it associates with a single LD motif across three oblique helices, whereas FAT accommodates two LD motifs in parallel fashion on opposite sites of its 4-helical bundle (Hoellerer et al., 2003).

0889
The structure of α-parvin-CHC is topologically distinct from other LD-binding domains, such as the FAT domain of FAK; it associates with a single LD motif across three oblique helices, whereas FAT accommodates two LD motifs in parallel fashion on opposite sites of its 4-helical bundle (Hoellerer et al., 2003).

0030
Notably, the role of electrostatic contacts in LD recognition might differ between the two proteins: while substitutions of the conserved aspartates (D+1) of LD2 and LD4 to alanine were found to abolish the interaction of N-terminal paxillin fragments with FAT (Brown et al., 1996; Scheswohl et al., 2008; Thomas et al., 1999), our study and others (Wang et al., 2008) show that the equivalent substitutions in the isolated LD1 peptides do not significantly perturb α-parvin-CHC binding in vitro.

0096
Notably, the role of electrostatic contacts in LD recognition might differ between the two proteins: while substitutions of the conserved aspartates (D+1) of LD2 and LD4 to alanine were found to abolish the interaction of N-terminal paxillin fragments with FAT (Brown et al., 1996; Scheswohl et al., 2008; Thomas et al., 1999), our study and others (Wang et al., 2008) show that the equivalent substitutions in the isolated LD1 peptides do not significantly perturb α-parvin-CHC binding in vitro.

0107
Notably, the role of electrostatic contacts in LD recognition might differ between the two proteins: while substitutions of the conserved aspartates (D+1) of LD2 and LD4 to alanine were found to abolish the interaction of N-terminal paxillin fragments with FAT (Brown et al., 1996; Scheswohl et al., 2008; Thomas et al., 1999), our study and others (Wang et al., 2008) show that the equivalent substitutions in the isolated LD1 peptides do not significantly perturb α-parvin-CHC binding in vitro.

0889
Notably, the role of electrostatic contacts in LD recognition might differ between the two proteins: while substitutions of the conserved aspartates (D+1) of LD2 and LD4 to alanine were found to abolish the interaction of N-terminal paxillin fragments with FAT (Brown et al., 1996; Scheswohl et al., 2008; Thomas et al., 1999), our study and others (Wang et al., 2008) show that the equivalent substitutions in the isolated LD1 peptides do not significantly perturb α-parvin-CHC binding in vitro.
0030
Bidirectional protein-ligand interactions are widespread among PPII (poly-proline type-2) helical ligands, as this conformation has a pseudo C2-rotational symmetry perpendicular to its long axis (Ball et al., 2005).

0889
Bidirectional protein-ligand interactions are widespread among PPII (poly-proline type-2) helical ligands, as this conformation has a pseudo C2-rotational symmetry perpendicular to its long axis (Ball et al., 2005).

0030
To our knowledge, this phenomenon has only been reported for the following: the recruitment of the histone deactelylase-associated Sin3 corepressor by the HBP1 and Mad1 repressors, respectively (Brubaker et al., 2000; Spronk et al., 2000; Swanson et al., 2004), the association of FAT with paxillin LD motifs and the endocytosis motif of CD4 (Garron et al., 2008), and the interaction between AKAP-IS and the RIIa subunit of PKA (Gold et al., 2006).

0107
To our knowledge, this phenomenon has only been reported for the following: the recruitment of the histone deactelylase-associated Sin3 corepressor by the HBP1 and Mad1 repressors, respectively (Brubaker et al., 2000; Spronk et al., 2000; Swanson et al., 2004), the association of FAT with paxillin LD motifs and the endocytosis motif of CD4 (Garron et al., 2008), and the interaction between AKAP-IS and the RIIa subunit of PKA (Gold et al., 2006).

0889
To our knowledge, this phenomenon has only been reported for the following: the recruitment of the histone deactelylase-associated Sin3 corepressor by the HBP1 and Mad1 repressors, respectively (Brubaker et al., 2000; Spronk et al., 2000; Swanson et al., 2004), the association of FAT with paxillin LD motifs and the endocytosis motif of CD4 (Garron et al., 2008), and the interaction between AKAP-IS and the RIIa subunit of PKA (Gold et al., 2006).
0077
Small changes in a variety of factors, such as experimental conditions or the length of the paxillin fragment studied, might influence the observed binding orientation in NMR, where a mean is detected.

0030
This could explain a similar PRE experiment performed with a shorter LD1 variant (residues 3–13), which was interpreted as unidirectional forward-type binding and used to restrain the solution structure of the LD1/α-parvin-CHC complex (Wang et al., 2008).

0889
This could explain a similar PRE experiment performed with a shorter LD1 variant (residues 3–13), which was interpreted as unidirectional forward-type binding and used to restrain the solution structure of the LD1/α-parvin-CHC complex (Wang et al., 2008).
0030
Although α−parvin has been shown to bind to F-actin with an affinity comparable to other CH domain-containing actin-binding proteins (Nikolopoulos and Turner, 2000; Olski et al., 2001), the canonical model of actin recognition may not apply to this protein. Usually CH domains bind F-actin through a tandem array of an N-terminal type-1 CH domain containing two actin-binding sites (ABS1 and ABS2) and a C-terminal type-2 CH domain contributing a third actin-binding site (ABS3) (Gimona et al., 2002).

0889
Although α−parvin has been shown to bind to F-actin with an affinity comparable to other CH domain-containing actin-binding proteins (Nikolopoulos and Turner, 2000; Olski et al., 2001), the canonical model of actin recognition may not apply to this protein. Usually CH domains bind F-actin through a tandem array of an N-terminal type-1 CH domain containing two actin-binding sites (ABS1 and ABS2) and a C-terminal type-2 CH domain contributing a third actin-binding site (ABS3) (Gimona et al., 2002).

0081
Usually CH domains bind F-actin through a tandem array of an N-terminal type-1 CH domain containing two actin-binding sites (ABS1 and ABS2) and a C-terminal type-2 CH domain contributing a third actin-binding site (ABS3) (Gimona et al., 2002).

0114
Interestingly, the crystal structure of α−parvin-CHC presented here shows that part of the putative ABS2 region is obstructed by the N-linker helix.
0030
CH domains in intact ABDs often interact across a sizeable interface, which can modulate their ability to interact with F-actin (Gimona et al., 2002). It remains to be established whether such intramolecular interactions occur to regulate the association of α-parvin with F-actin and other binding partners, such as paxillin, TESK1 (LaLonde et al., 2005), and ILK (Tu et al., 2001).

0889
CH domains in intact ABDs often interact across a sizeable interface, which can modulate their ability to interact with F-actin (Gimona et al., 2002). It remains to be established whether such intramolecular interactions occur to regulate the association of α-parvin with F-actin and other binding partners, such as paxillin, TESK1 (LaLonde et al., 2005), and ILK (Tu et al., 2001).
0030
Several ABD containing proteins are capable of forming dimers, thereby cross-linking actin filaments (Gimona et al., 2002). On the basis of gel filtration experiments with the N-terminal CH domain of α-parvin, Wang et al.

0889
Several ABD containing proteins are capable of forming dimers, thereby cross-linking actin filaments (Gimona et al., 2002). On the basis of gel filtration experiments with the N-terminal CH domain of α-parvin, Wang et al.

0071
On the basis of gel filtration experiments with the N-terminal CH domain of α-parvin, Wang et al.
0107
Taken together, we have presented a comprehensive structural characterization of the interaction between paxillin LD motifs and α-parvin, which has revealed a surprising degree of promiscuity, both in terms of LD motif selectivity and binding directionality.
0004
α-parvin-CHC was purified as follows: cleared cell lysate (in 75 mM Tris [pH 8.0], 200 mM NaCl, 5 mM β-mercaptoethanol, 0.4% Triton X-100, 2 mM EDTA, 5 mM benzamidine, and protease inhibitor cocktail [Roche]) was applied to glutathione sepharose 4B (GE Healthcare) in binding buffer (20 mM Tris [pH 8.0], 150 mM NaCl, and 2 mM DTT) washed with 20 mM Tris (pH 8.0), 1 M NaCl, 2 mM DTT, 2 mM EDTA, and 5 mM benzamidine, and was eluted with 50 mM glutathione in binding buffer (pH 8.0). After cleavage with recombinant human rhinovirus 3C-protease, the sample was subjected to size exclusion chromatography (Superdex 75, GE Healthcare) in 25 mM Tris (pH 8.0), 150 mM NaCl, 2 mM DTT, and 2 mM EDTA.

0071
After cleavage with recombinant human rhinovirus 3C-protease, the sample was subjected to size exclusion chromatography (Superdex 75, GE Healthcare) in 25 mM Tris (pH 8.0), 150 mM NaCl, 2 mM DTT, and 2 mM EDTA.
0004
To purify full-length α-parvin, cleared cell lysate in 200 mM potassium phosphate (pH 8.0), 10 mM NaCl, 5 mM β-mercaptoethanol, 0.4% Triton X-100, 5 mM benzamidine, 2 mM EDTA, and protease inhibitor cocktail (Roche) was applied to glutathione sepharose 4B (GE Healthcare), washed with 200 mM potassium phosphate, 10 mM NaCl, and 4 mM DTT, and α−parvin was released by cleavage with recombinant human rhinovirus 3C-protease at 4°C over night. After elution with 200 mM potassium phosphate, 10 mM NaCl, 4 mM DTT, and 2.5% glycerol, the protein was dialyzed into 25 mM potassium phosphate (pH 8.0), 1.5 mM NaCl, 2 mM DTT, and 2.5% (v/v) glycerol for subsequent anion exchange chromatography (MonoQ, GE Healthcare) and gradient elution with 250 mM potassium phosphate (pH 8.0), 15 mM NaCl, 250 mM KCl, 2 mM DTT, and 2.5% glycerol.
0053
For fluorescence anisotropy studies, LD1 with 5-carboxyfluorescein (5-FAM) attached to the ɛ-amino group of the C-terminal lysine was used.
0030
Manual model building was performed with COOT (Emsley and Cowtan, 2004), and refinement was performed with REFMAC5 (Murshudov et al., 1997); occupancies of alternate conformers were refined with PHENIX (Afonine et al., 2007).

0889
Manual model building was performed with COOT (Emsley and Cowtan, 2004), and refinement was performed with REFMAC5 (Murshudov et al., 1997); occupancies of alternate conformers were refined with PHENIX (Afonine et al., 2007).
0004
For PRE measurements, 1H-15N HSQC experiments were recorded on 230 μM 15N-enriched α-parvin-CHC in the presence of 250 μM PROXYL-labeled LD1 peptide in 50 mM sodium phosphate and 100 mM NaCl (pH 6.9) in the absence and presence of 5 mM ascorbate at 25°C.

0030
For the simulation of PRE effects in both unidirectional binding modes, the crystal structures of α-parvin-CHC bound to LD1 and LD4 were protonated with the program PDB 2PQR (Dolinsky et al., 2004).

0889
For the simulation of PRE effects in both unidirectional binding modes, the crystal structures of α-parvin-CHC bound to LD1 and LD4 were protonated with the program PDB 2PQR (Dolinsky et al., 2004).

0030
The resulting values were used as approximation of the distances, r, of the unpaired electron of the PROXYL moiety to the backbone amide protons and to derive residue-specific PRE-values, I/I0, according to (Jain et al., 2001; Johnson et al., 1999) (for details, see Figure S5).

0889
The resulting values were used as approximation of the distances, r, of the unpaired electron of the PROXYL moiety to the backbone amide protons and to derive residue-specific PRE-values, I/I0, according to (Jain et al., 2001; Johnson et al., 1999) (for details, see Figure S5).
0114
The binding orientation of LD1 seen in the crystal structure is denoted “forward.”.
0019
Chromatin immunoprecipitation (ChIP) experiments show that Ctk1 associates with RNApII throughout elongation (Kim et al, 2004a).
0676
Consistent with this, when ChIP was carried out using strains deleted for the other subunits of the Ctk1 complex, including Ctk2 (cyclin subunit) or Ctk3 (accessory factor), TBP occupancy was again increased in the coding region of PMA1 (Supplementary Figure S1).
0030
The upstream activating sequence (UAS; Figure 1A) of the three genes used in our studies (PMA1, ADH1, and PYK1) contain a binding site for repressor-activator protein 1 (Rap 1), a transactivator for many promoters in exponentially growing cells (Rao et al, 1993; Lieb et al, 2001).

0426
The upstream activating sequence (UAS; Figure 1A) of the three genes used in our studies (PMA1, ADH1, and PYK1) contain a binding site for repressor-activator protein 1 (Rap 1), a transactivator for many promoters in exponentially growing cells (Rao et al, 1993; Lieb et al, 2001). The UAS from all three genes show strong Rap1 cross-linking, and this signal remains appropriately localized in ctk1Δ cells (Figure 3C).
0081
As serine 2 phosphorylation is known to be required for cotranscriptional 3′-end processing (Ahn et al, 2004), we also tested for the polyadenylation factor Rna14 as a positive control (Figure 4B).
0081
Two mutant alleles were employed: ctk1-D324N is a catalytic site mutant that shows no kinase activity in vitro and confers a slow-growth, cold-sensitive phenotype in vivo, whereas ctk1-T338A is a weakened kinase mutated in the T-loop threonine that accepts an activating phosphorylation (Ostapenko and Solomon, 2003).
0411
Cells deficient in Ctk1 were deleted at the genomic locus but covered by pCTK1, a low-copy URA3 plasmid containing WT Ctk1. Before our analyses, cells were grown overnight on YPD, streaked onto 5-FOA to select for those who lost the covering plasmid (confirmed by slow growth) and immediately used for subsequent experiments.

0426
Before our analyses, cells were grown overnight on YPD, streaked onto 5-FOA to select for those who lost the covering plasmid (confirmed by slow growth) and immediately used for subsequent experiments.
0019
Chromatin immunoprecipitation was performed as described previously (Ahn et al, 2004).

0019
For Rpb3 immunoprecipitation, antibody (1Y26, NeoClone) was preincubated with protein G-sepharose (Amersham).

0019
The signal for each specific gene primer in the immunoprecipitation was then divided by this ratio to convert the signal to normalized units. This value was divided by the immunoprecipitation signal of the non-transcribed control product to determine the fold enrichment of the ChIP over background signals.
0004
Dynabeads M-280 Streptavidin (Dynal) were concentrated with a magnetic particle concentrator (MPC) (Dynal) and washed twice in Buffer T (10 mM Tris (pH 7.5), 1 mM EDTA, 1 M NaCl).
0004
The supernatant was then removed and incubated with protein G-sepharose (Amersham) overnight at 4°C with anti-Rpb3 antibody in buffer E (20 mM HEPES (pH 8), 350 mM NaCl, 10% glycerol, 0.1% Tween 20).
0019
The abnormal cross-linking of basal transcription factors in ctk1Δ cells is confirmed by independent immunoprecipitation of TAP-tagged TFIIF subunits.

0019
IgG agarose was used for immunoprecipitation of TAP-tagged proteins.
0019
Protein A- or protein G-sepharose was used for TBP or Rpb3 immunoprecipitation and rabbit IgG agarose was used for RNA14–TAP pull-down.
0412
(D) Efficiency of scaffold formation was measured by quantitation of band intensities from lane 3 or 7 in WT and ctk1Δ cells (ImageJ v1.32).

0006
To measure the association between RNApII and the basal transcription factors after termination, the supernatant was removed after incubation with NTPs for 2 min and immunoprecipitated with anti-Rpb3 antibody.
