0006
The MACS epitope tagged protein isolation kit (Miltenyi Biotec) was used to precipitate candidate partner proteins via the HA epitope. Eluted protein samples were split (1/3 and 2/3), size-fractionated in parallel on two polyacrylamide SDS-gels (concentration adjusted to protein size), electroblotted onto Immobilon-P membrane (Millipore) and subjected to antibody detection. HA-tagged (1/3 aliquot) partner proteins were identified using an anti-HA horseradish peroxidase (HRP)-conjugated antibody (clone 3F10; Roche 2012819). The co-precipitated 6× his-tagged STM protein was visualized by a primary penta-His mouse antibody [α-(H)5; Qiagen 34660] and secondary HRP-coupled goat anti-mouse IGG (Dianova 115-035-062). Peroxidase activity was detected non-radioactively via chemiluminescence (ECL PLUS kit; Amersham Biosciences) and documented on Kodak-X-omat AR films. 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. GFP fusion proteins were detected after transfer to Immobilon-P membrane (Millipore) by a mouse monoclonal anti-GFP HRP-conjugated antibody (IgG1; Milteny Biotec 130-091-833).
0809
As a potential transcription factor, STM was expected to direct GFP fluorescence into the plant cell nucleus. However, both fusion proteins, GFP-STM and STM-GFP remained in the cytoplasm (Figure 1B and C).
0007
In addition to the interactions in yeast, we performed co-immunoprecipitation experiments with epitope-tagged full-length proteins. The HA-tagged BLH proteins shown in Figure 2B were used to co-precipitate epitope-tagged STM-His (Figure 2C). The detection of the STM-His protein was strictly dependent on the presence of BLH partner proteins. 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.
0071
Various forms of gE and the gE-gI heterodimer were subcloned, expressed, and purified from baculovirus-infected insect cell supernatants by nickel affinity and/or IgG affinity and gel-filtration chromatography as described previously [12]. Two recombinant forms of the Fc fragment of IgG1, wtFc and heterodimeric Fc, which contain two and one gE-gI binding sites, respectively, were also produced in CHO cells and purified as described previously [12].
0114
Crystallization trials were conducted for various forms of CgE, gE, and gE-gI (including CgE [residues 213–390], gE [residues 21–419], gE2 [residues 21–390], gE-gI [gE plus gI residues 21–266], gE2-gI2A [gE2 plus gI residues 21–208], and gE2-gI2B [gE2 plus gI residues 21–201]) both alone and complexed with wtFc or heterodimeric Fc. The only isolated protein to crystallize was CgE (described above), and the only complex of the six possible gE-gI/Fc complexes that crystallized was one that contained gE residues 21–419 and gI residues 21–266 and wtFc (residues 223–447). The complex crystals grew from drops containing a 2:1 molar ratio of gE-gI and wtFc mixed with an equal volume of well solution (0.1 M MES [pH 6.0] or 0.1 M HEPES [pH 7.0] and 0.9–1.1 M sodium malonate), resulting in a final pH of approximately 7.5. Microseeding increased the reproducibility of crystal growth.
0077
(E) Superposition of the structure of the RIM2α ZF domain observed in the heterodimer (blue) and its solution structure determined in isolation by NMR spectroscopy (red) [20].
0018
Mutagenesis of residue R1161 (in α2AA and α2AAKYA, removing another conserved charged residue) to alanine significantly reduced α2 tail association with Rab21 judged by yeast mating tests and immunoprecipitations (Fig. 1, D and E). In addition, F1159A mutation (α2AARA, possibly creating a conformational change) showed reduced association in the yeast assays, whereas in immunoprecipitations the reduction was not significant. These data on the mutant integrins suggest that the conformation of the cytoplasmic domain and residue R1161 of α2-integrin are important for the Rab21 association.
0019
Mutagenesis of residue R1161 (in α2AA and α2AAKYA, removing another conserved charged residue) to alanine significantly reduced α2 tail association with Rab21 judged by yeast mating tests and immunoprecipitations (Fig. 1, D and E). In addition, F1159A mutation (α2AARA, possibly creating a conformational change) showed reduced association in the yeast assays, whereas in immunoprecipitations the reduction was not significant. These data on the mutant integrins suggest that the conformation of the cytoplasmic domain and residue R1161 of α2-integrin are important for the Rab21 association.
0006
(A) Nontransfected MDA-MB-231 cells were surface labeled with cleavable biotin and lysed immediately or allowed to internalize cell surface proteins for 15 min. Immunoprecipitations (IPs) were performed as indicated, and the Rab coprecipitating proteins were detected first with anti-biotin antibody followed by stripping and reprobing with anti–β1-integrin antibody. The quantification shows the amount of coprecipitated biotinylated protein relative to total precipitated integrin (means ± range; n = 2).
0029
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. 3 D). A further shift in the endogenous integrin pool (to fractions 5–11) was observed upon expression of GFP-Rab21GTP, and GFP-Rab21GDP was also observed in the denser fractions (Fig. 3 D). 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. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200509019/DC1). This data, together with the abundance of integrin vesicles observed in Rab21-expressing cells (Fig. 2), suggests that Rab21 targets integrins to the endocytic fraction in human cells.
0416
The effect of depleting RCK/p54 on localization of Ago2 was next examined by immunofluorescence analysis of HeLa cells expressing Myc-Ago2 and siRNAs against RCK/p54. As shown in Figure 4B, 4C, 4F, and 4G, depleting RCK/p54 disrupted the cellular P-body structures. In these P-body-deficient cells, Ago2 proteins were diffused throughout the cytoplasm and no longer accumulated at specific foci (Figure 4A and 4E). This cytoplasmic redistribution of Ago2 suggests that its localization to P-bodies is driven in mammalian cells by factors such as RCK/p54.
0416
We next visualized Lsm1 and Ago2 by immunofluorescence using antibodies against Lsm1 and Myc tag and found that Lsm1 and Ago2 co-localized in P-bodies (Figure 5B). To determine the status of P-body structures and RISC localization in cells after Lsm1 knockdown, Lsm1-depleted cells were analyzed by immunofluorescence. We found that P-body structures were drastically disrupted, Ago2 was diffused throughout the cytoplasm, and Lsm1 and Ago2 were minimally co-localized (Figure 5B). These results show that P-body structures were drastically disrupted and Ago2 was diffuse throughout the cytoplasm when Lsm1 was depleted by RNAi.
0004
To dissect and understand the relationship between RNAi function and P-bodies, we affinity-purified RISC using Myc-Ago2 and expression vectors of the YFP-tagged P-body proteins, Lsm1, RCK/p54, Dcp2, and eIF4E. Ago2 interacted with these various P-body components in ways that were RNA-dependent or RNA-independent (Figure 1A).
0019
Ago1 and RCK/p54 immunoprecipitated with Ago2 after RNase A treatment of HeLa cell extracts, suggesting that these proteins directly interact with Ago2. Interestingly, RCK/p54, Ago1, and Ago2 were also identified as a component of active RISC programmed with siRNA or miRNA and purified by biotin affinity to streptavidin-conjugated magnetic beads (Figures 2 and 3). We examined the P-body localization of Ago2 with Lsm1 and RCK/p54 by co-expressing YFP-tagged Lsm1 and RCK/p54 with CFP-Ago2. Interestingly, overexpressing YFP-RCK/p54 in HeLa cells increased the number of P-bodies (from ∼ 8 to ∼ 20 foci/cell). The number of P-bodies containing CFP-Ago2 also increased (Figure 1B). These results suggested a functional relationship between RCK/p54-Ago interactions and their localization to P-bodies.
0007
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. Myc-tagged proteins were precipitated by incubating overnight with polyclonal rabbit anti-Myc antibodies directly conjugated to agarose beads (Santa Cruz Biotech, California, United States).
0004
(A) Affinity-purified miRISCs associated with PCK/p54 retain cleavage activity. To purify miRISC associated with RCK/p54, magnetic protein A beads coupled with rabbit IgG, rabbit anti-Ago2, or rabbit anti-RCK/p54 antibodies were incubated with HeLa cytoplasmic extracts. 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. Cleavage products were resolved on 6% denaturing polyacrylamide gels. CE, cytoplasmic extract; PM, perfect match; MM, mismatch.
0019
(C) RCK/p54 interacts with Myc-Ago2 in Lsm1-depleted cells. HeLa cells were transfected for 48 h with Myc-Ago2 and control siRNA or siRNA against Lsm1, TCEs were prepared, and Myc-Ago2 was immunoprecipitated from an aliquot of TCE.
0006
In a converse experiment, ephrin-B1 was co-immunoprecipitated with an anti-Cx43 antibody (Figure 6Cb).
0096
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.
0006
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. For immunoprecipitations: rat Bim antibody (Oncogene, San Diego, California, United States) was coated to goat-anti-rat immunoglobulin-agarose; rabbit Puma antibody was coated to protein A-sepharose; mouse NHE-1 antibody was coated to protein G-sepharose; rabbit Bcl-xL antibody was coated to goat-anti-rabbit immunoglobulin-agarose. Lysates were precleared with the appropriate agarose.
0019
(A) Bim binds to the native (Asn-Asn) but not deamidated forms of Bcl-xL. Wild-type (C57BL/6) thymocytes (1.5 × 107) were exposed to 5 Gy irradiation (IR) and then maintained in culture for the times shown, after which cells were lysed and either separated as whole cell lysates (WCL) or as Bim immunoprecipitates, followed by immunoblotting for either Bcl-xL or for Bim. Bim migrates as “extra-long” (EL) or “long” (L) forms.
0096
To assess which of the PDE4 subtypes contribute to the activity recovered in the β1AR IP, cardiomyocytes deficient in PDE4A, PDE4B, and PDE4D were subjected to pull-down experiments.
0014
Importantly, upon stimulation with the adenylyl cyclase activator, Forskolin, all PDE4D isoforms show the same increase in activity in both cell types (Figure 5C), suggesting that loss of one βAR subtype or the other has not perturbed overall cAMP signaling. It also demonstrates that the spatial dimension of cAMP signaling is lost when generalized adenylyl cyclase activation is induced with Forskolin.
0007
(B) Co-IP of β1AR and PDE activity from cardiomyocytes deficient in PDE4A, PDE4B, or PDE4D, and wild-type controls.
0007
(A, B) Co-IP of exogenous β1AR and Myc-tagged PDE4D splice variants expressed in HEK293 cells. The efficiency with which β1AR pulls down the different PDE4D splice variants is quantified in (B). (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). (D, E) PDE4D3, and Flag-tagged receptors, β1AR and β2AR, were affinity purified after baculovirus expression (see Supplementary Figure 1). Purified PDE and (βARs) were then combined and the βARs immunoprecipitated.
0019
It remains to be determined to what extent PDE4D9, which is activated after both β1AR and β2AR stimulation (Figure 5) and which also showed interaction with β1AR in co-IPs of exogenous proteins (Figure 3A and B), can substitute for interaction with the βARs in vivo.
0017
The greater binding affinity of AIRE–PHD1 for H3K4me0 peptides was confirmed by both tryptophan fluorescence spectroscopy and isothermal titration calorimetry (ITC), yielding dissociation constants of ∼4 μM, ∼20 μM and >0.5 mM for H3K4me0, H3K4me1 and H3K4me2, respectively (supplementary Fig S2C online; Table 1).
0017
In agreement with the GST fusion pull-down experiments, fluorescence spectroscopy showed no binding of H3K4me0 to AIRE–PHD1 containing the APECED-causing C311Y mutation (Bjorses et al, 2000). Nevertheless, a second pathological mutant, V301M (Soderbergh et al, 2000), was still able to bind to H3K4me0, indicating that this mutation is not located in the H3 interaction site (Table 1).
0017
Indeed, fluorescence spectroscopy and ITC assays showed that the alanine mutations R2A in the H3 peptide and D312A in AIRE–PHD1 markedly reduced the binding affinity (Table 1; Fig 4C) without affecting the protein fold (supplementary Fig S3 online).
0096
Similarly, pull-down experiments with whole histones and the H3K4me0 peptide, together with fluorescence spectroscopy and ITC measurements performed on AIRE–PHD1-D297A showed reduced binding (Table 1; Fig 4).
0017
Similarly, pull-down experiments with whole histones and the H3K4me0 peptide, together with fluorescence spectroscopy and ITC measurements performed on AIRE–PHD1-D297A showed reduced binding (Table 1; Fig 4).
0096
(A) Pull-down assay of AIRE–PHD1 (PHD1) and AIRE–PHD1-D297A (D297A) mutant proteins with histones.
0019
To identify the mechanism of Plx1 recruitment to chromatin, we tested the interaction of Plx1 with the Mcm complex. We found that Plx1 co-precipitated with Mcm7 and that its binding was enhanced by activation of the checkpoint induced by pApT (Figure 5C).
0276
The proteins in the supernatant were subjected for SDS-PAGE, which was visualized by Coomassie Blue staining.
0276
The samples were fractionated in SDS-PAGE and stained with Coomassie Blue.
0019
(B–D) Mutations prevent the binding of Bcl-XL to tBid, Bax, or both. (B and D) Bcl-XL (20 nM), or the indicated Bcl-XL mutants, (C) Bcl-XL Y101K or (D) Bcl-XL ΔBH4, were incubated with (C and D) 20 nM tBid or (B) tBid-mt1 with or without Bax (100 nM) and with liposomes. Immunoprecipitations and immunoblotting were performed as in (A).
0030
To determine whether the Bcl-XL/Bax heterodimer also prevented the subsequent oligomerization of Bax, we examined oligomerization by cross-linking. In these experiments, the cross-linker was added to reactions containing an equal amount of membrane-bound Bax in the absence or presence of Bcl-XL (Figure S3). In these reactions, membrane-bound Bcl-XL inhibited Bax oligomerization, as detected by cross-linking concomitant with inhibition of dye release from liposomes. Taken together, these results suggest that, when bound to Bcl-XL, Bax function is neutralized, both in recruitment of other Bax molecules through autoactivation and in oligomerization to permeabilize membranes (Figure 6C, step 6).
0019
(B) Bax was incubated in the presence of liposomes, tBid, and increasing concentrations of Bcl-XL. Immunoprecipitations and immunoblotting were performed as in (A) without the addition of 2% CHAPS.
0019
(C) Bax was incubated for 2 h with liposomes (without tBid) at increasing concentrations of Bcl-XL or Bcl-XL Y101K. Immunoprecipitations and immunoblotting were performed as in (B).
0030
(D and E) Bcl-XL inhibits liposome-induced cross-linking of Bax. (D) Bax (100 nM) was incubated with liposomes for 2 h either alone (left panel), with 20 nM tBid (middle panel), or with 20 nM tBid and 100 nM Bcl-XL (right panel). Cross-linking with DSS was performed for 30 min at room temperature with or without 2% CHAPS to solubilize the liposomes prior to cross-linking, as indicated. Results were analyzed by immunoblotting. (E) Bax (100 nM) was incubated with or without liposomes for 2 h. Cross-linking and immunoblotting were performed as in (D).
0019
(F) Membrane-bound Bcl-XL inhibits the liposome-induced Bax conformational change with 50 μM m1Bid but not with 50 μM Bak BH3 peptide. Immunoprecipitations and immunoblotting were performed as in (B).
0030
To investigate further the effects of the membrane surface on Bax and the inhibition of these effects by Bcl-XL, cross-linking experiments using disuccinimidyl suberate (DSS) were performed. Cross-linking of Bax into higher-order structures after Bax binds to membranes has been observed previously [18].
0030
Nevertheless, incubation with liposomes did result in the cross-linking of Bax into higher-order complexes (Figure 5D, left panel). As expected from previous results [7,9], the interactions between Bax monomers induced by incubation with membranes were not resistant to detergent solubilization prior to cross-linking. The Bax–Bax cross-links were reduced in the absence of liposomes (Figure 5E), suggesting that, similar to binding by the 6A7 antibody, they result from a liposome-induced conformational change in Bax. Addition of tBid to Bax and liposomes resulted in a similar cross-linking pattern, but these Bax oligomers were resistant to solubilization of the membrane with detergent (Figure 5D, middle panel). Membrane-bound Bcl-XL not only prevented the formation of detergent-resistant Bax cross-links but also prevented the cross-linking of Bax that resulted when Bax contacted the membrane surface (Figure 5D, right panel).
0096
In contrast, previous data based on less-sensitive pull-down experiments suggested that α-parvin binding is limited to LD1 and LD4 (Nikolopoulos and Turner, 2000).
0077
Data were recorded on home-built or Bruker spectrometers with 11.7, 14.1, 17.6, and 22.3 T field strengths and processed with NMRPipe (Delaglio et al., 1995). Backbone chemical shift assignments were obtained using standard triple resonance experiments. Peptide titration experiments were performed by mixing two stock solutions (in 50 mM sodium phosphate, 100 mM NaCl, 2 mM DTT, 5% D2O, and 30 μM DSS [pH 6.9]) containing 235 μM 15N-enriched α-parvin-CHC and either no or a maximum concentration of LD peptide at the required protein/ligand ratios (Figure S2). Phase-sensitive gradient-enhanced 1H-15N HSQC spectra (Kay et al., 1992) were recorded at 25°C.
0402
To determine whether Ctk1 regulates the disposition of basal transcription factors, ChIP experiments on three constitutively expressed genes (PMA1, ADH1, and PYK1) were performed as described previously (Kim et al, 2004a). ChIP analysis using polyclonal antibodies against TBP (TATA-binding protein), TFIIB (Sua7), TFIIE (Tfa2) and TFIIH (Tfb1 and Kin28) generally confirmed the localization of these basal transcription factors at promoter regions in a wild-type (WT) background.
0402
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).
0402
To exclude the possibility that the abnormal cross-linking of basal transcription factors in ctk1Δ cells was due to issues with the specific antibodies, the experiment was repeated with strains carrying TAP-tagged Tfg1, Tfg2, or Tfg3 (all subunits of TFIIF). ChIP was carried out with IgG-agarose as described previously (Kim et al, 2004a). Again, spreading of TFIIF from the promoter throughout the transcribed region was seen in ctk1Δ cells (Figure 2, Tfg1–TAP result shown is representative). Some promoter preference was still seen, but the qualitative difference upon Ctk1 deletion was clear.
0402
A second model that could explain the cross-linking of basal factors throughout a gene is that they fail to release from RNApII as it moves into productive elongation. To test whether the extended pattern of basal factor cross-linking was completely coincident with RNApII, ChIP experiments were carried out at the termination site for PMA1 (Figure 3). RNApII levels decreased between primer sets 7 and 9 in WT and ctk1Δ strains, indicative of transcription termination in both backgrounds (Ahn et al, 2004; Kim et al, 2004b). TBP and TFIIH (Kin28) decrease in parallel with RNApII in ctk1Δ cells (Figure 3A), supporting the idea that these basal transcription factors travel with elongating RNApII.
0402
ChIP analysis for TBP was performed in these strains to see whether aberrant cross-linking was observed. 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). In accordance with our previous report, Rna14 cross-linking at the 3′-end of PMA1 was reduced to about one-third of WT levels.
0402
As in vitro experiments with mammalian Cdk9 (Wada et al, 1998; Chao and Price, 2001) and ChIP experiments with Ctk1 (Cho et al, 2001) also suggest that these kinases function after initiation, we modified the protocol.
0402
There are at least two possible variations of this model that could explain the ChIP results (Figure 5G). In the first, basal factors release from the promoter in ctk1Δ cells, but remain associated with the polymerase as it moves through the gene body. In the second, RNApII actually remains tethered to the basal factors at the promoter while transcribing. This would result in the transcribing polymerase ‘pulling' the transcribed region past basal factors, thereby allowing cross-linking with downstream sequences.
0402
Ctk1 is necessary for the dissociation of basal transcription factors from elongating RNA polymerase II (RNApII). (A) Schematic of the PMA1, ADH1, and PYK1 genes. The UAS of each gene is indicated by an open box (see Figure 3C). The TATA/promoter region and open reading frames are represented by black and grey boxes, respectively. Arrows indicate the position of the major polyadenylation sites reported previously (Kim et al, 2004a) and bars below the genes show the relative positions of PCR products in ChIP analysis. (B) Occupancy of Rpb1 and basal transcription factors (Sua7, TBP, Kin28, Tfb1, Tfa2) at the indicated regions in WT (YSB726) or ctk1Δ (YSB854) cells. INPUT was used to normalize the PCR amplification and the asterisk marks a non-transcribed PCR fragment indicated in all reactions as a background control. (C) Quantitation of the ChIP experiments in (B), with PMA1 as representative. The x axis indicates the specific primer pair used in each PCR. The y axis shows the specific signal relative to the negative control (i.e., a ratio of one is equivalent to background).
0007
The abnormal cross-linking of basal transcription factors in ctk1Δ cells is confirmed by independent immunoprecipitation of TAP-tagged TFIIF subunits.
0402
(A) ChIP analysis was carried out using Rpb3–TAP (YSB956) or Tfg1–TAP tagged strains (YSB925) +/− Ctk1. Similar cross-linking patterns were seen with Tfg2–TAP (YSB926) or Tfg3–TAP (YSB927) strains (not shown).
0402
Basal transcription factors coincide with elongating RNApII in ctk1Δ cells. (A) ChIP analyses were carried out with antibodies against Rpb3, TBP, or Kin28 in WT (YSB726) and ctk1Δ (YSB854) backgrounds. Numbers (6–9) correspond to PMA1 primer locations in Figure 1A. PCR products from (A) and (C) are shown in Figure 1B. (B) Quantitation of results in (A). (C) Occupancies of Rap1, Rpb1, and TBP at the indicated regions of genes were determined using the indicated polyclonal antibodies in both WT (YSB726) and ctk1Δ (YSB854) backgrounds. The upstream activating sequence (UAS) regions of each gene are depicted in Figure 1A.
