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).
0663
The cytoplasmic retention of the chimeric GFP-STM protein was further substantiated by confocal laser microscopy scanning and staining of nuclei with propidium iodide. The comparison of a median Z-stack projection through the nucleus either analysed for GFP-STM fluorescence (Figure 1F), the corresponding propidium iodine stains (Figure 1G) and their overlay in Figure 1H confirm exclusion of GFP fluorescence from the nuclear compartment.
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.
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.
0114
The structural information available on protein–protein interactions involving ZF domains is also limited [25]. The X-ray structure of the Munc13–1/RIM2α heterodimer described here provides the first high-resolution view of a complex directly involving a member from the family of ZF domains that includes α-RIMs and other Rab effectors. The structure shows that two surfaces at opposite sides of the RIM2α ZF domain interact with two different structural motifs of Munc13–13–150. Thus, the N-terminal loop region of the RIM2α ZF domain binds to the tip of the C2A-domain β-sandwich, whereas the crevice formed by the C-terminal β-hairpin and helix a2 binds the C-terminal helix of Munc13–13–150. Interestingly, in the complex between the α subunit of Hif-1 (Hif-1α) and the TAZ1 domain of CREB-binding protein (a ZF domain unrelated to the RIM2α ZF domain), Hif-1α also wraps around the TAZ1 domain, interacting with surfaces at opposite sides of the domain [31]. An emerging theme from these observations is that, because of their small size, ZF domains may often use multiple surfaces to increase the affinity and specificity of interactions with target proteins, although further research will be necessary to assess the generality of this notion.
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.
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).
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.
0006
In a converse experiment, ephrin-B1 was co-immunoprecipitated with an anti-Cx43 antibody (Figure 6Cb).
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.
0007
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. A significant amount of endogenous PDE activity was recovered in the β1AR immunoprecipitation (IP) pellet (Figure 1A).
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.
0007
(A, B) Neonatal cardiac myocytes expressing a Flag-tagged β1AR were treated for 3 min with 100 nM Norepinephrine before cell lysis and IP with M1 (α-Flag) resin. The effect of a 5 min pre-treatment with 10 μM of the PDE4-specific inhibitor, Rolipram, or the PDE3-selective inhibitor, Cilostamide, on PKA-phosphorylation of the β1AR is detected in IBs using a PKA-site-specific antibody. (C, D) MEFs derived from mice deficient in PDE4D or wild-type controls were infected with adenovirus to express a Flag-tagged β1AR construct. At 40 h post-infection, cells were treated for 3 min with 100 nM Norepinephrine (NorEpi) before cell lysis and IP with M1 (α-Flag) resin. PKA-phosphorylation of the β1AR is detected in IB using a PKA-site-specific antibody. (E, F) Neonatal cardiac myocytes coexpressing a Flag-tagged β1AR and either GFP, a catalytically inactive PDE4D8 construct (PDE4D-DN; see also Supplementary Figure 5), or a catalytically inactive PDE3A1 (PDE3A1-DN) were subjected to α-Flag(M1)-IP, and the phosphorylation of the β1AR was subsequently detected in IB using a PKA-substrate-specific antibody.
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).
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).
0077
The model of AIRE–PHD1 complexed with H3K4me0 was in perfect agreement with the experimental chemical shift perturbation data, as the peptide-binding region coincided with the binding surface identified by NMR spectroscopy (Fig 3A). In fact, the H3K4me0 peptide induced chemical shift changes in AIRE–PHD1 residues that map only on one side of the protein surface, involving residues in the N terminus of the PHD finger, the first β-strand, and the loop connecting the first and the second β-strands (D297, G305, G306, L308, C310, D312 and G313; Fig 2; supplementary Fig S5 online). A similar pattern of chemical shift changes indicated the same binding site for H3K4me1.
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 samples were fractionated in SDS-PAGE and stained with Coomassie Blue.
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).
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).
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].
0007
The abnormal cross-linking of basal transcription factors in ctk1Δ cells is confirmed by independent immunoprecipitation of TAP-tagged TFIIF subunits.
