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• W2017813215 abstract "The transcription factor Stat6 plays a critical role in interleukin-4-dependent gene activation. To mediate this function, Stat6 recruits canonical transcriptional co-activators including the histone acetyl transferases CREB-binding protein and NCoA-1 and other proteins such as a p100 co-factor. However, much remains unknown regarding the constituents of Stat6 enhancer complexes, and the exact molecular events that modulate Stat6-dependent gene activation are not fully understood. Recently, we identified a novel co-factor, CoaSt6 (collaborator of Stat6), which associates with Stat6 and enhances its transcriptional activity. Sequence homologies place CoaSt6 in a superfamily of poly(ADP-ribosyl)polymerase (PARP)-like proteins. We have demonstrated here that PARP enzymatic activity is associated with CoaSt6, and this function of CoaSt6 can append ADP-ribose to itself and p100. Further, we show that a catalytically inactive mutant of CoaSt6 was unable to enhance Stat6-mediated transcription of a test promoter. Consistent with these findings, chemical inhibition of PARP activity blocked interleukin-4-dependent transcription from target promoters in vivo. Taken together, we have identified a CoaSt6-associated PARP activity and provided evidence for a role of poly(ADP ribosyl)ation in Stat-mediated transcriptional responses involving a novel PARP. The transcription factor Stat6 plays a critical role in interleukin-4-dependent gene activation. To mediate this function, Stat6 recruits canonical transcriptional co-activators including the histone acetyl transferases CREB-binding protein and NCoA-1 and other proteins such as a p100 co-factor. However, much remains unknown regarding the constituents of Stat6 enhancer complexes, and the exact molecular events that modulate Stat6-dependent gene activation are not fully understood. Recently, we identified a novel co-factor, CoaSt6 (collaborator of Stat6), which associates with Stat6 and enhances its transcriptional activity. Sequence homologies place CoaSt6 in a superfamily of poly(ADP-ribosyl)polymerase (PARP)-like proteins. We have demonstrated here that PARP enzymatic activity is associated with CoaSt6, and this function of CoaSt6 can append ADP-ribose to itself and p100. Further, we show that a catalytically inactive mutant of CoaSt6 was unable to enhance Stat6-mediated transcription of a test promoter. Consistent with these findings, chemical inhibition of PARP activity blocked interleukin-4-dependent transcription from target promoters in vivo. Taken together, we have identified a CoaSt6-associated PARP activity and provided evidence for a role of poly(ADP ribosyl)ation in Stat-mediated transcriptional responses involving a novel PARP. Covalent protein modification by poly(ADP-ribose) (PAR) 2The abbreviations used are: PAR, poly(ADP-ribose); PARP, poly(ADP-ribose) polymerase; CREB, cAMP-response element-binding protein; BAL, B aggressive lymphoma; CMV, cytomegalovirus; IP, immunoprecipitation; 3-ABA, 3-aminobenzamide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin. 2The abbreviations used are: PAR, poly(ADP-ribose); PARP, poly(ADP-ribose) polymerase; CREB, cAMP-response element-binding protein; BAL, B aggressive lymphoma; CMV, cytomegalovirus; IP, immunoprecipitation; 3-ABA, 3-aminobenzamide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin. appears to be involved in a wide array of biological processes (1Smith S. Trends Biochem. Sci. 2001; 26: 174-179Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 2Schreiber V. Dantzer F. Ame J.C. de Murcia G. Nat. Rev. Mol. Cell Biol. 2006; 7: 517-528Crossref PubMed Scopus (1551) Google Scholar, 3Kim M.Y. Zhang T. Kraus W.L. Genes Dev. 2005; 19: 1951-1967Crossref PubMed Scopus (645) Google Scholar), and sustained inhibition of poly(ADP-ribosyl)ation is highly toxic to cells (2Schreiber V. Dantzer F. Ame J.C. de Murcia G. Nat. Rev. Mol. Cell Biol. 2006; 7: 517-528Crossref PubMed Scopus (1551) Google Scholar). A major source of poly(ADP-ribose) polymerase (PARP) activity is encoded by PARP-1 (3Kim M.Y. Zhang T. Kraus W.L. Genes Dev. 2005; 19: 1951-1967Crossref PubMed Scopus (645) Google Scholar), which can engage in heterotypic interactions with transcription factors (4Kraus W.L. Lis J.T. Cell. 2003; 113: 677-683Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar), components of the DNA repair complexes (2Schreiber V. Dantzer F. Ame J.C. de Murcia G. Nat. Rev. Mol. Cell Biol. 2006; 7: 517-528Crossref PubMed Scopus (1551) Google Scholar, 3Kim M.Y. Zhang T. Kraus W.L. Genes Dev. 2005; 19: 1951-1967Crossref PubMed Scopus (645) Google Scholar), and at least one other PARP, PARP-2 (5Schreiber V. Ame J.C. Dolle P. Schultz I. Rinaldi B. Fraulob V. Menissier-de Murcia J. de Murcia G. J. Biol. Chem. 2002; 277: 23028-23036Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar). Analyses of PARP-1-deficient cells indicate that this protein constitutes a major portion of the global PARP activity in some cell types (3Kim M.Y. Zhang T. Kraus W.L. Genes Dev. 2005; 19: 1951-1967Crossref PubMed Scopus (645) Google Scholar, 6Shall S. de Murcia G. Mutat. Res. 2000; 460: 1-15Crossref PubMed Scopus (471) Google Scholar). However, it is clear that PARP-1 selectively interacts with some proteins but not others (2Schreiber V. Dantzer F. Ame J.C. de Murcia G. Nat. Rev. Mol. Cell Biol. 2006; 7: 517-528Crossref PubMed Scopus (1551) Google Scholar, 3Kim M.Y. Zhang T. Kraus W.L. Genes Dev. 2005; 19: 1951-1967Crossref PubMed Scopus (645) Google Scholar), that a substantial fraction of poly(ADP-ribosyl)ation is maintained in PARP-1 knock-out cells, and that a complete lack of PARP-1 is compatible with normal mouse development (6Shall S. de Murcia G. Mutat. Res. 2000; 460: 1-15Crossref PubMed Scopus (471) Google Scholar). Moreover, most cellular functions are maintained in PARP-1-deficient cells (6Shall S. de Murcia G. Mutat. Res. 2000; 460: 1-15Crossref PubMed Scopus (471) Google Scholar), which grow normally in the absence of genotoxic stress (3Kim M.Y. Zhang T. Kraus W.L. Genes Dev. 2005; 19: 1951-1967Crossref PubMed Scopus (645) Google Scholar). These findings indicate that functions of other PARP enzymatic activities likely compensate for the absence of PARP-1.Analyses of genomic sequences indicate that 15 mammalian proteins contain a segment with significant homology to the PARP catalytic domain of PARP-1 (reviewed in Ref. 2Schreiber V. Dantzer F. Ame J.C. de Murcia G. Nat. Rev. Mol. Cell Biol. 2006; 7: 517-528Crossref PubMed Scopus (1551) Google Scholar). These proteins in a PARP-like superfamily have been divided into subgroups based on analyses of their primary structures in silico. Prior cloning work and enzymatic analyses have indicated that several other members of the PARP superfamily are enzymatically active, but relatively little is known about their function (2Schreiber V. Dantzer F. Ame J.C. de Murcia G. Nat. Rev. Mol. Cell Biol. 2006; 7: 517-528Crossref PubMed Scopus (1551) Google Scholar). Members of one of the least-characterized branches of the PARP superfamily are termed the macro-PARP proteins (2Schreiber V. Dantzer F. Ame J.C. de Murcia G. Nat. Rev. Mol. Cell Biol. 2006; 7: 517-528Crossref PubMed Scopus (1551) Google Scholar). In these polypeptides, the portion exhibiting homology to the PARP catalytic region is preceded by one or several iterations of a domain homologous to a unique component of an atypical histone, macroH2a (7Pehrson J.R. Fried V.A. Science. 1992; 257: 1398-1400Crossref PubMed Scopus (278) Google Scholar). Various functions have been ascribed to macrodomains taken from among the set of different proteins containing them, including binding of PAR polymers (8Karras G.I. Kustatscher G. Buhecha H.R. Allen M.D. Pugieux C. Sait F. Bycroft M. Ladurner A.G. EMBO J. 2005; 24: 1911-1920Crossref PubMed Scopus (382) Google Scholar) or phosphoesterase activity (8Karras G.I. Kustatscher G. Buhecha H.R. Allen M.D. Pugieux C. Sait F. Bycroft M. Ladurner A.G. EMBO J. 2005; 24: 1911-1920Crossref PubMed Scopus (382) Google Scholar, 9Martzen M.R. McCraith S.M. Spinelli S.L. Torres F.M. Fields S. Grayhack E.J. Phizicky E.M. Science. 1999; 286: 1153-1155Crossref PubMed Scopus (353) Google Scholar). The macro-PARP subfamily is of particular interest, because one of its members, termed BAL (B aggressive lymphoma), was highlighted in a differential display analysis of mRNAs in comparisons of diffuse large B cell lymphomas of greater versus less clinical aggressiveness (10Aguiar R.C. Yakushijin Y. Kharbanda S. Salgia R. Fletcher J.A. Shipp M.A. Blood. 2000; 96: 4328-4334Crossref PubMed Google Scholar). However, it has been noted that, despite the homology of its PARP-like domain, this enzymatic activity could not be detected in association with BAL1 (11Aguiar R.C. Takeyama K. He C. Kreinbrink K. Shipp M.A. J. Biol. Chem. 2005; 280: 33756-33765Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar).Another member of the macro-PARP subfamily was identified recently by a search for additional proteins that could serve as co-factors for or modifiers of Stat6-mediated transcriptional regulation in cellular responses to the immunoregulatory cytokine interleukin (IL)-4 (12Goenka S. Boothby M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4210-4215Crossref PubMed Scopus (84) Google Scholar). A cytosolic 2-hybrid screen in yeast seeking Stat6-interacting proteins isolated a cDNA from mouse splenocytes that encodes a macro-PARP (12Goenka S. Boothby M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4210-4215Crossref PubMed Scopus (84) Google Scholar). Additional studies validated the interaction between this polypeptide, which was termed CoaSt6 (collaborator of Stat6) and Stat6. Functional analyses provided evidence that CoaSt6 can amplify the function of Stat6 and thereby modulate the induction of target genes by IL-4. However, it is not known whether the PARP sequence homology within CoaSt6 would, similar to BAL, fail to encode a functional ribosyl transferase or whether it instead is a functional PARP. Furthermore, it is not clear whether the PARP domain is important for any of the transcriptional functions of CoaSt6.Using single-point substitutions as well as assay of purified bacterially produced recombinant protein, we have shown here that CoaSt6 is associated with PARP catalytic activity that can ADP-ribosyl)ate itself as well as p100, a co-activator recruited by Stat6. Many transcriptional functions of PARP-1 appear not to require its PARP enzymatic activity. For instance, PARP-1 serves as a co-factor for NF-κB transcription factors in a manner independent from its PARP catalytic activity (13Hassa P.O. Covic M. Hasan S. Imhof R. Hottiger M.O. J. Biol. Chem. 2001; 276: 45588-45597Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). The role of PARP-1 instead is to promote multiprotein interactions (14Hassa P.O. Buerki C. Lombardi C. Imhof R. Hottiger M.O. J. Biol. Chem. 2003; 278: 45145-45153Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Our data indicate that a catalytically inactive point mutant of CoaSt6 failed to potentiate the Stat6-dependent activation of a reporter. Moreover, pharmacological inhibition of PARP activity blocked IL-4 induction of endogenous genes as well as a Stat6 and CoaSt6 responsive reporter. We conclude that CoaSt6-associated PARP function plays a role in IL-4 induced, Stat6-mediated gene regulation.EXPERIMENTAL PROCEDURESCell Lines and Recombinant DNAs—Cell lines 293T (derived from human embryonic kidney), ΦNX (derived from 293), HepG2 (human hepatocellular carcinoma), and M12 (mouse B lymphoma) and their culture in standard media (Dulbecco's modified Eagle's and RPMI 1640 media) containing 10% fetal bovine serum were as described previously (12Goenka S. Boothby M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4210-4215Crossref PubMed Scopus (84) Google Scholar, 15Goenka S. Marlar C. Schindler U. Boothby M. J. Biol. Chem. 2003; 278: 50362-50370Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 16Goenka S. Youn J. Dzurek L.M. Schindler U. Yu-Lee L.Y. Boothby M. J. Immunol. 1999; 163: 4663-4672PubMed Google Scholar). Wild-type CoaSt6 in the expression vectors pCMV-Tag2 and pcDNA3 were generated as described previously (12Goenka S. Boothby M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4210-4215Crossref PubMed Scopus (84) Google Scholar). Mutated cDNAs encoding portions of CoaSt6 (N-terminal, middle, C-terminal, and CoaSt6-(1216–1817) were generated by PCR with Pfu polymerase and cloned into the pCMV-Tag2 vector in-frame with the FLAG epitope tag. CoaSt6-(1216–1817) was also cloned in-frame with a His tag in the bacterial expression vector pET28. Point mutations were engineered in the PARP domain of CoaSt6 using the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA). PCR primers were designed containing the point mutation and were used in PCR reactions with wild type CoaSt6 in pCMV-Tag2 as a template. The methylated template DNA containing wild type CoaSt6 sequences was fragmented using DpnI. After bacterial transformation, clones containing the desired point mutations were verified by DNA sequencing. Inserts in pCMV-Tag2 were excised and subcloned into pcDNA3. FLAG epitope-tagged p100 expression constructs were generous gifts of E. Kieff (17Tong X. Drapkin R. Yalamanchili R. Mosialos G. Kieff E. Mol. Cell. Biol. 1995; 15: 4735-4744Crossref PubMed Scopus (208) Google Scholar) and O. Silvennoinen (18Yang J. Aittomaki S. Pesu M. Carter K. Saarinen J. Kalkkinen N. Kieff E. Silvennoinen O. EMBO J. 2002; 21: 4950-4958Crossref PubMed Scopus (143) Google Scholar).Immunoprecipitation, PARP Assays, and Immunoblotting—293T cells were transfected with expression plasmids, and whole cell lysates were prepared as described previously (12Goenka S. Boothby M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4210-4215Crossref PubMed Scopus (84) Google Scholar, 15Goenka S. Marlar C. Schindler U. Boothby M. J. Biol. Chem. 2003; 278: 50362-50370Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 16Goenka S. Youn J. Dzurek L.M. Schindler U. Yu-Lee L.Y. Boothby M. J. Immunol. 1999; 163: 4663-4672PubMed Google Scholar). Immunoprecipitations (IPs) were performed using 5 μg of monoclonal anti-FLAG (M2) (Sigma-Aldrich) alone or with 1 μg of anti-Stat6 (M-20) (Santa Cruz Biotechnology, Santa Cruz, CA). Alternatively, 293-derived ΦNX cells (19Ouyang W. Ranganath S.H. Weindel K. Bhattacharya D. Murphy T.L. Sha W.C. Murphy K.M. Immunity. 1998; 9: 745-755Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar) were transfected with untagged CoaSt6 in pcDNA3 and epitope-tagged p100 in pSG5. Lysates were then subjected to immunoprecipitation with anti-FLAG. In each case, the immune complexes were collected using protein G beads (Santa Cruz Biotechnology), rinsed, and divided equally. One portion was eluted and used for Western blotting. The other fraction was incubated for 30 min at room temperature in PARP assay buffer (20 μl) containing 5 μCi (0.25 μm) 32P-labeled NAD+ (1000 Ci/mmol) (GE Healthcare), 12.5 μm cold NAD+, 50 mm Tris-Cl (pH 8.0), 4 mm MgCl2, and 0.2 mm dithiothreitol (20Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (895) Google Scholar). Where indicated, PARP activity was inhibited with 1 mm 3-aminobenzamide (3-ABA) (Sigma-Aldrich). After completion of the reaction, the beads were washed twice with phosphate-buffered saline to remove excess substrate, and the bound proteins were eluted, resolved on SDS-PAGE, and autoradiographed. PARP assays were also performed with cold NAD+ only and probed with the monoclonal anti-PAR antibody 10H (Alexis Biochemicals, San Diego, CA). For detection of PAR-modified CoaSt6 in cell lysates, extracts were prepared and analyzed by Western blotting using 10H anti-PAR antibody as above. His-tagged CoaSt6-(1216–1817) was expressed in Escherichia coli, and proteins expressed in inclusion bodies were extracted under denaturing conditions and refolded by sequential dialysis against buffer containing decreasing amounts of urea. Refolded CoaSt6-(1216–1817) was purified using Talon beads (Clontech, Mountain View, CA) and used in PARP assays and Western blotting.Northern Blotting—M12 B cells were incubated with increasing concentrations of 3-ABA and a combination of IL-4 (5 ng/ml) and lipopolysaccharide (5 μg/ml). Northern blots of total RNA isolated from these cultured cells were probed with cDNAs for CD23, Gϵ, and GAPDH, subjected to autoradiography, and quantified using a phosphorimaging device (Fuji FLA-2000).Reporter Assays—Using a constant total DNA for each sample, CMV-β-galactosidase (100 ng), and the indicated expression constructs in pCMV-Tag2 or pcDNA3 (1 μg), along with a CD23b-(N3)3-Luc (21Dent A.L. Shaffer A.L. Yu X. Allman D. Staudt L.M. Science. 1997; 276: 589-592Crossref PubMed Scopus (756) Google Scholar) or Gϵ-(N4)3-Luc (22Litterst C.M. Pfitzner E. J. Biol. Chem. 2001; 276: 45713-45721Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) reporter construct (1 μg) were co-transfected into HepG2 cells using Transfectin reagent (Bio-Rad) according to the manufacturer's protocol. After 24 h, the cells were divided equally and either left untreated or treated with 5 ng/ml of IL-4 for an additional 24 h. For assays after inhibition of PARP activity, the cells were divided 24 h after transfection, recultured for 12 h, and then treated with IL-4 along with increasing concentrations of 3-ABA for a final 12 h. Firefly luciferase and β-galactosidase assays were performed on extracts of the transfected cells as described (12Goenka S. Boothby M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4210-4215Crossref PubMed Scopus (84) Google Scholar).RESULTSPoly(ADP-ribosyl)ation of CoaSt6 in the Presence of NAD+—We recently identified a novel co-factor, CoaSt6, which interacts with Stat6 and amplifies its activity in mediating transcriptional responses to IL-4 (12Goenka S. Boothby M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4210-4215Crossref PubMed Scopus (84) Google Scholar). To understand better the mechanism(s) by which CoaSt6 modulates Stat6 function, we focused on a region toward the C terminus that exhibited sequence homology with the catalytic domain of poly(ADP-ribose) polymerases (PARP) (Fig. 1a). Because the prototypical PARP-1 and PARP-2 each can serve as a substrate for poly(ADP-ribosyl)ation, we first tested whether CoaSt6 underwent a similar modification. Epitope-tagged CoaSt6 was expressed in 293T cells, immunoprecipitated using anti-FLAG antibodies, and incubated with [32P]NAD+ as a donor of ADP-ribose moieties. IPs that contained CoaSt6 showed a radiolabeled signal migrating at the same position as CoaSt6, only in samples transfected with the CoaSt6 cDNA; these data strongly indicate that CoaSt6 underwent poly(ADP-ribosyl)ation (Fig. 1b). To test whether this post-translational modification of CoaSt6 was a result of a typical PARP catalytic activity, the transfer reaction was carried out in the presence of 3-ABA, a broad-spectrum inhibitor of PARP catalytic activity. Virtually all 32P modification of CoaSt6 was abrogated by 3-ABA, further indicating that a PARP catalytic activity was responsible for the post-translational modification of CoaSt6 (Fig. 1c). To extend these findings, we probed blots of proteins with antibodies against the PAR polymer. In experiments where CoaSt6 PARP reactions with exogenous NAD were carried out in the presence or absence of 3-ABA and then subjected to Western blot analysis, a band co-migrating with CoaSt6 was recognized by the anti-PAR antibody (Fig. 1d) but eliminated when the reaction was carried out with 3-ABA present. Furthermore, we analyzed extracts of cells transfected with CoaSt6 and controls. These experiments showed that a protein strongly reactive with anti-PAR antibodies and co-migrating with CoaSt6 was observed only in CoaSt6-transfected cells (Fig. 1e). To determine whether CoaSt6 endogenous to mouse lymphoid cells similarly confers a PARP function, we immunoprecipitated CoaSt6 from M12 B cells and subjected the IPs to PARP reaction conditions in the absence or presence of 3-ABA. Consistent with the preceding data, endogenously expressed CoaSt6 was able to confer PARP modification (Fig. 1f). Endogenously expressed CoaSt6 also showed evidence of PAR modification as detected by an antibody specific for this post-translational modification (Fig. 1g). These results collectively indicate that CoaSt6 is an in vivo target for ADP-ribosylation.The observed poly(ADP-ribosyl)ation of CoaSt6 could arise because the protein participates in a heterotypic interaction with a PARP such as PARP-1, which is known to form various heterodimers. Alternatively, the PARP-like region of CoaSt6 could encode a bona fide poly(ADP-ribose) polymerase. To test whether CoaSt6 sequences were enzymatically active independent of other protein interactions, we expressed His-tagged CoaSt6-(1216–1817) in E. coli, purified the protein, and performed PARP assays. Purified CoaSt6-(1216–1817) catalyzed the addition of NAD+ to the protein in vitro (Fig. 1h), a finding most consistent with intrinsic PARP activity residing in the PARP-like domain independent of other heterotypic interactions.Role of PARP Domain Residues in CoaSt6-associated PARP Activity—To determine whether the PARP-like domain contained in CoaSt6 was crucial for poly(ADP-ribosyl)ation, we tested a variety of mutated cDNAs. Several deletion mutants of CoaSt6 (Fig. 2a) were tested for their ability to self-poly(ADPribosyl)ate in immunoprecipitates incubated with radiolabeled NAD+. Each version of CoaSt6 that contained the PARP-like domain was poly(ADP-ribosyl)ated (Fig. 2b). These included the C-terminal only, a combination of the C and middle portion of CoaSt6, and CoaSt6-(1216–1817), which is the version of CoaSt6 that contains the C-terminal and one hismacro domain (Fig. 2b). In contrast, no labeling by [32P]NAD+ was observed for the CoaSt6 N-terminal portion or the triplicate hismacro domains, which lack the PARP-like region. These results suggest that the PARP-like domain of CoaSt6 is responsible for adding the ADP-ribose moieties. To extend these results, we identified several amino acid residues within the PARP-like domain of CoaSt6 that are conserved among active PARPs, several of which are essential for catalytic activity in PARP-1 (Fig. 2c). When substitution mutants were tested for their ability to undergo poly(ADP-ribosyl)ation in immune precipitates (as described above), we observed a dramatic decrease in their capacity to self-poly(ADP-ribosyl)ate as compared with their wild type counterpart (Fig. 2d).FIGURE 2The CoaSt6 PARP-like sequence encodes the catalytically active polymerase. a, schematic diagram of expression constructs encoding FLAG-tagged portions of CoaSt6, which were transfected into 293T cells and assayed for [32P]NAD addition to proteins as described for Fig. 1a. Shown in b is a representative autoradiograph of SDS-PAGE-resolved products of these poly(ADP-ribosyl)ation reactions (upper panel) and control immunoblots of the anti-FLAG reactive proteins (lower panel). ○ indicates the positions of the CoaSt6 polypeptides. As indicated, the C-terminal portion of CoaSt6 co-migrated with a nonspecific (N.S.) band. c, alignment of the PARP catalytic domain of CoaSt6 (uppermost line, from amino acid residues 1695 to 1817) and PARP-1 (lowest line, residues 859–997) with identical residues and conservative substitutions (+). The conserved amino acid residues that were mutated in CoaSt6 (H1698, Y1730, and E1810) are indicated by bold letters, an asterisk, and underlining. d, wild type and mutated CoaSt6 were expressed in 293T cells and analyzed for PARP activity by [32P]NAD+ transfer assays and Western blotting as described for Fig. 1a.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Enhancement of the Poly(ADP-ribosyl)ating Function of CoaSt6 in the Presence of IL-4-activated Stat6—CoaSt6 was originally identified as a co-factor that associated with Stat6 and enhanced its transcription function (12Goenka S. Boothby M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4210-4215Crossref PubMed Scopus (84) Google Scholar). To explore the interplay between the poly(ADP-ribosyl)ating activity of CoaSt6 and Stat6, FLAG-tagged CoaSt6 and Stat6 were co-expressed, Stat6 was induced by IL-4, and the resultant cell extracts were subjected to co-immunoprecipitation of Stat6 and CoaSt6. The resultant IPs were then assayed with radiolabeled NAD+. As above, CoaSt6 poly(ADP-ribosyl)ated itself in cells with little endogenous Stat6 and untreated with IL-4 (Fig. 3, lanes 5 and 6). Although there were abundant protein levels of Stat6 and CoaSt6 in the IPs, no positive signal for the poly(ADP-ribosyl)-ation of Stat6 by CoaSt6 was observed (Fig. 3, lanes 7 and 8). These results suggest that, although Stat6 associates with CoaSt6, Stat6 is not an efficient substrate for the PARP catalytic activity of CoaSt6. However, the ability of CoaSt6 to poly(ADP-ribosyl)ate itself was substantially increased in the presence of Stat6 after induction by IL-4 (Fig. 3, lanes 7 and 8). These observations indicate that the co-association of CoaSt6 with IL-4-activated Stat6 enhances the auto-PARylation activity of CoaSt6.FIGURE 3IL-4 enhances the poly(ADP-ribosyl)ating function of CoaSt6 in the presence of Stat6. 293T cells were transiently transfected with the indicated expression plasmids and were left untreated or treated with IL-4 for 1 h. Immunoprecipitated extracts from these cells were divided and either subjected to poly(ADP-ribosyl)ation reactions with 32P-labeled NAD+ and autoradiographed (upper panel, with quantitation by phosphorimaging indicated in the bar graph beneath) or immunoblotted and probed for Stat6 and FLAG as indicated (lower two panels). The two-lane side panel is a shorter exposure of the same lanes 7 and 8 from the same gel to allow better visualization of the ∼200 kDa CoaSt6 band.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Allomodification of an Exogenous Protein Substrate by CoaSt6/PARP-14—We wanted to determine whether CoaSt6 is able to target heterologous proteins for poly(ADP-ribosyl)ation, or if, instead, it is only efficient at automodification. The preceding data indicate that Stat6, despite its physical interaction with CoaSt6, is not an efficient substrate for CoaSt6-mediated poly(ADP-ribosyl)ation, but CoaSt6 might modify other proteins that are involved in enhancer complexes with Stat6. When CoaSt6 endogenous to B cells was immunoprecipitated using anti-CoaSt6 and used in PARP assays, a protein migrating at the 100-kDa position was detected in addition to the ∼200-kDa CoaSt6 signal (Fig. 4a). A nuclear protein p100 is a Stat6-associated transcriptional co-factor thought to enhance interactions between Stat6 and RNA polymerase II as part of the basal transcriptional machinery (18Yang J. Aittomaki S. Pesu M. Carter K. Saarinen J. Kalkkinen N. Kieff E. Silvennoinen O. EMBO J. 2002; 21: 4950-4958Crossref PubMed Scopus (143) Google Scholar). To test whether this p100 was a target for poly(ADP ribosyl)ation, we immunoprecipitated endogenously expressed p100 and probed the precipitate with an anti-PAR antibody. Consistent with the in vitro PARP reaction, we observed a signal for PAR at the 100-kDa position which co-migrated with the signal observed with anti-p100 (Fig. 4b). Therefore, we evaluated whether CoaSt6 can target p100 for poly(ADP-ribosyl)ation. First, we tested whether p100 and CoaSt6 associate with one another independently of Stat6. Untagged CoaSt6 and epitope-tagged p100 were co-expressed and subjected to immunoprecipitation with anti-FLAG followed by immunoblotting with anti-CoaSt6 antibodies. A CoaSt6-specific signal was observed in anti-FLAG immune complexes only when both the proteins were expressed (Fig. 4c), indicating that CoaSt6 associated with p100. Similar results were obtained when cells were separately transfected with individual expression constructs (CoaSt6; p100) and cell-free extracts were mixed prior to IP (data not shown).FIGURE 4p100 can associate with and be poly(ADP-ribosyl)ated by CoaSt6. a, extracts from M12 cells were subjected to immunoprecipitation with either normal rabbit IgG or anti-CoaSt6. The resultant precipitate was used in PARP reactions as in Fig. 1a. b, BJAB cell extracts were used for immunoprecipitation with either a control antibody or one specific for p100. The precipitated proteins were then probed with anti-PAR or anti-p100. c, ΦNX cells were transiently transfected with combinations of expression vectors containing FLAG-tagged p100, CoaSt6, and empty vector controls. Extracts of transfected cells were subjected to immunoprecipitation with anti-FLAG antibodies and analyzed by Western blotting with either anti-peptide antibodies specific for CoaSt6 (12Goenka S. Boothby M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4210-4215Crossref PubMed Scopus (84) Google Scholar) or anti-FLAG. In parallel, portions of each extract prior to IP were probed with the indicated antibodies. d, FLAG-CoaSt6 (or the Coast6(E1810K) mutant) and FLAG-p100 were transfected into ΦNX cells followed by immunoprecipitation of cell extracts with anti-FLAG antibodies. Shown are the results of autoradiography of the products after PARP assays using [32P]NAD+ (upper panel), as described for Fig. 1a, and of anti-FLAG We" @default.
• W2017813215 created "2016-06-24" @default.
• W2017813215 creator A5033161671 @default.
• W2017813215 creator A5043054491 @default.
• W2017813215 creator A5069140129 @default.
• W2017813215 date "2007-06-01" @default.
• W2017813215 modified "2023-09-29" @default.
• W2017813215 title "Collaborator of Stat6 (CoaSt6)-associated Poly(ADP-ribose) Polymerase Activity Modulates Stat6-dependent Gene Transcription" @default.
• W2017813215 cites W1991311051 @default.
• W2017813215 cites W1991742606 @default.
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