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• W2004408181 abstract "The largest subunit of RNA polymerase II contains a unique C-terminal domain (CTD) consisting of tandem repeats of the consensus heptapeptide sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Two forms of the largest subunit can be separated by SDS-polyacrylamide gel electrophoresis. The faster migrating form termed IIA contains little or no phosphate on the CTD, whereas the slower migrating II0 form is multiply phosphorylated. CTD kinases with different phosphoryl acceptor specificities are able to convert IIA to II0 in vitro, and different phosphoisomers have been identified in vivo. In this paper we report the binding specificities of a set of monoclonal antibodies that recognize different phosphoepitopes on the CTD. Monoclonal antibodies like H5 recognize phosphoserine in position 2, whereas monoclonal antibodies like H14 recognize phosphoserine in position 5. The relative abundance of these phosphoepitopes changes when growing yeast enter stationary phase or are heat-shocked. These results indicate that phosphorylation of different CTD phosphoacceptor sites are independently regulated in response to environmental signals. The largest subunit of RNA polymerase II contains a unique C-terminal domain (CTD) consisting of tandem repeats of the consensus heptapeptide sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Two forms of the largest subunit can be separated by SDS-polyacrylamide gel electrophoresis. The faster migrating form termed IIA contains little or no phosphate on the CTD, whereas the slower migrating II0 form is multiply phosphorylated. CTD kinases with different phosphoryl acceptor specificities are able to convert IIA to II0 in vitro, and different phosphoisomers have been identified in vivo. In this paper we report the binding specificities of a set of monoclonal antibodies that recognize different phosphoepitopes on the CTD. Monoclonal antibodies like H5 recognize phosphoserine in position 2, whereas monoclonal antibodies like H14 recognize phosphoserine in position 5. The relative abundance of these phosphoepitopes changes when growing yeast enter stationary phase or are heat-shocked. These results indicate that phosphorylation of different CTD phosphoacceptor sites are independently regulated in response to environmental signals. The largest subunit of RNA polymerase II (pol II) 1The abbreviations used are: pol, polymerase; CTD, C-terminal domain; TF, transcription factor; mAb, monoclonal antibody; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; WT, wild type. 1The abbreviations used are: pol, polymerase; CTD, C-terminal domain; TF, transcription factor; mAb, monoclonal antibody; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; WT, wild type. contains a repetitive C-terminal domain (CTD) consisting of tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser (1Allison L.A. Moyle M. Shales M. Ingles C.J. Cell. 1985; 42: 599-610Abstract Full Text PDF PubMed Scopus (442) Google Scholar, 2Corden J.L. Cadena D.L. Ahearn Jr., J. Dahmus M.E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7934-7938Crossref PubMed Scopus (236) Google Scholar). The CTD plays an essential (3Nonet M. Sweetser D. Young R.A. 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Nakayasu H. Du L. Warren S.L. Sharp P.A. Berezney R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8253-8257Crossref PubMed Scopus (282) Google Scholar, 18Du L. Warren S.L. J. Cell Biol. 1997; 136: 5-18Crossref PubMed Scopus (123) Google Scholar, 19McCracken S. Fong N. Yankulov K. Ballantyne S. Pan G. Greenblatt J. Patterson S.D. Wickens M. Bentley D.L. Nature. 1997; 385: 357-361Crossref PubMed Scopus (734) Google Scholar, 20Corden J.L. Patturajan M. Trends Biochem. Sci. 1997; 22: 413-416Abstract Full Text PDF PubMed Scopus (148) Google Scholar). Phosphorylation of the CTD is a key feature of CTD function. SDS gel electrophoresis separates the largest subunit into two species as follows: IIA contains a hypophosphorylated CTD and pol II0 is hyperphosphorylated on the CTD (21Cadena D.L. Dahmus M.E. J. Biol. Chem. 1987; 262: 12468-12474Abstract Full Text PDF PubMed Google Scholar). Serine is the predominant in vivo phosphoacceptor with minor amounts of phosphothreonine and phosphotyrosine detected (22Zhang J. Corden J.L. J. Biol. Chem. 1991; 266: 2290-2296Abstract Full Text PDF PubMed Google Scholar, 23Baskaran R. Dahmus M.E. Wang J.Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11167-11171Crossref PubMed Scopus (187) Google Scholar). Although in vivophosphorylation sites have not been mapped, in vitro studies have identified serines in both positions 2 and 5 (22Zhang J. Corden J.L. J. Biol. Chem. 1991; 266: 2290-2296Abstract Full Text PDF PubMed Google Scholar, 24Stone N. Reinberg D. J. Biol. Chem. 1992; 267: 6353-6360Abstract Full Text PDF PubMed Google Scholar, 25Gebara M. Sayre M.H. Corden J.L. J. Cell. Biochem. 1997; 64: 390-402Crossref PubMed Scopus (69) Google Scholar) and tyrosine in position 1 (23Baskaran R. Dahmus M.E. Wang J.Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11167-11171Crossref PubMed Scopus (187) Google Scholar) as potential phosphoryl acceptors. Mutation of these sites to unphosphorylatable alanine or phenylalanine residues in each yeast CTD repeat is lethal, suggesting a requirement for CTD phosphorylation in vivo (26West M.L. Corden J.L. Genetics. 1995; 140: 1223-1233Crossref PubMed Google Scholar). The preferential inclusion of pol IIA into preinitiation complexes (27Laybourn P.J. Dahmus M.E. J. Biol. Chem. 1990; 265: 13165-13173Abstract Full Text PDF PubMed Google Scholar, 28Lu H. Flores O. Weinmann R. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10004-10008Crossref PubMed Scopus (248) Google Scholar, 29Chesnut J.D. Stephens J.H. Dahmus M.E. J. Biol. Chem. 1992; 267: 10500-10506Abstract Full Text PDF PubMed Google Scholar, 30Kang M.E. Dahmus M.E. J. Biol. Chem. 1993; 268: 25033-25040Abstract Full Text PDF PubMed Google Scholar) together with the observation that elongating pol II is phosphorylated on the CTD (31Bartholomew B. Dahmus M.E. Meares C.F. J. Biol. Chem. 1986; 261: 14226-14231Abstract Full Text PDF PubMed Google Scholar) led to the hypothesis that the CTD is reversibly phosphorylated with each transcription cycle (8Dahmus M.E. J. Biol. Chem. 1996; 271: 19009-19012Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). The unphosphorylated CTD has been shown to contact basal transcription factors TATA binding protein (32Usheva A. Maldonado E. Goldring A. Lu H. Houbavi C. Reinberg D. Aloni Y. Cell. 1992; 69: 871-881Abstract Full Text PDF PubMed Scopus (179) Google Scholar), TFIIE, and TFIIF (33Kang M.E. Dahmus M.E. J. Biol. Chem. 1995; 270: 23390-23397Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), and these contacts, together with as yet undefined interactions withSRBs (34Nonet M.L. Young R.A. Genetics. 1989; 123: 715-724Crossref PubMed Google Scholar, 35Koleske A.J. Buratowski S. Nonet M. Young R.A. Cell. 1992; 69: 883-894Abstract Full Text PDF PubMed Scopus (139) Google Scholar, 36Thompson C.M. Koleske A.J. Chao D.M. Young R.A. Cell. 1993; 73: 1361-1375Abstract Full Text PDF PubMed Scopus (388) Google Scholar, 37Koleske A.J. Young R.A. Nature. 1994; 368: 466-469Crossref PubMed Scopus (529) Google Scholar), suggest that the CTD acts as a structural framework for the preinitiation complex (38Koleske A.J. Young R.A. Trends Biochem. Sci. 1995; 20: 113-116Abstract Full Text PDF PubMed Scopus (266) Google Scholar). The pol II preinitiation complex also contains several protein kinases that are capable of phosphorylating the CTD (39Feaver W.J. Svejstrup J.Q. Henry N.L. Kornberg R.D. Cell. 1994; 79: 1103-1109Abstract Full Text PDF PubMed Scopus (357) Google Scholar, 40Liao S.M. Zhang J. Jeffery D.A. Koleske A.J. Thompson C.M. Chao D.M. Viljoen M. van Vuuren H.J. Young R.A. Nature. 1995; 374: 193-196Crossref PubMed Scopus (364) Google Scholar, 41Shiekhattar R. Mermelstein F. Fisher R.P. Drapkin R. Dynlacht B. Wessling H.C. Morgan D.O. Reinberg D. Nature. 1995; 374: 283-287Crossref PubMed Scopus (359) Google Scholar, 42Serizawa H. Makela T.P. Conaway J.W. Conaway R.C. Weinberg R.A. Young R.A. Nature. 1995; 374: 280-282Crossref PubMed Scopus (307) Google Scholar, 43Valay J.G. Simon M. Dubois M.F. Bensaude O. Facca C. Faye G. J. Mol. Biol. 1995; 249: 535-544Crossref PubMed Scopus (167) Google Scholar, 44Svejstrup J.Q. Feaver W.J. Kornberg R.D. J. Biol. Chem. 1996; 271: 643-645Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 45Rickert P. Seghezzi W. Shanahan F. Cho H. Lees E. Oncogene. 1996; 12: 2631-2640PubMed Google Scholar) suggesting that one role of this complex is to effect the conversion of pol IIA to pol II0 thereby releasing pol II from the initiation complex. Finally, CTD phosphatase is required to dephosphorylate pol II0 thus completing the CTD phosphorylation cycle (46Chambers R.S. Dahmus M.E. J. Biol. Chem. 1994; 269: 26243-26248Abstract Full Text PDF PubMed Google Scholar, 47Chambers R.S. Wang B.Q. Burton Z.F. Dahmus M.E. J. Biol. Chem. 1995; 270: 14962-14969Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Several observations complicate this simple two-state CTD phosphorylation cycle. Transcription of some promoters in vitro and in vivo does not require the CTD (30Kang M.E. Dahmus M.E. J. Biol. Chem. 1993; 268: 25033-25040Abstract Full Text PDF PubMed Google Scholar,48Buratowski S. Sharp P.A. Mol. Cell. Biol. 1990; 10: 5562-5564Crossref PubMed Scopus (36) Google Scholar, 49Serizawa H. Conaway J.W. Conaway R.C. Nature. 1993; 363: 371-374Crossref PubMed Scopus (138) Google Scholar, 50Gerber H.P. Hagmann M. Seipel K. Georgiev O. West M.A. Litingtung Y. Schaffner W. Corden J.L. Nature. 1995; 374: 660-662Crossref PubMed Scopus (136) Google Scholar). In addition, CTD phosphorylation can be inhibited without blocking activated transcription in vitro (49Serizawa H. Conaway J.W. Conaway R.C. Nature. 1993; 363: 371-374Crossref PubMed Scopus (138) Google Scholar, 51Jiang Y. Gralla J.D. Nucleic Acids Res. 1994; 22: 4958-4962Crossref PubMed Scopus (10) Google Scholar, 52Makela T.P. Parvin J.D. Kim J. Huber L.J. Sharp P.A. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5174-5178Crossref PubMed Scopus (111) Google Scholar). Bentley and colleagues (53Akhtar A. Faye G. Bentley D.L. EMBO J. 1996; 15: 4654-4664Crossref PubMed Scopus (59) Google Scholar) have shown that deleting the CTD or blocking CTD phosphorylation by Kin28p does not alter the synthesis of promoter proximal transcripts. Thus, at least for some promoters, transcription initiation can occur in the absence of a CTD or a CTD phosphorylation cycle. Finally, hyperphosphorylation of the CTD does not correlate with pol II's transcriptional activity in vivo (54Kim E. Du L. Bregman D.B. Warren S.L. J. Cell Biol. 1997; 136: 19-28Crossref PubMed Scopus (213) Google Scholar). Another complication in understanding the role of CTD phosphorylation is the multiplicity of CTD kinases and the diversity of possible phosphate acceptors in the CTD (8Dahmus M.E. J. Biol. Chem. 1996; 271: 19009-19012Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Although in vivo CTD phosphorylation sites have not been mapped, the in vitrotargets of several serine/threonine-specific CTD kinases have been determined. Cdc2 kinase phosphorylates both serine 2 and serine 5 (22Zhang J. Corden J.L. J. Biol. Chem. 1991; 266: 2290-2296Abstract Full Text PDF PubMed Google Scholar), whereas the TFIIH-associated CTD kinase (Cdk7/cyclin H) phosphorylates serine 5 (25Gebara M. Sayre M.H. Corden J.L. J. Cell. Biochem. 1997; 64: 390-402Crossref PubMed Scopus (69) Google Scholar). CTD kinases induced by heat shock or arsenite also preferentially phosphorylate serine 5 (55Trigon S. Morange M. J. Biol. Chem. 1995; 270: 13091-13098Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Genetic evidence indicates that the roles of serines in positions 2 and 5 are different. First, partial substitutions of serines in either position 2 or 5 have different effects on viability (26West M.L. Corden J.L. Genetics. 1995; 140: 1223-1233Crossref PubMed Google Scholar). Second, mutations inSRB genes suppress position 2 substitutions but not position 5 substitutions (56Yuryev A. Corden J.L. Genetics. 1996; 143: 661-671Crossref PubMed Google Scholar). Taken together with the differences in CTD kinase specificity, these results suggest that pol II0 may be a collection of different phosphoisomers. In this paper we describe the results of experiments testing the interaction specificity of different anti-pol II mAbs for a set of phosphorylated CTD binding sites. These mAbs were isolated in screens for antibodies directed against proteins involved in a range of different cell functions (17Mortillaro M.J. Blencowe B.J. Wei X.Y. Nakayasu H. Du L. Warren S.L. Sharp P.A. Berezney R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8253-8257Crossref PubMed Scopus (282) Google Scholar, 57Warren S.L. Landolfi A.S. Curtis C. Morrow J.S. J. Cell Sci. 1992; 103: 381-388Crossref PubMed Google Scholar, 58Bregman D.B. Du L. Li Y. Ribisi S. Warren S.L. J. Cell Sci. 1994; 107: 387-396PubMed Google Scholar, 59Bregman D.B. Du L. Vanderzee S. Warren S.L. J. Cell Biol. 1995; 129: 287-298Crossref PubMed Scopus (309) Google Scholar, 60Thibodeau A. Vincent M. Exp. Cell Res. 1991; 195: 145-153Crossref PubMed Scopus (27) Google Scholar, 61Chabot B. Bisotto S. Vincent M. Nucleic Acids Res. 1995; 23: 3206-3213Crossref PubMed Scopus (38) Google Scholar). We show here that each of these antibodies is capable of recognizing wild-type heptapeptide repeats phosphorylated by Cdc2 kinase. The specificities of the different antibodies for mutant CTDs are different, however, indicating that they recognize different phosphoepitopes. Nutritional stress and heat shock result in higher levels of serine 2 phosphorylation suggesting that the serine 2 and serine 5 phosphoepitopes are functionally different. In an earlier study we described the construction and characterization of a set of CTD phosphorylation site substitution mutations (26West M.L. Corden J.L. Genetics. 1995; 140: 1223-1233Crossref PubMed Google Scholar). These mutations focus on serines in positions 2 and 5 of the consensus heptapeptide YSPTSPS which were shown to be phosphorylated by Cdc2 kinase (22Zhang J. Corden J.L. J. Biol. Chem. 1991; 266: 2290-2296Abstract Full Text PDF PubMed Google Scholar). Each reconstructed CTD consists of multiple wild-type or mutant heptapeptide repeats and a 9-amino acid epitope derived from the influenza virus hemagglutinin gene that is recognized by mAb 12CA5 (62Wilson I.A. Niman H.L. Houghten R.A. Cherenson A.R. Connolly M.L. Lerner R.A. Cell. 1984; 37: 767-778Abstract Full Text PDF PubMed Scopus (651) Google Scholar). The mutant yeast CTD sequences were excised and cloned into the pGEX2 vector (Pharmacia Biotech Inc.) allowing the expression of glutathioneS-transferase-CTD fusions in Escherichia coli(Fig. 1 A). We have also expressed the mammalian CTD as a GST fusion protein. The plasmid expressing this protein was a gift of Dr. David Bentley (Amgen Institute, Toronto). E. coli (DH5α) strains expressing GST-CTD fusion proteins were grown overnight to saturation. Cultures were diluted 10-fold in fresh L broth containing 100 μg/ml ampicillin and grown for 1 h before addition of isopropyl-1-thio-β-d-galactopyranoside to 0.1 mm. After 4 h of induced expression cells were collected by centrifugation and sonicated, and the fusion protein was purified by glutathione-agarose (Pharmacia) affinity chromatography as described (64Smith D.B. Corcoran L.M. Ausubel F.M.E.A. Current Protocols in Molecular Biology. 2. John Wiley & Sons, Inc., New York1995: 16.7.1-16.7.7Google Scholar). Purified GST fusions containing 16 copies of the wild-type heptapeptide YSPTSPS, 18 copies of YSPTEPS, 15 copies of YESPTSP, 15 copies of YSPTAPS, or 12 copies of YAPTSPS were separated by SDS-PAGE (10%) and stained with Coomassie Blue dye (Fig. 1 B). CTD fusion proteins were phosphorylated in vitro with baculovirus-expressed epitope-tagged Cdc2 kinase (65Desai D. Gu Y. Morgan D.O. Mol. Biol. Cell. 1992; 3: 571-582Crossref PubMed Scopus (191) Google Scholar) as described previously for RNA pol II (25Gebara M. Sayre M.H. Corden J.L. J. Cell. Biochem. 1997; 64: 390-402Crossref PubMed Scopus (69) Google Scholar) but with minor modifications. For each phosphorylation reaction, 10 μl of Cdc2 kinase-bound 12CA5 Affi-Gel beads (∼5 μg of Cdc2 kinase) were incubated with 2.5 μg of fusion protein in 60 mm KCl, 50 mm Tris-HCl, pH 7.8, 10 mm MgCl2, 0.5 mm dithiothreitol, 1 mm ATP for 20 min at 30 °C. For labeling, the reaction was pulsed with 20 μCi of [γ-32P-]ATP for 5 min and then chased with 1 mm ATP for 20 min at 30 °C. Phosphorylated GST-CTD fusion protein was removed from the beads by spinning the supernatant through a bovine serum albumin-treated filter (UFC3 OHV 00; Millipore). The level of phosphorylation was assessed by electrophoresis in a 5% polyacrylamide-SDS gel followed by direct autoradiography. Phosphorylation of the mouse CTD fusion protein with c-ABL kinase was essentially as described (23Baskaran R. Dahmus M.E. Wang J.Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11167-11171Crossref PubMed Scopus (187) Google Scholar). mAb 8WG16 is an anti-CTD IgG described by Thompson et al. (66Thompson N.E. Steinberg T.H. Aronson D.B. Burgess R.R. J. Biol. Chem. 1989; 264: 11511-11520Abstract Full Text PDF PubMed Google Scholar). mAbs H5 and H14 are IgMs directed against phosphoepitopes on the CTD (57Warren S.L. Landolfi A.S. Curtis C. Morrow J.S. J. Cell Sci. 1992; 103: 381-388Crossref PubMed Google Scholar, 58Bregman D.B. Du L. Li Y. Ribisi S. Warren S.L. J. Cell Sci. 1994; 107: 387-396PubMed Google Scholar, 59Bregman D.B. Du L. Vanderzee S. Warren S.L. J. Cell Biol. 1995; 129: 287-298Crossref PubMed Scopus (309) Google Scholar). mAb MARA 3 is an anti-pol II IgM isolated in a screen for mAbs against tyrosine-phosphorylated B cell proteins. 2R. J. Schulte and B. M. Sefton, submitted for publication. mAb CC-3 is an IgG isolated in a screen for chicken proteins with developmentally regulated expression (60Thibodeau A. Vincent M. Exp. Cell Res. 1991; 195: 145-153Crossref PubMed Scopus (27) Google Scholar). mAb B3 is an IgM directed against nuclear matrix components (17Mortillaro M.J. Blencowe B.J. Wei X.Y. Nakayasu H. Du L. Warren S.L. Sharp P.A. Berezney R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8253-8257Crossref PubMed Scopus (282) Google Scholar). An overnight culture ofSaccharomyces cerevisiae YPH499 (ATCC 76625) was used to inoculate YEPD (2% bactopeptone, 2% glucose, 1% yeast extract) to anA600 of 0.2. Aliquots (50 ml) were withdrawn at intervals of 2 h and cells harvested by centrifugation at 4000 × g for 5 min. The yeast cell pellet was washed in ice-cold water and stored at −80 °C. Cells were suspended in 200 μl of buffer A (200 mm Tris-HCl, pH 8, 320 mmammonium sulfate, 5 mm MgCl2, 10 mmEGTA, pH 8, 20 mm EDTA, pH 8, 1 mmdithiothreitol, 20% glycerol, 1 mm phenylmethylsulfonyl fluoride, 2 mm pepstatin, 0.6 mm leupeptin, 2 mm benzamidine HCl). Acid-washed glass beads (425–600 μm; Sigma) were added to the meniscus and vortexed 10 times for 10 s with 30-s intervals on ice. The lysate was cleared by centrifugation at 10,000 × g for 10 min, and supernatant was used for SDS-PAGE analysis. The high ionic strength of the grinding buffer allows extraction of almost all of the pol II in the cell. We see no difference when cells are extracted in denaturing buffer (not shown). Yeast cells (YPH499) were grown at 30 °C for 7 h after inoculation (starting A600 = 0.2). The culture was diluted 1:1 with fresh medium pre-warmed to 55 °C and then maintained at 39 °C for the indicated times. Control cells were diluted 1:1 using fresh medium at room temperature and then incubated at 30 °C for the indicated times. The heat shock response was terminated by further diluting the culture (1:1) with ice-cold water. The cells were harvested and the extract prepared as described above. Phosphorylated and unphosphorylated CTD fusion proteins (250 ng) were subjected to SDS-PAGE (10%) followed by electrophoretic transfer to nitrocellulose paper (Protran, Schleicher & Schuell). The blots were probed with mAb tissue culture supernatants at a dilution of 1:10 (12CA5, 8WG16, H5, and H14) or ascites fluid at a dilution of 1:1000 (CC3 and B3). Immunoreactive proteins were detected using either anti-mouse IgG (Amersham Corp.) or anti-mouse IgM (Kirkegaard & Perry Laboratories) at a dilution of 1:4000. Supersignal substrate (Pierce) was used to illuminate the reactive bands. To ensure that all proteins transferred efficiently, blots probed with anti-phospho-CTD antibodies were stripped and reprobed with mAb 12CA5 which recognizes an epitope present in each fusion protein. In each case all lanes contained 12CA5 immunoreactive bands. Fig. 1 shows the purified GST-CTD fusion proteins used in this study. From examination of the relative mobility of these proteins (Fig. 1 B), it is obvious that the sequence of the unphosphorylated heptapeptide repeat confers aberrant mobility. In particular, glutamate substitution at position 5 seems to increase mobility, whereas glutamate at position 2 results in a marked retardation. The physical basis of this difference is likely due to differences in the ability of SDS to bind to these mutant proteins (22Zhang J. Corden J.L. J. Biol. Chem. 1991; 266: 2290-2296Abstract Full Text PDF PubMed Google Scholar). Phosphorylation of these fusion proteins with Cdc2 kinase results in similar levels of phosphorylation of all proteins with the exception of the E5 mutant (Fig. 1 C). The top panel of Fig. 2 shows that phosphorylation causes additional shifts in electrophoretic mobility of these proteins in SDS-PAGE. mAb 12CA5 was used to detect the fusion proteins (see “Materials and Methods”). The lower intensity of some phosphorylated species indicates that phosphorylation may alter the binding of mAb 12CA5. For the wild-type and alanine-substituted proteins phosphorylation causes a marked retardation in mobility, much as seen with pol II0 in vivo. Phosphorylation of the CTD with glutamate in position 2 causes an increase in mobility. This result suggests that little if any SDS is bound to the unphosphorylated mutant CTD, and phosphorylation thus causes an increase in the charge to mass ratio of the fusion protein in SDS. Phosphorylation of the E5 mutant causes no change in mobility, a result which may be due to the fact that E5 is a poor substrate for Cdc2 phosphorylation as seen in experiments using [γ-32P]ATP (Fig. 1 C). From the >10-fold molar excess of kinase to substrate used in the phosphorylation reaction and the resulting mobility shift on substrates other than E5, we conclude that the CTD is maximally phosphorylated on serines 2 and 5, the known Cdc2 kinase target sites (22Zhang J. Corden J.L. J. Biol. Chem. 1991; 266: 2290-2296Abstract Full Text PDF PubMed Google Scholar). The mAb 8WG16 was raised against wheat germ RNA polymerase II and found to recognize epitopes conserved among the CTDs of pol IIs derived from a variety of different eucaryotes (66Thompson N.E. Steinberg T.H. Aronson D.B. Burgess R.R. J. Biol. Chem. 1989; 264: 11511-11520Abstract Full Text PDF PubMed Google Scholar). The 2nd panel in Fig. 2 shows that mAb 8WG16 recognizes primarily unphosphorylated CTD. Unphosphorylated WT, A5, and E5 interact more strongly than unphosphorylated E2, A2, and phosphorylated A5 and E5. The failure to bind unphosphorylated E2 and weak interaction with unphosphorylated A2 suggests that serine 2 (S2) is an important feature of the 8WG16 epitope. Weak binding to phosphorylated A5 and E5 could mean that position 5 partially overlaps the 8WG16 recognition site. Alternatively, mutation of S5 could interfere with phosphorylation of S2 yielding more unphosphorylated S2 sites and thus partial reactivity with 8WG16. The remainder of the mAbs tested bind only to phosphorylated CTD (Fig. 2). With the exception of B3, the strongest binding is to the phosphorylated WT sequence. From previous studies we know that Cdc2 kinase phosphorylates the consensus heptapeptide repeat on both S2 and S5 (22Zhang J. Corden J.L. J. Biol. Chem. 1991; 266: 2290-2296Abstract Full Text PDF PubMed Google Scholar). Binding to phosphorylated mutant CTD targets reveals interesting differences in the specificities of the different mAbs. mAb H14 recognizes both phosphorylated A2 and E2 but not A5 or E5. This result indicates that H14 recognizes phosphorylated S5. In contrast, mAb H5 recognizes phosphorylated A5 suggesting that phosphoserine in position 2 is a critical feature recognized by mAb H5. Consistent with this interpretation mAb H5 does not recognize phosphorylated A2. Weak interaction between H5 and phosphorylated E2 indicates that substitution of the charged side chain at this position mimics the effect of phosphorylation. Note also that the mobility of unphosphorylated E2 is greatly retarded. Glutamate substitution at position 2 is insufficient for H5 recognition, however, indicating that position 5 phosphorylation may induce a conformational change that enables a weak interaction at position 2. CC3 shows a weak recognition of phosphorylated A5 in addition to recognizing the phosphorylated WT. Thus, like mAb H5, phosphoserine in position 2 appears to be part of the CC3 epitope. None of the other phosphorylated CTDs are recognized by CC3, although in the case of E5 this may be due to the low level of phosphorylation. MARA 3 recognizes phosphorylated A2, A5, and E2. This could mean that both phosphoserines S2 and S5 are recognized or, alternatively, that the epitope is distinct from either site but requires at least one of the serines to be phosphorylated to achieve the proper conformation. The strong interaction of MARA 3 with phosphorylated E2 suggests that the charged residue in position 2 mimics phosphoserine. The fact that phosphorylated A2 and E2 interact better than A5 suggests that MARA 3 is related to H14. MARA 3 recognizes phosphorylated A5, however, whereas H14 does not recognize it at all. Furthermore, MARA 3 recognizes WT better than A2, whereas H14 interacts with both phosphorylated substrates to the same extent. mAb B3 is unique in interacting with phosphorylated A2 more strongly than with WT and the other mutants. This could indicate that the epitope recognized by B3 is phosphorylated at position 5. In this respect B3 is like H14. B3's weak interaction with A5, however, suggests a more complicated situation. For example, B3 may prefer a phosphoepitope in the distal nonconsensus repeats of the CTD which are only approximated by the A2 mutation. To verify that the mAbs used in this study also recognize the mammalian CTD, we generated several phosphorylated mouse CTD fusion proteins. Fig. 3 shows the interaction with unphosphorylated, Cdc2, and c-ABL phosphorylated mouse CTD fusion proteins. All of the anti-phosphorylated CTD mAbs tested interact solely with the Cdc2 phosphorylated molecules and not with unphosphorylated or tyrosine-phosphorylated CTD. mAb 8WG16 shows a very weak recognition of the CTD phosphorylated by c-ABLsuggesting that not all repeats have been phosphorylated. Our earlier studies showed that different CTD kinases can recognize different serine phosphoacceptors in the CTD (25Gebara M. Sayre M.H. Corden J.L. J. Cell. Biochem. 1997; 64: 390-402Crossref PubMed Scopus (69) Google Scholar). In vivo, the plethora of CTD kinases could give rise to a family of pol II phosphoisomers whose populations may change under different growth conditions. To test this hypothesis we examined CTD phosphorylation in cells in different stages of growth. Fig. 4 A shows a typical yeast growth curve, and Fig. 4 B shows the results of Western blot analysis using different anti-CTD" @default.
• W2004408181 created "2016-06-24" @default.
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• W2004408181 date "1998-02-01" @default.
• W2004408181 modified "2023-10-12" @default.
• W2004408181 title "Growth-related Changes in Phosphorylation of Yeast RNA Polymerase II" @default.
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• W2004408181 doi "https://doi.org/10.1074/jbc.273.8.4689" @default.
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