Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

TFIIF-associating Carboxyl-terminal Domain Phosphatase Dephosphorylates Phosphoserines 2 and 5 of RNA Polymerase II

2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês

10.1074/jbc.m208588200

ISSN

1083-351X

Autores

Patrick S. Lin, Marie‐Françoise Dubois, Michael Dahmus,

Tópico(s)

RNA and protein synthesis mechanisms

Resumo

The carboxyl-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit undergoes reversible phosphorylation throughout the transcription cycle. The unphosphorylated form of RNAP II is referred to as IIA, whereas the hyperphosphorylated form is known as IIO. Phosphorylation occurs predominantly at serine 2 and serine 5 within the CTD heptapeptide repeat and has functional implications for RNAP II with respect to initiation, elongation, and transcription-coupled RNA processing. In an effort to determine the role of the major CTD phosphatase (FCP1) in regulating events in transcription that appear to be influenced by serine 2 and serine 5 phosphorylation, the specificity of FCP1 was examined. FCP1 is capable of dephosphorylating heterogeneous RNAP IIO populations of HeLa nuclear extracts. The extent of dephosphorylation at specific positions was assessed by immunoreactivity with monoclonal antibodies specific for phosphoserine 2 or phosphoserine 5. As an alternative method to assess FCP1 specificity, RNAP IIO isozymes were prepared in vitroby the phosphorylation of purified calf thymus RNAP IIA with specific CTD kinases and used as substrates for FCP1. FCP1 dephosphorylates serine 2 and serine 5 with comparable efficiency. Accordingly, the specificity of FCP1 is sufficiently broad to dephosphorylate RNAP IIO at any point in the transcription cycle irrespective of the site of serine phosphorylation within the consensus repeat. The carboxyl-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit undergoes reversible phosphorylation throughout the transcription cycle. The unphosphorylated form of RNAP II is referred to as IIA, whereas the hyperphosphorylated form is known as IIO. Phosphorylation occurs predominantly at serine 2 and serine 5 within the CTD heptapeptide repeat and has functional implications for RNAP II with respect to initiation, elongation, and transcription-coupled RNA processing. In an effort to determine the role of the major CTD phosphatase (FCP1) in regulating events in transcription that appear to be influenced by serine 2 and serine 5 phosphorylation, the specificity of FCP1 was examined. FCP1 is capable of dephosphorylating heterogeneous RNAP IIO populations of HeLa nuclear extracts. The extent of dephosphorylation at specific positions was assessed by immunoreactivity with monoclonal antibodies specific for phosphoserine 2 or phosphoserine 5. As an alternative method to assess FCP1 specificity, RNAP IIO isozymes were prepared in vitroby the phosphorylation of purified calf thymus RNAP IIA with specific CTD kinases and used as substrates for FCP1. FCP1 dephosphorylates serine 2 and serine 5 with comparable efficiency. Accordingly, the specificity of FCP1 is sufficiently broad to dephosphorylate RNAP IIO at any point in the transcription cycle irrespective of the site of serine phosphorylation within the consensus repeat. Reversible phosphorylation of the carboxyl-terminal domain (CTD) 1The abbreviations used for: CTD, carboxyl-terminal domain; CTDa and CTDo, unphosphorylated and hyperphosphorylated CTD, respectively; RNAP II, RNA polymerase II; FCP, TFIIF-associating CTD phosphatase; TFII, general transcription factor for RNA polymerase II; RAP, RNA polymerase II-associating protein; GST, glutathione S-transferase; P-TEF, positive transcription elongation factor; MAP, mitogen-activated protein; MAPK2/ERK2, MAP kinase 2/extracellular signal-regulated kinase 2; DTT, dithiothreitol. of the largest RNA polymerase (RNAP) II subunit plays an important role in the regulation of gene expression. The CTD of mammalian RNAP II is comprised of 52 repeats of the consensus sequence 1The abbreviations used for: CTD, carboxyl-terminal domain; CTDa and CTDo, unphosphorylated and hyperphosphorylated CTD, respectively; RNAP II, RNA polymerase II; FCP, TFIIF-associating CTD phosphatase; TFII, general transcription factor for RNA polymerase II; RAP, RNA polymerase II-associating protein; GST, glutathione S-transferase; P-TEF, positive transcription elongation factor; MAP, mitogen-activated protein; MAPK2/ERK2, MAP kinase 2/extracellular signal-regulated kinase 2; DTT, dithiothreitol.YSPTSPS7(for a review, see Ref. 1Dahmus M.E. J. Biol. Chem. 1996; 271: 19009-19012Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). RNAP IIA, which contains an unmodified CTD, is actively recruited to the promoter as part of the preinitiation complex (2Laybourn P.J. Dahmus M.E. J. Biol. Chem. 1990; 265: 13165-13173Abstract Full Text PDF PubMed Google Scholar, 3Lu H. Flores O. Weinmann R. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10004-10008Crossref PubMed Scopus (248) Google Scholar, 4Chesnut J.D. Stephens J.H. Dahmus M.E. J. Biol. Chem. 1992; 267: 10500-10506Abstract Full Text PDF PubMed Google Scholar, 5Kang M.E. Dahmus M.E. J. Biol. Chem. 1993; 268: 25033-25040Abstract Full Text PDF PubMed Google Scholar), whereas RNAP IIO, which contains a hyperphosphorylated CTD, is responsible for transcript elongation (6Bartholomew B. Dahmus M.E. Meares C.F. J. Biol. Chem. 1986; 261: 14226-14231Abstract Full Text PDF PubMed Google Scholar, 7Cadena D.L. Dahmus M.E. J. Biol. Chem. 1987; 262: 12468-12474Abstract Full Text PDF PubMed Google Scholar). Therefore, protein kinases and phosphatases that alter the state of CTD phosphorylation can serve as transcriptional activators or repressors depending on the point in the transcription cycle at which they function. CTD phosphorylation occurs predominantly at serines 2 and 5 within the heptapeptide repeat. Genetic evidence indicates that the roles of serines in positions 2 and 5 are different. First, the partial substitution of serines in either position 2 or 5 have different effects on viability (8West M.L. Corden J.L. Genetics. 1995; 140: 1223-1233Crossref PubMed Google Scholar). Second, SRB (suppressors ofRNA Polymerase IIB) mutations suppress the lethal effect of position 2 substitutions but not position 5 substitutions (9Yuryev A. Corden J.L. Genetics. 1996; 143: 661-671Crossref PubMed Google Scholar). Biochemical evidence has also confirmed differences between the two predominant serine positions. Serine 5 but not serine 2 phosphorylation recruits and activates the 5′-capping machinery (10Ho C.K. Shuman S. Mol. Cell. 1999; 3: 405-411Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar,11Rodriguez C.R. Cho E.-J. Keogh M.-C. Moore C.L. Greenleaf A.L. Buratowski S. Mol. Cell. Biol. 2000; 20: 104-112Crossref PubMed Scopus (161) Google Scholar). Furthermore, nutritional stress and heat shock can independently alter the pattern of CTD phosphorylation, indicating that phosphoserine 2 and phosphoserine 5 are functionally different (12Dubois M.F. Vincent M. Vigneron M. Adamczewski J. Egly J.M. Bensaude O. Nucleic Acids Res. 1997; 25: 694-700Crossref PubMed Scopus (55) Google Scholar, 13Patturajan M. Schulte R.J. Sefton B.M. Berezney R. Vincent M. Bensaude O. Warren S.L. Corden J.L. J. Biol. Chem. 1998; 273: 4689-4694Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 14Lavoie S.B. Albert A.L. Thibodeau A. Vincent M. Biochem. Cell Biol. 1999; 77: 367-374Crossref PubMed Scopus (7) Google Scholar). A recent study using chromatin immunoprecipitation inSaccharomyces cerevisiae demonstrates that the pattern of CTD phosphorylation changes as RNAP II transcribes a given gene (15Komarnitsky P. Cho E.J. Buratowski S. Genes Dev. 2000; 14: 2452-2460Crossref PubMed Scopus (802) Google Scholar). Serine 5 phosphorylation is detected at the promoter regions, whereas serine 2 phosphorylation is increased as RNAP II leaves the promoter and transcribes the body of the gene. This dynamic phosphorylation of the CTD has functional consequences for the synthesis and processing of the primary transcript. During initiation, TFIIH phosphorylates the CTD at serine 5 (16Lu H. Zawel L. Fisher L. Egly J.M. Reinberg D. Nature. 1992; 358: 641-645Crossref PubMed Scopus (330) Google Scholar, 17Hengartner C.J. Myer V.E. Liao S.M. Wilson C.J. Koh S.S. Young R.A. Mol. Cell. 1998; 2: 43-53Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). Phosphorylation presumably disrupts various protein-protein interactions important in the formation of the preinitiation complex (18Svejstrup J.Q. Li Y. Fellows J. Gnatt A. Bjorklund S. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6075-6078Crossref PubMed Scopus (101) Google Scholar). Serine 5 phosphorylation specifically facilitates the recruitment and activation of the mammalian capping enzyme (10Ho C.K. Shuman S. Mol. Cell. 1999; 3: 405-411Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 19McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (433) Google Scholar). Capping of the 5′ end of the RNA occurs as soon as the nascent transcript becomes accessible, usually at an RNA length of 25–30 nucleotides (20Rasmussen E.B. Lis J.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7923-7927Crossref PubMed Scopus (284) Google Scholar). Similarly in yeast, the recruitment and activation of Ceg1 (guanylyltransferase) and Abd1 (methyltransferase) are dependent on serine 5 phosphorylation (11Rodriguez C.R. Cho E.-J. Keogh M.-C. Moore C.L. Greenleaf A.L. Buratowski S. Mol. Cell. Biol. 2000; 20: 104-112Crossref PubMed Scopus (161) Google Scholar, 19McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (433) Google Scholar, 21Cho E.J. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (373) Google Scholar, 22Schroeder S.C. Schwer B. Shuman S. Bentley D. Genes Dev. 2000; 14: 2435-2440Crossref PubMed Scopus (302) Google Scholar). The retention of these capping enzymes displays a 5′-3′ polarity on the gene (22Schroeder S.C. Schwer B. Shuman S. Bentley D. Genes Dev. 2000; 14: 2435-2440Crossref PubMed Scopus (302) Google Scholar). Ceg1 is released early in elongation, whereas Abd1 is released toward the 3′ end of the gene. Although it is unclear what happens to phosphoserine 5, an increase in serine 2 phosphorylation is observed as RNAP II elongates past position 200 (15Komarnitsky P. Cho E.J. Buratowski S. Genes Dev. 2000; 14: 2452-2460Crossref PubMed Scopus (802) Google Scholar). P-TEFb is the most likely candidate for serine 2 phosphorylation. P-TEFb preferentially phosphorylates serine 2 in early elongation complexes (23Zhou M. Halanski M.A. Radonovich M.F. Kashanchi F. Peng J. Price D.H. Brady J.N. Mol. Cell. Biol. 2000; 20: 5077-5086Crossref PubMed Scopus (220) Google Scholar) and promotes processive transcript elongation by alleviating the negative effects of DSIF and NELF (24Wada T. Takagi T. Yamaguchi Y. Watanabe D. Handa H. EMBO J. 1998; 17: 7395-7403Crossref PubMed Scopus (282) Google Scholar). The dynamic phosphorylation of the CTD and the preferential phosphorylation of serine 2 and serine 5 can be viewed as molecular switches that control the progression of RNAP II and the recruitment of factors involved in the synthesis and processing of the primary transcript. The extent and specificity of CTD phosphorylation is maintained by the opposing actions of CTD kinases and CTD phosphatase(s). For example, Ctk1 (a putative P-TEFb homolog in yeast) and FCP1 (TFIIF-associating CTDphosphatase) appear to modulate the level of serine 2 phosphorylation in the RNAP II elongation complex (25Cho E.-J. Kobor M.S. Kim M. Greenblatt J. Buratowski S. Genes Dev. 2001; 15: 3319-3329Crossref PubMed Scopus (340) Google Scholar). In addition, the level of phosphorylation of nontranscribing RNAP IIO inXenopus laevis early embryos is maintained by MAP kinase Xp42 and FCP1 (26Palancade B. Dubois M.F. Dahmus M.E. Bensaude O. Mol. Cell. Biol. 2001; 21: 6359-6368Crossref PubMed Scopus (26) Google Scholar). Unlike many CTD kinases that have been discovered and characterized, a single CTD phosphatase has been reported to date (for a review, see Ref. 27Lin P.S. Marshall N.F. Dahmus M.E. Prog. Nucleic Acid Res. Mol. Biol. 2002; 72: 333-365Crossref PubMed Google Scholar). Genetic studies have demonstrated that FCP1 is required for transcription in vivo, and its inactivation leads to a global defect in mRNA synthesis (28Kobor M.S. Archambault J. Lester W. Holstege F.C. Gileadi O. Jansma D.B. Jennings E.G. Kouyoumdjian F. Davidson A.R. Young R.A. Greenblatt J. Mol. Cell. 1999; 4: 55-62Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The dephosphorylation of RNAP II is dependent on the interaction of FCP1 with a site on RNAP II that is outside of the CTD (29Chambers 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, 30Kimura M. Suzuki H. Ishihama A. Mol. Cell. Biol. 2002; 22: 1577-1588Crossref PubMed Scopus (84) Google Scholar). FCP1 activity is stimulated by RAP74, the larger of the two subunits of TFIIF (29Chambers 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). TFIIB abrogates the stimulatory activity of TFIIF but has no influence on FCP1 activity in the absence of TFIIF. FCP1 dephosphorylates RNAP IIO generated by serine/threonine CTD kinases but is not sensitive to vanadate, a tyrosine phosphatase inhibitor (31Chambers R.S. Dahmus M.E. J. Biol. Chem. 1994; 269: 26243-26248Abstract Full Text PDF PubMed Google Scholar). Furthermore, the sensitivity of RNAP IIO in an elongation complex to FCP1 is dependent on its position with respect to the transcriptional start site (32Lehman A.L. Dahmus M.E. J. Biol. Chem. 2000; 275: 14923-14932Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 33Marshall N.F. Dahmus M.E. J. Biol. Chem. 2000; 275: 32430-32437Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Although it has been established that FCP1 dephosphorylates phosphoserine 2 at the 3′ end of the gene (25Cho E.-J. Kobor M.S. Kim M. Greenblatt J. Buratowski S. Genes Dev. 2001; 15: 3319-3329Crossref PubMed Scopus (340) Google Scholar) and can recycle RNAP IIO to RNAP IIA (34Cho H. Kim T.K. Mancebo H. Lane W.S. Flores O. Reinberg D. Genes Dev. 1999; 13: 1540-1552Crossref PubMed Scopus (172) Google Scholar), it is unclear if FCP1 can dephosphorylate phosphoserine 5 during transcript elongation (15Komarnitsky P. Cho E.J. Buratowski S. Genes Dev. 2000; 14: 2452-2460Crossref PubMed Scopus (802) Google Scholar). To understand the involvement of FCP1 at discrete stages in the transcription cycle, it is necessary to determine its substrate specificity. To examine FCP1 specificity, two independent experimental approaches were used in this study. First, FCP1 activity was assayed toward endogenous RNAP IIO populations contained in HeLa nuclear extracts. Second, FCP1 activity was assessed using purified calf thymus RNAP IIO substrates prepared in vitro by the phosphorylation of RNAP IIA with different CTD kinases. [γ-32P]ATP (6000 Ci/mmol) was purchased from PerkinElmer Life Sciences. Human recombinant casein kinase II and mouse recombinant MAP kinase 2/Erk 2 (MAPK2/ERK2) were obtained from Upstate Biotechnology, and human recombinant Cdc2 kinase was purchased from New England Biolabs. Human CTDK1/CTDK2 were partially purified as previously described (35Payne J.M. Dahmus M.E. J. Biol. Chem. 1993; 268: 80-87Abstract Full Text PDF PubMed Google Scholar). Human TFIIH was generously provided by Dr. Jean-Marc Egly (36Gerard M. Fischer L. Moncollin V. Chipoulet J.M. Chambon P. Egly J.M. J. Biol. Chem. 1991; 266: 20940-20945Abstract Full Text PDF PubMed Google Scholar). Human P-TEFb was partially purified from HeLa S-100 extract by chromatography on heparin-Sepharose (Amersham Biosciences), DEAE 15HR (Millipore), HiTrap S, and Phenyl-Superose (both from Amersham Biosciences). P-TEFb was dialyzed against buffer A (25 mm Hepes, pH 7.9, 20% glycerol, 25 mm KCl, 0.1 mm EDTA, 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride). Monoclonal antibody POL3/3 recognizes a conserved epitope within the largest RNAP II subunit that is distinct from the CTD (37Kramer A. Haars R. Kabisch R. Will H. Bautz F.A. Bautz E.K. Mol. Gen. Genet. 1980; 180: 193-199Crossref PubMed Scopus (80) Google Scholar, 38Kontermann R.E. Liu Z. Schulze R.A. Sommer K.A. Queitsch I. Duebel S. Kipriyanov S.M. Breitling F. Bautz E.K.F. Biol. Chem. Hoppe-Seyler. 1995; 376: 473-481Crossref PubMed Scopus (31) Google Scholar). H5 and H14 are IgMs directed against phospho-epitopes within the CTD (39Warren S.L. Landolfi A.S. Curtis C. Morrow J.S. J. Cell Sci. 1992; 103: 381-388Crossref PubMed Google Scholar, 40Bregman D.B. Du L. Li Y. Ribisi S. Warren S.L. J. Cell Sci. 1994; 107: 387-396PubMed Google Scholar, 41Bregman D.B. Du L. van der Zee S. Warren S.L. J. Cell Biol. 1995; 129: 287-298Crossref PubMed Scopus (311) Google Scholar) and were obtained from Covance. CC3 is an IgG isolated in a screen for chicken proteins with developmentally regulated expression (42Thibodeau A. Vincent M. Exp. Cell Res. 1991; 195: 145-153Crossref PubMed Scopus (27) Google Scholar). B3 is an IgM directed against nuclear matrix components (43Mortillaro M.J. Blencowe B.J. Wei X. 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). HeLa nuclear extracts were prepared from control cells and cells treated with actinomycin D at 1 μg/ml for 1 h or serum-deprived for 24 h and stimulated with 20% serum for 1 h. After their respective treatments, the cell monolayers grown in 150-cm2 dishes were washed with cold phosphate-buffered saline and gently removed by scrapping in buffer B (10 mm Hepes, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm DTT). Cells were centrifuged at 1,000 ×g for 10 min, and the pellets were resuspended in buffer B on ice. The cells were then homogenized with a Dounce homogenizer (10 times), and the lysates were centrifuged at 10,000 × gfor 10 min and fractionated into nuclear pellets and cytosolic supernatants. The nuclear pellets were resuspended in buffer C (20 mm Hepes, pH 7.9, 1.5 mm MgCl2, 25% glycerol, 420 mm NaCl, 0.2 mm EDTA, 0.5 mm DTT) and centrifuged at 15,000 × g for 20 min. Pellets were resuspended in buffer D (20 mm Hepes, pH 7.9, 1.5 mm MgCl2, 25% glycerol, 1m NaCl, 0.2 mm EDTA, 0.5 mm DTT) through a syringe to fragment the DNA and centrifuged at 15,000 ×g for 20 min. The nuclear extracts were dialyzed against buffer E (50 mm Tris, pH 7.9, 20% glycerol, 120 mm KCl, 0.1 mm EDTA, 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride). Calf thymus RNAP IIA was purified by the method of Hodo and Blatti (44Hodo H.D. Blatti S.P. Biochemistry. 1977; 16: 2334-2343Crossref PubMed Scopus (129) Google Scholar) with modifications as described by Kang and Dahmus (5Kang M.E. Dahmus M.E. J. Biol. Chem. 1993; 268: 25033-25040Abstract Full Text PDF PubMed Google Scholar). Specific isozymes of 32P-labeled RNAP IIO were prepared by phosphorylation of purified RNAP IIA with recombinant casein kinase II and [γ-32P]ATP followed by phosphorylation in the presence of excess unlabeled ATP (2 mm) with either purified CTDK1/CTDK2, TFIIH, P-TEFb, recombinant MAPK2/ERK2, or Cdc2 kinase. Each RNAP IIO isozyme was purified by step elution from DE53 as previously described (4Chesnut J.D. Stephens J.H. Dahmus M.E. J. Biol. Chem. 1992; 267: 10500-10506Abstract Full Text PDF PubMed Google Scholar). Because only the most carboxyl-terminal serine (casein kinase II site) is labeled with 32P and lies outside the consensus repeat, dephosphorylation by CTD phosphatase results in an electrophoretic mobility shift of subunit IIo to the position of subunit IIa without loss of label. CTD kinase assays were performed as described previously (35Payne J.M. Dahmus M.E. J. Biol. Chem. 1993; 268: 80-87Abstract Full Text PDF PubMed Google Scholar). Reactions were performed in 20 μl of CTD kinase buffer (20 mm Hepes, pH 7.9, 8 mmMgCl2, 0.5% glycerol, 0.1% Triton X-100, 1 mmDTT). Each reaction contained 2.75 fmol of 32P-labeled GST-CTDa and an equivalent molar amount of 32P-labeled RNAP IIA. Reactions were initiated by the addition of either TFIIH, P-TEFb, or MAPK2/ERK2 and incubated at 30 °C for 30 min. Assays were terminated by the addition of 5× Laemmli buffer, and RNAP II subunits were resolved on a 5% SDS-PAGE gel. The gel image was scanned in a Molecular Dynamics Image Scanner Storm 860 in the phosphor screen mode and analyzed by ImageQuant software. Both purified and recombinant human FCP1 were used in these studies. Human FCP1 was purified from HeLa cells as described previously (45Marshall N.F. Dahmus G.K. Dahmus M.E. J. Biol. Chem. 1998; 273: 31726-31730Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and used in assays involving endogenous RNAP IIO substrates. Recombinant human FCP1 was expressed in Sf21 cells, purified to homogeneity, and used in assays involving RNAP IIO substrates prepared in vitro by CTD kinases. Recombinant FCP1 was purified by chromatography on Ni2+-nitrilotriacetic acid-agarose (Qiagen) and HiTrap SP and Q in series (both from Amersham Biosciences) and HiTrapN-hydroxysuccinimide-activated column that had been coupled with recombinant RAP74 (Amersham Biosciences). Purified FCP1 had a specific activity of 40,000 units/mg, whereas recombinant FCP1 had a specific activity of 270,000 units/mg. One unit of CTD phosphatase corresponds to the activity required to convert 1 pmol of free RNAP IIO to RNAP IIA in 1 min in the presence of a saturating amount of RAP74. CTD phosphatase assays were performed as described previously (31Chambers R.S. Dahmus M.E. J. Biol. Chem. 1994; 269: 26243-26248Abstract Full Text PDF PubMed Google Scholar) with minor modifications. Reactions were performed in 20 μl of CTD phosphatase buffer (50 mm Tris, pH 7.9, 10 mmMgCl2, 20% glycerol, 0.025% Tween 80, 0.1 mmEDTA, 5 mm DTT) in the presence of 20 mm KCl. Reactions involving endogenous RNAP II in HeLa nuclear extracts contained ∼200–250 fmol of RNAP IIO. Reactions involving purified RNAP II contained 18 fmol of RNAP IIO. Both reactions were carried out in the presence of 7 pmol of RAP74. Reactions were initiated by the addition of FCP1 and incubated at 30 °C for 30 min. Assays were terminated by the addition of 5× Laemmli buffer, and RNAP II subunits were resolved on a 5 or 6% SDS-PAGE gel. CTD phosphatase assays of endogenous RNAP IIO contained in nuclear extracts were analyzed by Western blots using dilutions of 1:1,000 POL3/3, 1:250 H5, 1:250 H14, 1:1,000 CC3, or 1:1,000 B3 followed by 1:10,000 anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (Promega). The blots were visualized by ECL Plus detection system (AmershamBiosciences). CTD phosphatase assays of FCP1 on purified casein kinase II-labeled RNAP IIO were analyzed by autoradiography. The corresponding blot and gel images were scanned on a Molecular Dynamics Image Scanner Storm 860 in blue fluorescence mode and phosphor screen mode, respectively. FCP1 specificity was initially examined with in vivo populations of heterogeneous RNAP IIO contained in HeLa nuclear extracts. The reactivity of endogenous RNAP IIO with phosphoserine-specific monoclonal antibodies was assessed before and after dephosphorylation with FCP1. Based on reactivity with synthetic peptides, monoclonal antibodies H5 and CC3 recognize phosphoserine in position 2 within the heptapeptide repeat, whereas H14 and B3 recognize phosphoserine in position 5 (Fig.1 A) (13Patturajan M. Schulte R.J. Sefton B.M. Berezney R. Vincent M. Bensaude O. Warren S.L. Corden J.L. J. Biol. Chem. 1998; 273: 4689-4694Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). POL3/3, directed against an epitope in the largest RNAP II subunit that is outside of the CTD (37Kramer A. Haars R. Kabisch R. Will H. Bautz F.A. Bautz E.K. Mol. Gen. Genet. 1980; 180: 193-199Crossref PubMed Scopus (80) Google Scholar, 38Kontermann R.E. Liu Z. Schulze R.A. Sommer K.A. Queitsch I. Duebel S. Kipriyanov S.M. Breitling F. Bautz E.K.F. Biol. Chem. Hoppe-Seyler. 1995; 376: 473-481Crossref PubMed Scopus (31) Google Scholar), permits the detection of the largest subunit irrespective of the state of CTD phosphorylation. The degree and the specificity by which FCP1 dephosphorylates endogenous RNAP IIO were measured by the disappearance of immunoreactivity. Because a variety of studies have shown that changes in growth conditions can give rise to changes in the level and pattern of CTD phosphorylation, HeLa nuclear extracts from differentially treated cells provide a source of heterogeneous RNAP IIO. The FCP1 sensitivity of RNAP IIO present in HeLa nuclear extracts of control cells, cells treated with actinomycin D, and cells stimulated with serum was examined. Treatment of cells with the transcription inhibitor actinomycin D or α-amanitin increases the ratio of RNAP IIO/IIA (46Cassé C. Giannoni F. Nguyen V.T. Dubois M.F. Bensaude O. J. Biol. Chem. 1999; 274: 16097-16106Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Serum stimulation and heat shock increase the level of RNAP IIO via the cellular activation of MAPK2/ERK2 (47Dubois M.F. Nguyen V.T. Dahmus M.E. Pages G. Pouyssegur J. Bensaude O. EMBO J. 1994; 13: 4787-4797Crossref PubMed Scopus (72) Google Scholar, 48Dubois M.F. Bellier S. Seo S.J. Bensaude O. J. Cell. Physiol. 1994; 158: 417-426Crossref PubMed Scopus (49) Google Scholar, 49Venetianer A. Dubois M.-F. Nguyen V.T. Bellier S. Seo S.-J. Bensaude O. Eur. J. Biochem. 1995; 233: 83-92Crossref PubMed Scopus (42) Google Scholar) and change the pattern of CTD phosphorylation (12Dubois M.F. Vincent M. Vigneron M. Adamczewski J. Egly J.M. Bensaude O. Nucleic Acids Res. 1997; 25: 694-700Crossref PubMed Scopus (55) Google Scholar, 13Patturajan M. Schulte R.J. Sefton B.M. Berezney R. Vincent M. Bensaude O. Warren S.L. Corden J.L. J. Biol. Chem. 1998; 273: 4689-4694Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 14Lavoie S.B. Albert A.L. Thibodeau A. Vincent M. Biochem. Cell Biol. 1999; 77: 367-374Crossref PubMed Scopus (7) Google Scholar). The increase in IIo signals, as indicated by increases in POL3/3 immunoreactivity relative to control extracts, confirms the above-mentioned studies that RNAP IIO levels are elevated in response to environmental stimuli (Fig. 1 B, panel POL3/3, compare lane 1 with lanes 3 and5). Upon treatment of the three HeLa nuclear extracts with 100 milliunits of FCP1, the major fraction of RNAP IIO is converted to RNAP IIA (Fig.1 B, panel POL3/3, lanes 2,4, and 6). FCP1 processively dephosphorylates endogenous RNAP IIO. As indicated by the disappearance of immunoreactivity of H5 and H14, FCP1 is capable of removing phosphates from serine 2 and serine 5 in control HeLa nuclear extract (Fig.1 B, panels H5 and H14, lanes 1 and 2). Likewise, FCP1 shows similar specificity toward RNAP IIO in HeLa nuclear extract from cells treated with actinomycin D (Fig. 1 B, panels H5 andH14, lanes 3 and 4). Interestingly, phosphoserine 2 is relatively resistant to FCP1 dephosphorylation in HeLa nuclear extract from cells stimulated with serum (Fig.1 B, panels H5 and H14, lanes 5 and 6). These observations are confirmed by the use of CC3 and B3 in parallel Western blots (Fig. 1 B,panels CC3 and B3). In the presence of higher concentrations of FCP1, the complete dephosphorylation of phosphoserine 2 in serum-stimulated HeLa nuclear extract is observed (data not shown). These results indicate that FCP1 is capable of removing phosphates from serine positions 2 and 5. To investigate whether the resistance of phosphoserine 2 to FCP1 dephosphorylation is conferred with increasing time of serum exposure, HeLa cells were serum-starved for 24 h, and nuclear extracts were prepared from cells treated with 20% serum for 0, 10, 30, and 60 min. The percentage of RNAP IIO that remained relatively resistant to dephosphorylation did not change as a function of time, suggesting that resistance of phosphoserine 2 to FCP1 dephosphorylation is conferred by serum starvation rather than serum stimulation (data not shown). An alternative approach to establishing the specificity of FCP1 is to examine the ability of FCP1 to dephosphorylate RNAP IIO prepared in vitro by the phosphorylation of RNAP IIA with distinct CTD kinases. The phosphorylation of purified calf thymus RNAP IIA and GST-CTDa in the presence of increasing amounts of TFIIH (lanes 1–5), P-TEFb (lanes 6–10), and MAPK2/ERK2 (lanes 11–15) is shown in Fig. 2. Both RNAP IIA and GST-CTDa are labeled with 32P at their terminal serine by phosphorylation with casein kinase II. Because RNAP IIA and GST-CTDa are present in equimolar amounts in the same reaction, the efficiency with which each is shifted to the phosphorylated form is a measure of the substrate specificity of the CTD kinase present. The relative difference in the intensity of radiolabeled GST-CTDa and radiolabeled subunit IIa is a consequence of the difference in the efficiency of32P incorporation by casein kinase II. TFIIH and P-TEFb efficiently convert RNAP IIA to RNAP IIO in a processive manner but show no or marginal activity toward GST-CTDa, respectively (Fig. 2, lanes 1–5 and lanes 6–10). However, MAPK2/ERK2 converts both RNAP IIA and GST-CTDa to their phosphorylated forms in a distributive manner and with comparable efficiency (Fig. 2, lanes 11–15). This result indicates that the activities of TFIIH and P-TEFb are strongly dependent on the context in which the CTD is presented. In contrast, the activity of MAPK2/ERK2 appears to be insensitive to context. These findings suggest that the activities of TFIIH and P-TEFb are dependent on factors that are extrinsic to the CTD. Accordingly, reacti

Referência(s)