Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 Protein Kinase
2003; Elsevier BV; Volume: 278; Issue: 44 Linguagem: Inglês
10.1074/jbc.m307319200
ISSN1083-351X
Autores Tópico(s)Bacterial Genetics and Biotechnology
ResumoSchizosaccharomyces pombe Cdk9/Pch1 protein kinase is a functional ortholog of the essential Saccharomyces cerevisiae Bur1/Bur2 kinase and a putative ortholog of metazoan P-TEFb (Cdk9/cyclin T). SpCdk9/Pch1 phosphorylates of the carboxyl-terminal domain (CTD) of the S. pombe transcription elongation factor Spt5, which consists of 18 tandem repeats of a nonapeptide of consensus sequence 1TPAWNSGSK9. We document the divalent cation dependence and specificity of SpCdk9/Pch1, its NTP dependence and specificity, the dependence of Spt5-CTD phosphorylation on the number of tandem nonamer repeats, and the specificity for phosphorylation of the Spt5-CTD on threonine at position 1 within the nonamer element. SpCdk9/Pch1 also phosphorylates the CTD heptaptide repeat array of the largest subunit of S. pombe RNA polymerase II (consensus sequence YSPTSPS) and does so exclusively on serine. SpCdk9/Pch1 catalyzes autophosphorylation of the kinase and cyclin subunits of the kinase complex. The distribution of phosphorylation sites on SpCdk9 (86% Ser(P), 11% Thr(P), 3% Tyr(P)) is distinct from that on Pch1 (2% Ser(P), 98% Thr(P)). We conducted a structure-guided mutational analysis of SpCdk9, whereby a total of 29 new mutations of 12 conserved residues were tested for in vivo function by complementation of a yeast bur1Δ mutant. We identified many lethal and conditional mutations of side chains implicated in binding ATP and the divalent cation cofactor, phosphoacceptor substrate recognition, and T-loop dynamics. We surmise that the lethality of the of T212A mutation in the T-loop reflects an essential phosphorylation event, insofar as the conservative T212S change rescued wild-type growth; the phosphomimetic T212E change rescued growth at 30 °C; and the effects of mutating the T-loop threonine were phenocopied by mutations in the three conserved arginines predicted to chelate the phosphate on the T-loop threonine. Schizosaccharomyces pombe Cdk9/Pch1 protein kinase is a functional ortholog of the essential Saccharomyces cerevisiae Bur1/Bur2 kinase and a putative ortholog of metazoan P-TEFb (Cdk9/cyclin T). SpCdk9/Pch1 phosphorylates of the carboxyl-terminal domain (CTD) of the S. pombe transcription elongation factor Spt5, which consists of 18 tandem repeats of a nonapeptide of consensus sequence 1TPAWNSGSK9. We document the divalent cation dependence and specificity of SpCdk9/Pch1, its NTP dependence and specificity, the dependence of Spt5-CTD phosphorylation on the number of tandem nonamer repeats, and the specificity for phosphorylation of the Spt5-CTD on threonine at position 1 within the nonamer element. SpCdk9/Pch1 also phosphorylates the CTD heptaptide repeat array of the largest subunit of S. pombe RNA polymerase II (consensus sequence YSPTSPS) and does so exclusively on serine. SpCdk9/Pch1 catalyzes autophosphorylation of the kinase and cyclin subunits of the kinase complex. The distribution of phosphorylation sites on SpCdk9 (86% Ser(P), 11% Thr(P), 3% Tyr(P)) is distinct from that on Pch1 (2% Ser(P), 98% Thr(P)). We conducted a structure-guided mutational analysis of SpCdk9, whereby a total of 29 new mutations of 12 conserved residues were tested for in vivo function by complementation of a yeast bur1Δ mutant. We identified many lethal and conditional mutations of side chains implicated in binding ATP and the divalent cation cofactor, phosphoacceptor substrate recognition, and T-loop dynamics. We surmise that the lethality of the of T212A mutation in the T-loop reflects an essential phosphorylation event, insofar as the conservative T212S change rescued wild-type growth; the phosphomimetic T212E change rescued growth at 30 °C; and the effects of mutating the T-loop threonine were phenocopied by mutations in the three conserved arginines predicted to chelate the phosphate on the T-loop threonine. mRNA synthesis by RNA polymerase (pol) 1The abbreviations used are: pol, polymerase; Cdk, cyclin-dependent kinase; CTD, carboxyl-terminal domain; DTT, dithiothreitol; 5-FOA, 5-fluoroorotic acid; GST, glutathione S-transferase; TEF, transcriptional elongation factor.1The abbreviations used are: pol, polymerase; Cdk, cyclin-dependent kinase; CTD, carboxyl-terminal domain; DTT, dithiothreitol; 5-FOA, 5-fluoroorotic acid; GST, glutathione S-transferase; TEF, transcriptional elongation factor. II is regulated by phosphorylation of several of the protein components of the transcription apparatus (1Kobor M.S. Greenblatt J. Biochim. Biophys. Acta. 2002; 1577: 261-275Crossref PubMed Scopus (161) Google Scholar). Phosphorylation of the carboxyl-terminal domain (CTD) of the largest subunit of pol II has been the focus of much attention because the CTD acts as an essential scaffold for the binding of macromolecular assemblies that regulate mRNA synthesis and cotranscriptional mRNA processing (2Hirose Y. Manley J.L. Genes Dev. 2000; 14: 1415-1429PubMed Google Scholar). The pol II CTD is composed of a heptapeptide motif of consensus sequence YSPTSPS repeated in tandem. The mammalian pol II CTD has 52 heptad repeats, the fission yeast Schizosaccharomyces pombe CTD has 29 repeats, and the budding yeast Saccharomyces cerevisiae CTD has 26 copies. The pol II CTD undergoes a cycle of extensive phosphorylation and dephosphorylation at positions Ser5 and Ser2, which is coordinated with the transcription cycle (3Prelich G. Eukaryotic Cell. 2002; 1: 153-162Crossref PubMed Scopus (98) Google Scholar). Multiple CTD kinases are present in eukaryotic cells, e.g. budding yeast has four CTD kinases, two of which (Kin28 and Bur1) are essential (3Prelich G. Eukaryotic Cell. 2002; 1: 153-162Crossref PubMed Scopus (98) Google Scholar). CTD kinases are heteromeric enzymes consisting of a cyclin subunit plus a cyclin-dependent kinase (Cdk) subunit. The essential Bur1 Cdk of budding yeast and its cyclin partner Bur2 are putative orthologs of the Cdk9 and cyclin T subunits of the metazoan protein kinase P-TEFb (4Yao S. Neimann A. Prelich G. Mol. Cell. Biol. 2000; 20: 7080-7087Crossref PubMed Scopus (57) Google Scholar, 5Murray S. Udupam R. Yao S. Hartzog G. Prelich G. Mol. Cell. Biol. 2001; 21: 4089-4096Crossref PubMed Scopus (85) Google Scholar). P-TEFb is a transcription elongation factor that overrides the negative actions of Spt5 and its associated factors. Metazoan Spt5, with its binding partner Spt4 and a second factor NELF, arrests pol II elongation at promoter-proximal positions (6Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (548) Google Scholar, 7Yamaguchi Y. Wada T. Watanabe D. Takagi T. Hasegawa J. Handa H. J. Biol. Chem. 1999; 274: 8085-8092Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 8Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Escape from the elongation delay depends on the kinase activity of P-TEFb, which phosphorylates both the pol II CTD and Spt5 (8Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 9Wada T. Takagi T. Yamaguchi Y. Watanabe D. Handa H. EMBO J. 1998; 17: 7395-7403Crossref PubMed Scopus (278) Google Scholar, 10Price D.H. Mol. Cell. Biol. 2000; 20: 2629-2634Crossref PubMed Scopus (562) Google Scholar, 11Ivanov D. Kwak Y.T. Guo J. Gaynor R.B. Mol. Cell. Biol. 2000; 20: 2970-2983Crossref PubMed Scopus (173) Google Scholar, 12Ping Y.H. Rana T.M. J. Biol. Chem. 2001; 276: 12951-12958Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 13Lavoie S.B. Albert A.L. Handa H. Vincent M. Bensaude O. J. Mol. Biol. 2001; 312: 675-685Crossref PubMed Scopus (50) Google Scholar). Spt5 was initially suggested to have negative and positive effects on elongation (6Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. 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One potential role for phosphorylation of Spt5 by Cdk9 may be to modulate Spt5 function, e.g. converting it from a negative elongation factor into a positive factor. Although the rationale for the arrest and Cdk9-dependent restart of pol II elongation is still not clear, recent studies suggest a connection to mRNA 5′-capping, which is coupled to transcription elongation via physical and functional interactions among the cap-forming enzymes, the phosphorylated pol II CTD, and Spt5 (18McCracken 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 (428) Google Scholar, 19Cho E. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (368) Google Scholar, 20Yue Z. Maldonado E. Pillutla R. Cho H. Reinberg D. Shatkin A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12898-12903Crossref PubMed Scopus (196) Google Scholar, 21Schroeder S. Schwer B. Shuman S. 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Spt5 binds directly to the triphosphatase and guanylyltransferase components of the capping apparatus in mammals and in the fission yeast S. pombe (27Wen Y. Shatkin A.J. Genes Dev. 1999; 13: 1774-1779Crossref PubMed Scopus (163) Google Scholar, 28Pei Y. Shuman S. J. Biol. Chem. 2002; 277: 19639-19648Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Connections between capping enzymes and Spt5 have also been reported in S. cerevisiae (29Lindstrom D.L. Squazzo S.L. Muster N. Burckin T.A. Wachter K.C. Emigh C.A. McCleery J.A. Yates J.R. Hartzog G.A. Mol. Cell. Biol. 2003; 23: 1368-1378Crossref PubMed Scopus (200) Google Scholar). The interactions of the S. pombe triphosphatase (Pct1) and guanylyltransferase (Pce1) enzymes have been studied in vivo and in vitro using two-hybrid assays and purified recombinant proteins, respectively (28Pei Y. Shuman S. J. Biol. Chem. 2002; 277: 19639-19648Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Pce1 and Pct1 bind directly and independently to the unmodified CTD domain of Spt5, which consists of tandem repeats of a nonapeptide motif of consensus sequence TPAWNSGSK (28Pei Y. Shuman S. J. Biol. Chem. 2002; 277: 19639-19648Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). We recently described a physical interaction between S. pombe RNA triphosphatase and SpCdk9, a fission yeast homolog of metazoan Cdk9 and S. cerevisiae Bur1 (30Pei Y. Schwer B. Shuman S. J. Biol. Chem. 2003; 278: 7180-7188Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Initial studies of SpCdk9 included the identification of the essential S. pombe cyclin Pch1 as its binding partner. Pch1 is a homolog of metazoan cyclin T and S. cerevisiae cyclin Bur2. Complementation of the S. cerevisiae bur1Δ and bur2Δ mutants by coexpression of SpCdk9 and Pch1 showed that the fission yeast proteins are genuine orthologs of Bur1/Bur2, a putative fungal P-TEFb. Analysis of the recombinant SpCdk9/Pch1 complex produced in baculovirus-infected insect cells showed that the S. pombe proteins comprise a bona fide protein kinase with a heterodimeric quaternary structure (30Pei Y. Schwer B. Shuman S. J. Biol. Chem. 2003; 278: 7180-7188Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The capacity of SpCdk9/Pch1 to phosphorylate the CTD arrays of both pol II and Spt5 in vitro echoed the substrate specificity of metazoan P-TEFb (11Ivanov D. Kwak Y.T. Guo J. Gaynor R.B. Mol. Cell. Biol. 2000; 20: 2970-2983Crossref PubMed Scopus (173) Google Scholar, 12Ping Y.H. Rana T.M. J. Biol. Chem. 2001; 276: 12951-12958Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 31Kim J.B. Sharp P.A. J. Biol. Chem. 2001; 276: 12317-12323Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 32Garber M.E. Mayall T.P. Suess E.M. Meisenhelder J. Thompson N.E. Jones K.A. Mol. Cell. Biol. 2000; 20: 6958-6969Crossref PubMed Scopus (140) Google Scholar). These findings suggested a model whereby Spt5-induced arrest of early elongation ensures a temporal window for recruitment of the capping enzymes, which in turn attract Cdk9 to alleviate the arrest via phosphorylation of one or more components of the pol II elongation complex (30Pei Y. Schwer B. Shuman S. J. Biol. Chem. 2003; 278: 7180-7188Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Here, we present a biochemical and molecular genetic characterization of the fission yeast SpCdk9/Pch1 kinase complex. The biochemical characterization focuses on delineation of the reaction requirements, optima, and kinetic parameters for SpCdk9/Pch1-mediated phosphorylation of the Spt5-CTD. This event has received less attention from the enzymatic perspective than pol II CTD phosphorylation and is of interest in light of the unique structure of the CTD nonamer array of S. pombe Spt5 and its interaction with mRNA processing enzymes. We exploit budding yeast as a surrogate genetic system to perform an extensive mutational analysis of SpCdk9 guided by the crystal structure of the activated Cdk2/cyclin A-substrate complex (33Brown N.R. Noble M.E.M. Endicott J.A. Johnson L.N. Nat. Cell Biol. 1999; 1: 438-443Crossref PubMed Scopus (469) Google Scholar). Our results illuminate structure-activity relationships at the active site and a requirement for phosphorylation of the T-loop for SpCdk9 function in yeast. Protein Kinase Assay—The SpCdk9/His-Pch1 kinase complex was isolated from Sf9 insect cells coinfected with recombinant baculoviruses expressing SpCdk9 or His-Pch1 by nickel-nitrilotriacetic acid-agarose affinity chromatography as described previously (30Pei Y. Schwer B. Shuman S. J. Biol. Chem. 2003; 278: 7180-7188Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Kinase reaction mixtures (20 μl) containing 50 mm Tris acetate (pH 6.0), 1 mm DTT, 2.5 mm MnCl2 or 10 mm MgCl2, 50 μm [γ-32P]ATP, 4 μg of GST-Spt5(801–990) containing the C-terminal nonapeptide repeat array of S. pombe Spt5, and SpCdk9/His-Pch1 were incubated for 60 min at 22 °C. The reactions were halted by adding SDS to 1% final concentration. The products were analyzed by electrophoresis through a 10% polyacrylamide gel containing 0.1% SDS. Phosphorylated polypeptides were visualized by autoradiographic exposure of the dried gel and quantified by scanning the gel with a Fujix BAS2500 PhosphorImager. Phosphoamino Acid Analysis—A kinase reaction mixture (200 μl) containing 50 mm Tris acetate (pH 6.0), 1 mm DTT, 2.5 mm MnCl2 or 10 mm MgCl2, 50 μm [γ-32P]ATP, 60 μg of GST-Spt5(801–898), and 3 μg of SpCdk9/Pch1 complex was incubated for 2 h at 22 °C, then supplemented with 300 μl of binding buffer A (50 mm Tris-HCl (pH 8.0), 50 mm NaCl, 1 mm DTT, 5% glycerol, 0.03% Triton X-100) and 50 μl of GSH-Sepharose beads. The 32P-labeled GST-Spt5(801–989) was adsorbed to the beads during a 1.5-h incubation at 4 °C. The beads were then washed three times with 1 ml of binding buffer A to remove the free [γ-32P]ATP. After the third wash, the bound protein was eluted with 100 μl of 10 mm glutathione. The eluted protein was hydrolyzed by adding 100 μl of concentrated HCl and then heating the mixture at 110 °C for 2 h. The acid hydrolysate was evaporated to dryness in a vacuum centrifuge. The sample was resuspended in 10 μl of water. An aliquot was mixed with unlabeled Tyr(P), Thr(P), and Ser(P) markers (Sigma) and spotted onto a cellulose thin layer plate. The phosphoamino acids were separated by high voltage electrophoresis in pyridine acetate (pH 3.5) (34van der Geer P. Hunter T. Electrophoresis. 1994; 15: 544-554Crossref PubMed Scopus (125) Google Scholar). The unlabeled phosphoamino acid standards were visualized by staining the plate with ninhydrin; 32P-labeled material was visualized by autoradiography. 32P-Labeled phosphoamino acid analysis of autophosphorylated SpCdk9 and Pch1 was performed as follows. Reaction mixtures (200 μl) containing 50 mm Tris acetate (pH 6.0), 1 mm DTT, 2.5 mm MnCl2, 50 μm [γ-32P]ATP, and 10 μg of SpCdk9/Pch1 complex were incubated for 2 h at 22 °C, then supplemented with 300 μl of binding buffer B (50 mm NaH2PO4 (pH 8.0), 50 mm NaCl, 7.5 mm imidazole, and 0.0025% Tween 20) and 50 μl of nickel-agarose beads. The phosphorylated products were absorbed to the beads during a 1.5-h incubation at 4 °C. The beads were washed three times with 1 ml of binding buffer B. After the third wash, the bound protein was eluted with 30 μl of binding buffer B containing 250 mm imidazole. The eluted phosphoproteins were resolved by SDS-PAGE and then transferred electrophoretically to a polyvinylidene difluoride membrane. The membrane-bound radiolabeled SpCdk9 and Pch1 polypeptides were localized by autoradiography and excised. The membrane slices were incubated in 6 n HCl for 2 h at 110 °C and then analyzed by high voltage thin layer electrophoresis. Site-directed Mutagenesis of SpCdk9 —Plasmid pYX-SpCDK9 (CEN TRP1) contains the cDNA encoding full-length wild-type SpCdk9 under the control of the S. cerevisiae TPI1 promoter (30Pei Y. Schwer B. Shuman S. J. Biol. Chem. 2003; 278: 7180-7188Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Amino acid substitution mutations were introduced into the SpCDK9 gene by two-stage overlap extension PCR. The mutated PCR products were digested with NcoI and HindIII and inserted into NcoI/HindIII-digested pYX-SpCDK9 in lieu of the wild-type gene fragment. The inserts of the mutant plasmids were sequenced completely to confirm the presence of the desired mutation and exclude the acquisition of unwanted coding changes during amplification or cloning. Test of SpCdk9 Activity in Vivo by Plasmid Shuffle in S. cerevisiae— The BUR1 gene was deleted in the S. cerevisiae diploid strain W303 and replaced with a cassette specifying kanamycin resistance as described previously (30Pei Y. Schwer B. Shuman S. J. Biol. Chem. 2003; 278: 7180-7188Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Sporulation of the BUR1 bur1::kanR diploid that had been transformed with plasmid p360-BUR1 (BUR1 URA3 CEN) yielded viable bur1::kanR haploids that were incapable of growth in the presence of 5-fluoroorotic acid (5-FOA), a drug that selects against the BUR1 URA3 plasmid. A Mat a haploid of the bur1Δ strain was cotransformed with pYX-SpCDK9 (CEN TRP1 SpCDK9) containing wild-type or mutant SpCDK9 alleles and with plasmid pYX-PCH1 (CEN ADE2 PCH1) containing the full-length cDNA encoding Pch1 driven by the TP11 promoter (30Pei Y. Schwer B. Shuman S. J. Biol. Chem. 2003; 278: 7180-7188Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Individual transformants were selected on medium lacking tryptophan and adenine. Colonies were patched on drop-out medium and then streaked on agar plates containing 0.75 mg/ml 5-FOA. Lethal mutations were those that failed to support growth after incubation on 5-FOA agar for 7 days 30 °C. The viable 5-FOA-resistant colonies containing mutated SpCDK9 genes were patched to YPD agar at 30 °C and then tested for growth on YPD agar at 18, 23, 30, and 37 °C. Growth was assessed as follows: +++ indicates colony size indistinguishable from strains bearing wild-type SpCDK9; ++ denotes reduced colony size; + indicates that only pinpoint colonies were formed.; – indicates no growth. Spt5-CTD Kinase Activity of the Fission Yeast Cdk9/Pch1 Complex—Recombinant SpCdk9/Pch1 catalyzed the transfer of 32Pi from [γ-32P]ATP to the CTD of S. pombe Spt5, which spans amino acids 801–990 and consists of 18 tandem repeats of a nonapeptide motif (consensus sequence TPAWNSGSK) (Fig. 1A). SpCdk9/Pch1 phosphorylated the nonamer array of Spt5 in the context of a GST-Spt5 fusion protein (Fig. 1C) or as tag-free Spt5(801–990) (30Pei Y. Schwer B. Shuman S. J. Biol. Chem. 2003; 278: 7180-7188Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). No phosphoryl transfer was detected to GST alone (Fig. 1C) or to histone H1 (30Pei Y. Schwer B. Shuman S. J. Biol. Chem. 2003; 278: 7180-7188Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The radiolabeled GST-Spt5-CTD-PO4 product is detectable by SDS-PAGE analysis and can be quantified by scanning the gel with a PhosphorImager. Initial experiments showed that the extent of the kinase reaction was severalfold higher in the presence of MnCl2 than MgCl2 (Fig. 1C). Divalent Cation Dependence and Specificity—Phosphorylation of Spt5-CTD by SpCdk9/Pch1 required a divalent cation cofactor (Fig. 2A). Protein kinase activity was proportional to magnesium concentration in the range of 0.6–2.5 mm and plateaued at 5–10 mm. Manganese was a superior cofactor at all concentrations tested; activity was optimal at 0.6–10 mm MnCl2 (Fig. 2A). Cobalt (10 mm) supported kinase activity, albeit less effectively than manganese or magnesium (Fig. 2B). Other divalent cations were tested for cofactor activity at 10 mm concentration (Fig. 2B) and for their effects on activity in reactions containing 2.5 mm manganese (Fig. 2C). Whereas calcium neither activated nor inhibited SpCdk9/Pch1, copper and zinc were unable to support activity and were profoundly inhibitory in the presence of manganese. Effect of pH—The phosphorylation of Spt5-CTD by SpCdk9/Pch1 in the presence of manganese was optimal at pH 6.0–7.0 in Tris acetate buffer and at pH 6.0–8.0 in Tris-HCl buffer (Fig. 3A). We calculated that 0.6 pmol of phosphate was incorporated per pmol of input Spt5 protein at optimal pH. The manganese-dependent kinase activity was abolished at pH ≤4.5 or ≥9.0. The activity of SpCdk9/Pch1 in magnesium was also optimal at pH 6.0–7.0 in Tris acetate buffer and declined as the pH was incrementally acidified (Fig. 3B), just as was seen in the manganese-dependent reaction. However, the magnesium-dependent reaction was apparently insensitive to inhibition at alkaline pH, insofar as activity in Tris-HCl buffer at pH 9.5 was ∼65% of the activity in this buffer at the optimal pH of 6.0–6.5 (Fig. 3B). Kinetic Parameters, Nucleotide Specificity, and Inhibition by Salt—The transfer of 32Pi from [γ-32P]ATP to the Spt5-CTD increased with reaction time up to 80 min; the initial rate was severalfold higher in manganese than magnesium, and the extent of product formation remained severalfold higher in manganese at all times tested (Fig. 4A). Kinase activity was proportional to the amount of Spt5(801–990) phosphate acceptor protein included in the reaction in the range of 0.06–2 μg of Spt5-CTD and began to level off at higher Spt5-CTD concentrations (Fig. 4B). From a double-reciprocal plot of the data in Fig. 4B, we calculated a Km value of 3 μm Spt5-CTD. The kinase activity was proportional to the ATP concentration in the range of 2–12 μm and saturated at 40–50 μm (Fig. 5A). A double-reciprocal plot of the data yielded a Km of 10 μm ATP (Fig. 5B). SpCdk9/Pch1 was also capable of catalyzing the transfer of 32Pi from [γ-32P]GTP to the Spt5-CTD (Fig. 5C). The extent of 32P incorporation was comparable with 50 μm GTP and 50 μm ATP in the presence of manganese. Although both the GTP- and ATP-dependent kinase activities were reduced when magnesium replaced manganese, the GTP-dependent activity was more sensitive to the switch in metal cofactor (Fig. 5C). Phosphorylation of Spt5 was inhibited by increasing the ionic strength of the reaction mixture with either NaCl or NH4Cl (Fig. 4C). Activity was inhibited by 50% at ∼150 mm concentration of either salt; there was 20% residual kinase activity in a reaction containing 500 mm added NaCl and 10% activity in 500 mm NH4Cl (Fig. 4C).Fig. 5ATP dependence and nucleotide specificity of SpCdk9/Pch1 kinase activity. A, 20-μl reaction mixtures containing 50 mm Tris acetate (pH 6.0), 1 mm DTT, 2.5 mm MnCl2, 4 μg of GST-Spt5(801–990), 80 ng of SpCdk9/Pch1, and [γ-32P]ATP as specified were incubated for 60 min at 22 °C. Phosphoprotein formation is plotted as a function of ATP concentration. B, a double-reciprocal plot of phosphoprotein formation (v = pmol of phosphoprotein/3,600) versus [ATP]. C, 20-μl reaction mixtures containing 50 mm Tris acetate (pH 6.0), 1 mm DTT, 2.5 mm MnCl2 (▪) or 10 mm MgCl2 (□), 50 μm [γ-32P]ATP or [γ-32P]GTP, 4 μg of GST-Spt5(801–990), and 80 ng of SpCdk9/Pch1 were incubated for 60 min at 22 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Six Nonamer Repeats of the Spt5-CTD Suffice for Effective Phosphorylation—We tested the kinase activity of SpCdk9/Pch1 with a series of GST-Spt5 fusion proteins containing either the full-length array of 18 nonamer repeats (amino acids 801–990) or truncated segments containing 10 repeats (amino acids 801–898), 6 repeats (amino acids 845–898), or 4 repeats (amino acids 845–880). SDS-PAGE analysis of the purified GST-Spt5 substrates is shown in Fig. 1B and reveals the expected decrements in electrophoretic mobility as the CTD was truncated incrementally. The extent of manganese-dependent phosphorylation of the 10-repeat substrate (43 pmol) was similar to that of the full CTD array (45 pmol), which implies that the amino-terminal half of the CTD sufficed as a phosphate acceptor (Fig. 1C). The extent of phosphorylation of the 6-repeat array (22 pmol) was half that of the 10-repeat array. Taking into account that the number of potential phosphorylation sites within the 10-repeat CTD (10 threonines and 11 serines) is nearly twice that of the 6-repeat array (6 threonines and 6 serines), we surmise that the 6-repeat CTD is also an effective phosphate acceptor on a molar basis. The steep decline in the extent of phosphorylation of the 4-repeat CTD substrate (4 pmol of 32P incorporation) was disproportionate to the reduction in the number of potential phosphorylation sites (4 threonines and 5 serines) compared with the 6-repeat CTD. Thus, we conclude that 6 nonamer repeats suffice for effective phosphorylation of the Spt5-CTD in the presence of manganese. A similar relationship between kinase activity and CTD length was observed with magnesium as the cofactor, except that the decrement in activity between the 10-repeat and 6-repeat substrates was 3-fold instead of 2-fold as with manganese (Fig. 1C). SpCdk9/Pch1 Phosphorylates Spt5 on Threonine within the CTD Nonamer Repeat—GST-Spt5(801–898) phosphorylated in vitro by SpCdk9/Pch1 in the presence of 2.5 mm manganese was isolated free of [γ-32P]ATP and the kinase by adsorption to GSH-Sepharose and elution with GSH. The protein was subjected to acid hydrolysis, and the 32P-labeled phosphoamino acid content of the hydrolysate was gauged by high voltage electrophoresis under conditions designed to separate Tyr(P), Thr(P), and Ser(P) (Fig. 1D). This analysis showed that SpCdk9/Pch1 phosphorylated Spt5 exclusively on threonine and not on serine. SpCdk9/Pch1 also phosphorylated Spt5 exclusively on threonine when the kinase reactions were performed with 10 mm magnesium as the cofactor (data not shown). As noted above, the CTD substrate composed of 10 nonamer repeats contains 10 threonines and 11 serines, but no tyrosines. We surmise that Thr1 of the nonamer consensus sequence 1TPAWNSGSK9 is the target of the SpCdk9/Pch1 kinase. SpCdk9/Pch1 Phosphorylation of the S. pombe pol II CTD on Serine—We compared the kinase activity of SpCdk9/Pch1 with GST-fused phosphate acceptors containing either the CTD nonamer array of S. pombe Spt5 or the complete CTD of S. pombe pol II, which consists of 29 heptapeptide repeats. The extent of phosphorylation of the Spt5-CTD was 5–8-fold higher than that of the S. pombe pol II CTD in the presence of manganese or magnesium (data not shown). Phosphorylation of the S. pombe pol II CTD by SpCdk9/Pch1 was 2-fold more effective with 2.5 mm manganese than with 10 mm magnesium (data not sh
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