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• W2011946029 abstract "The MCK1 gene of Saccharomyces cerevisiae encodes a protein kinase homologous to metazoan glycogen synthase kinase-3. Previous studies implicated Mck1p in negative regulation of pyruvate kinase. In this study we find that purified Mck1p does not phosphorylate pyruvate kinase, suggesting that the link is indirect. We find that purified Tpk1p, a cAMP-dependent protein kinase catalytic subunit, phosphorylates purified pyruvate kinase in vitro, and that loss of the cAMP-dependent protein kinase regulatory subunit, Bcy1p, increases pyruvate kinase activity in vivo. We find that purified Mck1p inhibits purified Tpk1p in vitro, in the presence or absence of Bcy1p. Mck1p must be catalytically active to inhibit Tpk1p, but Mck1p does not phosphorylate this target. We find that abolition of Mck1p autophosphorylation on tyrosine prevents the kinase from efficiently phosphorylating exogenous substrates, but does not block its ability to inhibit Tpk1p in vitro. We find that this mutant form of Mck1p appears to retain the ability to negatively regulate cAMP-dependent protein kinase in vivo. We propose that Mck1p, in addition to phosphorylating some target proteins, also acts by a separate, novel mechanism: autophosphorylated Mck1p binds to and directly inhibits, but does not phosphorylate, the catalytic subunits of cAMP-dependent protein kinase. The MCK1 gene of Saccharomyces cerevisiae encodes a protein kinase homologous to metazoan glycogen synthase kinase-3. Previous studies implicated Mck1p in negative regulation of pyruvate kinase. In this study we find that purified Mck1p does not phosphorylate pyruvate kinase, suggesting that the link is indirect. We find that purified Tpk1p, a cAMP-dependent protein kinase catalytic subunit, phosphorylates purified pyruvate kinase in vitro, and that loss of the cAMP-dependent protein kinase regulatory subunit, Bcy1p, increases pyruvate kinase activity in vivo. We find that purified Mck1p inhibits purified Tpk1p in vitro, in the presence or absence of Bcy1p. Mck1p must be catalytically active to inhibit Tpk1p, but Mck1p does not phosphorylate this target. We find that abolition of Mck1p autophosphorylation on tyrosine prevents the kinase from efficiently phosphorylating exogenous substrates, but does not block its ability to inhibit Tpk1p in vitro. We find that this mutant form of Mck1p appears to retain the ability to negatively regulate cAMP-dependent protein kinase in vivo. We propose that Mck1p, in addition to phosphorylating some target proteins, also acts by a separate, novel mechanism: autophosphorylated Mck1p binds to and directly inhibits, but does not phosphorylate, the catalytic subunits of cAMP-dependent protein kinase. The genome of Saccharomyces cerevisiae encodes four different protein kinases highly similar (greater than 40% identity) to mammalian glycogen synthase kinase-3 (GSK-3) 1The abbreviations used are: GSK-3glycogen synthase kinase-3PKAcAMP-dependent protein kinaseMBPmyelin basic proteinPKImammalian protein kinase inhibitor peptideORFopen reading frameHis6hexahistidineMES4-morpholineethanesulfonic acidMOPS4-morpholinepropanesulfonic acid (1.Hunter T. Plowman G.D. Trends Biochem. Sci. 1997; 22: 18-22Abstract Full Text PDF PubMed Scopus (404) Google Scholar). One of these kinases is encoded by MCK1 (2.Dailey D. Schieven G.L. Lim M.Y. Marquardt H. Gilmore T. Thorner J. Martin G.S. Mol. Cell. Biol. 1990; 10: 6244-6256Crossref PubMed Google Scholar, 3.Lim M.Y. Dailey D. Martin G.S. Thorner J. J. Biol. Chem. 1993; 268: 21155-21164Abstract Full Text PDF PubMed Google Scholar). Like its vertebrate homologues, Mck1p displays dual specificity in vitro, in that it is capable of autophosphorylating on serine and tyrosine residues, but only phosphorylates exogenous substrates on serine and threonine (3.Lim M.Y. Dailey D. Martin G.S. Thorner J. J. Biol. Chem. 1993; 268: 21155-21164Abstract Full Text PDF PubMed Google Scholar). glycogen synthase kinase-3 cAMP-dependent protein kinase myelin basic protein mammalian protein kinase inhibitor peptide open reading frame hexahistidine 4-morpholineethanesulfonic acid 4-morpholinepropanesulfonic acid The protein kinases of the glycogen synthase kinase-3 (GSK-3) sub-family have been implicated in a variety of regulatory processes. In mammals, the prototypic members, GSK-3α and GSK-3β, phosphorylate and are thought to regulate a number of metabolic enzymes (4.Plyte S.E. Hughes K. Nikolakaki E. Pulverer B.J. Woodgett J.R. Biochim. Biophys. Acta. 1992; 1114: 147-162Crossref PubMed Scopus (336) Google Scholar, 5.Woodgett J. Hardie G. Hanks S. The Protein Kinase Factsbook. I. Academic Press Inc., San Diego, CA1995: 231-233Crossref Google Scholar, 6.Welsh G.I. Wilson C. Proud C.G. Trends Cell Biol. 1996; 6: 274-279Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 7.Hoshi M. Takashima A. Noguchi K. Murayama M. Sato M. Kondo S. Saitoh Y. Ishiguro K. Hoshino T. Imahori K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2719-2723Crossref PubMed Scopus (228) Google Scholar). GSK-3 enzymes are intimately involved in the insulin signaling pathway and in key developmental pathways in a number of organisms (8.Avruch J. Mol. Cell. Biochem. 1998; 182: 31-48Crossref PubMed Scopus (324) Google Scholar,9.Kim L. Kimmel A.R. Curr. Opin. Genet. Dev. 2000; 10: 508-514Crossref PubMed Scopus (211) Google Scholar). The development of the dorsal-ventral axis in Drosophilaand Xenopus embryos relies on the function of theWingless/Wnt pathway. GSK-3 is inhibited by this pathway, allowing the expression of crucial developmental genes (9.Kim L. Kimmel A.R. Curr. Opin. Genet. Dev. 2000; 10: 508-514Crossref PubMed Scopus (211) Google Scholar). Yeast cells lacking Mck1p display a wide range of phenotypes, indicating that Mck1p may have multiple targets. Deletion of theMCK1 locus results in various defects in carbon metabolism, including reduced glycogen accumulation and poor growth on non-fermentable carbon sources (10.Puziss J.W. Hardy T.A. Johnson R.B. Roach P.J. Hieter P. Mol. Cell. Biol. 1994; 14: 831-839Crossref PubMed Google Scholar, 11.Brazill D.T. Thorner J. Martin G.S. J. Bacteriol. 1997; 179: 4415-4418Crossref PubMed Google Scholar). Other phenotypes resulting from deletion of MCK1 include heat and cold sensitivity (10.Puziss J.W. Hardy T.A. Johnson R.B. Roach P.J. Hieter P. Mol. Cell. Biol. 1994; 14: 831-839Crossref PubMed Google Scholar,12.Shero J.H. Hieter P. Genes Dev. 1991; 5: 549-560Crossref PubMed Scopus (89) Google Scholar), delayed sporulation (13.Neigeborn L. Mitchell A.P. Genes Dev. 1991; 5: 533-548Crossref PubMed Scopus (113) Google Scholar) and sensitivity to the microtubule-destabilizing drug benomyl (12.Shero J.H. Hieter P. Genes Dev. 1991; 5: 549-560Crossref PubMed Scopus (89) Google Scholar). In this work we show thatmck1Δ cells are also sensitive to caffeine. Overexpression of MCK1 has been shown to suppress temperature-sensitive mutations in CBF2 and CBF5, which encode essential centromere-binding proteins. Mck1p was found to bind to and phosphorylate Cbf2p in vitro (14.Jiang W. Lim M.Y. Yoon H.J. Thorner J. Martin G.S. Carbon J. Mol. Gen. Genet. 1995; 246: 360-366Crossref PubMed Scopus (26) Google Scholar, 15.Jiang W. Koltin Y. Mol. Gen. Genet. 1996; 251: 153-160PubMed Google Scholar). More recently,MCK1 and RIM11 have been implicated in the protein ubiquitination pathway (16.Andoh T. Hirata Y. Kikuchi A. Mol. Cell. Biol. 2000; 20: 6712-6720Crossref PubMed Scopus (52) Google Scholar). Finally, MCK1 has been implicated in the response to high concentrations of NaCl in growth medium (17.Piao H.L. Pih K.T. Lim J.H. Kang S.G. Jin J.B. Kim S.H. Hwang I. Plant Physiol. 1999; 119: 1527-1534Crossref PubMed Scopus (83) Google Scholar). Previous work has demonstrated that Mck1p down-regulates pyruvate kinase (EC 2.7.1.40), encoded by the CDC19/PYK1gene, in vivo (11.Brazill D.T. Thorner J. Martin G.S. J. Bacteriol. 1997; 179: 4415-4418Crossref PubMed Google Scholar). Like mck1Δ mutants, strains overproducing pyruvate kinase fail to grow at 37 °C and do not accumulate normal amounts of glycogen. Cells overexpressingCDC19, like mck1Δ, adapt poorly to growth on non-fermentable carbon sources (11.Brazill D.T. Thorner J. Martin G.S. J. Bacteriol. 1997; 179: 4415-4418Crossref PubMed Google Scholar, 18.Rosenzweig R.F. Genet. Res. 1992; 59: 35-48Crossref PubMed Scopus (20) Google Scholar, 19.Rosenzweig R.F. Genet. Res. 1992; 59: 167-177Crossref PubMed Scopus (4) Google Scholar). Finally, diploid cells overexpressing CDC19 show markedly diminished sporulation efficiency (11.Brazill D.T. Thorner J. Martin G.S. J. Bacteriol. 1997; 179: 4415-4418Crossref PubMed Google Scholar), similar to that observed inmck1Δ/mck1Δ diploids. Deletion ofMCK1 was found to exacerbate each of these CDC19overexpression phenotypes (11.Brazill D.T. Thorner J. Martin G.S. J. Bacteriol. 1997; 179: 4415-4418Crossref PubMed Google Scholar). The findings of Brazill et al. (11.Brazill D.T. Thorner J. Martin G.S. J. Bacteriol. 1997; 179: 4415-4418Crossref PubMed Google Scholar) suggested that Mck1p might directly phosphorylate pyruvate kinase, thereby down-regulating the activity of this enzyme. In this report we investigate this possibility. We find that Mck1p does not in fact phosphorylate pyruvate kinase but instead acts to inhibit an intermediary kinase, which would otherwise phosphorylate the glycolytic enzyme. We identify this intermediary kinase as cAMP-dependent protein kinase (PKA). The genome of S. cerevisiae contains three genes encoding PKA catalytic subunits, TPK1, TPK2, andTPK3, and one gene, BCY1, encoding the cAMP binding and negative regulatory subunit of PKA. A purified Tpk1p·Bcy1p complex phosphorylates purified pyruvate kinase, and this phosphorylation is stimulated by cAMP. Consistent with this result, we find that loss of Bcy1p increases pyruvate kinase activityin vivo. Phosphorylation of pyruvate kinase by Tpk1p·Bcy1p is inhibited by the addition of purified Mck1p in vitro. Mck1p also inhibits purified Tpk1p alone. This inhibition is dependent on Mck1p catalytic activity, but Mck1p does not phosphorylate Tpk1p (or indeed Bcy1p) in vitro. Mck1p autophosphorylation on tyrosine-199 is required for efficient phosphorylation of exogenous substrates but not for Mck1p autophosphorylation on other residues or for inhibition of Tpk1p in vitro. Finally, autophosphorylation by Mck1p on tyrosine (and hence phosphorylation of exogenous substrates) is not required for the regulation of PKA by Mck1p in vivo. We propose that, in addition to phosphorylating some target proteins, Mck1p also directly binds to and inhibits, but does not phosphorylate, the catalytic subunits of PKA. Purified bovine brain myelin basic protein (MBP), cAMP, LRRASLG (Kemptide), PKI, l-lactate dehydrogenase, DEAE-Sephadex, CM-Sephadex, and cAMP-agarose were purchased from Sigma-Aldrich Co. (St. Louis, MO). S-Sepharose was purchased from Amersham Biosciences, Inc. (Peapack, NJ). Nickel-agarose was purchased from Novagen (Madison, WI) and prepared according to the manufacturer's instructions. Anti-His6 tag antibodies were purchased from Covance Research Products (Richmond, CA). Anti-phosphotyrosine monoclonal antibody clone 4G10 was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Modified Bradford protein concentration assay reagent was purchased from Bio-Rad Laboratories Inc. (Hercules, CA). Partially purified pyruvate kinase was a gift of Tom Nowak (University of Notre Dame, Notre Dame, IN) and fully purified pyruvate kinase was generously provided by Barry Stoddard (Fred Hutchinson Cancer Research Center, Seattle, WA). Yeast strains were grown in YPD medium (1% yeast extract, 2% peptone, 2% glucose) (20.Sherman F. Fink G.R. Hicks J.B. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar) where appropriate. Selection for plasmid maintenance was provided where necessary by growing strains in synthetic minimal medium supplemented with the appropriate nutrients (20.Sherman F. Fink G.R. Hicks J.B. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar). Media containing non-fermentable carbon sources (acetate, glycerol, dl-lactate, or ethanol) were made by adding the relevant compound to suitable medium to a final concentration of 2%. In the cases of lactate and acetate, agar plates were made using unbuffered acids at 0.2%. All other plates contained 2% glucose as the carbon source. Agar plates contained 10 mm caffeine where indicated. Standard molecular biology techniques were used in the construction of plasmids (21.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). All plasmids were sequenced to check for errors introduced in PCR and DNA ligation steps. Plasmid pDD7 is an MCK1 deletion construct in which theMCK1 open reading frame (ORF) has been disrupted with theURA3 gene flanked by direct repeats of the hisGgene (3.Lim M.Y. Dailey D. Martin G.S. Thorner J. J. Biol. Chem. 1993; 268: 21155-21164Abstract Full Text PDF PubMed Google Scholar). Plasmid pDD4 is a 2-μm plasmid based on YEp352 (22.Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1083) Google Scholar) encoding MCK1 under the control of its own promoter (3.Lim M.Y. Dailey D. Martin G.S. Thorner J. J. Biol. Chem. 1993; 268: 21155-21164Abstract Full Text PDF PubMed Google Scholar). The ORF encoding C-terminal hexahistidine (His6)-tagged Mck1p was constructed using a PCR-based approach consisting of two steps. The first step amplified the regions immediately flanking the 3′-end of the MCK1 ORF using plasmid pDD4 as a template. These reactions used two pairs of primers. Primer pUCforward (5′-GTT TTC CCA GTC ACG AC-3′) was used with primer A (5′-CTC GAG TTA CGC GTG ATG ATG ATG ATG ATG TGA CGC GTC AGC AAC TTT CGT AGG TTT AAT TTT ATC-3′) in one reaction, whereas the other reaction contained primer pUCreverse (5′-AGC GGA TAA CAA TTT CAC ACA GGA-3′) and primer B (5′-CAT CAT CAC GCG TAA CTC GAG TAA TTT TCT ATT AAT TTG TTC TCT TTC C-3′). The second step in the construction of the His6-tagged Mck1p construct combined the products of the first step and re-amplified the whole region using primers pUCforward and pUCreverse. The resultant full-length PCR product was cleaved with BstXI andPstI and subcloned into the corresponding sites in pDD4, producing plasmid pTRYMHU. The DNA sequence of this plasmid was confirmed to encode a protein incorporating the amino acid sequence Ser(His)6Ala-CO2H in place of the terminal glutamate residue of wild-type Mck1p. This plasmid was found to fully complement the mck1Δ mutation in vivo. 2T. F. R., unpublished observation. Insertion of a BamHI site at the 5′-end of theMCK1 ORF was necessary to subclone the ORF downstream of theGAL promoter. The approach taken was similar to that for the insertion of the His6 tag (above). The outlying primers in this case were primer pUCforward and primer C (5′-TTT ATT ATC TTG CGG GGA C-3′). The BamHI site was introduced using a primer with the sequence 5′-CTA GTA GGA TCC AAT ATG TCT ACG GAA GAG CAG AAT GGT GTT CC-3′ and another primer complementary to this sequence. PCR reactions were performed as described for the generation of the His6-tagged construct above. The final PCR product was digested with HindIII and BsrGI and subcloned into the corresponding sites in pTRYMHU to yield plasmid pTRYBMHU. TheMCK1 ORF was then subcloned into plasmid YEp352GAL (23.Benton B.M. Zang J.H. Thorner J. J. Cell Biol. 1994; 127: 623-639Crossref PubMed Scopus (76) Google Scholar) by cleaving plasmid pTRYBMHU with BamHI and subcloning the 2-kb fragment bearing the MCK1 ORF into the BamHI site of YEp352GAL. The resulting plasmid was named pTRYGMHU. The BCY1 ORF was cloned by PCR amplification using yeast genomic DNA as template and primers flanking the BCY1 locus (5′-TTA TCT CTC TCT GAT GAC GTG-3′ and 5′-ATA TCA CGA TTA TAG TCG CAG C-3′). The resulting PCR product was cleaved with EcoRV andScaI and subcloned into the EcoRV site of pRS423 (24.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) to generate plasmid YEpBCY1H. The BCY1 ORF was transferred to a 2-μm plasmid bearing the TRP1 marker by digesting the YEpBCY1H plasmid with XhoI andEcoRI and subcloning it into the corresponding sites of plasmid pRS424. The plasmid was finally cut with BamHI andSmaI, blunt-ended using Klenow DNA polymerase, and re-ligated to destroy these unwanted sites, yielding plasmid YEpBCY1. A vector constitutively overexpressing histidine-tagged TPK1was constructed in the following manner. The TPK1 locus was PCR-amplified from genomic DNA with the primers 5′-TGG ATC CAA TAT GTC GAC TGA AGA ACA AAA TGG AGG-3′ and 5′-TAC TCG AGT TAC GCG TGA TGA TGA TGA TGA TGT GAC GCG AAG TCC CGG AAA AGA TCA GCA TAT GGG-3′. The ends of the PCR product were trimmed with BamHI, blunted with Klenow DNA polymerase, and cut again with XhoI. The resulting fragment was then subcloned into the SmaI-SalI sites of plasmid pAD4M (25.Martin G.A. Viskochil D. Bollag G. McCabe P.C. Crosier W.J. Haubruck H. Conroy L. Clark R. O'Connell P. Cawthon R.M. Cell. 1990; 63: 843-849Abstract Full Text PDF PubMed Scopus (743) Google Scholar), producing plasmid pTRYAT1HL. Standard molecular biology techniques were used in the construction of yeast strains (21.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). S. cerevisiae strains used in this work include YPH500 (MATα ura3–52 lys2–801 ade2–101 trp1-Δ63 his3-Δ200 leu2-Δ1(24.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar)), BJ2168 (MAT a leu2 trp1 ura3–52 prb1–1122 pep4–3 pre1–451 (26.Jones E.W. Methods Enzymol. 1991; 194: 428-453Crossref PubMed Scopus (367) Google Scholar)), DBY1 (MATαura3–52 lys2–801 ade2–101 trp1-Δ63 his3-Δ200 leu2-Δ1 mck1Δ::hisG (11.Brazill D.T. Thorner J. Martin G.S. J. Bacteriol. 1997; 179: 4415-4418Crossref PubMed Google Scholar)), and JCY100 (MAT a ura3–52 leu2::hisG trp1::hisG his3::hisG (27.Cook J.G. Bardwell L. Thorner J. Nature. 1997; 390: 85-88Crossref PubMed Scopus (222) Google Scholar, 28.Madhani H.D. Fink G.R. Science. 1997; 275: 1314-1317Crossref PubMed Scopus (321) Google Scholar)). Strains TRYBJm (MAT a leu2 trp1 ura3–52 prb1–1122 pep4–3 pre1–451 mck1Δ::hisG) and JCYmck1Δ (MAT a ura3–52 leu2::hisG trp1::hisG his3::hisG mck1Δ::hisG) were constructed by transforming strains BJ2168 and JCY100, respectively, with the 6.3-kbXhoI-XbaI fragment derived from pDD7. The resulting transformants were grown on medium containing 5-fluoroorotic acid to select for the recombination of the hisG repeats and consequent loss of the URA3 marker gene (29.Alani E. Cao L. Kleckner N. Genetics. 1987; 116: 541-545Crossref PubMed Scopus (752) Google Scholar). Successful deletion of the MCK1 locus was confirmed by PCR. Strain Y27300 (MAT a/αhis3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/LYS2 MET15/met15Δ0 ura3Δ0/ura3Δ0 bcy1Δ::kanMX4/BCY1) and its corresponding wild-type strain, BY4743 (MAT a/αhis3Δ1/his3Δ1 leu2Δ0/leu2Δ0 met15Δ0/MET15 LYS2/lys2Δ0 ura3Δ0/ura3Δ0) were obtained from EUROSCARF (Frankfurt, Germany) (30.Brachmann C.B. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Yeast. 1998; 14: 115-132Crossref PubMed Scopus (2645) Google Scholar). Mutations were introduced into the MCK1 coding region using a similar approach to that adopted for the original His6 tagging of this ORF (see above). In each case a pair of PCR primers flanking the ORF were used (5′-ACA GCG GAT CAA AGG TGA-3′ and 5′-TAG GAG TTA AGC CCA AG-3′). The various mutants were created using the following PCR primers in conjunction with primers having complementary sequence. The D164A primer was 5′-CGT TTG TCA TCG TGC TAT CAA ACC ATC C-3′ and the Y199F primer was 5′-GCC TTC AAT TAG TTT CAT CTG TTC AAG-3′. In each case the PCR product was cleaved with BsaBI and BsrGI and subcloned into the corresponding site in either pDD4 (giving rise to pTRYMU-D164A and pTRYMU-Y199F) or pTRYGMHU (giving rise to pTRYGMHU-D164A and pTRYGMHU-Y199F). C-terminally His6-tagged Mck1p was overproduced from plasmid pTRYGMHU transformed into strain TRYBJm. Expression was induced by growing cells for 24 h at 30 °C in 2-liter synthetic complete medium containing 2% galactose. To purify mutant Mck1p, the plasmids pTRYGMHU-D164A or pTRYGMHU-Y199F were substituted for plasmid pTRYGMHU in this initial induction step. Cells were collected by centrifugation, washed, rapidly frozen in liquid nitrogen, and subsequently lysed as described by Kellogg et al. (31.Kellogg D.R. Kikuchi A. Fujiinakata T. Turck C.W. Murray A.W. J. Cell Biol. 1995; 130: 661-673Crossref PubMed Scopus (159) Google Scholar). The lysis buffer (buffer A) consisted of 50 mm sodium phosphate, pH 7.5, 145 mm NaCl, and 5 mm imidazole. Following clarification of the lysate by ultracentrifugation, Tween 20 was added to a final concentration of 0.1%. The lysate was passed through a nickel-agarose column (1-ml bed volume) previously equilibrated in buffer A. The flow-through from the column was discarded, and the column was washed with ten volumes of buffer A. A second wash was performed with six column volumes of buffer A containing 20 mm imidazole. The Mck1 protein was then eluted with three column volumes each of buffer A containing 50 and 100 mmimidazole. These final fractions were assayed for protein concentration using a Bradford-based assay (32.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217508) Google Scholar) and checked for Mck1p content by SDS-PAGE (33.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207523) Google Scholar) and Western blotting (34.Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar). Peak fractions were pooled and dialyzed overnight against buffer B (50 mm MES, pH 6.5, 1 mm EDTA, 25% glycerol). The protein was then further purified by running the dialysate over an S-Sepharose column (1-ml bed volume) equilibrated in buffer B. The column was washed with ten volumes of buffer B and a further five volumes of buffer B containing 100 mm NaCl. Purified Mck1p was then eluted using buffer B containing increasing concentrations of NaCl. Mck1p typically eluted in the range of 300–400 mm NaCl. Fractions were assayed by SDS-PAGE followed by silver staining or Western blotting. Western blotted membranes were probed with anti-Mck1p (2.Dailey D. Schieven G.L. Lim M.Y. Marquardt H. Gilmore T. Thorner J. Martin G.S. Mol. Cell. Biol. 1990; 10: 6244-6256Crossref PubMed Google Scholar) or anti-His6 tag antibodies. The procedure for purifying PKA subunits from yeast was based on the work of Hixson and Krebs (35.Hixson C.S. Krebs E.G. J. Biol. Chem. 1980; 255: 2137-2145Abstract Full Text PDF PubMed Google Scholar). Tpk1p, His6-tagged at its C terminus, was overproduced in strain BJ2168 using the 2-μm plasmid pTRYAT1HL, which expresses His6-tagged Tpk1p under the control of the constitutive ADH1 promoter. The regulatory subunit Bcy1p was co-overproduced with His6-tagged Tpk1p using a second 2-μm plasmid, YEpBCY1, which expresses BCY1 under the control of its own promoter. Such co-overexpression has been previously found to enhance TPK1 expression (36.Zoller M.J. Kuret J. Cameron S. Levin L. Johnson K.E. J. Biol. Chem. 1988; 263: 9142-9148Abstract Full Text PDF PubMed Google Scholar). Cells were harvested from a 2-liter culture and lysed as described for the purification of Mck1p. The cells were lysed into 50 mm MOPS, pH 7.5, 1 mm EDTA. The lysate was fractionated by adding ammonium sulfate to 40% saturation. The precipitate was resuspended in 50 mm MOPS, pH 7.5, with 0.1% Tween 20. This fraction was loaded onto a nickel-agarose column (1-ml bed volume) equilibrated in 50 mm MOPS, pH 7.5. This buffer was used throughout the nickel-agarose column chromatography with increasing amounts of imidazole added. The column was washed with ten volumes of buffer containing 5 mm imidazole followed by a further six column volumes with 20 mm imidazole. The proteins were eluted in buffer containing 50–100 mm imidazole. Both here and subsequently, fractions were assayed for Tpk1p content by Western blotting with an anti-His6 tag antibody. Peak fractions were pooled and loaded onto a DEAE-Sephadex column (0.5-ml bed volume) equilibrated with 50 mm MOPS, pH 7.5, 1 mmEDTA. Again, this buffer was used throughout this anion exchange chromatography step, with varying concentrations of NaCl added. The column was washed with 10 volumes of buffer, and the protein was eluted with increasing concentrations of NaCl in the same MOPS buffer. Fractions were assayed by immunoblotting for the His6 tag, and peak fractions (typically in the range 100–200 mmNaCl) were pooled. The pooled fractions were dialyzed overnight in buffer B (50 mm MES, pH 6.5, 1 mm EDTA, 25% glycerol, as for the purification of Mck1p) and applied to a CM-Sephadex column (0.5-ml bed volume) equilibrated in the same buffer. The column was washed in 10 volumes of buffer before the protein was eluted with increasing concentrations of NaCl in buffer B. The 100–200 mm NaCl fractions were found to contain two SDS-PAGE bands by silver staining (Fig. 1). The higher molecular weight band was found to cross-react with the anti-His6 tag antibody. The lower band could be removed from the fraction by a brief binding step to cAMP-agarose (50-μl bed volume per 200-μl sample volume). These observations, combined with subsequent kinase assays using Kemptide as a substrate, identified these two bands as Tpk1p and Bcy1p, respectively. Mck1p autophosphorylation was assayed using the procedure described by Lim et al. (3.Lim M.Y. Dailey D. Martin G.S. Thorner J. J. Biol. Chem. 1993; 268: 21155-21164Abstract Full Text PDF PubMed Google Scholar). Mck1p tyrosine phosphorylation was assayed by Western blotting using the anti-phosphotyrosine monoclonal antibody clone 4G10 as described by Zhan et al. (37.Zhan X.L. Hong Y. Zhu T. Mitchell A.P. Deschenes R.J. Guan K.L. Mol. Biol. Cell. 2000; 11: 663-676Crossref PubMed Scopus (25) Google Scholar). Phosphotransferase activity of Mck1p or Tpk1p was measured in a similar manner to that described by Limet al. (3.Lim M.Y. Dailey D. Martin G.S. Thorner J. J. Biol. Chem. 1993; 268: 21155-21164Abstract Full Text PDF PubMed Google Scholar), with minor modifications. To measure Mck1p activity toward exogenous substrates, purified Mck1p was incubated in reaction buffer (10 mm MgCl2, 50 mmTris-HCl) with 20 μm [γ-32P]ATP (approximately 800 Ci/mol) and 1 μg of myelin basic protein (MBP). The reactions were incubated for between 15 and 30 min at 30 °C before being quenched by addition of sample buffer and boiling, prior to analysis by SDS-PAGE. The assay for Tpk1p activity toward pyruvate kinase was conducted in an essentially identical fashion to the assay for Mck1p activity toward MBP. Between 2 and 5 μg of purified pyruvate kinase was used as a substrate in these assays, in place of MBP. Approximately 5 ng of Tpk1p was used per reaction, and 170 ng of Mck1p was added during studies of its inhibitory activity. The total reaction volume was 10 μl. Reactions were quenched by addition of an equal volume of reaction buffer, and half of the total reaction volume was analyzed by SDS-PAGE. Adenosine 3′:5′ cyclic monophosphate (cAMP) was added to reactions as indicated to a final concentration of 10 μm. PKA phosphorylation of its synthetic substrate, Kemptide (amino acid sequence LRRASLG), was assayed as described by Toda et al.(38.Toda T. Cameron S. Sass P. Zoller M. Scott J.D. McMullen B. Hurwitz M. Krebs E.G. Wigler M. Mol. Cell. Biol. 1987; 7: 1371-1377Crossref PubMed Scopus (381) Google Scholar). Synthetic mammalian protein kinase inhibitor peptide (PKI, amino acid sequence TTYADFIASGRTGRRNAIHD) was added to reactions where indicated (1 μg per reaction). Assays of pyruvate kinase activity were performed using an assay coupled to l-lactate dehydrogenase as described by Juricaet al. (39.Jurica M.S. Mesecar A. Heath P.J. Shi W. Nowak T. Stoddard B.L. Structure. 1998; 6: 195-210Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). Cell extracts were assayed for protein concentration using a Bradford-based assay (32.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217508) Google Scholar). Invasive growth of yeast cells on agar plates was measured using a plate adhesion assay (40.Roberts R.L. Fink G.R. Genes Dev. 1994; 8: 2974-2985Crossref PubMed Scopus (529) Google Scholar). Cells were streaked onto YPD or synthetic minimal solid medium and allowed to grow for 3–5 days. The plates were photographed before and after washing under a stream of running water. The results of Brazill et al. (11.Brazill D.T. Thorner J. Martin G.S. J. Bacteriol. 1997; 179: 4415-4418Crossref PubMed Google Scholar) suggested that Mck1p might down-regulate pyruvate kinase by direct phosphorylation. We set out to determine if purified Mck1p phosphorylates purified pyruvate kinasein vitro. Mck1p was purified to homogeneity (Fig. 1) using a C-terminal His6tag, a nickel-agarose column, and cation exchange chromatography. The purified enzyme retained catalytic activity toward myelin basic protein (MBP) (Fig. 4) and had a K m for ATP of 70 μm, comparable to that previously determined for the untagged enzyme (27 μm in Dailey et al. (2.Dailey D. Schieven G.L. Lim M.Y. Marquardt H. Gilmore T. Thorner J. Martin G.S. Mol. Cell. Biol. 1990; 10: 6244-6256Crossref PubMed Google Scholar); 70 μm in Lim et al. (3.Lim M.Y. Dailey D. Martin G.S. Thorner J. J. Biol. Chem. 1993; 268: 21155-21164Abstract F" @default.
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• W2011946029 title "Direct and Novel Regulation of cAMP-dependent Protein Kinase by Mck1p, a Yeast Glycogen Synthase Kinase-3" @default.
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