Phosphorylation of the Yeast Choline Kinase by Protein Kinase C
2005; Elsevier BV; Volume: 280; Issue: 28 Linguagem: Inglês
10.1074/jbc.m503551200
ISSN1083-351X
AutoresMal‐Gi Choi, Vladlen Kurnov, Michael C. Kersting, Avula Sreenivas, George Carman,
Tópico(s)Biotin and Related Studies
ResumoThe Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μm). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSSQRRHS (V5max/Km = 17.5 mm–1 μmol min–1 mg–1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway, whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Although the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHSLTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mm–1 μmol min–1 mg–1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μm). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSSQRRHS (V5max/Km = 17.5 mm–1 μmol min–1 mg–1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway, whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Although the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHSLTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mm–1 μmol min–1 mg–1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PC 1The abbreviation used is: PC, phosphatidylcholine. is the major phospholipid in the membranes of eukaryotic cells (1Vance D.E. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science Publishers B. V., Amsterdam1996: 153-181Google Scholar, 2Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (192) Google Scholar, 3Kent C. Carman G.M. Trends Biochem. Sci. 1999; 24: 146-150Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (264) Google Scholar). It is a structural component of cell membranes and a source of lipid molecules (e.g. lyso-PC, phosphatidate, diacylglycerol, lysophosphatidate, platelet activating factor, arachidonic acid) involved in cell signaling (1Vance D.E. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science Publishers B. V., Amsterdam1996: 153-181Google Scholar, 2Kent C. Biochim. 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In the Kennedy pathway PC is derived from choline via phosphocholine and CDP-choline, whereas in the CDP-diacylglycerol pathway, PC is derived from CDP-diacylglycerol via the major phospholipids phosphatidylserine and phosphatidylethanolamine (1Vance D.E. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science Publishers B. V., Amsterdam1996: 153-181Google Scholar, 2Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (192) Google Scholar, 4Carman G.M. Henry S.A. Prog. Lipid Res. 1999; 38: 361-399Crossref PubMed Scopus (264) Google Scholar, 16Paltauf F. Kohlwein S.D. Henry S.A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 415-500Google Scholar). When grown in the presence of choline, wild type S. cerevisiae primarily synthesizes PC via the Kennedy pathway (17McMaster C.R. Bell R.M. J. Biol. 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Chem. 2002; 277: 34978-34986Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Moreover, phosphorylation at these sites stimulates PC synthesis via the Kennedy pathway. In the present work we addressed the hypothesis that phosphorylation of choline kinase was also mediated by protein kinase C. Protein kinase C is a lipid-dependent protein kinase required for S. cerevisiae cell cycle (50Nishizuka Y. Nature. 1984; 308: 693-698Crossref PubMed Scopus (5764) Google Scholar, 51Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4232) Google Scholar, 52Bell R.M. Burns D.J. J. Biol. Chem. 1991; 266: 4661-4664Abstract Full Text PDF PubMed Google Scholar, 53Levin D.E. Fields F.O. Kunisawa R. Bishop J.M. Thorner J. Cell. 1990; 62: 213-224Abstract Full Text PDF PubMed Scopus (312) Google Scholar, 54Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1361) Google Scholar) and plays a role maintaining cell wall integrity (55Levin D.E. Bartlett-Heubusch E. J. Cell Biol. 1992; 116: 1221-1229Crossref PubMed Scopus (303) Google Scholar). The rationale for this hypothesis was based on the presence of potential protein kinase C target sites in the choline kinase enzyme (Fig. 1). We showed here that protein kinase C phosphorylated and stimulated choline kinase and identified Ser25 and Ser30 as major sites of phosphorylation. We also showed a S25A mutation in choline kinase correlated with a decrease in PC synthesis by the Kennedy pathway. Materials—All chemicals were reagent grade. Difco was the source of growth medium supplies. Nucleotides, ammonium reinecke, phenylmethylsulfonyl fluoride, benzamidine, aprotinin, leupeptin, pepstatin, choline, l-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin, standard phosphoamino acids, and bovine serum albumin were purchased from Sigma. Protein kinase C (rat brain) and protein kinase A (bovine heart) were purchased from Promega. Bio-Rad was the source of the protein assay reagent, electrophoresis reagents, and protein molecular mass markers. Protein A-Sepharose CL-4B beads, polyvinylidene difluoride membrane, the enhanced chemifluorescence Western blotting detection kit, and [methyl-14C]choline were purchased from Amersham Biosciences. Phospholipids were purchased from Avanti Polar Lipids. Silica Gel 60 thin-layer chromatography plates and cellulose thin-layer glass plates were purchased from EM Science. Peptides were synthesized and purified commercially by Bio-Synthesis, Inc. Phosphocellulose filters were supplied by Pierce. [γ-32P]ATP was purchased from PerkinElmer Life Sciences. Scintillation counting supplies were from National Diagnostics. Restriction enzymes, modifying enzymes, and Vent DNA polymerase were from New England Biolabs. Polymerase chain reaction and sequencing primers were prepared commercially by Genosys Biotechnologies, Inc. The QuikChange site-directed mutagenesis kit was purchased from Stratagene. The Yeastmaker yeast transformation system was from Clontech. The DNA size ladder used for agarose gel electrophoresis was from Invitrogen. The plasmid DNA purification and DNA gel extraction kits were from Qiagen, Inc. Strains and Growth Conditions—The strains used in this work are listed in Table I. The growth and analysis of yeast were performed by standard methods (56Rose M.D. Winston F. Heiter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar, 57Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Yeast cultures were grown in synthetic complete medium (56Rose M.D. Winston F. Heiter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar) containing 2% glucose at 30 °C. Plasmid maintenance and amplifications were performed in Escherichia coli strain DH5α. Bacteria were cultured in LB medium (1% Tryptone, 0.5% yeast extract, 1% NaCl (pH 7.4)) at 37 °C. Ampicillin (100 μg/ml) was added to the growth medium to select bacterial cells that carried plasmids. Growth media were supplemented with either 2% (yeast) or 1.5% (E. coli) agar for growth on plates. Yeast growth in liquid media was monitored spectrophotometrically (A600 nm).Table IStrains and plasmids used in this workStrain or plasmidGenotype or relevant characteristicsSource or referenceE. coliDH5αF- ϕ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rk- mk+) phoA supE44 λ-thi-1 gyrA96 relA157Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google ScholarS. cerevisiaeKS106MATα ade2-1 can1-100 his3-11,15 leu2-3 112, trp1-1 ura3-1 cki1Δ::HIS3 eki1Δ::TRP161Kim K. Kim K.-H. Storey M.K. Voelker D.R. Carman G.M. J. Biol. Chem. 1999; 274: 14857-14866Abstract Full Text Full Text PDF PubMed Scopus (67) Google ScholarPlasmidsYEp351Multicopy E. coli/yeast shuttle vector containing LEU291Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1083) Google ScholarpYY264CKI1 derivative of YEp35149Yu Y. Sreenivas A. Ostrander D.B. Carman G.M. J. Biol. Chem. 2002; 277: 34978-34986Abstract Full Text Full Text PDF PubMed Scopus (36) Google ScholarpYY265CKI1S30A derivative of pYY26449Yu Y. Sreenivas A. Ostrander D.B. Carman G.M. J. Biol. Chem. 2002; 277: 34978-34986Abstract Full Text Full Text PDF PubMed Scopus (36) Google ScholarpYY266CKI1S85A derivative of pYY26449Yu Y. Sreenivas A. Ostrander D.B. Carman G.M. J. Biol. Chem. 2002; 277: 34978-34986Abstract Full Text Full Text PDF PubMed Scopus (36) Google ScholarpCK25ACKI1S25A derivative of pYY264This studypCK25DCKI1S25D derivative of pYY264This studypCK37ACKI1S37A derivative of pYY264This studypCK25A/37ACKI1S25A,S37A derivative of pCK25AThis studypCK25A/30ACKI1S25A,S30A derivative of pCK25AThis study Open table in a new tab DNA Manipulations, Amplification of DNA by PCR, and DNA Sequencing—Standard methods were used to prepare genomic and plasmid DNA, to digest DNA with restriction enzymes, and to ligate DNA (57Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Transformation of yeast (58Ito H. Yasuki F. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar, 59Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1776) Google Scholar) and E. coli (57Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) were performed by standard methods. PCR reactions were optimized as described by Innis and Gelfand (60Innis M.A. Gelfand D.H. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA1990: 3-12Google Scholar). DNA sequencing reactions were performed by the dideoxy method using Taq polymerase (57Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and analyzed with an automated DNA sequencer. Construction of Plasmids and Expression of Wild Type and Mutant CKI1 Alleles—The plasmids used in this work are listed in Table I. The CKI1S25A (primer 5′-GCAAGGTCCAGATCGAGTGCTCAAAGAAGACATTC-3′ and its complement), CKI1S25D (primer 5′-GTCCAGATCGAGTGATCAAAGAAGAC-3′ and its complement), and CKI1S37A (primer 5′-CACGCCAACGTTCCGCTCAAAGACTGATTAG-3′ and its complement) alleles were constructed by PCR with the QuikChange site-directed mutagenesis kit using plasmid pYY264 (49Yu Y. Sreenivas A. Ostrander D.B. Carman G.M. J. Biol. Chem. 2002; 277: 34978-34986Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) as the template. The CKI1S25A,S37A and CKI1S25A,S30A mutant alleles were constructed with the primers for the S37A and S30A mutations using plasmid pCK25A as the template, respectively. The correct mutations in the CKI1 alleles were confirmed by DNA sequencing. The eki1Δ cki1Δ mutant strain KS106 (61Kim K. Kim K.-H. Storey M.K. Voelker D.R. Carman G.M. J. Biol. Chem. 1999; 274: 14857-14866Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) was transformed with plasmids containing the wild type and mutant alleles of the CKI1 gene. Preparation of Enzymes—Cell extracts were prepared by disruption of cells with glass beads using a Mini-BeadBeater-8 (Biospec Products, Inc.) as described previously (49Yu Y. Sreenivas A. Ostrander D.B. Carman G.M. J. Biol. Chem. 2002; 277: 34978-34986Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 62Klig L.S. Homann M.J. Carman G.M. Henry S.A. J. Bacteriol. 1985; 162: 1135-1141Crossref PubMed Google Scholar). The cell disruption buffer contained 50 mm Tris-HCl (pH 8.0), 1 mm EDTA, 0.3 m sucrose, 10 mm 2-mercaptoethanol, and a protease and phosphatase inhibitor mixture. The protease and phosphatase inhibitor mixture contained 0.5 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 5 μg/ml pepstatin, 10 mm NaF, and 5 mm β-glycerophosphate. The CKI1-encoded choline kinase was expressed in Sf9 insect cells and purified to homogeneity from the cytosolic fraction by chromatography with Con A, Affi-Gel Blue, and Mono Q (28Kim K.-H. Voelker D.R. Flocco M.T. Carman G.M. J. Biol. Chem. 1998; 273: 6844-6852Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Immunoprecipitation and Immunoblotting—The IgG fraction of rabbit anti-choline kinase antibodies (49Yu Y. Sreenivas A. Ostrander D.B. Carman G.M. J. Biol. Chem. 2002; 277: 34978-34986Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) was isolated from antisera by protein A-Sepharose chromatography (63Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988Google Scholar) and used for immunoprecipitation and immunoblotting experiments. Cell extracts (0.5 mg of protein) were incubated for 1 h with 10 μg of anti-choline kinase antibodies in a total volume of 0.5 ml followed by incubation with 100 μl of protein A-Sepharose CL-4B beads (10% slurry, w/v) for 1 h at 4 °C. Immune complexes were collected by centrifugation at 1500 × g for 30 s and washed 3 times with phosphorylation reaction buffer (50 mm Tris-HCl (pH 8.0), 10 mm MgCl2, 10 mm 2-mercaptoethanol, 0.375 mm EDTA, 0.375 mm EGTA, and 1.7 mm CaCl2). After the washing steps the buffer was removed by aspiration, and the choline kinase attached to the protein A-Sepharose CL-4B beads was used as the substrate for protein kinase C phosphorylation. The reaction mixtures were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membrane was probed with anti-choline kinase antibodies (0.6 μg/ml) as described previously (63Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988Google Scholar). The choline kinase protein was detected on immunoblots using the ECF Western blotting chemifluorescence detection kit as described by the manufacturer. Fluorescent signals on the immunoblots were acquired by fluorescence imaging analysis. The relative densities of the proteins were analyzed using ImageQuant software. Immunoblot signals were in the linear range of detectability. Phosphorylation Reactions—Pure choline kinase, immunoprecipitated choline kinase, and choline kinase synthetic peptides were phosphorylated with rat brain protein kinase C. This enzyme preparation contains a mixture of the α, β, and γ isoforms of the enzyme. We used the rat brain protein kinase C in our studies because the S. cerevisiae protein kinase C (64Ogita K. Miyamoto S. Koide H. Iwai T. Oka M. Ando K. Kishimoto A. Ikeda K. Fukami Y. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5011-5015Crossref PubMed Scopus (73) Google Scholar, 65Simon A.J. Milner Y. Saville S.P. Dvir A. Mochly-Rosen D. Orr E. Proc. Biol. Sci. 1991; 243: 165-171Crossref PubMed Scopus (26) Google Scholar) has catalytic properties characteristic of the α, β, and γ isoforms of the rat brain enzyme (50Nishizuka Y. Nature. 1984; 308: 693-698Crossref PubMed Scopus (5764) Google Scholar, 66Nishizuka Y. Nature. 1988; 334: 661-665Crossref PubMed Scopus (3537) Google Scholar). In addition, the rat brain enzyme has been shown to phosphorylate other yeast proteins with the same efficiency as a partially purified preparation of yeast protein kinase C (67Yang W.-L. Carman G.M. J. Biol. Chem. 1995; 270: 14983-14988Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 68Yang W.-L. Bruno M. E.C. Carman G.M. J. Biol. Chem. 1996; 271: 11113-11119Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 69Sreenivas A. Villa-Garcia M.J. Henry S.A. Carman G.M. J. Biol. Chem. 2001; 276: 29915-29923Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Phosphorylation reactions were performed in a total volume of 25 μl at 30 °C. Choline kinase was incubated for 10 min with 50 mm Tris-HCl (pH 8.0), 50 μm [γ-32P]ATP (4 μCi/nmol), 10 mm MgCl2, 10 mm 2-mercaptoethanol, 0.375 mm EDTA, 0.375 mm EGTA, 1.7 mm CaCl2, 20 μm diacylglycerol, 50 μm phosphatidylserine, and the indicated amounts of protein kinase C. At the end of the phosphorylation reactions samples were treated with 4× Laemm
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