Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

Abstract

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. 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. 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, 5Spector A.A. Yorek M.A. J. Lipid Res. 1985; 26: 1015-1035Abstract Full Text PDF PubMed Google Scholar). PC is also a component of pulmonary surfactant (6Rooney S.A. Am. Rev. Respir. Dis. 1985; 131: 439-460PubMed Google Scholar), serum lipoproteins (7Vance J.E. Vance D.E. Can. J. Biochem. Cell Biol. 1985; 63: 870-881Crossref PubMed Scopus (85) Google Scholar), and bile (8Agellon L.B. Walkey C.J. Vance D.E. Kuipers F. Verkade H.J. Hepatology. 1999; 30: 725-729Crossref PubMed Scopus (27) Google Scholar). The importance of PC to cell physiology is highlighted by the fact that alterations in its metabolism are linked to programmed cell death (9Cui Z. Houweling M. M. Vance D.E. F. J. 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Google Scholar, J. Google Scholar), and the enzyme is a target for J. M.A. A. PubMed Scopus Google Scholar, A. Biochem. Biophys. Res. PubMed Scopus Google Scholar, A. M. M.A. A. PubMed Scopus Google Scholar). is a major by which enzymes are Rev. Biochem. PubMed Scopus Google Scholar, Rev. Biochem. PubMed Scopus Google Scholar), and the choline kinase cerevisiae is by phosphorylation Carman G.M. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). kinase is phosphorylated on multiple serine in and some of this phosphorylation is mediated by protein kinase A Carman G.M. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). kinase A and choline kinase Ser30 and with the site the major on enzyme Carman G.M. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). phosphorylation PC synthesis via the Kennedy pathway. 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A Scholar). in synthetic F. in A containing and in in μg/ml) was to the to cells that with for on in was and used in this J. A Carman G.M. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google containing J.E. A. PubMed Scopus Google of A. Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google of A. Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google of A. Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google of of of of of in a of by and used to and to with and to J. A Scholar). of F. A. J. PubMed Google Scholar, PubMed Scopus Google and J. A by as by and M.A. M.A. A to and Scholar). by the J. A and with an of and of and used in this work are in The and its and its and and its by with the A. Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google as the The and mutant with the for the and S30A as the The in the confirmed by The mutant Carman G.M. J. 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This enzyme contains a of the and of the enzyme. used the protein kinase C in the cerevisiae protein kinase C M. A. Sci. A. PubMed Scopus Google Scholar, A. Biol. Sci. PubMed Scopus Google of the and of the enzyme PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). In the enzyme to with the as a of protein kinase C Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Henry S.A. Carman G.M. J. Biol. 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Full Text PDF PubMed Google Scholar). for and for on to and kinase activity was in a that and enzyme protein in a of Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The was the substrate by of choline as a C. PubMed Scopus Google Scholar). the was choline by on the J. Biol. Full Text PDF PubMed Google Scholar). The of the enzyme in was with and protein A of choline kinase activity was as the of enzyme that the of of activity was as of was by the of Biochem. PubMed Scopus Google serum as the and of cells containing the wild type and mutant choline kinase enzymes for with the the cells Can. J. Biochem. PubMed Scopus Google Scholar, Carman G.M. J. PubMed Google Scholar), and PC was on the PC was by The of the PC on was with PC to The of PC was by of with the enzyme F. Biochem. PubMed Scopus Google Scholar). with of by choline kinase was a target for protein kinase C phosphorylation, we examined protein kinase C the of the of ATP choline the to by to analysis of the that choline kinase was a substrate for protein kinase C The phosphorylation of choline kinase was dependent on the of protein kinase C of the of protein kinase C activity that the enzyme and of and with to the concentrations of choline kinase and ATP The to the the enzyme F. Biochem. PubMed Scopus Google Scholar). The for choline kinase and ATP 27 and 15 and the for and The of protein kinase C activity choline kinase a of the of the choline kinase enzyme Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). which of choline kinase was a target for phosphorylation, choline kinase was phosphorylated with protein kinase and the enzyme was to kinase C phosphorylated choline kinase on a serine Phosphopeptide mapping analysis of the phosphorylated choline kinase that the enzyme was phosphorylated on multiple serine The of protein kinase C phosphorylation on choline kinase activity was this the choline kinase enzyme was phosphorylated with protein kinase C and the phosphorylation choline kinase activity was by the of by protein kinase C resulted in a stimulation of choline kinase activity This of stimulation an of the protein kinase C on activity the choline kinase is phosphorylated Carman G.M. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). a C for of choline kinase serine and phosphorylation a protein kinase C motif and containing serine target on the of choline kinase examined as for protein kinase C. as with the activity the peptide by the peptide and also the serine the protein kinase C motif to did not as for protein kinase C. the serine the protein kinase C motif of the and was the target site for for protein kinase C choline kinase synthetic μmol kinase C kinase A activity. in a A analysis was to the choline kinase synthetic as for protein kinase C activity. The of protein kinase C activity on of the peptide The to the and the are in on the of and the peptide was by the substrate by the that Ser25 and major protein kinase C phosphorylation in the choline kinase enzyme. of C on the of by C and by of Ser25 and the choline kinase was to the hypothesis that are for protein kinase C. with serine to and by protein kinase C phosphorylation site mutant a in a mutant to to the choline kinase by the J. Biol. Full Text PDF PubMed Google and Carman G.M. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google A was used for to of the mutant enzymes by cell analysis that the did not affect the of the choline kinase enzyme. In we that the phosphorylation of the wild type choline kinase was dependent on the of protein kinase C and the of the The mutant choline kinase enzymes with protein kinase C and the phosphorylation to to and for choline The of phosphorylation of the S25A and mutant enzymes was by and with the wild type enzyme. also a mutant choline kinase enzyme and as a substrate for protein kinase C. The of the mutation on the protein kinase C phosphorylation of the enzyme was to that of the S25A The wild type and protein kinase C phosphorylation site mutant choline kinase enzymes also to phosphorylation with protein kinase A Carman G.M. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). a the of phosphorylation was examined as The S25A and did not affect the phosphorylation of the choline kinase by protein kinase A. of A on the of by and are target for protein kinase A phosphorylation in the choline kinase enzyme A. Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the S30A and phosphorylation of the enzyme by protein kinase C. The S30A mutation caused a decrease in protein kinase C phosphorylation of choline kinase by whereas the mutation did not affect the phosphorylation of the enzyme. A Ser30 a for choline kinase and which Ser30 and are for protein kinase A phosphorylation A. Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). examined also as for protein kinase C. The peptide was a substrate for protein kinase C phosphorylation, whereas the peptide did not as a substrate for this kinase The which an for the serine the protein kinase A was a substrate for protein kinase C. This that the serine the protein kinase A motif of peptide was the target site for protein kinase C The phosphorylation of the peptide by protein kinase C and the are in on the the peptide was as of a substrate for protein kinase C as the peptide the for the peptide was the for the the for the peptide was that of the peptide The for the peptide for protein kinase A phosphorylation is A. Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). on this Ser30 was a target site for protein kinase A phosphorylation with protein kinase C. of and on the Phosphopeptide of by of the S25A and S30A on the phosphorylation of choline kinase by protein kinase C examined in The phosphorylation of the S25A and S30A mutant choline kinase enzymes was the the of phosphorylation was and with the wild type enzyme A mutant was also and examined for its phosphorylation by protein kinase C. The phosphorylation of this mutant was with the phosphorylation site examined the of the S25A and S30A on the phosphopeptide of the choline kinase was cells the wild S30A mutant The phosphorylated with protein kinase C and ATP and to phosphopeptide mapping The phosphopeptide of the choline kinase phosphorylated with protein kinase C was to that of the choline kinase phosphorylated with protein kinase C with The and in the phosphopeptide of the wild type choline kinase enzyme in the of the S25A mutant enzyme The S30A mutation resulted in the of phosphopeptide In the phosphopeptide of the phosphopeptide was and and that Ser25 was in and and that Ser30 was in phosphopeptide for the of Ser25 in of the phosphorylated enzyme by of the S25A and S25D on and and on the of PC by the Kennedy analysis of the cell cells the wild type choline kinase that the enzyme as a This choline kinase is also the enzyme is in cells Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). with the S25A mutant the of the was with the is a by some phosphorylated enzymes C. J. Biol. Full Text Full Text PDF Scopus Google Scholar, A. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). and a choline kinase with a Ser25 to mutation to the of a phosphorylation mimic on the enzyme. analysis that the S25D mutant enzyme as a the wild type enzyme that a of phosphorylated and of choline kinase in The of the S25A and S25D on the activity of choline kinase Cell cells the mutant enzymes and for choline kinase activity. The activity of the S25A mutant choline kinase was by with the wild type enzyme The activity of the S25D mutant enzyme was that of the wild type enzyme the of phosphorylation of choline kinase Ser25 on the synthesis of PC, cells the S25A mutant enzyme with choline is PC that is by the Kennedy J. Biol. Full Text PDF PubMed Google Scholar, Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the and PC was by The of the choline PC in cells the S25A mutant enzyme was by with cells the wild type enzyme also examined the of the S25D which the phosphorylation on PC synthesis. The S25D mutation in choline kinase resulted in a increase in the of choline PC the synthesis of PC in cells the S25D mutant choline kinase was that of cells the S25A mutant enzyme. The CKI1-encoded choline kinase is an enzyme that catalyzes the committed step in PC synthesis via the of the Kennedy in cerevisiae 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, J. Biol. Full Text PDF PubMed Google Scholar, B. and and Elsevier Science Publishers B. V., Scholar, G.M. Vance D.E. Scholar). by phosphorylation is an by which the activity of an enzyme a Rev. Biochem. PubMed Scopus Google Scholar, Rev. Biochem. PubMed Scopus Google Scholar). that the choline kinase is phosphorylated and by protein kinase A Carman G.M. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Phosphopeptide mapping analysis of choline kinase phosphorylated in vivo and phosphorylated in by protein kinase A that protein are for the phosphorylation of choline kinase Carman G.M. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In the work we the hypothesis that choline kinase phosphorylation was also mediated by protein kinase C. In protein kinase C phosphorylated choline kinase on serine residues, in the stimulation of activity. The phosphorylation of choline kinase was and dependent on the concentrations of choline kinase and that the choline kinase was a substrate for protein kinase C. this is the of protein kinase C phosphorylation of a choline kinase of the protein kinase C phosphorylation in choline kinase was to the of this a analysis we protein kinase C target in choline kinase with for and examined for to as for protein kinase C. Although as the containing a protein kinase C phosphorylation motif Ser25 and the This that major phosphorylation for protein kinase C. for this S25A and in choline kinase and in The S25A mutation a major on the of choline kinase to phosphorylated by protein kinase C. The of phosphorylation of this mutant was by 60% with the wild type enzyme. analysis of protein kinase choline kinase that in the wild type enzyme in the S25A mutant enzyme. that Ser25 was a target for protein kinase C cells the S25A mutant choline kinase activity. phosphorylation Ser25 to the of choline kinase activity. correlated with the stimulation of choline kinase activity protein kinase C phosphorylation in The mutant did not a major on the protein kinase C phosphorylation of choline The phosphopeptide of the mutant was that of the wild type and the phosphopeptide of the mutant enzyme did not that of the S25A mutant not we did not as a major protein kinase C phosphorylation site in choline the of the phosphorylation of choline kinase by protein kinase C the phosphorylation of the enzyme by protein kinase A and phosphorylation J. Biol. Full Text PDF PubMed Google Scholar, J. PubMed Scopus Google 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. This to a decrease in the of for the protein kinase C phosphorylation of the S30A mutant choline kinase was that Ser30 was a target site for protein A and C. The choline kinase synthetic peptide containing which is a substrate for protein kinase A A. Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), was also a substrate for protein kinase C. a major phosphopeptide in the phosphopeptide of the wild type protein kinase enzyme was in the phosphopeptide of the S30A mutant enzyme. This phosphopeptide the protein kinase A phosphorylation of choline kinase A. Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). site is phosphorylated by multiple protein is not J. PubMed Scopus Google Scholar, Z. F. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the is phosphorylated by protein kinase A and protein kinase C Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Carman G.M. 1999; 38: PubMed Scopus Google Scholar). The choline kinase in cerevisiae is an step in the synthesis of PC via the Kennedy J. Biol. Full Text PDF PubMed Google Scholar). we the of the protein kinase C phosphorylation of Ser25 with to PC synthesis in cells the S25A and S25D mutant choline kinase The in vivo that cells the S25A mutation a decrease in PC whereas cells the S25D mutation an increase in PC synthesis. the that of choline kinase activity by protein kinase C phosphorylation Ser25 resulted in the stimulation of PC synthesis via the Kennedy pathway. the of choline kinase activity by phosphorylation is The enzyme is also phosphorylated by protein kinase A, and phosphorylated by protein we phosphorylation that affect PC synthesis in cells the S25A and S25D mutant choline kinase that the S30A (protein kinase A site) mutation in choline kinase in a decrease in PC synthesis A. Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). This to the that protein kinase A phosphorylation Ser30 PC synthesis via the Kennedy A. Carman G.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). in the the phosphorylation of choline kinase Ser30 was also mediated by protein kinase C. the phosphorylation Ser30 by protein a in the of PC synthesis via the Kennedy pathway. to the that protein kinase C choline kinase Ser25 and Ser30 and the that protein kinase A the enzyme In cells protein kinase C a in the of lipid by of (e.g. PC and Rev. Biochem. PubMed Scopus Google Scholar, J. Biol. Full Text PDF PubMed Google Scholar, J. Biochem. PubMed Scopus Google Scholar). of choline kinase by protein kinase C in cerevisiae a by which lipid signaling are to PC synthesis and cell for the of this