Evidence for phosphorylation of CTP:phosphocholine cytidylyltransferase by multiple proline-directed protein kinases.
Reversible phosphorylation of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of phosphatidylcholine biosynthesis, is thought to play a role in regulating its activity. In the present study, the hypothesis that proline-directed kinases play a major role in phosphorylating cytidylyltransferase is substantiated using a c-Ha-ras-transfected clone of the human keratinocyte cell line HaCaT. Cellular extracts from epidermal growth factor-stimulated HaCaT cells and from ras-transfected HaCaT cells phosphorylated cytidylyltransferase much stronger as compared with extracts from quiescent HaCaT cells. The tryptic phosphopeptide pattern of cytidylyltransferase phosphorylated by cell-free extracts from ras-transfected HaCaT cells was similar compared with the patterns of cytidylyltransferase phosphorylated by p44mpkmitogen-activated protein kinase and p34cdc2 kinase in vitro, whereas in the case of casein kinase II the pattern was different. Furthermore, in c-Ha-ras-transfected HaCaT cells the in vivo phosphorylation state of cytidylyltransferase was 2-fold higher as compared with untransfected HaCaT cells. This higher phosphorylation of cytidylyltransferase in the ras-transfected clone was reduced to a level below the phosphorylation of cytidylyltransferase in untransfected cells, using olomoucine, a specific inhibitor of proline-directed kinases. The reduced phosphorylation of cytidylyltransferase in olomoucine-treated cells correlated with an enhanced stimulation of enzyme activity by oleic acid.
Wieprecht
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Institute of Molecular Biology and Biochemistry, University Medical Center Benjamin Franklin, Free University of Berlin, Berlin D-12200, Germany.
Wieder
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Paul
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Geilen
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Orfanos
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D016203:CDC2 Protein Kinase / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D002474:Cell-Free System; D019916:Choline-Phosphate Cytidylyltransferase; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007701:Kinetin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009713:Nucleotidyltransferases / Q000378:metabolism*; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D011687:Purines / Q000494:pharmacology; D014162:Transfection
D004791:Enzyme Inhibitors; D009829:Oleic Acids; D010748:Phosphopeptides; D011687:Purines; D019301:Oleic Acid; C090046:olomoucine; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016203:CDC2 Protein Kinase; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D007701:Kinetin