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 thou ght to play a role in regulating its activity. In the present study, t he hypothesis that proline-directed kinases play a major role in phosp horylating cytidylyltransferase is substantiated using a c-Ha-ras-tran sfected clone of the human keratinocyte cell line HaCaT. Cellular extr acts from epidermal growth factor-stimulated HaCaT cells and from ras- transfected HaCaT cells phosphorylated cytidylyltransferase much stron ger 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 wi th the patterns of cytidylyltransferase phosphorylated by p44(mpk) mit ogen-activated protein kinase and p34(cdc2) kinase in vitro, whereas i n the case of casein kinase II the pattern was different, Furthermore, in c-Ha-ras-transfected HaCaT cells the in. vivo phosphorylation stat e of cytidylyltransferase was 2-fold higher as compared with untransfe cted HaCaT cells, This higher phosphorylation of cytidylyltransferase in the ras-transfected clone was reduced to a level below the phosphor ylation of cytidylyltransferase in untransfected cells, using olomouci ne, a specific inhibitor of proline-directed kinases. The reduced phos phorylation of cytidylyltransferase in olomoucine-treated cells correl ated with an enhanced stimulation of enzyme activity by oleic acid.