CHARACTERIZATION OF TYROSINE-PHOSPHORYLATED DELTA-ISOFORM OF PROTEIN-KINASE-C ISOLATED FROM CHINESE-HAMSTER OVARY CELLS

Phorbol ester treatment of Chinese hamster ovary cells stably overexpr essing the delta isoform of protein kinase C induced the association o f the isoform with the particulate fraction and the tyrosine phosphory lation of a small portion of the delta isoform, The delta isoform with out tyrosine phosphorylation was recovered as an enzyme dependent on p hospholipid and diacylglycerol, whereas the tyrosine-phosphorylated de lta isoform was recovered in two fractions, one dependent on, and the other independent of, phospholipid and diacylglycerol. The tyrosine-ph osphorylated delta isoform independent of lipid activators might be as sociated with phorbol ester and phospholipids. Immunoblot analysis rev ealed that the delta isoform is a doublet protein of 76 and 78 kDa, an d that the delta isoform fraction without tyrosine phosphorylation con tained 76- and 78-kDa proteins, whereas the tyrosine-phosphorylated de lta isoform contained the 78-kDa protein but not the 76-kDa protein, I n vitro analysis showed that the 78-kDa protein of the delta isoform w ithout tyrosine phosphorylation is an efficient substrate of tyrosine kinase only when phosphatidylserine and either diacylglycerol or phorb ol ester are present; however, the 76-kDa protein can not be tyrosine- phosphorylated even in the presence of these lipid activators, The pho spholipid and diacylglycerol-dependent form of the tyrosine-phosphoryl ated enzyme isolated from the cell line required lower concentrations of phosphatidylserine and phorbol ester for its activity in. vitro as compared with the enzyme without tyrosine phosphorylation, These resul ts suggest that the tyrosine-phosphorylated enzyme generated upon stim ulation of the cells may associate with membranes and exert its full a ctivity even with the lower concentrations of the lipid activators.