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 overexpressing the delta isoform of protein kinase C induced the association of the isoform with the particulate fraction and the tyrosine phosphorylation of a small portion of the delta isoform. The delta isoform without tyrosine phosphorylation was recovered as an enzyme dependent on phospholipid and diacylglycerol, whereas the tyrosine-phosphorylated delta isoform was recovered in two fractions, one dependent on, and the other independent of, phospholipid and diacylglycerol. The tyrosine-phosphorylated delta isoform independent of lipid activators might be associated with phorbol ester and phospholipids. Immunoblot analysis revealed that the delta isoform is a doublet protein of 76 and 78 kDa, and that the delta isoform fraction without tyrosine phosphorylation contained 76- and 78-kDa proteins, whereas the tyrosine-phosphorylated delta isoform contained the 78-kDa protein but not the 76-kDa protein. In vitro analysis showed that the 78-kDa protein of the delta isoform without tyrosine phosphorylation is an efficient substrate of tyrosine kinase only when phosphatidylserine and either diacylglycerol or phorbol ester are present; however, the 76-kDa protein can not be tyrosine-phosphorylated even in the presence of these lipid activators. The phospholipid and diacylglycerol-dependent form of the tyrosine-phosphorylated 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 results suggest that the tyrosine-phosphorylated enzyme generated upon stimulation of the cells may associate with membranes and exert its full activity even with the lower concentrations of the lipid activators.
Kadotani
M
M
Department of Biochemistry, Kobe University School of Medicine, Hyogo.
Nishiuma
T
T
Nanahoshi
M
M
Tsujishita
Y
Y
Ogita
K
K
Nakamura
S
S
Kikkawa
U
U
Asaoka
Y
Y
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D007527:Isoenzymes / Q000302:isolation & purification*; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000302:isolation & purification*; D051745:Protein Kinase C-delta; D014443:Tyrosine / Q000378:metabolism*
D007527:Isoenzymes; D010703:Phorbol Esters; D014443:Tyrosine; D011493:Protein Kinase C; D051745:Protein Kinase C-delta