Protein kinase C-alpha is multiply phosphorylated in response to phorbol ester stimulation of PC12 cells.
The protein kinase C (PKC) family consists of a number of closely related isotypes, whose in vivo phosphorylation state is regulated in a dynamic fashion by the enzyme's activators. We have investigated here the changes in PKC phosphorylation in response to phorbol ester. Using a combination of hydroxylapatite chromatography and immunoblot with isotype-specific antibodies, we identified PKC-alpha, -delta, -epsilon, and -zeta as the isotypes expressed in PC12 cells. A two-dimensional immunoblot approach was then developed to measure the changes in the phosphorylation state of PKC-alpha before and after exposure of intact PC12 cells to phorbol ester. We found a pool of four differentially migrating PKC-alpha forms in untreated cells, which undergoes an acidic shift after phorbol ester. Furthermore, a similar shift in the two-dimensional immunoblot profile of PKC-alpha was the result of the enzyme autophosphorylation upon in vitro treatment with a combination of phosphatidylserine and phorbol ester, an effect which was enhanced by co-application of purified bovine lung cGMP-dependent protein kinase-I (PKG-I). These results demonstrate a multiple phosphorylation of PKC-alpha in untreated PC12 cells and suggest that various levels of autophosphorylation and trans-phosphorylation of this isoenzyme may occur in response to phorbol ester.
Gatti
A
A
Endocrinology Unit, John Hunter Hospital, Newcastle, N.S.W., Australia.
Wang
X
X
Robinson
P J
PJ
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism*; D008722:Methods; D016716:PC12 Cells; D010703:Phorbol Esters / Q000494:pharmacology*; D010718:Phosphatidylserines / Q000502:physiology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051381:Rats; D015398:Signal Transduction
D007527:Isoenzymes; D010703:Phorbol Esters; D010718:Phosphatidylserines; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha