Protein kinase C-a is multiply phosphorylated in response to phorbol ester stimulation of PC 12 cells

摘要

The protein kinase C (PKC) family consists of a number of closely related isotypes, whose in vivo phosphorylation state is regulated in u 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-a, -8, -e, and -? as (tie isotypes expressed in PC12 cells. A two-dimensional immunoblot approach was then developed to measure the changes in Ihe phosphorylation state of PKC-M before and after exposure of intact PC12 cells to phorbol ester. We found a pool of four differentially migrating PKC-a forms in untreated cells, which undergoes an acidic shift after phorbol ester. Furthermore, a similar shift in Ihe iwo-dimensional immunoblot profile of PKC-" 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-depeiulenl protein kinase-I (PKG-I). These results demonstrate a multiple phosphorylation of PKC-a in untreated PC12 cells and suggest that various levels of autophosphorylation and /ra/w-phosphorylation of this isoenzyme may occur in response to phorbol ester.