The phosphorylation of protein kinase C

The calcium and phospholipid dependent protein kinase C (PKC) is the primary mediator of the diacylglycerol (DG) limb of the phosphatidylinositol second messenger signalling pathway. In response to an increase in DG, PKC phosphorylates substrates as well as phosphorylating itself by an intrapeptide mechanism.; To examine the phosphorylation of PKC in greater detail, a convenient source of a pure form of the enzyme was sought. Antibodies were raised against the catalytic and regulatory domains of PKC {dollar}beta{dollar}II to facilitate its detection. Bacterially-expressed protein kinase C was inactive; however, a baculovirus vector enabled fully active protein to be successfully produced in insect cells.; This purified enzyme was found to be a metalloprotein containing two atoms of zinc per protein molecule. The zinc is probably bound to the cysteine-rich, phorbol ester-binding sequences in the N-terminal half of the protein. Enzyme which had been activated for autophosphorylation in the absence of calcium and phospholipid by a brief exposure to pH 4.5 contained no zinc.; Six major autophosphorylation sites were identified in PKC {dollar}beta{dollar}II and were found to lie in three widely separated regions of the primary sequence. In the folded structure, however, these regions were probably close to the active site to allow their modification by the intrapeptide mechanism.; Most of the autophosphorylation could be reversed in vitro with the addition of several partially purified and classified phosphoprotein phosphatases from rat brain. Presumably this self phosphorylation can be reversed in cells and may be used to reprime PKC for a later response to another stimulus.; An intracellular inhibitor of PKC activity was also characterized. Its mechanism of action was through competitive binding with ATP and it was shown to also inhibit other kinases.; Finally, an 88 kDa protein resembling a PKC {dollar}varepsilon{dollar} isozyme was discovered using anti-PKC monoclonal antibody #1.4. It was also detected by the anti-catalytic domain antiserum suggesting that it has kinase domain epitopes. Analysis of extracts from cells expressing PKC {dollar}varepsilon{dollar} showed that the 88 kDa protein is not PKC {dollar}varepsilon{dollar}, but may be another isozyme of protein kinase C.