We have investigated protein kinase C (PKC) in skeletal muscle cytosol and demonstrated the presence of two major activities. These did not correspond to different PKC isoenzymes but seemed to represent two species of PKC α as deduced by: elution during hydroxyapatite chromatography at KH2PO4 concentrations expected of PKC α; detection of the two species by three specific but unrelated anti-(PKC α) antibodies; immunodepletion of both activities with anti-(PKC α) antibody; and demonstration of identical requirements of both Ca2+ ions and lipid for activation. These species, termed PKC α1 and PKC α2, phosphorylated the modified conventional PKC pseudosubstrate peptide (19–31, Ser-25) equally well. Importantly, however, the activities differed in that PKC α1 phosphorylated histone IIIS, and also peptides derived from the EGF receptor and glycogen synthase, to a much greater extent than did PKC α2. Similarly, incubation of crude muscle extracts with either PKC α1 or α2 gave rise to different protein phosphorylation patterns. The involvement of proteolysis, dephosphorylation or oxidative modification in the interconversion of PKC α1 and PKC α2 during preparation was ruled out. Although some PKC-binding proteins were detected in overlay assays, their presence did not explain the anomalous PKC α2 activity. The results suggest that a modification of PKC α in situ limits its substrate specificity, and indicate an additional level of control of the kinase that may be a site for modulation of PKC-mediated signal transduction.

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