CHARACTERIZATION OF 2 FORMS OF PROTEIN-KINASE-C-ALPHA, WITH DIFFERENTSUBSTRATE SPECIFICITIES, FROM SKELETAL-MUSCLE

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 spe cies of PKC alpha as deduced by: elution during hydroxyapatite chromat ography at KH2PO4 concentrations expected of PKC alpha; detection of t he two species by three specific but unrelated anti-(PKC alpha) antibo dies; immunodepletion of both activities with anti-(PKC alpha) antibod y; and demonstration of identical requirements of both Ca2+ ions and l ipid for activation. These species, termed PKC alpha 1 and PKC alpha 2 , phosphorylated the modified conventional PKC pseudosubstrate peptide (19-31, Ser-25) equally well. Importantly, however, the activities di ffered in that PKC alpha 1 phosphorylated histone IIIS, and also pepti des derived from the EGF receptor and glycogen synthase, to a much gre ater extent than did PKC alpha 2. Similarly, incubation of crude muscl e extracts with either PKC alpha 1 or alpha 2 gave rise to different p rotein phosphorylation patterns. The involvement of proteolysis, depho sphorylation or oxidative modification in the interconversion of PKC a lpha 1 and PKC alpha 2 during preparation was ruled out. Although some PKC-binding proteins were detected in overlay assays, their presence did not explain the anomalous PKC alpha 2 activity. The results sugges t that a modification of PKC alpha in situ limits its substrate specif icity, and indicate an additional level of control of the kinase that may be a site for modulation of PKC-mediated signal transduction.