Characterization of two forms of protein kinase C alpha, with different substrate 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 species of PKC alpha as deduced by: elution during hydroxyapatite chromatography at KH2PO4 concentrations expected of PKC alpha; detection of the two species by three specific but unrelated anti-(PKC alpha) antibodies; immunodepletion of both activities with anti-(PKC alpha) antibody; and demonstration of identical requirements of both Ca2+ ions and lipid 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 differed in that PKC alpha 1 phosphorylated histone IIIS, and also peptides derived from the EGF receptor and glycogen synthase, to a much greater extent than did PKC alpha 2. Similarly, incubation of crude muscle extracts with either PKC alpha 1 or alpha 2 gave rise to different protein phosphorylation patterns. The involvement of proteolysis, dephosphorylation or oxidative modification in the interconversion of PKC alpha 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 suggest that a modification of PKC alpha 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.
Schmitz-Peiffer
C
C
Garvan Institute of Medical Research, St. Vincent's Hospital, Darlinghurst, Australia.
Browne
C L
CL
Biden
T J
TJ
D000818:Animals; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D018482:Muscle, Skeletal / Q000201:enzymology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity
D007527:Isoenzymes; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha