IN-VIVO REGULATION OF PROTEIN-KINASE-C BY TRANS-PHOSPHORYLATION FOLLOWED BY AUTOPHOSPHORYLATION

Dephosphorylation by the catalytic subunits of protein phosphatases 1 (CS1) and 2A (CS2) reveals that mature protein kinase C is phosphoryla ted at two distinct sites. Treatment of protein kinase C beta II with CS1 causes a significant increase in the protein's electrophoretic mob ility (approximately 4 kDa) and a coincident loss in catalytic activit y. The CS1-dephosphorylated enzyme cannot autophosphorylate or be phos phorylated by mature protein kinase C, indicating that a different kin ase catalyzes the phosphorylation at this site. The loss of activity i s consistent with dephosphorylation on protein kinase C's activation l oop (Orr, J. W., and Newton, A. C., (1994) J. Biol. Chem. 269, 27715-2 7718). Treatment with CS2 results in a smaller shift in electrophoreti c mobility (approximately 2 kDa) and no loss in catalytic activity. Fu rthermore, the CS2-dephosphorylated form can autophosphorylate and thu s regain the electrophoretic mobility of mature enzyme, consistent wit h dephosphorylation at protein kinase C's carboxyl-terminal autophosph orylation site, which is modified in vivo (Flint, A. J., Paladini, R. D., and Koshland, D. E., Jr. (1990) Science 249, 408-411). In summary, two phosphorylations process protein kinase C to generate the mature form: a transphosphorylation that renders the kinase catalytically com petent and an autophosphorylation that may be important for the subcel lular localization of the enzyme.