Bryostatin produces nonphosphorylated protein kinase c (pkc) from autophosphorylated enzyme in vivo

Bryostatins are novel antineoplastic compounds that acutely activate and then downrcgulate PKC by inducing its ubiquitination and degradation by the proteasome (H.-W. Lee et al.; J.B. Smith et al., this meeting). In the LLCMK2 line of renal epithelial cells, bryostatin 1 (Btyo) potently activated PKC as shewn by autophosphorylation and translocation of PKCo, the predominant diacylglycerol-sensitive isofonn expressed by the cells. PKCa was immunoprecipitated from Biyo-treated cells and fractionated by gel electrophoiesis in the presence of sodium dodecylsulphate. Following 32P labeling of -80 kDa PKCa by autophosphorylation in vivo, Bryo produced a 76 kDa form of PKCa that lacked detectable 32P. The alkaline phosphatase treatment of immunoprecipitated PKCa converted the 80 kDa form to 76 kDa, but had no effect on the mobility of the 76 kDa form confirming that it was not phosphorylated. Pulse-chase labeling of PKCa with 3'S-Met/Cys indicated that there is a precursor-product relationship between the 80 and 76 kDa forms, respectively. Inhibition of protein synthesis had no effect on the production of 76 kDa PKCa by Bryo, which excludes de novo synthesis as the source of the 76 kDa form. Phorbol myristate acetate (PMA) also produced 76 kDa PKCa, but was less potent than Bryo. Bryo produced a more rapid loss of 80 kDa PKC a protein and Ca²⁺ and lipid-dependent kinase activity than PMA. Production of nonphosphorylated PKCa contributes to PKC downregulation because nonphosphorylated PKC is known be inactive. Nonphosphorylated PKC may predispose it to ubiquitination and degradation by the proteasome. Increased dephosphorylation of PKC appears to cause the more efficient downregulation of the kinase by Bryo versus PMA.