Ubiquitination of protein kinase C-α and degradation by the proteasome

Bryostatins and phorbol esters acutely activate and subsequently down- regulate protein kinase C (PKC) by inducing its proteolysis via an unknown pathway. Here we show that treatment of renal epithelial cells with bryostatin 1 (Bryo) produced novel PKC-α species, which were larger than the native protein (80 kDa). The >80 kDa PKC-α species contained Ubi as indicated by immunostaining and accumulated in the presence of lactacystin, a selective inhibitor of proteolysis by the proteasome. In vitro experiments with ¹²⁵I-ubiquitin and membranes from Bryo-treated cells showed that PKC- α became ubiquitinated by a reaction that depended on ATP and a cytosolic fraction. Lactacystin or a peptidyl aldehyde, Bz-Gly-Leu-Ala-leucinal, which inhibits certain proteinase activities of the proteasome, inhibited Bryo- evoked disappearance of PKC-α protein from the cells. Lacta preserved Bryo- induced ³²P-labeled PKC-α indicating that the proteasome inhibitor spared activated enzyme from down-regulation in vivo. These findings show that Bryo induces the degradation of PKC-α by the ubiquitin-proteasome complex.