bryostatin-1 and leucinal

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-alpha species, which were larger than the native protein (80 kDa). The >80 kDa PKC-alpha 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-alpha 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-alpha protein from the cells. Lacta preserved Bryo-induced 32P-labeled PKC-alpha indicating that the proteasome inhibitor spared activated enzyme from down-regulation in vivo. These findings show that Bryo induces the degradation of PKC-alpha by the ubiquitin-proteasome complex.

Topics: Acetylcysteine; Adenosine Triphosphate; Amino Acid Sequence; Animals; Bryostatins; Cells, Cultured; Cysteine Endopeptidases; Cytosol; Down-Regulation; Hydrolysis; Isoenzymes; Lactones; Leucine; Macaca mulatta; Macrolides; Molecular Sequence Data; Multienzyme Complexes; Proteasome Endopeptidase Complex; Protein Kinase C; Protein Kinase C-alpha; Ubiquitins