Production of nonphosphorylated, ubiquitinated protein kinase c (pkc) from autophosphorylated enzyme in human fibroblasts

We show that bryostatin 1 (Bryo) or phorbol myristate (PMA) acutely activates and then downregulates PKC by inducing its degradation by the ubiquitin-proteasome pathway. Incubation of human skin fibroblasts with 50 nM Bryo or 0. l UM PMA almost completely downregulated PKCa protein in 20 or 40 h, respectively. Lactacystin (Lacta), which specifically blocks proteolysis mediated by the 26S proteasome, strongly preserved PKCa protein and Ca- plus lipid-dependent kinase activity, which was assayed after partial purification of PKC. Incubation with Bryo plus Lacta produced a ladder of PKCa bands that were ubiquitinated as shown by western analysis. A peptidyl aldehyde (Bz-G-L-A-L-al), which specifically inhibits proteolytic activities of the proteasome, preserved PKCa protein and activity from downregulation by Bryo or PMA, similarly to Lacta. The corresponding peptidyl alcohol, which does not inhibit the proteasome, had no effect on Bryo- or PMA-induced loss of PKCa protein and PKC activity. Lacta preserved 32p-iabeled PKCa that was produced by Bryo in [32P]orthophosphate labeled cells. Interestingly the ubiquitinated PKCa bands lacked detectable 32p even though they were derived from highly radioactive 32PPKCa. Production of non-phosphorylated PKC from autophosphorylated active enzyme appears to be a prerequisite for ubiquitination because the ladder of ubiquitinated PKC bands lacked detectable 32P even though they were produced from highly 32P-labeled PKCa. The present findings support the idea that dephosphorylation predisposes PKC to ubiquitination which targets it to the proteasome. Lee et al. (this meeting) show that Bryo induces PKCa degradation by the ubiquitin-proteasome system in epithelial cells.