We evaluated the possibility that distinct proteolytic pathways contribute to the down-regulation of a novel (ε) or conventional (α) isoform of protein kinase C (PKC) in nonimmortalized human fibroblasts. Inhibitors of calpains and other cysteine proteinases, vesicle trafficking, or lysosomal proteolysis did not affect the down-regulation of PKC-α or -ε produced by bryostatin 1 (Bryo). Lactacystin (Lacta) and certain terminal aidehyde tripeptides or tetrapeptides, which selectively inhibit the proteasome, preserved substantial PKC-α and -ε protein from down-regulation by Bryo or phorbol-12-myristate-13-acetate. Lacta preserved active kinase in vivo, as shown by the retention of Bryo-induced autophosphorylated PKC-α. Concomitant with down-regulation, Bryo produced PKC-α and -ε species that were larger than the native proteins (80 and 90 kDa, respectively). Western blot analysis showed that the larger PKC-α species were ubiquitinylated. Treatment with Bryo plus Lacta synergistically increased multiubiquitinylated PKC-α, as expected if Bryo induces ubiquitinylation of PKC-α and Lacta blocks its degradation. Bryo also produced a 76-kDa, nonphosphorylated form of PKC-α and an 86-kDa form of PKC-ε. Phosphatase inhibitors decreased production of 76- and 86-kDa PKC-α and -ε by Bryo and preserved 80- and 90- kDa PKC-α and -ε, respectively. Our results suggest that the down- modulation of PKC-α and -ε occurs principally via the ubiquitin/proteasome pathway. Dephosphorylation seems to predispose PKC to ubiquitinylation.
|Original language||English (US)|
|Number of pages||9|
|State||Published - Mar 1997|
ASJC Scopus subject areas
- Molecular Medicine