Dephosphorylation of activated protein kinase C contributes to downregulation by bryostatin

Abstract

We show that bryostatin 1 (Bryo) rapidly produces an inactive, incompetent 76-kDa form of protein kinase C-alpha (PKC-alpha) in the LLC-MK2 line of renal epithelial cells. Bryo, like phorbol 12-myristate 13-acetate (PMA), acutely activated PKC, as indicated by autophosphorylation and translocation of PKC-alpha, the predominant PMA-sensitive isoform expressed by the cells. Bryo concomitantly increased the 32P labeling of 80-kDa PKC-alpha by autophosphorylation and produced a 76-kDa form of PKC-alpha that lacked detectable 32P. The 76-kDa form was in the particulate rather than the cytosolic fraction, which suggests that it was produced from activated kinase. Alkaline phosphatase treatment of immunoprecipitated PKC-alpha converted the 80-kDa form to 76 kDa, but it had no effect on the mobility of the 76-kDa form, suggesting that it was not phosphorylated. Pulse-chase labeling of PKC-alpha with [35S]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 PKC-alpha by Bryo. PMA also produced 76-kDa PKC-alpha but was less potent and efficacious than Bryo. Bryo produced a more rapid loss of 80-kDa PKC-alpha protein and total Ca(2+)- and phospholipid-dependent PKC activity than PMA. The 76-kDa form is inactive and incompetent because it lacked detectable 32P under conditions that strongly autophosphorylated the 80-kDa form. We suggest that dephosphorylation predisposes PKC to proteolysis, and greater production of the 76-kDa form explains the more efficient downregulation of the kinase by Bryo vs. PMA.

Bryostatin-1 Enhances the Maturation and Antigen-Presenting Ability of Murine and Human Dendritic Cells

“…A previous study showed that Bryo-1 substantially regulated PKC-␣, -␦, and -⑀ in NIH mouse fibroblast cells, whereas PMA did not distinguish between these isozymes (40). Other studies showed that Bryo-1 down-regulates PKC-␣ in epithelial cells and PKC-␣ and -␤ in human T cells (41,16). Bryo-1 also induces a unique biphasic response in PKC-␦, depending on concentrations leading to its degradation in NIH mouse fibroblast cells, B16F10 melanocytes, and HeLa cells (40,42,43).…”

Heat Shock Proteins Regulate Activation-induced Proteasomal Degradation of the Mature Phosphorylated Form of Protein Kinase C

“…Extensive evidence points to proteolytic degradation as a major mechanism for desensitization of PKC signaling following prolonged activation. A widely accepted desensitization pathway involves proteasomal degradation of dephosphorylated protein that has accumulated in an intracellular detergent-insoluble compartment, with priming site dephosphorylation being the rate-limiting step for degradation (4,11,14,26,30,31,36,38,40). Although our findings confirm the existence of this pathway for endogenous PKC␣, the data point to the fully phosphorylated form of the activated protein as a major target for degradation in multiple cell types.…”

The Phosphatase PHLPP Controls the Cellular Levels of Protein Kinase C

“…However, because the endogenous activators of PKC, diacylglycerol, and Ca 2ϩ are only transiently elevated following agonist stimulation, only a small population of PKC may undergo dephosphorylation at a given time. The accumulation of dephosphorylated PKC is more readily observed with long lasting pharmacological treatments such as those from phorbol esters or the anti-cancer drug bryostatin (32).…”