Protein Kinase C (PKC) Is Resistant to Long Term Desensitization/Down-regulation by Prolonged Diacylglycerol Stimulation

Sustained activation of PKC is required for long term physiological responses, such as growth arrest and differentiation. However, studies with pharmacological agonists (e.g. phorbol 12-myristate 13-acetate (PMA)) indicate that prolonged stimulation leads to PKC desensitization via dephosphorylation and/or degradation. The current study analyzed effects of chronic stimulation with the physiological agonist diacylglycerol. Repeated addition of 1,2-dioctanoyl-sn-glycerol (DiC(8)) resulted in sustained plasma membrane association of PKC in a pattern comparable with that induced by PMA. However, although PMA potently down-regulated PKC, prolonged activation by DiC(8) failed to engage known desensitization mechanisms, with the enzyme remaining membrane-associated and able to support sustained downstream signaling. DiC(8)-activated PKC did not undergo dephosphorylation, ubiquitination, or internalization, early events in PKC desensitization. Although DiC(8) efficiently down-regulated novel PKCs PKC and PKCE, differences in Ca2+ sensitivity and diacylglycerol affinity were excluded as mediators of the selective resistance of PKC. Roles for Hsp/Hsc70 and Hsp90 were also excluded. PMA, but not DiC(8), targeted PKC to detergent-resistant membranes, and disruption of these domains with cholesterol-binding agents demonstrated a role for differential membrane compartmentalization in selective agonist-induced degradation. Chronic DiC(8) treatment failed to desensitize PKC in several cell types and did not affect PKCI; thus, conventional PKCs appear generally insensitive to desensitization by sustained diacylglycerol stimulation. Consistent with this conclusion, prolonged (several-day) membrane association/activation of PKC is seen in self-renewing epithelium of the intestine, cervix, and skin. PKC deficiency affects gene expression, differentiation, and tumorigenesis in these tissues, highlighting the importance of mechanisms that protect PKC from desensitization in vivo.