Autophosphorylation of Protein Kinase C Facilitates its Ubiquitination and Down‐regulation

Protein kinase C (PKC) is a Ser/Thr kinase that tranduces signals generated from the second messengers, diacylglycerol and calcium, the products of receptor‐mediated lipid hydrolysis. PKC has three modes of regulation: phosphorylation, cofactor binding, and localization. Phosphorylation of PKC regulates its maturation, activation, and signaling properties. Autophosphorylation at two conserved sites in the carboxyl‐terminus of PKC is a major step in the generation of catalytically competent enzyme. PKC has been show to readily autophosphorylate at additional sites in vitro , and their physiological role is unknown. Chronic activation of PKC that occurs with phorbol esters, functional analogues of diacylglycerol, results in the degradation and “downregulation” of the enzyme. Here, we examine the role of autophosphorylation at two sites preceding the autoinhibitory pseudosubstrate sequence in the phorbol ester‐mediated downregulation of PKC βII. We find that mutating the in vitro ‐identified autophosphorylation residues, Ser16 and Thr17, to generate a phospho‐mimic (S16E/T17D) increases the rate of PKC degradation following phorbol ester stimulation. This mutant was more rapidly dephosphorylated and ubiquitinated in comparison to wildtype PKC. In contrast to its altered degradation, the phospho‐mimic was processed by phosphorylation similarly to wildtype and had similar activity. Generation of a phospho‐specific antibody to Thr17 indicated that PKC autophosphorylates in vitro as well as in vivo in response to phorbol esters in both an overexpression system as well as with endogenous PKC in the K562 lymphona cell line. Based on these data, we propose that PKC is additionally autophosphorylated following activation by phorbol esters, an event that promotes the downregulation of the enzyme. This work was supported by NIHGM43154.