Autophosphorylation of Protein Kinase C facilitates its ubiquitination and downregulation

Abstract

Protein kinase C (PKC) is a ubiquitous enzyme involved in a wide variety of cellular functions. One of the factors controlling the maturation, activation, and signaling properties of PKC is phosphorylation. A major step in the generation of a competent, functional enzyme is autophosphorylation at two conserved sites in the carboxyl-terminus. In addition, the enzyme has been show to readily autophosphorylate at additional sites in vitro. The role of these additional autophosphorylations is unknown. Chronic activation of PKC by phorbol ester stimulation ultimately results its degradation and “downregulation”. Here, we examine the role of autophosphorylation at two sites directly 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 (S16ET17D) increase the rate of PKC degradation following phorbol ester stimulation. This phospho-mimic mutant is more rapidly dephosphorylated, ubiquitinated, and partitioned to a greater extent in the detergent-insoluble pellet compared to wildtype PKC. In contrast to its degradation properties, the phospho-mimic was processed similarly to wildtype enzyme and had similar activity. Based on this 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 P01 DK54441 (ACN) and 2T32 GM07752 (CMG).