Activation-dependent degradation of protein kinase Cη

Abstract

Prolonged activation of protein kinase Cs (PKCs) by long-term treatment of cells with phorbol ester tumor promoters down-regulates the expression of many PKCs. To investigate the molecular mechanisms involved in the down-regulation of PKCη, we expressed the novel PKCs η and θ and various mutant forms in baby hamster kidney cells. Upon overexpression, constitutively active PKCη, but not wild type or kinase-dead PKCη, underwent rapid degradation to generate several lower molecular weight polypeptides. When co-expressed with active kinases, kinase-dead PKCη with a pseudosubstrate site mutation designed to give an active conformation was down-regulated while the wild type PKCη was not. These results suggest requirements for kinase activity and an active conformation for down-regulation of PKCη. Treatment with the proteasome inhibitors N-Ac-Leu-Leu-norleucinal and lactacystin led to accumulation of PKCη proteolytic products and potentially ubiquitinated forms. While wild type PKCη localizes mostly to the detergent-soluble fraction of the cell, a significant portion of full-length constitutively active PKCη and of kinase-dead, active conformation PKCη were found in the detergent-insoluble fraction. Several proteolytic fragments of constitutively active PKCη also were found in the detergent insoluble fraction. These full-length and proteolytic fragments of PKCη in the detergent-insoluble fraction accumulated further in the presence of proteasome inhibitors. These data suggest that active conformation PKCη accumulates in the detergent-insoluble compartment, is degraded by proteolysis in the presence of kinase activity, and that the cleavage products undergo further degradation via ubiquitin-mediated degradation in the proteasome.