Activation-dependent degradation of protein kinase C eta.

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 PKC eta, we expressed the novel PKCs eta and θ and various mutant forms in baby hamster kidney cells. Upon overexpression, constitutively active PKC eta, but not wild type or kinase-dead PKC eta, underwent rapid degradation to generate several lower molecular weight polypeptides. When co-expressed with active kinases, kinase-dead PKC eta with a pseudosubstrate site mutation designed to give an active conformation was down-regulated while the wild type PKC eta was not. These results suggest requirements for kinase activity and an active conformation for down-regulation of PKC eta. Treatment with the proteasome inhibitors N-Ac-Leu-Leu-norleucinal and lactacystin led to accumulation of PKC eta proteolytic products and potentially ubiquitinated forms. While wild type PKC eta localizes mostly to the detergent-soluble fraction of the cell, a significant portion of full-length constitutively active PKC eta and of kinase-dead, active conformation PKC eta were found in the detergent-insoluble fraction. Several proteolytic fragments of constitutively active PKC eta also were found in the detergent insoluble fraction. These full-length and proteolytic fragments of PKC eta in the detergent-insoluble fraction accumulated further in the presence of proteasome inhibitors. These data suggest that active conformation PKC eta 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. Oncogene (2000) 19, 4263 - 4272