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 m any PKCs, To investigate the molecular mechanisms involved in the down-regu lation of PKC eta, we expressed the novel PKCs eta and theta and various mu tant forms in baby hamster kidney cells. Upon overexpression, constitutivel y 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 pseudosubstrat e site mutation designed to give an active conformation was dawn-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 lact acystin led to accumulation of PKC eta proteolytic products and potentially ubiquitinated forms, While wild type PKC eta localizes mostly to the deter gent-soluble fraction of the cell, a significant portion of full-length con stitutively active PKC eta and of kinase-dead, active conformation PKC eta were found in the detergent-insoluble fraction. Several proteolytic fragmen ts of constitutively active PKC eta also were found in the detergent insolu ble fraction. These full-length and proteolytic fragments of PKC eta in the detergent-insoluble fraction accumulated further in the presence of protea some inhibitors. These data suggest that active conformation PKC eta accumu lates in the detergent-insoluble compartment, is degraded by proteolysis in the presence of kinase activity, and that the cleavage products undergo fu rther degradation via ubiquitin-mediated degradation in the proteasome.