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Activation-dependent degradation of protein kinase C eta
B S Kang1, O G French, J J Sando
1Department of Microbiology, Beirne B. Carter Center for Immunology Research, University of Virginia Health Sciences Center, Charlottesville, Virginia, VA 22908, USA.
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 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
Insights
Prolonged activation of protein kinase C eta (PKC eta) requires kinase activity and an active conformation for its degradation. This process involves proteolysis and ubiquitin-mediated proteasome degradation, particularly for PKC eta in detergent-insoluble cellular fractions.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Prolonged activation of protein kinase Cs (PKCs) via tumor promoters leads to PKC downregulation.
- Understanding the molecular mechanisms of PKC eta downregulation is crucial.
Purpose of the Study:
- To investigate the molecular mechanisms underlying the downregulation of protein kinase C eta (PKC eta).
- To determine the roles of kinase activity and active conformation in PKC eta degradation.
Main Methods:
- Overexpression of wild type, constitutively active, and kinase-dead mutant forms of PKC eta and PKC theta in baby hamster kidney cells.
- Treatment with proteasome inhibitors (N-Ac-Leu-Leu-norleucinal and lactacystin).
- Fractionation of cell lysates into detergent-soluble and detergent-insoluble components.
Main Results:
- Constitutively active PKC eta, but not wild type or kinase-dead forms, rapidly degraded upon overexpression.
- Kinase-dead PKC eta with an active conformation mutation underwent downregulation, unlike wild type PKC eta.
- Proteasome inhibitors caused accumulation of PKC eta proteolytic and ubiquitinated products.
- Active conformation PKC eta and its fragments accumulated in the detergent-insoluble fraction, with further accumulation in the presence of proteasome inhibitors.
Conclusions:
- PKC eta downregulation necessitates kinase activity and an active conformation.
- PKC eta in an active conformation localizes to the detergent-insoluble compartment.
- Degradation involves proteolysis and subsequent ubiquitin-mediated proteasome degradation of PKC eta and its fragments.