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 &theta; 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
Kang
B S
BS
Department of Microbiology, Beirne B. Carter Center for Immunology Research, University of Virginia Health Sciences Center, Charlottesville, Virginia, VA 22908, USA.
French
O G
OG
Sando
J J
JJ
Hahn
C S
CS
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line / Q000201:enzymology; D002642:Chick Embryo; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000187:drug effects; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007668:Kidney; D007976:Leupeptins / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D008647:Mesocricetus; D051379:Mice; D009097:Multienzyme Complexes / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000074121:Protein Kinase C-theta; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012845:Sindbis Virus / Q000235:genetics; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000378:metabolism
D003902:Detergents; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D007976:Leupeptins; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; C063261:acetylleucyl-leucyl-norleucinal; C067713:lactacystin; C081975:protein kinase C eta; C076233:PRKCQ protein, human; C489593:Prkcq protein, mouse; C489591:Prkcq protein, rat; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine