title: Caspase processing activates atypical protein kinase C zeta by relieving autoinhibition and destabilizes the protein.
abstract: Treatment of HeLa cells with tumour necrosis factor alpha (TNFalpha) induced caspase processing of ectopic PKC (protein kinase C) zeta, which converted most of the holoenzyme into the freed kinase domain and increased immune-complex kinase activity. The goal of the present study was to determine the basis for the increased kinase activity that is associated with caspase processing of PKC zeta. Atypical PKC iota is largely identical with PKC zeta, except for a 60-amino-acid segment that lacks the caspase-processing sites of the zeta isoform. Replacement of this segment of PKC zeta with the corresponding segment of PKC iota prevented caspase processing and activation of the kinase function. Processing of purified recombinant PKC zeta by caspase 3 in vitro markedly increased its kinase activity. Caspase processing activated PKC zeta in vitro or intracellularly without increasing the phosphorylation of Thr410 of PKC zeta, which is required for catalytic competency. The freed kinase domain of PKC zeta had a much shorter half-life than the holoenzyme in transfected HeLa cells and in non-transfected kidney epithelial cells. Treatment with TNF-alpha shortened the half-life of the kinase domain protein, and proteasome blockade stabilized the protein. Studies of kinase-domain mutants indicate that a lack of negative charge at Thr410 can shorten the half-life of the freed kinase domain. The present findings indicate that the freed kinase domain has substantially higher kinase activity and a much shorter half-life than the holoenzyme because of accelerated degradation by the ubiquitin-proteasome system.
authors.affiliation: Department of Pharmacology and Toxicology, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
authors.forename: Lucinda
authors.initials: L
authors.lastname: Smith
authors.forename: Zhi
authors.initials: Z
authors.lastname: Wang
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D004847:Epithelial Cells / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D013912:Threonine / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D000099221:Protein Kinase C zeta
chemical_list: D053148:Caspase 3; D020169:Caspases; D003513:Cycloheximide; D007527:Isoenzymes; D011493:Protein Kinase C; D011994:Recombinant Proteins; D013912:Threonine; D014409:Tumor Necrosis Factor-alpha; D000099221:Protein Kinase C zeta; C505407:CASP3 protein, human