Treatment of HeLa cells with tumour necrosis factor α (TNFα) induced caspase processing of ectopic PKC (protein kinase C) ζ, 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 ζ. Atypical PKC ι is largely identical with PKC ζ, except for a 60-amino-acid segment that lacks the caspase-processing sites of the ζ isoform. Replacement of this segment of PKC ζ with the corresponding segment of PKC ι prevented caspase processing and activation of the kinase function. Processing of purified recombinant PKC ζ by caspase 3 in vitro markedly increased its kinase activity. Caspase processing activated PKC ζ in vitro or intracellularly without increasing the phosphorylation of Thr410 of PKC ζ, which is required for catalytic competency. The freed kinase domain of PKC ζ had a much shorter half-life than the holoenzyme in transfected HeLa cells and in non-transfected kidney epithelial cells. Treatment with TNF-α 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.

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