Biochemical Journal

Research article

Caspase processing activates atypical protein kinase C ζ by relieving autoinhibition and destabilizes the protein

Lucinda SMITH, Zhi WANG, Jeffrey B. SMITH

Abstract

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.

  • apoptosis
  • atypical protein kinase C
  • caspase
  • lactacystin
  • proteasome
  • tumour necrosis factor (TNF)

Footnotes

  • Abbreviations used: Ac-DEVDal, acetyl-Asp-Glu-Val-Asp-aldehyde; CAT ζ, catalytic domain of PKC ζ; CHX, cycloheximide; DAG, diacylglycerol; DTT, dithiothreitol; MBP, myelin basic protein; PDK-1, phosphoinositide-dependent kinase 1; PKC, protein kinase C; pThr, phosphothreonine; TNFα, tumour necrosis factor α; zIEALal, benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone.