Proteolytic Activation of Protein Kinase C-ε by Caspase-mediated Processing and Transduction of Antiapoptotic Signals

Abstract

Several novel protein kinase C (PKC) isozymes have been identified as substrates for caspase-3. We have previously shown that novel PKC⑀ is cleaved during apoptosis in MCF-7 cells that lack any functional caspase-3. In the present study, we show that in vitro-translated PKC⑀ is processed by human recombinant caspase-3, -7, and -9. Tumor necrosis factor-␣ (TNF) triggered processing of PKC⑀ to a 43-kDa carboxyl-terminal fragment, and cell-permeable caspase inhibitors prevented TNF-induced processing of PKC⑀ in MCF-7 cells. PKC⑀ was cleaved primarily at the SSPD2G site to generate two fragments with an approximate molecular mass of 43 kDa. It was also cleaved at the DDVD2C site to generate two fragments with molecular masses of 52 and 35 kDa. Treatment of MCF-7 cells with TNF resulted in the activation of PKC⑀, and mutation at the SSPD2G (D383A) site inhibited proteolytic activation of PKC⑀. Overexpression of wild-type but not dominant-negative PKC⑀ in MCF-7 cells delayed TNF-induced apoptosis, and mutation at the D383A site prevented antiapoptotic activity of PKC⑀. These results suggest that cleavage of PKC⑀ by caspase-7 at the SSPD2G site results in the activation of PKC⑀. Furthermore, activation of PKC⑀ was associated with its antiapoptotic function.

Downregulation of Bid is associated with PKCɛ-mediated TRAIL resistance

“…We have previously shown that PKCe protects cells against TNF-induced cell death in MCF-7 cells. 30 We determined whether PKCe overexpression would exert a protective effect against TRAIL-induced apoptosis. MCF-7 cells overexpressing PKCe (MCF-7/PKCe) or vectortransfected control cells (MCF-7/Neo) were treated with different concentrations of TRAIL, and cell viability was determined by the 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay.…”

“…MCF-7/Neo, MCF-7/PKCe and MCF-7/DN-PKCe cells were generated as described previously. 30 MCF-7/PKCe cells were co-transfected with either 2 mg of empty vector or Bid-pcDNA3.1 together with 2 mg SV40-b-gal plasmids using Fugene HD reagent (Roche Biomedicals) according to the manufacturer's instructions. Transfection efficiency was determined by b-galactosidase expression.…”

“…27,28 There is also considerable evidence that PKCe acts as an antiapoptotic protein. 23,29,30 Activation of PKC was shown to protect against TRAILinduced apoptosis in several cell lines, including cervical cancer, multiple myeloma, glioma and breast cancer. 19,20,22,23 There are conflicting reports as to the mechanisms by which PKC exerts its protective effect.…”

“…30 In this study, we have examined the mechanism(s) of the antiapoptotic effect of PKCe in TRAIL-induced cell death. We report for the first time that PKCe inhibited TRAIL-mediated apoptosis by simultaneously increasing Bcl-2 and decreasing Bid expression in MCF-7 cells.…”

Functions of epidermal growth factor receptor in cisplatin response of thyroid cells

“…PKC-ε acts a protective role against oxidative stress-induced injury in diverse cell types [49], for example, inhibiting mitochondrial permeability transition pore opening, which prevents apoptosis [50]. Over cleavage prevented its antiapoptotic signal [51]. A down-regulation of PKC- ε by chronic exposure to a phorbol ester was associated with resistance to drug-induced apoptosis in human prostatic carcinoma cells [52].…”