Bax activation and induction of apoptosis in human keratinocytes by the protein kinase C δ catalytic domain

Abstract

The constitutively active catalytic domain of protein kinase C (PKC)δ is an apoptotic effector generated by caspase-3 cleavage of full-length PKCδ in response to a wide variety of apoptotic stimuli, including UV radiation. The PKCδ catalytic domain induces apoptosis when ectopically expressed, however, the mechanism of apoptosis induction is unclear. We constructed a chimeric protein encoding the PKCδ catalytic domain fused to a mutated estrogen receptor ligand-binding domain in order to selectively activate the PKCδ catalytic domain. The enzymatic activity of the PKCδ catalytic domain fusion protein was induced in human keratinocytes treated with 4-hydroxytamoxifen, and its activation triggered loss of mitochondrial membrane potential and apoptosis. The apoptosis was associated with release of cytochrome c from the mitochondria and caspase activation, and was blocked by caspase inhibitors and the anti-apoptotic proteins Bcl-2, and Bcl-xL, suggesting a role for mitochondrial pore formation. Consistent with this, the activated PKCδ catalytic domain triggered the redistribution and activation of Bax, a Bcl-2 family protein that can directly induce cytochrome c release. In summary, despite being an apoptotic effector activated late in the apoptotic cascade, PKCδ also activates upstream components of the death effector pathway to insure the demise of cells committed to apoptosis.