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)delta is an apoptotic effector generated by caspase-3 cleavage of full-length PKCdelta in response to a wide variety of apoptotic stimuli, including UV radiation. The PKCdelta catalytic domain induces apoptosis when ectopically expressed, however, the mechanism of apoptosis induction is unclear. We constructed a chimeric protein encoding the PKCdelta catalytic domain fused to a mutated estrogen receptor ligand-binding domain in order to selectively activate the PKCdelta catalytic domain. The enzymatic activity of the PKCdelta 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-x(L), suggesting a role for mitochondrial pore formation. Consistent with this, the activated PKCdelta 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, PKCdelta also activates upstream components of the death effector pathway to insure the demise of cells committed to apoptosis.

Protein kinase C delta inhibits Caco-2 cell proliferation by selective changes in cell cycle and cell death regulators

“…These results are in agreement with previous studies that suggested this PKC isoform is a positive regulator of apoptosis in other colon cancer cells (Weller et al, 1999;McMillan et al, 2003;Perletti et al, 2005). Similar effects were recently reported by Sitailo et al (2004) in human keratinocytes, in which PKC-d-induced apoptosis was mediated by Bax upregulation.…”

The Protein Kinase Cδ Catalytic Fragment Is Critical for Maintenance of the G2/M DNA Damage Checkpoint

“…These constructs were subcloned in XhoI/NotI sites of the LZRS-Linker retroviral expression vector for infection of cultured KCs and mouse embryonic fibroblasts (MEFs). LZRS-PKC␦-cat-FLAG and the 4-hydroxytamoxifen-activatable LZRS-PKC␦-cat-estrogen receptor (ER) were previously described (18). Retroviral supernatant was generated by calcium phosphate-mediated transfection of Phoenix-Ampho packaging cells as described previously (30).…”

“…Spontaneously immortalized MEFs from wild-type or PKC␦ null mice were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum and were kindly provided by Dr. Anning Lin (University of Chicago). PKC␦-cat-ER was activated by treatment with 10 M 4-hydroxytamoxifen (Alexis Biochemicals) (18). In all experiments using PKC␦-cat-ER, control (Linker) transduced cells were also treated with 4-hydroxytamoxifen.…”

The Localization of Protein Kinase Cδ in Different Subcellular Sites Affects Its Proapoptotic and Antiapoptotic Functions and the Activation of Distinct Downstream Signaling Pathways

“…Different apoptotic stimuli induce caspase-dependent cleavage of PKCy, which result in the generation of a constitutively active catalytic fragment (10,11). The cleavage of PKCy has been implicated in its apoptotic function (12,13), and the expression of the catalytic fragment has been shown to induce cell apoptosis in different cellular systems (14,15). The apoptotic function of PKCy has been associated with the activation of multiple signaling proteins, such as c-Jun NH 2 -terminal kinase (JNK; ref.…”