Protein kinase C, a multigene family of phospholipiddependent and diacylglycerol-activated Ser/Thr protein kinases, is a key component in many signal transduction pathways. The kinase activity was thought to be essential for a plethora of biological processes attributed to these enzymes. Here we show that at least one protein kinase C function, the induction of apoptosis by protein kinase C␦, is independent of the kinase activity. Stimulation of green fluorescent protein-protein kinase C␦ fusion protein with phorbol ester or diacylglycerol led to its redistribution within seconds after the stimulus. Membrane blebbing, an early hallmark of apoptosis, was visible as early as 20 min after stimulation, and nuclear condensation was visible after 3-5 h. Apoptosis could be inhibited by expression of Bcl-2 but not by specific protein kinase C inhibitors. In addition, a kinase-negative mutant of protein kinase C␦ also induced apoptosis to the same extent as the wild type enzyme. Apoptosis was confined to the protein kinase C␦-overexpressing cells. Stimulation of overexpressed protein kinase C⑀ did not result in increased apoptosis. Our results indicate that distinct protein kinase C isozymes induce apoptosis in vascular smooth muscle cells. More importantly, they show that some protein kinase C effector functions are independent of the catalytic activity.

The catalytic domain of overexpressed protein kinase C (PKC)-δ mediates phorbol 12-myristate 13-acetate (PMA)-induced differentiation or apoptosis in appropriate model cell lines. To define the portions of the catalytic domain that are critical for these isozyme-specific functions, we constructed reciprocal chimeras, PKC-δ/εV5 and -ε/δV5, by swapping the V5 domains of PKC-δ and -ε. PKC-δ/εV5 failed to mediate PMA-induced differentiation of 32D cells, showing the essential nature of the V5 domain for PKC-δ's functionality. The other chimera, PKC-ε/δV5, endowed inactive PKC-ε with nearly all PKC-δ's apoptotic ability, confirming the importance of PKC-δ in this function. Green fluorescent protein (GFP)-tagged PKC-δV5 and -ε/δV5 in A7r5 cells showed substantial basal nuclear localization, while GFP-tagged PKC-ε and -δ/εV5 showed significantly less, indicating that the V5 region of PKC-δ contains determinants critical to its nuclear distribution. PKC-ε/δV5-GFP showed much slower kinetics of translocation to membranes in response to PMA than parental PKC-ε, implicating the PKC-εV5 domain in membrane targeting. Thus, the V5 domain is critical in several of the isozyme-specific functions of PKC-δ and -ε.

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