Vitamin E isoforms directly bind PKCα and differentially regulate activation of PKCα

Vitamin E isoforms have opposing regulatory effects on leucocyte recruitment during inflammation. Furthermore, in vitro, vitamin E isoforms have opposing effects on leucocyte migration across endothelial cells by regulating VCAM (vascular cell-adhesion molecule)-1 activation of endothelial cell PKC alpha (protein kinase C alpha). However, it is not known whether tocopherols directly regulate cofactor-dependent or oxidative activation of PKC alpha. We report in the present paper that cofactor-dependent activation of recombinant PKC alpha was increased by gamma-tocopherol and was inhibited by alpha-tocopherol. Oxidative activation of PKC alpha was inhibited by alpha-tocopherol at a 10-fold lower concentration than gamma-tocopherol. In binding studies, NBD (7-nitrobenz-2-oxa-1,3-diazole)-tagged alpha-tocopherol directly bound to full-length PKC alpha or the PKC alpha-C1a domain, but not PKC zeta. NBD-tagged alpha-tocopherol binding to PKC alpha or the PKC alpha-C1a domain was blocked by diacylglycerol, alpha-tocopherol, gamma-tocopherol and retinol, but not by cholesterol or PS (phosphatidylserine). Tocopherols enhanced PKC alpha-C2 domain binding to PS-containing lipid vesicles. In contrast, the PKC alpha-C2 domain did not bind to lipid vesicles containing tocopherol without PS. The PKC alpha-C1b domain did not bind to vesicles containing tocopherol and PS. In summary, alpha-tocopherol and gamma-tocopherol bind the diacylglycerol-binding site on PKC alpha-C1a and can enhance PKC alpha-C2 binding to PS-containing vesicles. Thus the tocopherols can function as agonists or antagonists for differential regulation of PKC alpha.