title: Vitamin E isoforms directly bind PKCα and differentially regulate activation of PKCα.
abstract: 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 PKCα (protein kinase Cα). However, it is not known whether tocopherols directly regulate cofactor-dependent or oxidative activation of PKCα. We report in the present paper that cofactor-dependent activation of recombinant PKCα was increased by γ-tocopherol and was inhibited by α-tocopherol. Oxidative activation of PKCα was inhibited by α-tocopherol at a 10-fold lower concentration than γ-tocopherol. In binding studies, NBD (7-nitrobenz-2-oxa-1,3-diazole)-tagged α-tocopherol directly bound to full-length PKCα or the PKCα-C1a domain, but not PKCζ. NBD-tagged α-tocopherol binding to PKCα or the PKCα-C1a domain was blocked by diacylglycerol, α-tocopherol, γ-tocopherol and retinol, but not by cholesterol or PS (phosphatidylserine). Tocopherols enhanced PKCα-C2 domain binding to PS-containing lipid vesicles. In contrast, the PKCα-C2 domain did not bind to lipid vesicles containing tocopherol without PS. The PKCα-C1b domain did not bind to vesicles containing tocopherol and PS. In summary, α-tocopherol and γ-tocopherol bind the diacylglycerol-binding site on PKCα-C1a and can enhance PKCα-C2 binding to PS-containing vesicles. Thus the tocopherols can function as agonists or antagonists for differential regulation of PKCα.
authors.affiliation: Allergy-Immunology Division, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
authors.forename: Christine A
authors.initials: CA
authors.lastname: McCary
authors.forename: Youngdae
authors.initials: Y
authors.lastname: Yoon
authors.forename: Candace
authors.initials: C
authors.lastname: Panagabko
authors.forename: Wonhwa
authors.initials: W
authors.lastname: Cho
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Atkinson
authors.forename: Joan M
authors.initials: JM
authors.lastname: Cook-Mills
mesh_terms: D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005456:Fluorescent Dyes; D010084:Oxidation-Reduction; D011485:Protein Binding; D051571:Protein Kinase C-alpha / Q000737:chemistry* / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D024502:alpha-Tocopherol / Q000737:chemistry / Q000378:metabolism*; D024504:gamma-Tocopherol / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D024504:gamma-Tocopherol; D051571:Protein Kinase C-alpha; D024502:alpha-Tocopherol