In these studies, we demonstrate that α-tocopherol and γ-tocopherol directly modulate cofactor-dependent activation of rPKCα and oxidative-activation of rPKCα. This regulation occurs through direct binding of tocopherol to PKCα’s C1a domain and through enhancement of PKCα binding to PS in tocopherol-containing lipid layers. These innovative studies are the first to demonstrate that tocopherols directly bind and modulate PKCα activity.
The PKCα-C1a regulatory domain contains a high-affinity binding site for DAG and retinol [33
]. Cofactor binding to PKCα-C1a is influenced by cofactor fatty acid chain length and saturation and by the hydroxyl group donation of a hydrogen to a recipient atom in the PKCα-C1a domain [23
]. We found that tocopherols, which have a reactive hydroxyl group on the chromanol head and an unsaturated lipid tail, compete with DAG binding to the PKCα-C1a domain. We also report that the binding of tocopherols regulate PKCα activity. γ-tocopherol elevates cofactor-dependent PKCα activity and α-tocopherol inhibits cofactor-dependent PKCα activity. Furthermore, 1 μM α-tocopherol blocks this enhancing effect of 1 μM γ-tocopherol. Therefore, γ-tocopherol serves as an agonist and α-tocopherol serves as an antagonist of cofactor-dependent PKCα activity. Without PS, γ-tocopherol does not increase the low PKCα activity (data not shown), suggesting that PS is necessary for γ-tocopherol enhancement of cofactor-dependent PKCα activity. At high PS concentrations (60 μg/ml) without tocopherol, PKCα activity is elevated to the level of PKCα activity with tocopherol and 30 μg/ml PS, suggesting that high PS induces maximal activation of PKCα activity without further agonist regulation through the PKCα-C1a domain.
Tocopherols also function as antioxidants. We report that both α-tocopherol (0.01 μM) and γ-tocopherol (0.1 μM) inhibited oxidative activation of rPKCα suggesting an antioxidant function for tocopherols. However, α-tocopherol significantly decreased peroxide activation of PKCα at a 10-fold lower dose compared to γ-tocopherol, even though α-tocopherol and γ-tocopherol have roughly equal anti-oxidant ability towards lipids in solution [38
] and equal ability to bind PKCα (). Therefore, tocopherol isoforms differ in their antioxidant capacity towards PKCα.
PKC activity is also reported to positively correlate with membrane bilayer curvature, nonbilayer phases, and dehydration of the membrane by DAGs in the presence of calcium [41
]. Using cell-free systems, it has been reported that the DAG polar head group spacing and degree of acylated chain saturation contribute to PKC activation [37
]. In our studies, α-tocopherol, at 5 fold lower concentrations than γ-tocopherol enhanced PKCα-C2 domain interaction with PS-containing lipid surfaces without direct tocopherol interaction with the PKCα-C2 domain. This may be consistent with α-tocopherol’s significantly greater partitioning than γ-tocopherol into polyunsaturated lipid-rich domains for differential regulation of membrane structure [44
]. Since it is reported that the PKCα-C2 domain binds to membranes and then, subsequently, the PKCα-C1a domain associates with DAG [27
], it suggests that tocopherols influence the association of the PKCα-C2 domain with PS in the membrane and then tocopherols in the membrane compete with membrane DAG for binding to the PKCα-C1a domain. Thus, α-tocopherol and γ-tocopherol directly bind the PKCα-C1a domain, regulate cofactor-dependent activation of PKCα, regulate oxidative activation of PKCα, and regulate recruitment of PKCα to lipid membranes This suggests that antioxidant and non-antioxidant effects of these tocopherols contribute to the overall regulation of PKCα activity in cells and tissues.
Tocopherols have been reported to modulate PKCα activation in cells [16
]. In cells, PKCα is recruited to the cell membrane where it interacts with PS and DAG [18
]. PKCα is also transiently activated by oxidation [10
]. We have previously reported that α-tocopherol pre-treatment of endothelial cells inhibits VCAM-1-induced oxidative activation of PKCα [6
]. However, during VCAM-1 activation of PKCα, in addition to oxidative activation of PKCα, there is also generation of calcium [26
] and consumption of the PKCα cofactor diacylglycerol [10
], suggesting a contribution of both oxidative activation of PKCα and cofactor-dependent activation of PKCα during VCAM-1 signaling in endothelial cells. Therefore, α-tocopherol may inhibit VCAM-1 signaling by functioning both as an antioxidant and as an antagonist of PKCα. In contrast, γ-tocopherol, which is at 1/10 the tissue concentration of α-tocopherol, elevates VCAM-1 activation of PKCα and ablates the inhibitory effects of α-tocopherol on VCAM-1 activation of PKCα in endothelial cells [6
]. Since in tissues, γ-tocopherol is at 1/10 the concentration of α-tocopherol [6
], but we report here that 10 times more γ-tocopherol than α-tocopherol was required to have equal antioxidant capacity towards PKCα, it suggest that, in cells γ-tocopherol has much lower total antioxidant capacity towards PKCα than α-tocopherol. Therefore, nonantioxidant functions for γ-tocopherol are consistent with the potent γ-tocopherol enhancement of VCAM-1 activation of PKCα in cells. This enhancing effect of γ-tocopherol in cells may occur through γ-tocopherol’s direct co-factor-dependent agonist activation PKCα and/or γ-tocopherol’s enhancement of PKCα recruitment to PS-containing membranes as observed in our studies in this report. An enhancing effect of γ-tocopherol on cofactor-dependent PKCα activity is consistent with a contribution of cofactor-dependent (calcium and DAG) activation of PKCα during VCAM-1 signaling [10
]. In vivo, the anti-inflammatory effect of α-tocopherol and pro-inflammatory effect of γ-tocopherol on leukocyte recruitment [6
] is the sum of tocopherol isoform antioxidant and agonist/antagonist functions.
In summary, α-tocopherol inhibits and γ-tocopherol elevates PKCα activity in the presence of PS. In contrast, both α-tocopherol and γ-tocopherol inhibit PS-independent oxidative activation of PKCα, although α-tocopherol significantly inhibits this oxidative PKCα activation at one-tenth the concentration required for γ-tocopherol inhibition. Alpha-tocopherol and γ-tocopherol modulate PKCα activity by enhancing association of the PKCα-C2 domain to PS-containing lipid layers and the tocopherols directly bind to the PKCα-C1a domain. Thus, tocopherols can function as antioxidants and function as PKCα agonists or antagonists for the regulation of PKCα activity in cells.