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

Area of Science:

• Biochemistry
• Cell Biology
• Immunology

Background:

• Vitamin E isoforms differentially regulate leucocyte recruitment in inflammation.
• In vitro studies show vitamin E isoforms affect leucocyte migration by modulating VCAM-1 activation of endothelial protein kinase C alpha (PKCα).

Purpose of the Study:

• To investigate whether tocopherols directly regulate cofactor-dependent or oxidative activation of PKCα.
• To elucidate the binding interactions of tocopherols with PKCα domains.

Main Methods:

• Cofactor-dependent and oxidative activation assays of recombinant PKCα.
• Binding studies using NBD-tagged α-tocopherol with full-length PKCα, PKCα domains, and PKCζ.
• Lipid vesicle binding assays with PKCα domains in the presence of phosphatidylserine (PS) and tocopherols.

Main Results:

• γ-tocopherol increased, while α-tocopherol inhibited cofactor-dependent PKCα activation.
• α-tocopherol inhibited oxidative PKCα activation at a 10-fold lower concentration than γ-tocopherol.
• α-tocopherol directly bound to the PKCα-C1a domain, with binding blocked by diacylglycerol and other tocopherols.
• Tocopherols enhanced PKCα-C2 domain binding to PS-containing lipid vesicles.

Conclusions:

• α-tocopherol and γ-tocopherol bind to the diacylglycerol-binding site on PKCα-C1a.
• Tocopherols can modulate PKCα activity by acting as agonists or antagonists, impacting differential regulation.
• These findings provide insights into the molecular mechanisms of vitamin E's role in inflammation and leucocyte regulation.