Modulation of Phosphorylation of Tocopherol and Phosphatidylinositol by hTAP1/SEC14L2-Mediated Lipid Exchange

Abstract

The vitamin E derivative, alpha-tocopheryl phosphate (αTP), is detectable in cultured cells, plasma and tissues in small amounts, suggesting the existence of enzyme(s) with α-tocopherol (αT) kinase activity. Here, we characterize the production of αTP from αT and [γ-32P]-ATP in primary human coronary artery smooth muscle cells (HCA-SMC) using separation by thin layer chromatography (TLC) and subsequent analysis by Ultra Performance Liquid Chromatography (UPLC). In addition to αT, although to a lower amount, also γT is phosphorylated. In THP-1 monocytes, γTP inhibits cell proliferation and reduces CD36 scavenger receptor expression more potently than αTP. Both αTP and γTP activate the promoter of the human vascular endothelial growth factor (VEGF) gene with similar potency, whereas αT and γT had no significant effect. The recombinant human tocopherol associated protein 1 (hTAP1, hSEC14L2) binds both αT and αTP and stimulates phosphorylation of αT possibly by facilitating its transport and presentation to a putative αT kinase. Recombinant hTAP1 reduces the in vitro activity of the phosphatidylinositol-3-kinase gamma (PI3Kγ) indicating the formation of a stalled/inactive hTAP1/PI3Kγ heterodimer. The addition of αT, βT, γT, δT or αTP differentially stimulates PI3Kγ, suggesting facilitated egress of sequestered PI from hTAP1 to the enzyme. It is suggested that the continuous competitive exchange of different lipophilic ligands in hTAPs with cell enzymes and membranes may be a way to make these lipophiles more accessible as substrates for enzymes and as components of specific membrane domains.

Modulation of Signal Transduction and Gene Expression by Vitamin E via PI3Kγ/PKB and hTAP1/SEC14L2-Mediated Lipid Exchange

“…A similar exchange of PC or vitamin E with PI has been observed for the three hTAPs (4,5). In addition, aT and more so phosphorylated aT (aTP) stimulate PI3Kgamma (PI3Kg) activity by facilitating hTAP1-mediated PI exchange and presentation to the enzyme and/or by dissociation of an inactive heterodimer (5). Activation of the PI3Kg/protein kinase B (PKB) signal transduction pathway by aTP induces the expression of a number of genes such as the vascular endothelial growth factor (VEGF) which is important for angiogenesis, vasculogenesis and cell survival (6,7).…”

“…Mutations in aTTP that impair PIP2 binding lead to ataxia with vitamin E deficiency (AVED) as result of insufficient enrichment of vitamin E by the liver (1, 3). A similar exchange of PC or vitamin E with PI has been observed for the three hTAPs (4,5). In addition, aT and more so phosphorylated aT (aTP) stimulate PI3Kgamma (PI3Kg) activity by facilitating hTAP1-mediated PI exchange and presentation to the enzyme and/or by dissociation of an inactive heterodimer (5).…”

Vitamin E: A Role in Signal Transduction

“…Another lipid kinase that is affected by vitamin E is PI3Kγ (113,279). Regulation of PI3Kγ activity by vitamin E occurs in an analogue-dependent manner and is modulated by tocopherolassociated proteins 1, 2, and 3 [TAP1, TAP2, and TAP3, also known as sec14-like (SEC14L) proteins 2, 3, and 4, respectively] (275,279).…”

“…Regulation of PI3Kγ activity by vitamin E occurs in an analogue-dependent manner and is modulated by tocopherolassociated proteins 1, 2, and 3 [TAP1, TAP2, and TAP3, also known as sec14-like (SEC14L) proteins 2, 3, and 4, respectively] (275,279). These proteins associate with and inhibit PI3Kγ activity in vitro (113) and become activated by vitamin E-mediated lipid exchange and/or dissociation of the inactive complex (275,279). Likewise, the mouse TAP1 protein competes with p85 for binding to PI3Kα, with consequent inhibition of PKB activity and prostate tumor formation (169); however, in this case αT was not able to further affect the inhibitory effect of TAP1 on PI3Kα and PKB.…”

“…The PKB activation and inhibition process offers several molecular targets that could be influenced by vitamin E (210,257). PKB translocation to the plasma membrane occurs by means of the pleckstrin homology domain, which binds PI34P and PI345P, produced by activated PI3K, an enzyme that is known to be modulated by vitamin E (113,275,279). A similar domain is found in PDK1, a kinase that phosphorylates Thr308, leading to partial activation of PKB.…”