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Characterization of tocopherol kinase activity in primary human coronary artery smooth muscle cells
Author(s) -
Zingg JeanMarc,
Meydani Mohsen,
Azzi Angelo
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1019.1
Subject(s) - phosphatidylinositol , kinase , chemistry , biochemistry , recombinant dna , in vitro , pi , enzyme , microbiology and biotechnology , biology , gene
The vitamin E derivative, alpha‐tocopheryl phosphate (αTP), is detectable in tissues in small quantity (30–300 nM) suggesting the existence of enzyme(s) with α‐tocopherol (αT) kinase activity. Here, we have characterized the production of αTP from αT and [γ‐ 32 P]‐ATP in primary human coronary artery smooth muscle cells (HCA‐SMC) using thin layer chromatography (TLC) followed by ultra performance liquid chromatography (UPLC). In addition to αT, γT is also phosphorylated. γTP inhibits proliferation of THP‐1 cells and reduces CD36 expression more potently than αTP (10 μM). The recombinant human tocopherol associated protein (TAP1/SEC14L2) binds both αT and αTP and stimulates phosphorylation of αT possibly by facilitating its transport and presentation to the putative αT kinase in the correct spatial orientation. Recombinant hTAP1 reduces the in vitro activity of the phosphatidylinositol‐3‐kinase gamma (PI3Kγ) potentially by sequestering the substrate phosphatidylinositol (PI). Indeed, the addition αT or αTP to the reaction stimulates PI3Kγ, most likely by facilitating egress of PI from hTAP1 and its binding to the enzyme. It is suggested that the continuous mutual exchange of different ligands in the metabolic nanoreactor formed by hTAPs may be a way to make these lipophiles more accessible as substrates to enzymes and as constituents to specific membrane domains. Supported by USDA Contract #58–1950‐0–014.