Cloning of novel human SEC14p-like proteins: ligand binding and functional properties.
We describe the cloning and expression of two novel genes highly similar to the tocopherol-associated protein (hTAP/SEC14L2/SPF). Immunoprecipitation of the three recombinant hTAPs and extraction of their associated lipid-soluble molecules indicates that they bind not just tocopherols, but also phosphatidylinositol, phosphatidylcholine, and phosphatidylglycerol. Ligand competition analysis by isoelectric point mobility shift assay indicates that phosphatidylcholine, tocopherols, and tocopheryl-succinate compete with phosphatidylinositol binding to hTAPs. To investigate a possible function of hTAPs on enzymes involved in phospholipids metabolism, the activity of recombinant phosphatidylinositol 3-kinase (PI3Kgamma/p110gamma) was tested. Recombinant hTAPs reduce in vitro the activity of the recombinant catalytic subunit of PI3Kgamma and stimulate it in the presence of alpha-tocopherol up to 5-fold. Immunoprecipitation of hTAP1 from cells results in co-precipitation of PI3-kinase activity, indicating a physical contact between the two proteins at a cellular level. In summary, hTAPs may modulate, in a tocopherol-sensitive manner, phosphatidylinositol-3-kinase, a central enzyme in signal transduction, cell proliferation, and apoptosis. It is possible that other phosphatidylinositol- and phosphatidylcholine-dependent signaling pathways are modulated by hTAPs and tocopherols, possibly by transporting and presenting these ligands to the corresponding enzymes.
Kempná
Petra
P
Institute of Biochemistry and Molecular Biology, University of Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland.
Zingg
Jean-Marc
JM
Ricciarelli
Roberta
R
Hierl
Markus
M
Saxena
Smita
S
Azzi
Angelo
A
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D005816:Genetic Complementation Test; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D049294:Phospholipid Transfer Proteins; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013379:Substrate Specificity; D024505:Tocopherols / Q000378:metabolism*
D002352:Carrier Proteins; D017931:DNA Primers; D008024:Ligands; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D019869:Phosphatidylinositol 3-Kinases; D010716:Phosphatidylinositols; D049294:Phospholipid Transfer Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D024505:Tocopherols; C118289:SEC24 protein, S cerevisiae; C017763:alpha-tocopherol transfer protein