PIKfyve, a mammalian ortholog of yeast Fab1p lipid kinase, synthesizes 5-phosphoinositides - Effect of insulin
PIKfyve, a mammalian ortholog of yeast Fab1p lipid kinase, synthesizes 5-phosphoinositides - Effect of insulin
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DOI：
10.1074/jbc.274.31.21589
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发表时间：
1999-07-30
影响因子：
4.8
通讯作者：
Shisheva, A
中科院分区：
文献类型：
--
作者：
Sbrissa, D;Ikonomov, OC;Shisheva, A
磷脂酰肌醇（PtdIns）头基的一个或多个游离羟基经历酶促磷酸化，产生在真核细胞调节中具有关键功能的磷酸肌醇（PI）。目前已在哺乳动物细胞中鉴定出两种这样的物种，PtdIns 5-P和PtdIns 3，5-P-2，但它们的生物合成仍不清楚。我们已经分离了一种新的哺乳动物PI激酶p235，其确切的底物特异性仍有待确定（Shisheva，A.，Sbrissa，D，，and Ikonomov，O.等（1999）Mol. Cell. 19，623-634）。在这里，我们报告的重组p235在COS细胞中表达，像真正的p235在脂肪细胞中，显示出惊人的特异性PtdIns超过PI底物，并产生两个产品，确定为PtdIns 5-P和PtdIns 3，5-P，通过HPLC分析。合成的PtdIns 3-P底物也被转化为PtdIns 3，5-P，但转化程度远低于从天然来源分离的PtdIns。P235 PI 5-激酶的重要特性包括对非离子去污剂的高敏感性和对渥曼青霉素和腺苷的相对抗性。通过分析异源细胞系统中的缺失突变体，我们确定除了预测的催化结构域之外，分子的其他区域对于p235酶活性是至关重要的。由来自用胰岛素急性刺激的3 T3-L1脂肪细胞的裂解物的p235免疫复合物产生的单磷酸肌醇产物的HPLC分辨率揭示了与静息细胞的相应p235免疫复合物基本上相同的PtdIns 5-P水平。然而，急性胰岛素作用导致渥曼青霉素敏感性PtdIns 3-P峰增加，提示渥曼青霉素敏感性PI 3-激酶可能募集至p235。总之，小鼠p235（在此重新命名为PIKfyve）显示出对PtdIns 5-P和PtdIns 3，5-P生成的强体外活性，这意味着PIKfyve在它们的生物合成中具有关键作用。
One or more free hydroxyls of the phosphatidylinositol (PtdIns) head group undergo enzymatic phosphorylation, yielding phosphoinositides (PIs) with key functions in eukaryotic cellular regulation. Two such species, PtdIns 5-P and PtdIns 3,5-P-2, have now been identified in mammalian cells, but their biosynthesis remains unclear. We have isolated a novel mammalian PI kinase, p235, whose exact substrate specificity remained to be determined (Shisheva, A., Sbrissa, D,, and Ikonomov, O. (1999) Mol. Cell. Biol. 19, 623-634). Here we report that recombinant p235 expressed in COS cells, like the authentic p235 in adipocytes, displays striking specificity for PtdIns over PI substrates and generates two products identified as PtdIns 5-P and PtdIns 3,5-P, by HPLC analyses. Synthetic PtdIns 3-P substrates were also converted to PtdIns 3,5-P, but to a substantially lesser extent than PtdIns isolated from natural sources. Important properties of the p235 PI 5-kinase include high sensitivity to nonionic detergents and relative resistance to wortmannin and adenosine. By analyzing deletion mutants in a heterologous cell system, we determined that in addition to the predicted catalytic domain other regions of the molecule are critical for the p235 enzymatic activity. HPLC resolution of monophosphoinositide products, generated by p235 immune complexes derived from lysates of 3T3-L1 adipocytes acutely stimulated with insulin, revealed essentially the same PtdIns 5-P levels as the corresponding p235 immune complexes of resting cells. However, the acute insulin action resulted in an increase of a wortmannin-sensitive PtdIns 3-P peak, suggestive of a plausible recruitment of wortmannin-sensitive PI 3-kinase(s) to p235. In conclusion, mouse p235 (renamed here PIKfyve) displays a strong in vitro activity for PtdIns 5-P and PtdIns 3,5-P, generation, implying PIKfyve has a key role in their biosynthesis.