PIKfyve lipid kinase is a protein kinase:: Downregulation of 5′-phosphoinositide product formation by autophosphorylation
PIKfyve lipid kinase is a protein kinase:: Downregulation of 5′-phosphoinositide product formation by autophosphorylation
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DOI：
10.1021/bi001897f
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发表时间：
2000-12-26
期刊：
影响因子：
2.9
通讯作者：
Shisheva, A
中科院分区：
文献类型：
--
作者：
Sbrissa, D;Ikonomov, OC;Shisheva, A
磷酸肌醇3-激酶家族成员的一个子集是双特异性酶;它们的蛋白激酶活性被认为对其细胞内调节带来额外的水平。在此，我们已经检查了先前报道的在体外催化PtdIns 5-P和PtdIns 3，5-P-2形成的5 ′-磷酸肌醇激酶PIKfyve [Sbrissa等（1999）J.Biol.Chem.274，21589-21597]是否显示双重特异性。我们现在报道PIKfyve具有与其脂质激酶活性不可分割的内在蛋白激酶活性，并且除了本身之外，还可以以底物特异性方式磷酸化外源蛋白。用从异源转染的COS细胞、感染的Sf 9细胞或天然3 T3-L1脂肪细胞免疫纯化或亲和纯化的PIKfyve证明了自磷酸化和转磷酸化。相反，没有蛋白激酶活性与免疫纯化的脂质激酶死点（K1831 E）或截短的（Delta 1812-2052）PIKfyve突变体相关。PIKfyve自身磷酸化或转磷酸化涉及Ser而非Thr或Tyr残基。在用PIKfyve脂质底物或磷酸酶预处理后，PIKfyve自身磷酸化在很大程度上被废除。用纯化的PIKfyve制剂进一步检查了自磷酸化对PIKfyve脂质激酶活性的影响。脂质产物形成减少70%与PIKfyve自磷酸化相关，其在用磷酸酶处理后逆转。在细胞环境中，PIKfyve或其一部分以磷酸化形式存在。总的来说，这些结果表明PIKfyve是一种双重特异性激酶，其可以在活细胞的背景下产生和传递蛋白磷酸化信号以调节其脂质产物的形成以及可能的其他事件。
A subset of phosphoinositide 3-kinase family members are dual specificity enzymes; their protein kinase activity is thought to bring about an additional level to their intracellular regulation. Here we have examined whether the 5'-phosphoinositide kinase PIKfyve, reported previously to catalyze the formation of PtdIns 5-P and PtdIns 3,5-P-2 in vitro [Sbrissa et al. (1999) J. Biol. Chem. 274, 21589-21597], displays dual specificity. We now report that PIKfyve possesses an intrinsic protein kinase activity inseparable from its lipid kinase activity and, besides itself, can phosphorylate exogenous proteins in a substrate-specific manner. Both the autophosphorylation and transphosphorylation were demonstrated with PIKfyve immunopurified or affinity-purified from heterologously transfected COS cells, infected Sf9 cells, or native 3T3-L1 adipocytes. Conversely, no protein kinase activity was associated with immunopurified lipid kinase dead point (K1831E) or truncated (Delta 1812-2052) PIKfyve mutants. PIKfyve autophosphorylation or transphosphorylation engaged Ser but not Thr or Tyr residues. PIKfyve autophosphorylation was largely abrogated upon pretreatment with PIKfyve lipid substrates or phosphatases. The impact of autophosphorylation on the PIKfyve lipid kinase activity was further examined with purified PIKfyve preparations. A decrease of 70% in the lipid product formation was associated with PIKfyve autophosphorylation, which was reversed upon treatment with phosphatases. In the cellular context, PIKfyve, or a fraction of it, was found in a phosphorylated form. Collectively, these results indicate that PIKfyve is a dual specificity kinase, which can generate and relay protein phosphorylation signals to regulate the formation of its lipid products, and possibly other events, in the context of living cells.