Activation loop sequences confer substrate specificity to phosphoinositide 3-kinase α (PI3Kα) -: Functions of lipid kinase-deficient PI3Kα in signaling
Activation loop sequences confer substrate specificity to phosphoinositide 3-kinase α (PI3Kα) -: Functions of lipid kinase-deficient PI3Kα in signaling
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DOI：
10.1074/jbc.m011330200
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发表时间：
2001-06-15
影响因子：
4.8
通讯作者：
Wymann, MP
中科院分区：
文献类型：
--
作者：
Pirola, L;Zvelebil, MJ;Wymann, MP
磷脂酰肌醇3-激酶(PI3Ks)是一种双重特异性的脂蛋白激酶。虽然脂质依赖的PI3K下游信号被很好地描述，但对PI3K蛋白激酶信号和各种PI3K类脂质底物专一性的结构决定因素知之甚少。在这里，我们证明了PI3K ATP结合位点的C末端序列决定了IA类PI3Kα(p85/p110α)的脂质底物专一性。这样的激活环序列从II类PI3K、III类PI3K和相关的哺乳动物雷帕霉素靶标(FRAP)转移到p110α中，使所产生的杂交蛋白的脂底物专一性变为供体蛋白的脂底物专一性，同时保持蛋白激酶活性不受影响。所有产生的杂交细胞都缺乏在完整细胞中产生磷脂酰肌醇3，4，5-三磷酸的能力。氨基酸取代和结构模拟表明，激活环中两个保守的正电荷残基(Lys和Arg)对于I类PI3K作为磷脂酰肌醇4，5-二磷酸激酶的功能至关重要。通过对293细胞的瞬时转染，我们发现p110α杂交体虽然不能支持脂依赖的PI3K信号转导，如蛋白激酶B/Akt和p70(S6K)的激活，但保留了与胰岛素受体底物1结合和磷酸化的能力，具有与野生型PI3Kα相同的特异性和更高的效率。我们的数据为理解I类PI3K底物的选择性和使用PI3Kα杂交物来剖析PI3Kα作为脂质和蛋白激酶的功能奠定了基础。
Phosphoinositide 3-kinases (PI3Ks) are dual specificity lipid and protein kinases. While the lipid-dependent PI3K downstream signaling is well characterized, little is known about PI3K protein kinase signaling and structural determinants of lipid substrate specificity across the various PI3K classes. Here we show that sequences C-terminal to the PI3K ATP-binding site determine the lipid substrate specificity of the class IA PI3K alpha (p85/p110 alpha). Transfer of such activation loop sequences from class II PI3Ks, class III PI3Ks, and a related mammalian target of rapamycin (FRaP) into p110 alpha turns the lipid substrate specificity of the resulting hybrid protein into that of the donor protein, while leaving the protein kinase activity unaffected. All resulting hybrids lacked the ability to produce phosphatidylinositol 3,4,5-trisphosphate in intact cells. Amino acid substitutions and structure modeling showed that two conserved positively charged (Lys and Arg) residues in the activation loop are crucial for the functionality of class I PI3Ks as phosphatidylinositol 4,5-bisphosphate kinases. By transient transfecion of 293 cells, we show that p110 alpha hybrids, although unable to support lipid-dependent PI3K signaling, such as activation of protein kinase B/Akt and p70(S6k), retain the capability to associate with and phosphorylate insulin receptor substrate-1, with the same specificity and higher efficacy than wild type PI3K alpha. Our data lay the basis for the understanding of the class I PI3K substrate selectivity and for the use of PI3K alpha hybrids to dissect PI3K alpha function as lipid and protein kinase.