Pirola L, Zvelebil M J, Bulgarelli-Leva G, Van Obberghen E, Waterfield M D, Wymann M P
Institute of Biochemistry, University of Fribourg, CH-1700 Fribourg, Switzerland.
J Biol Chem. 2001 Jun 15;276(24):21544-54. doi: 10.1074/jbc.M011330200. Epub 2001 Feb 13.
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 PI3Kalpha (p85/p110alpha). Transfer of such activation loop sequences from class II PI3Ks, class III PI3Ks, and a related mammalian target of rapamycin (FRAP) into p110alpha 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 p110alpha 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 PI3Kalpha. Our data lay the basis for the understanding of the class I PI3K substrate selectivity and for the use of PI3Kalpha hybrids to dissect PI3Kalpha function as lipid and protein kinase.
磷酸肌醇3激酶（PI3Ks）是具有双重特异性的脂质激酶和蛋白激酶。虽然脂质依赖性PI3K下游信号传导已得到充分表征，但对于PI3K蛋白激酶信号传导以及不同PI3K类别中脂质底物特异性的结构决定因素知之甚少。在此我们表明，PI3K ATP结合位点C端的序列决定了IA类PI3Kα（p85/p110α）的脂质底物特异性。将此类激活环序列从II类PI3K、III类PI3K以及雷帕霉素的相关哺乳动物靶点（FRAP）转移到p110α中，会使所得杂合蛋白的脂质底物特异性转变为供体蛋白的特异性，同时不影响蛋白激酶活性。所有所得杂合体均缺乏在完整细胞中产生磷脂酰肌醇3,4,5-三磷酸的能力。氨基酸取代和结构建模表明，激活环中两个保守的带正电荷（赖氨酸和精氨酸）残基对于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细胞进行瞬时转染，我们表明p110α杂合体虽然无法支持脂质依赖性PI3K信号传导，如蛋白激酶B/Akt和p70(S6k)的激活，但仍保留与胰岛素受体底物-1结合并使其磷酸化的能力，其特异性与野生型PI3Kα相同，但效率更高。我们的数据为理解I类PI3K底物选择性以及利用PI3Kα杂合体剖析PI3Kα作为脂质激酶和蛋白激酶的功能奠定了基础。
