Vainshtein I, Kovacina K S, Roth R A
Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5174, USA.
J Biol Chem. 2001 Mar 16;276(11):8073-8. doi: 10.1074/jbc.M008436200. Epub 2001 Jan 5.
The pleckstrin homology (PH) domain of the insulin receptor substrate-1 (IRS-1) plays a role in directing this molecule to the insulin receptor, thereby regulating its tyrosine phosphorylation. In this work, the role of the PH domain in subsequent signaling was studied by constructing constitutively active forms of IRS-1 in which the inter-SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase was fused to portions of the IRS-1 molecule. Chimeric molecules containing the PH domain were found to activate the downstream response of stimulating the Ser/Thr kinase Akt. A chimera containing point mutations in the PH domain that abolished the ability of this domain to bind phosphatidylinositol 4,5-bisphosphate prevented these molecules from activating Akt. These mutations also decreased by about 70% the amount of the constructs present in a particulate fraction of the cells. These results indicate that the PH domain of IRS-1, in addition to directing this protein to the receptor for tyrosine phosphorylation, functions in the ability of this molecule to stimulate subsequent responses. Thus, compromising the function of the PH domain, e.g. in insulin-resistant states, could decrease both the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor and to link to subsequent downstream targets.
胰岛素受体底物-1（IRS-1）的普列克底物蛋白同源（PH）结构域在将该分子导向胰岛素受体的过程中发挥作用，从而调节其酪氨酸磷酸化。在本研究中，通过构建组成型活性形式的IRS-1来研究PH结构域在后续信号传导中的作用，其中磷脂酰肌醇3激酶p85亚基的SH2结构域间区域与IRS-1分子的部分区域融合。发现含有PH结构域的嵌合分子可激活刺激丝氨酸/苏氨酸激酶Akt的下游反应。一种在PH结构域中含有点突变的嵌合体，该突变消除了该结构域结合磷脂酰肌醇4,5-二磷酸的能力，从而阻止这些分子激活Akt。这些突变还使细胞颗粒部分中存在的构建体数量减少了约70%。这些结果表明，IRS-1的PH结构域除了将该蛋白导向酪氨酸磷酸化受体外，还在该分子刺激后续反应的能力中发挥作用。因此，损害PH结构域的功能，例如在胰岛素抵抗状态下，可能会降低IRS-1被胰岛素受体酪氨酸磷酸化以及与后续下游靶点连接的能力。
