Dupont J, Derouet M, Simon J, Taouis M
Institut National de la Recherche Agronomique, Station de Recherches Avicoles, Endocrinologie Moléculaire et cellulaire du Métabolisme, Centre de Tours, Nouzilly 37380, France.
Biochem J. 1998 Oct 15;335 ( Pt 2)(Pt 2):293-300. doi: 10.1042/bj3350293.
The Src homology and collagen protein (Shc) is tyrosine phosphorylated in response to insulin; however, evidence for its interaction with insulin receptor (IR) in normal tissues is missing. Interactions between IR, Shc and regulatory subunits of the phosphatidylinositol 3'-kinase (PI 3'-kinase) were characterized in the present study in liver and muscles of chickens submitted to various nutritional states. A chicken liver Shc cDNA fragment encoding a 198 amino acid long fragment, including the phosphotyrosine binding domain was sequenced. It shows 89% homology with the corresponding human homologue. The amounts of the three Shc isoforms (66, 52 and 46 kDa) and Shc messenger were not altered by the nutritional state. Shc tyrosine phosphorylation was decreased by fasting in both liver and muscle. Importantly, Shc was immunoprecipitated by IR antibody (mostly the 52 kDa isoform) or by alphaIRS-1(mostly the 46 kDa isoform). IR-Shc association was decreased by fasting and restored by refeeding. In liver, alphaShc immunoprecipitated the three forms of regulatory subunits of PI 3'-kinase and a PI 3'-kinase activity which was decreased by fasting. In muscle, alphaShc immunoprecipitated only the p85 isoform; the associated PI 3'-kinase activity was not altered by the nutritional state. Conversely, in both tissues anti-p85 antibody precipitated only the 52 kDa Shc isoform. In liver, antibodies to insulin receptor substrate-1 (alphaIRS-1), Shc or IR immunoprecipitated the three regulatory subunits of PI 3'-kinase and an equal PI 3'-kinase activity, without any residual activity left in the supernatants, suggesting the presence of a large complex involving IR, IRS-1, Shc (mainly the 52 kDa isoform) and PI 3'-kinase activity. The presence of another complex containing IRS-1 and the 46 kDa Shc isoform, but no PI 3'-kinase activity, is suggested.
Src同源和胶原蛋白（Shc）会因胰岛素作用而发生酪氨酸磷酸化；然而，在正常组织中其与胰岛素受体（IR）相互作用的证据尚不存在。本研究对处于不同营养状态的鸡的肝脏和肌肉中IR、Shc与磷脂酰肌醇3'-激酶（PI 3'-激酶）调节亚基之间的相互作用进行了表征。对编码一个包含磷酸酪氨酸结合结构域的198个氨基酸长片段的鸡肝脏Shc cDNA片段进行了测序。它与相应的人类同源物显示出89%的同源性。三种Shc同工型（66、52和46 kDa）的量以及Shc信使核糖核酸并未因营养状态而改变。禁食会使肝脏和肌肉中的Shc酪氨酸磷酸化降低。重要的是，Shc可被IR抗体（主要是52 kDa同工型）或αIRS-1（主要是46 kDa同工型）免疫沉淀。禁食会降低IR与Shc的结合，重新喂食后可恢复。在肝脏中，αShc免疫沉淀了PI 3'-激酶的三种调节亚基形式以及一种因禁食而降低的PI 3'-激酶活性。在肌肉中，αShc仅免疫沉淀了p85同工型；相关的PI 3'-激酶活性并未因营养状态而改变。相反，在两种组织中，抗p85抗体仅沉淀了52 kDa的Shc同工型。在肝脏中，胰岛素受体底物-1（αIRS-1）、Shc或IR的抗体免疫沉淀了PI 3'-激酶的三种调节亚基以及等量的PI 3'-激酶活性，上清液中没有任何残留活性，这表明存在一个涉及IR、IRS-1、Shc（主要是52 kDa同工型）和PI 3'-激酶活性的大型复合物。提示存在另一种包含IRS-1和46 kDa Shc同工型但无PI 3'-激酶活性的复合物。
