Chuang L M, Myers M G, Backer J M, Shoelson S E, White M F, Birnbaum M J, Kahn C R
Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
Mol Cell Biol. 1993 Nov;13(11):6653-60. doi: 10.1128/mcb.13.11.6653-6660.1993.
Xenopus oocytes from unprimed frogs possess insulin-like growth factor I (IGF-I) receptors but lack insulin and IGF-I receptor substrate 1 (IRS-1), the endogenous substrate of this kinase, and fail to show downstream responses to hormonal stimulation. Microinjection of recombinant IRS-1 protein enhances insulin-stimulated phosphatidylinositol (PtdIns) 3-kinase activity and restores the germinal vesicle breakdown response. Activation of PtdIns 3-kinase results from formation of a complex between phosphorylated IRS-1 and the p85 subunit of PtdIns 3-kinase. Microinjection of a phosphonopeptide containing a pYMXM motif with high affinity for the src homology 2 (SH2) domain of PtdIns 3-kinase p85 inhibits IRS-1 association with and activation of the PtdIns 3-kinase. Formation of the IRS-1-PtdIns 3-kinase complex and insulin-stimulated PtdIns 3-kinase activation are also inhibited by microinjection of a glutathione S-transferase fusion protein containing the SH2 domain of p85. This effect occurs in a concentration-dependent fashion and results in a parallel loss of hormone-stimulated oocyte maturation. These inhibitory effects are specific and are not mimicked by glutathione S-transferase fusion proteins expressing the SH2 domains of ras-GAP or phospholipase C gamma. Moreover, injection of the SH2 domains of p85, ras-GAP, and phospholipase C gamma do not interfere with progesterone-induced oocyte maturation. These data demonstrate that phosphorylation of IRS-1 plays an essential role in IGF-I and insulin signaling in oocyte maturation and that this effect occurs through interactions of the phosphorylated YMXM/YXXM motifs of IRS-1 with SH2 domains of PtdIns 3-kinase or some related molecules.
未致敏青蛙的非洲爪蟾卵母细胞含有胰岛素样生长因子I（IGF-I）受体，但缺乏胰岛素和IGF-I受体底物1（IRS-1），这种激酶的内源性底物，并且对激素刺激不显示下游反应。显微注射重组IRS-1蛋白可增强胰岛素刺激的磷脂酰肌醇（PtdIns）3-激酶活性，并恢复生发泡破裂反应。PtdIns 3-激酶的激活源于磷酸化的IRS-1与PtdIns 3-激酶的p85亚基之间形成复合物。显微注射对PtdIns 3-激酶p85的src同源2（SH2）结构域具有高亲和力的含pYMXM基序的膦肽可抑制IRS-1与PtdIns 3-激酶的结合及激活。显微注射含有p85的SH2结构域的谷胱甘肽S-转移酶融合蛋白也可抑制IRS-1-PtdIns 3-激酶复合物的形成及胰岛素刺激的PtdIns 3-激酶激活。这种效应以浓度依赖的方式发生，并导致激素刺激的卵母细胞成熟平行丧失。这些抑制作用是特异性的，表达ras-GAP或磷脂酶Cγ的SH2结构域的谷胱甘肽S-转移酶融合蛋白不会模拟这种作用。此外，注射p85、ras-GAP和磷脂酶Cγ的SH2结构域不会干扰孕酮诱导的卵母细胞成熟。这些数据表明，IRS-1的磷酸化在卵母细胞成熟的IGF-I和胰岛素信号传导中起重要作用，并且这种效应是通过IRS-1的磷酸化YMXM/YXXM基序与PtdIns 3-激酶或一些相关分子的SH2结构域相互作用而发生的。
