Molecular Cloning of an Amphibian Insulin Receptor Substrate 1-Like cDNA and Involvement of Phosphatidylinositol 3-Kinase in Insulin-Induced Xenopus Oocyte Maturation

Abstract

An insulin receptor substrate 1 (IRS-1)-like cDNA was isolated from a Xenopus ovary cDNA library by low-stringency hybridization using rat IRS-1 cDNA as a probe. The deduced amino acid sequence encoded by this cDNA (termed XIRS-L) is 67% identical (77% similar) to that of rat IRS-1. Significantly, all the insulininduced tyrosine phosphorylation sites identified in rat IRS-1, including those responsible for binding to the IRS-1 does not possess any recognizable enzymatic activity as judged by its amino acid sequence (35). However, it is rapidly phosphorylated on multiple tyrosine residues following insulin stimulation (33). Tyrosine-phosphorylated IRS-1 forms physical complexes with several cellular proteins containing Src homology 2 (SH2) domains. These include phosphatidylinositol (PI) 3-kinase, the protein phosphotyrosine phosphatase Syp (also known as SH-PTP2 [22]), the Grb2 protein implicated in Ras regulation, and Nck, an adaptor protein of unknown function (3,14,15,28,29,35,37). Individual tyrosine phosphorylation sites on IRS-1 are apparently responsible for binding to specific SH2 domains, thereby coupling the relevant SH2 domain-containing proteins to the insulin receptor. For instance, tyrosine phosphorylation sites with the sequence pYMXM motifs (pY stands for phosphotyrosine) are binding sites for the SH2 domains of the p85 subunit of PI 3-kinase, and the p85 SH2 domains preferentially bind to Tyr-608 of IRS-1, with the sequence GpYMPMSG (33).PI 3-kinase is activated in insulin-stimulated Chinese hamster ovary cells overexpressing human insulin receptor, as indicated by an elevation of 3-phosphorylated phosphoinositides [PI(3,4)P 2 and PI(3,4,5) 3 ] (25). In vitro, binding of tyrosine phosphorylated IRS-1, or synthetic peptides containing pYMXM motifs, to PI 3-kinase results in a modest activation of PI 3-kinase activity (2). These results suggest that insulininduced PI 3-kinase activation is mediated in part by binding of tyrosine phosphorylated IRS-1 to the lipid kinase. An alternative mechanism in which a direct interaction between the autophosphorylated insulin receptor and PI 3-kinase may play a role was recently proposed (38).Fully grown, or stage VI, oocytes (8) isolated from Xenopus laevis, previously primed with pregnant-mare serum gonadotropin (PMSG), undergo meiotic maturation when exposed to mammalian insulin or insulin-like growth factor 1 (IGF-1) (9,17). Recent data suggest that while oocytes isolated from unprimed frogs did not respond to insulin, microinjection of recombinant rat IRS-1 restored their responsiveness (6). The injected IRS-1 also bound endogenous PI 3-kinase in an insulin-dependent fashion. A recombinant protein containing the N-terminal SH2 domain of the p85 subunit of PI 3-kinase disrupted this association and inhibited insulin-induced oocyte maturation (5). These results suggest that an endogenous IRS-

Progesterone and insulin stimulation of CPEB-dependent polyadenylation is regulated by Aurora A and glycogen synthase kinase-3

“…For example, although decreased cAMP levels are required for both progesterone-and insulin-mediated maturation (Sadler and Maller 1989), PI3 kinase is required only for the insulin pathway (Berra et al 1993;Liu et al 1995). Because it seemed likely that insulin-stimulated CPE-mediated and Aurora A-dependent mRNA polyadenylation and translation could also rely on PI3 kinase, oocytes were treated with wortmannin, a PI3 kinase inhibitor (Wymann et al 1996), prior to insulin or progesterone stimulation.…”

“…In vitro synthesis of mRNAs encoding myc-tagged CPEB and GSK-3 proteins has been described (Pierce and Kimelman 1995;Mendez et al 2000a). Oocytes were treated with rapamycin (2 µg/mL) or wortmannin (0.5 µM) as described (Liu et al 1995;Schwab et al 1999).…”

“…Under certain circumstances insulin can stimulate Xenopus oocyte maturation, which in contrast to progesterone, requires phosphoinositide 3-kinase (PI3 kinase) and protein kinase C (isoform zeta) activity (Sadler and Maller 1989;Dominguez et al 1992;Berra et al 1993;Liu et al 1995). At least in mammalian cells, insulin can also signal through glycogen synthase kinase 3 (GSK-3), whose activity is usually downregulated in response (Frame and Cohen 2001;Oriente et al 2001).…”

Xp42Mpk1 Activation Is Not Required for Germinal Vescicle Breakdown but for Raf Complete Phosphorylation in Insulin-stimulated Xenopus Oocytes

“…24). In Xenopus oocytes, the phosphoinositide 3-kinase pathway (25)(26)(27), which leads to activation of protein kinase B/Akt, has been well described because recent data demonstrate a crucial role for protein kinase B/Akt in the insulin-but not progesterone-stimulated resumption of meiosis (28,29).…”

Xenopus laevis insulin receptor substrate IRS‐1 is important for eye development

“…So far, no data are available concerning the function of IRSs during Xenopus embryogenesis. It has been shown, however, that IRS-1 mediates IGF-1-and insulin-induced oocyte maturation by means of the PI-3K pathway (Chuang et al, 1993;Liu et al, 1995;Yamamoto-Honda et al, 1996).…”