Insulin receptor substrate-1 (IRS-1) is phosphorylated on multiple tyrosine residues by ligand-activated insulin receptors. These tyrosine phosphorylation sites serve to dock several Src homology 2-containing signaling proteins. In addition, IRS-1 contains a pleckstrin homology domain and a phosphotyrosine binding domain (PTB) implicated in protein-protein and proteinlipid interactions. In a yeast two-hybrid screening using Xenopus IRS-1 (xIRS-1) pleckstrin homology-PTB domains as bait, we identified a Xenopus homolog of Rhoassociated kinase ␣ (xROK␣) as a potential xIRS-1-binding protein. The original clone contained the carboxyl terminus of xROK␣ (xROK-C) including the putative Rho binding domain but lacking the amino-terminal kinase domain. Further analyses in yeast indicated that xROK-C bound to the putative PTB domain of xIRS-1. Binding of xROK-C to xIRS-1 was confirmed in Xenopus oocytes after microinjection of mRNA corresponding to xROK-C. Furthermore, microinjection of xROK-C mRNA inhibited insulin-induced mitogen-activated protein kinase activation with a concomitant inhibition of oocyte maturation. In contrast, microinjection of xROK-C mRNA did not inhibit mitogen-activated protein kinase activation or oocyte maturation induced by progesterone or by microinjection of viral Ras (v-Ras) mRNA. These results suggest that xROK␣ may play a role in insulin signaling via a direct interaction with xIRS-1. Phosphorylation of insulin receptor substrate-1 (IRS-1)1 by ligand-activated insulin receptors serves to dock several Src homology 2 domain-containing proteins (1-4). In addition to the multiple tyrosine phosphorylation sites, IRS-1 contains an amino-terminal PH domain (5, 6) and a PTB domain (7,8) carboxyl-terminal to the PH domain (9, 10). Studies by White and co-workers (11, 12) have shown that the PH domain is required for efficient tyrosine phosphorylation of IRS-1 by the insulin receptor, although the mechanism by which the PH domain regulates this function is not clear. The presence of a PTB domain in IRS-1 explains the earlier observations that mutations in a human insulin receptor autophosphorylation site (NPEY 960 (13)) or the equivalent site in insulin-like growth factor I receptor (14) diminished its ability to phosphorylate IRS-1. Biochemical and structural studies indicate that amino acids 161-265 of rat IRS-1 include the required component of the PTB domain which binds the NPEpY motif of the insulin receptor (9, 10). However, using the yeast two-hybrid assay, Gustafson and colleagues have provided evidence that additional amino acids that are carboxyl-terminal to the PTB domain (termed the SAIN domain) are also necessary for binding to the NPEpY sequence of the insulin receptor (15, 16). IRS-1 also interacts with 14-3-3 protein, a process apparently dependent on serine phosphorylation of IRS-1 (17, 18).We have previously isolated a Xenopus cDNA encoding an IRS-1-like protein (termed xIRS-L) (19). Overall, xIRS-L exhibits 65% amino acid sequence identity to mammalian IRS-1 (1) but only 45% identi...

We have cloned a complementary DNA encoding the putative Xenopus insulin-like growth factor-1 (xIGF-1) receptor. Injection of messenger RNA derived from the cloned complementary DNA into Xenopus oocytes resulted in the expression and correct processing of the receptor's alpha- and beta-subunits. Using antibodies generated against protein expressed against the cloned sequence, we demonstrated that the endogenous xIGF-1 receptor in Xenopus oocytes was activated by nanomolar concentrations of mammalian IGF-1 and by insulin approximately 100-fold higher in concentration. This receptor activation profile correlated with hormone-induced Xenopus oocyte maturation. Furthermore, injection of a neutralizing antiinsulin receptor antibody into Xenopus oocytes inhibited hormone-induced xIGF-1 receptor activation. These results provide molecular and biochemical evidence supporting a role for xIGF-1 receptor in mediating insulin/IGF-1-induced Xenopus oocyte maturation. We also report here that embryonic transcription of xIGF-1 receptor is activated during the formation of the central nervous system in early Xenopus embryos.

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