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...

Stress-induced heat shock protein gene expression is developmentally regulated during early embryogenesis of the frog, Xenopus laevis. For example, a number of heat shock protein genes, such as hsp70, hsp90, and ubiquitin are not heat-inducible until after the midblastula stage of embryogenesis. Furthermore, the family of small heat shock protein genes, hsp30, are differentially expressed after the midblastula stage as well as being regulated at the level of mRNA stability. Many of these stress proteins are also synthesized constitutively during oogenesis and embryogenesis during which they may act as molecular chaperones as well as being involved in sequestering proteins in an inactive state until required by the developing embryo. Furthermore the induction of these stress protein genes has been correlated with enhanced thermoresistance. During stressful conditions heat shock proteins probably prevent aggregation or misfolding of damaged proteins within the embryo.

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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.

Four complete hsp 30 genes have been isolated from Xenopus laevis: hsp 30A, hsp 30B (a pseudogene), hsp 30C, and hsp 30D. The hsp 30A and hsp 30C genes are first heat inducible at the early tailbud stage, as determined by RNase protection and RT-PCR assays. In this study, we determined by RT-PCR that the hsp 30D gene was first heat inducible (33 degrees C for 1 h) at the mid-tailbud stage, approximately 1 day later in development than hsp 30A and hsp 30C. Furthermore, using Northern blot analysis, we detected the presence of very low levels of hsp 30 mRNA at the heat-shocked late blastula stage. The relative levels of these pre-tailbud (PTB) hsp 30 mRNAs increased at the gastrula and neurula stage followed by a dramatic enhancement in heat shocked tailbud and tadpole stage embryos (50- to 100- fold relative to late blastula). Interestingly, treatment of blastula or gastrula embryos at high temperatures (37 degrees C for 1 h) or with the protein synthesis inhibitor, cycloheximide, followed by heat shock, led to enhanced accumulation of the pre-tailbud (PTB) hsp 30 mRNAs. hsp 70, hsp 87, and actin messages were not stabilized at high temperatures or by cycloheximide treatment. Finally, hsp 30D mRNA was not detected by RT-PCR analysis of cycloheximide-treated, heat-shocked blastula stage embryos, confirming that it is not a member of the PTB hsp 30 mRNAs. This study indicates that differential gene expression and mRNA stability are involved in the regulation of hsp 30 gene expression during early Xenopus laevis development.