Molecular cloning of an amphibian insulin receptor substrate 1-like cDNA and involvement of phosphatidylinositol 3-kinase in insulin-induced Xenopus oocyte maturation.
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 insulin-induced tyrosine phosphorylation sites identified in rat IRS-1, including those responsible for binding to the Src homology domains of phosphatidylinositol (PI) 3-kinase, Syp and Grb2, are conserved in XIRS-L. Both mRNA and protein corresponding to the cloned XIRS-L can be detected in immature Xenopus oocytes. Recombinant XIRS-L protein produced in insect cells or a bacterial glutathione S-transferase fusion protein containing the putative PI 3-kinase binding site can be phosphorylated in vitro by purified insulin receptor kinase (IRK) domain, and the IRK-catalyzed phosphorylation renders both proteins capable of binding PI 3-kinase in Xenopus oocyte lysates. Another glutathione S-transferase fusion protein containing the C terminus of XIRS-L and including several putative tyrosine phosphorylation sites is also phosphorylated by IRK in vitro, but it failed to bind PI 3-kinase. Insulin stimulation of immature Xenopus oocytes activates PI 3-kinase in vivo [as indicated by an elevation of PI(3,4)P2 and PI(3,4,5)P3] as well as oocyte maturation (as indicated by germinal vesicle breakdown). Pretreatment of these oocytes with wortmannin inhibited insulin-induced activation of PI 3-kinase in vivo. The same treatment also abolished insulin-induced, but not progesterone-induced, germinal vesicle breakdown. These results (i) identify an IRS-1-like molecule in immature Xenopus oocytes, suggesting that the use of IRS-1-like Scr homology 2 domain-docking proteins in signal transduction is conserved in vertebrates, and (ii) strongly implicate PI 3-kinase as an essential effector of insulin-induced oocyte maturation.
Liu
X J
XJ
Loeb Medical Research Institute, Ottawa Civic Hospital, Ontario, Canada.
Sorisky
A
A
Zhu
L
L
Pawson
T
T
D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005260:Female; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D008969:Molecular Sequence Data; D009866:Oogenesis / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000502:physiology*; D013045:Species Specificity; D000077191:Wortmannin; D014981:Xenopus; D029867:Xenopus Proteins
D000730:Androstadienes; D018076:DNA, Complementary; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D010750:Phosphoproteins; D012333:RNA, Messenger; D029867:Xenopus Proteins; C526294:irs1 protein, Xenopus; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011505:Protein-Tyrosine Kinases; D000077191:Wortmannin