Functional interactions between isolated SH2 domains and insulin/Ras signaling pathways of Xenopus oocytes: opposite effects of the carboxy- and amino-terminal SH2 domains of p85 PI 3-kinase.
Purified amino-terminal Src homology 2 (SH2) domains of GAP, PLCgamma1 and the p85alpha subunit of PI 3-kinase, as well as the carboxy-terminal SH2 domain of the latter protein and the unique SH2 domain of Grb2, were injected into full grown, stage VI Xenopus laevis oocytes. None of the injected domains showed any effect when injected alone, nor did they affect the rate of GVBD induced by progesterone, an adenylate cyclase-dependent process. On the other hand, the unique Grb2 SH2 domain and all N-terminal SH2 domains injected inhibited to various degrees the rate of insulin-induced GVBD, a tyrosine kinase dependent pathway. Interestingly, and in contrast to the behavior shown by the N-terminal domain of the same molecule, the C-terminal SH2 domain of p85 did not inhibit, but slightly accelerated the rate of GVBD induced by insulin. Furthermore, whereas the Grb SH2 domain and all N-terminal SH2 domains tested failed to co-operate with normal Ras protein to induce GVBD, the C-terminal SH2 domain of p85alpha exhibited significant synergy when coinjected with normal Ras protein, indicating that the C- and N-terminal SH2 domains of p85alpha exert opposite (positive and negative, respectively) regulatory roles in the control of oocyte insulin/Ras signaling pathways. Our results demonstrate that the purified, isolated SH2 domains retain structural and functional specificity and that Xenopus oocytes constitute an useful biological system to analyse their functional role in tyrosine kinase signaling pathways.
Aroca
P
P
Laboratory of Cellular and Molecular Biology, DBS, National Cancer Institute, Bethesda, Maryland 20892, USA.
Mahadevan
D
D
Santos
E
E
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D005260:Female; D051380:GRB2 Adaptor Protein; D020690:GTPase-Activating Proteins; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008540:Meiosis / Q000187:drug effects; D008845:Microinjections; D015689:Oncogene Protein p21(ras) / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases; D051966:Phospholipase C gamma; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis / Q000378:metabolism; D020703:ras GTPase-Activating Proteins; D018909:src Homology Domains / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; D020690:GTPase-Activating Proteins; D007328:Insulin; D007527:Isoenzymes; D011506:Proteins; D011994:Recombinant Proteins; D020703:ras GTPase-Activating Proteins; D011374:Progesterone; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D015689:Oncogene Protein p21(ras)