DOMINANT-NEGATIVE MUTANTS OF THE SH2 SH3 ADAPTERS NCK AND GRB2 INHIBIT MAP KINASE ACTIVATION AND MESODERM-SPECIFIC GENE INDUCTION BY EFGF IN XENOPUS/

The SH2/SH3 adapters Nck, Grb2 and Crk promote the assembly of signali ng complexes by binding to tyrosine phosphorylated proteins using thei r SH2 domains and to proline-rich sequences on effector molecules usin g their SH3 domains. FGF, which activates a receptor tyrosine kinase, induces mesoderm formation in Xenopus embryos through activation of th e Ras/Raf/MAPK signaling pathway. We present evidence that dominant-ne gative mutants of Nck and Grb2, but not Crk1, can inhibit mesoderm-spe cific gene induction by eFGF in Xenopus animal cap explants. We also s how that dominant-negative mutants of Grb2 and Nck can inhibit eFGF-in duced Erk1 activation in Xenopus animal caps, and that targeting the f irst two SH3 domains of Nck to the membrane can activate Erk1 in the a bsence of eFGF.. Furthermore, combinations of the dominant-negative Gr b2 mutants with the inhibitory Nck mutant synergistically inhibited Er k1 activation by eFGF in Xenopus animal caps, suggesting that the domi nant-negative Nck and Grb2 mutants inhibit Erk1 activation by binding to different proteins. By contrast only Grb2 mutants could inhibit eFG F-induced Erk1 activation in human 293 cells, demonstrating diversity in the specific mechanisms of signaling from FGF to MAP kinases in dif ferent cells.