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 signaling complexes by binding to tyrosine phosphorylated proteins using their SH2 domains and to proline-rich sequences on effector molecules using their SH3 domains. FGF, which activates a receptor tyrosine kinase, induces mesoderm formation in Xenopus embryos through activation of the Ras/Raf/MAPK signaling pathway. We present evidence that dominant-negative mutants of Nck and Grb2, but not Crk1, can inhibit mesoderm-specific gene induction by eFGF in Xenopus animal cap explants. We also show that dominant-negative mutants of Grb2 and Nck can inhibit eFGF-induced Erk1 activation in Xenopus animal caps, and that targeting the first two SH3 domains of Nck to the membrane can activate Erk1 in the absence of eFGF. Furthermore, combinations of the dominant-negative Grb2 mutants with the inhibitory Nck mutant synergistically inhibited Erk1 activation by eFGF in Xenopus animal caps, suggesting that the dominant-negative Nck and Grb2 mutants inhibit Erk1 activation by binding to different proteins. By contrast only Grb2 mutants could inhibit eFGF-induced Erk1 activation in human 293 cells, demonstrating diversity in the specific mechanisms of signaling from FGF to MAP kinases in different cells.
Gupta
R W
RW
Howard Hughes Medical Institute, Children's Hospital, and Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mayer
B J
BJ
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000008:administration & dosage; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D004625:Embryo, Nonmammalian; D004789:Enzyme Activation; D005326:Fetal Proteins; D051525:Fibroblast Growth Factor 9; D005346:Fibroblast Growth Factors; D051380:GRB2 Adaptor Protein; D018507:Gene Expression Regulation, Developmental; D006133:Growth Substances / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008648:Mesoderm; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D015398:Signal Transduction; D020825:T-Box Domain Proteins; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014981:Xenopus / Q000196:embryology; D029867:Xenopus Proteins; D018909:src Homology Domains / Q000235:genetics; D000098908:Brachyury Protein
D048868:Adaptor Proteins, Signal Transducing; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004247:DNA; D004268:DNA-Binding Proteins; D005326:Fetal Proteins; D051525:Fibroblast Growth Factor 9; D005346:Fibroblast Growth Factors; D051380:GRB2 Adaptor Protein; D006133:Growth Substances; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D015513:Oncogene Proteins; D011506:Proteins; D012333:RNA, Messenger; D020825:T-Box Domain Proteins; D014157:Transcription Factors; D029867:Xenopus Proteins; D000098908:Brachyury Protein; C496459:FGF9 protein, human; C495948:GRB2 protein, human; C078332:Nck protein; C496462:fgf9 protein, Xenopus