Interaction of the Grb10 adapter protein with the Raf1 and MEK1 kinases.
Grb10 and its close homologues Grb7 and Grb14, belong to a family of adapter proteins characterized by a proline-rich region, a central PH domain, and a carboxyl-terminal Src homology 2 (SH2) domain. Their interaction with a variety of activated tyrosine kinase receptors is well documented, but their actual function remains ... a mystery. The Grb10 SH2 domain was isolated from a two-hybrid screen using the MEK1 kinase as a bait. We show that this unusual SH2 domain interacts, in a phosphotyrosine-independent manner, with both the Raf1 and MEK1 kinases. Mutation of the MEK1 Thr-386 residue, which is phosphorylated by mitogen-activated protein kinase in vitro, reduces binding to Grb10 in a two-hybrid assay. Interaction of Grb10 with Raf1 is constitutive, while interaction between Grb10 and MEK1 needs insulin treatment of the cells and follows mitogen-activated protein kinase activation. Random mutagenesis of the SH2 domain demonstrated that the Arg-betaB5 and Asp-EF2 residues are necessary for binding to the epidermal growth factor and insulin receptors as well as to the two kinases. In addition, we show that a mutation in Ser-betaB7 affects binding only to the receptors, while a mutation in Thr-betaC5 abrogates binding only to MEK1. Finally, transfection of Grb10 genes with specific mutations in their SH2 domains induces apoptosis in HTC-IR and COS-7 cells. These effects can be competed by co-expression of the wild type protein, suggesting that these mutants act by sequestering necessary signaling components.
Mesh Terms:
Amino Acid Sequence, Animals, Apoptosis, Base Sequence, Binding Sites, Cell Line, DNA, GRB10 Adaptor Protein, Humans, MAP Kinase Kinase 1, Mitogen-Activated Protein Kinase Kinases, Molecular Sequence Data, Phosphorylation, Point Mutation, Protein Binding, Protein-Serine-Threonine Kinases, Protein-Tyrosine Kinases, Proteins, Proto-Oncogene Proteins c-raf, Sequence Homology, Amino Acid, Sequence Homology, Nucleic Acid
Amino Acid Sequence, Animals, Apoptosis, Base Sequence, Binding Sites, Cell Line, DNA, GRB10 Adaptor Protein, Humans, MAP Kinase Kinase 1, Mitogen-Activated Protein Kinase Kinases, Molecular Sequence Data, Phosphorylation, Point Mutation, Protein Binding, Protein-Serine-Threonine Kinases, Protein-Tyrosine Kinases, Proteins, Proto-Oncogene Proteins c-raf, Sequence Homology, Amino Acid, Sequence Homology, Nucleic Acid
J. Biol. Chem.
Date: Apr. 24, 1998
PubMed ID: 9553107
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