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Interaction of the Grb10 adapter protein with the Raf1 and MEK1 kinases

From National Research Council Canada

DOIResolve DOI: https://doi.org/10.1074/jbc.273.17.10475
AuthorSearch for: 1; Search for: ; Search for: 1; Search for: ; Search for: 1
Affiliation
  1. National Research Council Canada. NRC Biotechnology Research Institute
FormatText, Article
ISSN0021-9258
0021-9258
1083-351X
0021-9258
1083-351X
Subjectamino acid sequence; apoptosis; base sequence; binding sites; cell line; DNA; GRB10 adaptor protein; MAP kinase kinase 1; mitogen-activated protein kinase kinases; molecular sequence data; phosphorylation; point mutation; protein binding; protein-serine-threonine kinases; protein-tyrosine kinases; protein; proto-oncogene proteins c-raf; sequence homology, amino acid; sequence homology, nucleic acid
Abstract
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-βB7 affects binding only to the receptors, while a mutation in Thr-βC5 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.
Publication date
PublisherAmerican Society for Biochemistry and Molecular Biology
In
  • The Journal of Biological Chemistry 273, no. 17: 10475–10484.
NoteUI - 98221184
LanguageEnglish
Peer reviewedYes
Identifier10008196
NRC numberNRC-BRI-41397
NPARC number12333673
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Record identifier9cc536fb-fad7-42a3-9954-d6cc91a56153
Record created2009-09-10
Record modified2020-03-20
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