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

DOITrouver le DOI : https://doi.org/10.1074/jbc.273.17.10475
AuteurRechercher : 1; Rechercher : ; Rechercher : 1; Rechercher : ; Rechercher : 1
Affiliation
  1. Conseil national de recherches Canada. Institut de recherche en biotechnologie du CNRC
FormatTexte, Article
ISSN0021-9258
0021-9258
1083-351X
0021-9258
1083-351X
Sujetamino 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
Résumé
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.
Date de publication
Maison d’éditionAmerican Society for Biochemistry and Molecular Biology
Dans
  • The Journal of Biological Chemistry 273, nº 17 : 10475–10484.
NoteUI - 98221184
Langueanglais
Publications évaluées par des pairsOui
Identificateur10008196
Numéro du CNRCNRC-BRI-41397
Numéro NPARC12333673
Exporter la noticeExporter en format RIS
Signaler une correctionSignaler une correction (s'ouvre dans un nouvel onglet)
Identificateur de l’enregistrement9cc536fb-fad7-42a3-9954-d6cc91a56153
Enregistrement créé2009-09-10
Enregistrement modifié2020-03-20

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