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 a dapter proteins characterized by a proline-rich region, a central PH d omain, and a carboxyl-terminal Src homology 2 (SH2) domain. Their inte raction 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 phosphot yrosine-independent manner, with both the Raf1 and MEK1 kinases. Mutat ion of the MEK1 Thr-386 residue, which is phosphorylated by mitogen-ac tivated protein kinase in vitro, reduces binding to Grb10 in a two-hyb rid assay. Interaction of Grb10 with Raf1 is constitutive, while inter action between Grb10 and MEK1 needs insulin treatment of the cells and follows mitogen-activated protein kinase activation. Random mutagenes is of the SH2 domain demonstrated that the Arg-beta B5 and Asp-EF2 res idues are necessary for binding to the epidermal growth factor and ins ulin receptors as well as to the two kinases. In addition, we show tha t a mutation in Ser-beta B7 affects binding only to the receptors, whi le a mutation in Thr-beta CB abrogates binding only to MEK1. Finally, transfection of Grb10 genes with specific mutations in their SH2 domai ns induces apoptosis in HTC-IR and COS-7 cells. These effects can be c ompeted by coexpression of the wild type protein, suggesting that thes e mutants act by sequestering necessary signaling components.