Gem is a Ras-related protein whose expression is induced in several cell types upon activation by extracellular stimuli. With the aim of isolating the cellular partners of Gem that mediate its biological activity we performed a yeast two-hybrid screen and identified a novel protein of 970 amino acids, Gmip, that interacts with Gem through its N-terminal half, and presents a cysteine-rich domain followed by a Rho GTPase-activating protein (RhoGAP) domain in its C-terminal half. The RhoGAP domain of Gmip stimulates in vitro the GTPase activity of RhoA, but is inactive towards other Rho family proteins such as Rac1 and Cdc42; it is also specific for RhoA in vivo. The same is true for the full-length protein, which is furthermore able to down-regulate RhoA-dependent stress fibres in Ref-52 rat fibroblasts. These findings suggest that the signalling pathways controlled by two proteins of the Ras superfamily, RhoA and Gem, are linked via the action of the RhoGAP protein Gmip (Gem-interacting protein).
Abbreviations used: CRD, cysteine-rich domain; DTT, dithiothreitol; GAP, GTPase-activating-protein; Gmip, Gem-interacting protein; GST, glutathione S-tranferase; PAK1, p21-activated kinase 1; PBD, p21-binding domain; PDZ, PSD95/Dlg/ZO-1 homology domain; PKC, protein kinase C; PTPL1, protein tyrosine phosphatase L1; RBD, Rho-binding domain; RGK, Rad/Gem/Kir; ROCK, RhoA-associated kinase.
Present address: Section of Cell and Molecular Biology, Biomedical Sciences Division, Sir Alexander Fleming Building, Imperial College Faculty of Medicine, South Kensington, London SW7 2AZ, U.K.
Present address: IGH du CNRS, UPR1142, 141, rue de la Cardonille, 34396 Montpellier Cedex 5, France.
The nucleotide sequence of human Gmip was deposited in the GenBank Nucleotide Sequence Database under accession no. AF132541.