Based on the in vitro binding data of PH domains, it is tempting to speculate that G subunits and/or specific phospholipids regulate the association of Tiam1 with the plasma membrane, as depicted in Fig. the DH-adjacent PH website, is essential for membrane association. This NH2-terminal PH website of Tiam1 can be functionally replaced from the myristoylated membrane localization website of c-Src, indicating that the primary function of this PH website is definitely to localize the protein in the membrane. After serum starvation, both membrane association of Tiam1 and ruffling can be induced by serum, suggesting that receptor activation induces membrane translocation of Tiam1. Much like V12Rac1, Tiam1 stimulates the activity of the c-Jun NH2-terminal kinase (JNK). This Rac-dependent activation of JNK also requires membrane association of Tiam1. We conclude the controlled membrane localization of Tiam1 through its NH2-terminal PH website decides the activation of unique Rac-mediated signaling pathways. Rho-like GTPases, which include Cdc42, Rac1, and RhoA, control unique transmission transduction pathways that GFAP determine the organization of the actin cytoskeleton in response to numerous extracellular stimuli. In fibroblast cells, activation of Cdc42 and Rac results in the formation of focal complexes and filopodia or lamellipodia, respectively, while Rho is definitely involved in the formation of stress materials and focal contacts (Ridley and Hall, 1992; Ridley et al., 1992; Kozma et al., 1995; Nobes and Hall, 1995was originally recognized in T-lymphoma cells as an invasion- and metastasis-inducing gene (Habets et al., 1994). The expected Tiam1 protein consists of 1,591 amino acids and contains a Dbl homology (DH) website. This website is present in GDS proteins for Rho-like GTPases and is considered to become the catalytic website (Hart et al., 1994). DH domains are found inside a rapidly expanding group of proteins, many of which are encoded by transforming genes that were recognized after transfection of genomic DNA or cDNA libraries into NIH3T3 cells (Cerione and Zheng, 1996; Collard, 1996). A number of these proteins, like Dbl, Ost, Cdc24, Lbc, and Vav were shown to activate Rho-like GTPases (Hart et al., 1991; Horii et al., 1994; Zheng et al., 1994, 1995; Olson et al., 1996). Tiam1 is the only GDS protein recognized that specifically activates Rac in vitro as well as with vivo (Michiels et al., 1995). In fibroblasts, Tiam1 induces the formation of membrane ruffles, similarly to constitutively triggered V12Rac1 (Michiels et al., 1995). The Tiam1-induced phenotype is definitely inhibited by coexpression of dominant-negative mutant (N17)Rac1, but not by coexpression of (N17)Cdc42 or by inactivation of RhoA with C3 transferase (Michiels et al., 1995; Collard, 1996). This indicates the Tiam1-induced membrane ruffling is definitely primarily caused by activation of Rac and that additional Rho-like GTPases are not involved. Moreover, V12Rac1, but not V14RhoA, induces an invasive phenotype in T-lymphoma cells, much like Tiam1, suggesting that Tiam1-induced invasiveness is also caused by activation of Rac (Michiels et al., 1995). Apart from the DH website, Tiam1 contains several other conserved motifs, including a consensus myristoylation sequence in the NH2 terminus, a Discs-large homology region (DHR), and two pleckstrin homology (PH) domains (Habets et al., 1994; Collard, 1996; observe Fig. ?Fig.11 depicts 7-Methyluric Acid the Tiam1 constructs encoding the COOH-terminal 1,199 amino acids (and LKB Biotechnology Inc., Uppsala, Sweden). Cells and Transfection Experiments NIH3T3 cells, stably expressing the C1199 Tiam1 protein and C682 Tiam1 protein, have been explained previously (Vehicle Leeuwen et al., 1995). NIH3T3 cells and COS-7 cells were cultured inside a humidified CO2 incubator in DME supplemented with 10% newborn calf serum or 10% fetal calf serum, respectively. For transient manifestation assays, NIH3T3 cells were seeded in dishes with or without coverslips (8,000 cells/cm2). 18 h after seeding, cells were transfected with lipofectamin (Existence Systems, Inc., Grand Island, NY). COS-7 cells were seeded in dishes with or without coverslips and transfected using DEAE dextran (Seed and Aruffo, 1987). 16 h after transfection, the medium was either 7-Methyluric Acid replaced and cells were incubated for an additional 30 h before washing and fixation, or cells were incubated for 24 h in serum-free medium, followed by the addition of 7-Methyluric Acid serum for 2 h. Western blot analyses were performed to monitor the size and manifestation levels of.