-terminal Pleckstrin Homology Domain, Is Required for Rac-dependent Membrane Ruffling and C-Jun NH

Rho-like GTPases, including Cdc42, Rac, and Rho, regulate signaling pathways that control actin cytoskeletal structures and transcriptional activation. The Tiam1 gene encodes an activator of Rac1, and similarly to constitutively activated (V12)Rac1, overexpression of Tiam1 in fibroblasts induces the formation of membrane ruffles. Tiam1 contains a Dbl homology (DH) domain and adjacent pleckstrin homology (PH) domain, hallmarks for activators of Rho-like GTPases. Unique for Tiam1 are an additional PH domain and a Discs-large homology region in the NH 2 -terminal part of the protein. Here we show that both in fibroblasts and COS cells, membrane localization of Tiam1 is required for the induction of membrane ruffling. A detailed mutational analysis, in combination with confocal laser scanning microscopy and immunoelectron microscopy, demonstrates that the NH 2 -terminal PH domain of Tiam1, but not the DH-adjacent PH domain, is essential for membrane association. This NH 2 -terminal PH domain of Tiam1 can be functionally replaced by the myristoylated membrane localization domain of c-Src, indicating that the primary function of this PH domain is to localize the protein at the membrane. After serum starvation, both membrane association of Tiam1 and ruffling can be induced by serum, suggesting that receptor stimulation induces membrane translocation of Tiam1. Similar to V12Rac1, Tiam1 stimulates the activity of the c-Jun NH 2 -terminal kinase (JNK). This Rac-dependent stimulation of JNK also requires membrane association of Tiam1. We conclude that the regulated membrane localization of Tiam1 through its NH 2 -terminal PH domain determines the activation of distinct Rac-mediated signaling pathways. R ho -like GTPases, which include Cdc42, Rac1, and RhoA, control distinct signal transduction pathways that determine the organization of the actin cytoskeleton in response to various 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 involved in the formation of stress fibers and focal contacts (Ridley and Hall, 1992; Ridley et al., 1992; Kozma et al., 1995; Nobes and Hall, 1995 a ). Each of these GTPases can be activated by specific growth factors, and they may also constitute a hierarchical signaling cascade (Kozma et al., 1995; Nobes and Hall, 1995 a ). The resulting cytoskeletal rearrangements influence both adhesion and motility of cells, implicating Rholike GTPases as key regulators of these processes (Nobes and Hall, 1995 b ). Apart from their effects on the cytoskeleton, both Cdc42 and Rac1 can activate the c-Jun NH 2 -terminal kinase (JNK) 1 pathway, while all three Rho-like GTPases activate serum response factor (Coso et al., 1995; Hill et al., 1995; Minden et al., 1995; Olson et al., 1995). Rho-like proteins thus may also play a role in transcriptional activation of genes. Moreover, both Rac1 and RhoA are involved in the transformation of cells by Ras (Khosravi-Far et al., 1995; Qiu et al., 1995 a , b ), and constitutively active mutants of Rac1 Address all correspondence to Dr. J.G. Collard, The Netherlands Cancer Institute, Division of Cell Biology (H1), 121 Plesmanlaan, 1066 CX Amsterdam, The Netherlands. Fax: (31) 20 5121944. E-mail: [email protected] 1. Abbreviations used in this paper : aa, amino acids; DH, Dbl homology; DHR, Discs-large homology region; GDI, guanine nucleotide dissociation inhibitors; GDS, guanine nucleotide dissociation stimulator; GRF, guanine nucleotide release factor; GST, glutathione S-transferase; HA, hemagglutinin; IP3, inositol (3,4,5)trisphosphate; JNK, c-Jun NH 2 -terminal kinase; MAPK, mitogen-activated protein kinase; PH, pleckstrin homology; PIP2 and PIP3, phosphatidylinositol (4,5)bisphosphate and (3,4,5)trisphosphate. on M ay 1, 2017 D ow nladed fom Published April 21, 1997