Regulated Membrane Localization of Tiam1, Mediated by the NH2-terminal Pleckstrin Homology Domain, Is Required for Rac-dependent Membrane Ruffling and C-Jun NH2-terminal Kinase Activation

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 NH2-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 NH2-terminal PH domain of Tiam1, but not the DH-adjacent PH domain, is essential for membrane association. This NH2-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 NH2-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 NH2-terminal PH domain determines the activation of distinct Rac-mediated signaling pathways.

[1]  M. Olson,et al.  Faciogenital dysplasia protein (FGD1) and Vav, two related proteins required for normal embryonic development, are upstream regulators of Rho GTPases , 1996, Current Biology.

[2]  C. Nobes,et al.  Phosphatidylinositol 3-kinase signals activate a selective subset of Rac/Rho-dependent effector pathways , 1996, Current Biology.

[3]  R. Buchsbaum,et al.  The N-terminal pleckstrin, coiled-coil, and IQ domains of the exchange factor Ras-GRF act cooperatively to facilitate activation by calcium , 1996, Molecular and cellular biology.

[4]  Yi Zheng,et al.  The Pleckstrin Homology Domain Mediates Transformation by Oncogenic Dbl through Specific Intracellular Targeting* , 1996, The Journal of Biological Chemistry.

[5]  B. Seed,et al.  αLβ2 Integrin/LFA-1 Binding to ICAM-1 Induced by Cytohesin-1, a Cytoplasmic Regulatory Molecule , 1996, Cell.

[6]  J. Schlessinger,et al.  PH Domains: Diverse Sequences with a Common Fold Recruit Signaling Molecules to the Cell Surface , 1996, Cell.

[7]  Y. Zheng,et al.  The Dbl family of oncogenes. , 1996, Current opinion in cell biology.

[8]  J. Collard.  Signaling pathways regulated by Rho-like proteins. , 1996, International journal of oncology.

[9]  P. Sigler,et al.  Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain , 1995, Cell.

[10]  John G. Collard,et al.  Oncogenic activity of Tiam1 and Rac1 in NIH3T3 cells. , 1995, Oncogene.

[11]  F. McCormick,et al.  A role for Rho in Ras transformation. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[12]  C. Der,et al.  Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation , 1995, Molecular and cellular biology.

[13]  A. Ashworth,et al.  An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1 , 1995, Science.

[14]  C. Tognon,et al.  Expression Cloning of lfc, a Novel Oncogene with Structural Similarities to Guanine Nucleotide Exchange Factors and to the Regulatory Region of Protein Kinase C (*) , 1995, The Journal of Biological Chemistry.

[15]  C. Nobes,et al.  Rho, rac and cdc42 GTPases: regulators of actin structures, cell adhesion and motility. , 1995, Biochemical Society transactions.

[16]  N. Thanki,et al.  Structural studies on the PH domains of Db1, Sos1, IRS-1, and beta ARK1 and their differential binding to G beta gamma subunits. , 1995, Biochemistry.

[17]  R. Lefkowitz,et al.  Mutational Analysis of the Pleckstrin Homology Domain of the β-Adrenergic Receptor Kinase. , 1995, The Journal of Biological Chemistry.

[18]  T. Sasaki,et al.  Translocation of activated Rho from the cytoplasm to membrane ruffling area, cell-cell adhesion sites and cleavage furrows. , 1995, Oncogene.

[19]  R. Treisman,et al.  The Rho family GTPases RhoA, Racl , and CDC42Hsregulate transcriptional activation by SRF , 1995, Cell.

[20]  M. Karin,et al.  Selective activation of the JNK signaling cascadeand c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs , 1995, Cell.

[21]  P. Crespo,et al.  The small GTP-binding proteins Rac1 and Cdc42regulate the activity of the JNK/SAPK signaling pathway , 1995, Cell.

[22]  John G. Collard,et al.  A role for Rac in Tiaml-induced membrane ruffling and invasion , 1995, Nature.

[23]  P. Adamson,et al.  Ultrastructural localization of ras-related proteins using epitope-tagged plasmids. , 1995, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[24]  M. Olson,et al.  Direct Involvement of the Small GTP-binding Protein Rho in lbc Oncogene Function (*) , 1995, The Journal of Biological Chemistry.

[25]  C. Nobes,et al.  Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia , 1995, Cell.

[26]  P. Hawkins,et al.  PDGF stimulates an increase in GTP–Rac via activation of phosphoinositide 3-kinase , 1995, Current Biology.

[27]  L. Lim,et al.  The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts , 1995, Molecular and cellular biology.

[28]  Giulio Superti‐Furga,et al.  Structure‐function relationships in Src family and related protein tyrosine kinases , 1995, BioEssays : news and reviews in molecular, cellular and developmental biology.

[29]  F. McCormick,et al.  An essential role for Rac in Ras transformation , 1995, Nature.

[30]  C P Ponting,et al.  DHR domains in syntrophins, neuronal NO synthases and other intracellular proteins. , 1995, Trends in biochemical sciences.

[31]  R. Kay,et al.  Retroviral transduction and oncogenic selection of a cDNA encoding Dbs, a homolog of the Dbl guanine nucleotide exchange factor. , 1995, Oncogene.

[32]  R. Lefkowitz,et al.  Mutational analysis of the pleckstrin homology domain of the beta-adrenergic receptor kinase. Differential effects on G beta gamma and phosphatidylinositol 4,5-bisphosphate binding. , 1995, The Journal of biological chemistry.

[33]  K. Sakaguchi,et al.  A novel oncogene, ost, encodes a guanine nucleotide exchange factor that potentially links Rho and Rac signaling pathways. , 1994, The EMBO journal.

[34]  P. Hajduk,et al.  Pleckstrin homology domains bind to phosphatidylinositol-4,5-bisphosphate , 1994, Nature.

[35]  T J Gibson,et al.  PH domain: the first anniversary. , 1994, Trends in biochemical sciences.

[36]  Michael J. Fry,et al.  Phosphatidylinositol-3-OH kinase direct target of Ras , 1994, Nature.

[37]  John G. Collard,et al.  Identification of an invasion-inducing gene, Tiam-1, that encodes a protein with homology to GDP-GTP exchangers for Rho-like proteins , 1994, Cell.

[38]  M. Kasuga,et al.  Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling , 1994, Current Biology.

[39]  G M Bokoch,et al.  Regulation of the human neutrophil NADPH oxidase by the Rac GTP-binding proteins. , 1994, Current opinion in cell biology.

[40]  Y. Zheng,et al.  Control of the yeast bud-site assembly GTPase Cdc42. Catalysis of guanine nucleotide exchange by Cdc24 and stimulation of GTPase activity by Bem3. , 1994, The Journal of biological chemistry.

[41]  S. Aaronson,et al.  Cellular transformation and guanine nucleotide exchange activity are catalyzed by a common domain on the dbl oncogene product. , 1994, The Journal of biological chemistry.

[42]  Mark S. Boguski,et al.  Proteins regulating Ras and its relatives , 1993, Nature.

[43]  S. Y. Cajal,et al.  Tumorigenic activity of rho genes from Aplysia californica. , 1993, Oncogene.

[44]  P. Adamson,et al.  Intracellular localization of the P21rho proteins , 1992, The Journal of cell biology.

[45]  Anne J. Ridley,et al.  The small GTP-binding protein rac regulates growth factor-induced membrane ruffling , 1992, Cell.

[46]  S. Volinia,et al.  Phosphatidylinositol 3-kinase: Structure and expression of the 110 kd catalytic subunit , 1992, Cell.

[47]  Anne J. Ridley,et al.  The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors , 1992, Cell.

[48]  P. Adamson,et al.  Intracellular Localization of the P 2 lrh ~ Proteins , 1992 .

[49]  S. Aaronson,et al.  Catalysis of guanine nucleotide exchange on the CDC42Hs protein by the dbloncogene product , 1991, Nature.

[50]  L. Hunt,et al.  A region of proto-dbl essential for its transforming activity shows sequence similarity to a yeast cell cycle gene, CDC24, and the human breakpoint cluster gene, bcr. , 1991, The New biologist.

[51]  B. Seed,et al.  Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. , 1987, Proceedings of the National Academy of Sciences of the United States of America.