Endogenous RhoG is dispensable for integrin-mediated cell spreading but contributes to Rac-independent migration

Rac activation by integrins is essential for cell spreading, migration, growth and survival. Based mainly on overexpression of dominant-negative mutants, RhoG has been proposed to mediate integrin-dependent Rac activation upstream of ELMO and Dock180. RhoG-knockout mice, however, display no significant developmental or functional abnormalities. To clarify the role of RhoG in integrin-mediated signaling, we developed a RhoG-specific antibody, which, together with shRNA-mediated knockdown, allowed analysis of the endogenous protein. Despite dramatic effects of dominant-negative constructs, nearly complete RhoG depletion did not substantially inhibit cell adhesion, spreading, migration or Rac activation. Additionally, RhoG was not detectably activated by adhesion to fibronectin. Using Rac1–/– cells, we found that constitutively active RhoG induced membrane ruffling via both Rac-dependent and -independent pathways. Additionally, endogenous RhoG was important for Rac-independent cell migration. However, RhoG did not significantly contribute to cell spreading even in these cells. These data therefore clarify the role of RhoG in integrin signaling and cell motility.

[1]  S. Aresta,et al.  Interaction of ezrin with the novel guanine nucleotide exchange factor PLEKHG6 promotes RhoG-dependent apical cytoskeleton rearrangements in epithelial cells. , 2007, Molecular biology of the cell.

[2]  Michael J. Allingham,et al.  RhoG regulates endothelial apical cup assembly downstream from ICAM1 engagement and is involved in leukocyte trans-endothelial migration , 2007, The Journal of cell biology.

[3]  M. Katan,et al.  Role of Phospholipase Cγ1 in Cell Spreading Requires Association with a β-Pix/GIT1-Containing Complex, Leading to Activation of Cdc42 and Rac1 , 2007, Molecular and Cellular Biology.

[4]  X. Bustelo,et al.  GTP‐binding proteins of the Rho/Rac family: regulation, effectors and functions in vivo , 2007, BioEssays : news and reviews in molecular, cellular and developmental biology.

[5]  Lei Wang,et al.  Cell type-specific functions of Rho GTPases revealed by gene targeting in mice. , 2007, Trends in cell biology.

[6]  Nicholas H Brown,et al.  Integrins and the actin cytoskeleton. , 2007, Current opinion in cell biology.

[7]  J. Galán,et al.  Differential activation and function of Rho GTPases during Salmonella–host cell interactions , 2006, The Journal of cell biology.

[8]  N. Cordes.  Integrin-mediated cell-matrix interactions for prosurvival and antiapoptotic signaling after genotoxic injury. , 2006, Cancer letters.

[9]  A. Ridley.  Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. , 2006, Trends in cell biology.

[10]  Stephen D. Smith,et al.  Rac1 and Rac2 regulate macrophage morphology but are not essential for migration , 2006, Journal of Cell Science.

[11]  Feng Chen,et al.  Rac1-null mouse embryonic fibroblasts are motile and respond to platelet-derived growth factor. , 2006, Molecular biology of the cell.

[12]  S. Nagata,et al.  Opposite Effects of Rho Family GTPases on Engulfment of Apoptotic Cells by Macrophages* , 2006, Journal of Biological Chemistry.

[13]  P. Hordijk,et al.  Jcb: Article Introduction , 2002 .

[14]  B. Kaina,et al.  Rho GTPases: promising cellular targets for novel anticancer drugs. , 2006, Current cancer drug targets.

[15]  Hironori Katoh,et al.  Activation of Rac1 by RhoG regulates cell migration , 2006, Journal of Cell Science.

[16]  Chittibabu Guda,et al.  CZH proteins: a new family of Rho-GEFs , 2005, Journal of Cell Science.

[17]  A. Hall,et al.  Rho GTPases and the control of cell behaviour. , 2005, Biochemical Society transactions.

[18]  Alan Hall,et al.  Rho GTPases: biochemistry and biology. , 2005, Annual review of cell and developmental biology.

[19]  R. Juliano,et al.  Clinging to life: cell to matrix adhesion and cell survival , 2005, Cancer and Metastasis Reviews.

[20]  C. Rabiner,et al.  Kalirin: A Dual Rho Guanine Nucleotide Exchange Factor That Is So Much More Than the Sum of Its Many Parts , 2005, The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry.

[21]  C. Der,et al.  GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors , 2005, Nature Reviews Molecular Cell Biology.

[22]  M. Hengartner,et al.  Phagocytosis of Apoptotic Cells Is Regulated by a UNC-73/TRIO-MIG-2/RhoG Signaling Module and Armadillo Repeats of CED-12/ELMO , 2004, Current Biology.

[23]  Bryce M Paschal,et al.  The Novel Cdc42 Guanine Nucleotide Exchange Factor, Zizimin1, Dimerizes via the Cdc42-binding CZH2 Domain* , 2004, Journal of Biological Chemistry.

[24]  K. Wennerberg,et al.  SGEF, a RhoG guanine nucleotide exchange factor that stimulates macropinocytosis. , 2004, Molecular biology of the cell.

[25]  P. Emson,et al.  Immunological Function in Mice Lacking the Rac-Related GTPase RhoG , 2004, Molecular and Cellular Biology.

[26]  K. Kaibuchi,et al.  Positive role of IQGAP1, an effector of Rac1, in actin-meshwork formation at sites of cell-cell contact. , 2003, Molecular biology of the cell.

[27]  G. Borisy,et al.  Cell Migration: Integrating Signals from Front to Back , 2003, Science.

[28]  X. Bustelo,et al.  Structural Basis for the Signaling Specificity of RhoG and Rac1 GTPases* , 2003, Journal of Biological Chemistry.

[29]  Hironori Katoh,et al.  RhoG activates Rac1 by direct interaction with the Dock180-binding protein Elmo , 2003, Nature.

[30]  V. Tybulewicz,et al.  Vav1: a key signal transducer downstream of the TCR , 2003, Immunological reviews.

[31]  K. Wennerberg,et al.  RhoG Signals in Parallel with Rac1 and Cdc42* , 2002, The Journal of Biological Chemistry.

[32]  S. Diriong,et al.  The Human Rho-GEF Trio and Its Target GTPase RhoG Are Involved in the NGF Pathway, Leading to Neurite Outgrowth , 2002, Current Biology.

[33]  Sheila M. Thomas,et al.  The Adaptor Protein Paxillin Is Essential for Normal Development in the Mouse and Is a Critical Transducer of Fibronectin Signaling , 2002, Molecular and Cellular Biology.

[34]  M. Schwartz,et al.  Integrins and cell proliferation: regulation of cyclin-dependent kinases via cytoplasmic signaling pathways. , 2001, Journal of cell science.

[35]  P. Marignani,et al.  Vav2 is required for cell spreading , 2001, The Journal of cell biology.

[36]  M. Symons,et al.  The Small GTPase Rac Suppresses Apoptosis Caused by Serum Deprivation in Fibroblasts , 2001, Molecular medicine.

[37]  Y. Yamaguchi,et al.  Small GTPase RhoG Is a Key Regulator for Neurite Outgrowth in PC12 Cells , 2000, Molecular and Cellular Biology.

[38]  F. Alt,et al.  Cdc42 is required for PIP2-induced actin polymerization and early development but not for cell viability , 2000, Current Biology.

[39]  A. Hall,et al.  Rho GTPases and their effector proteins. , 2000, The Biochemical journal.

[40]  M. Schwartz,et al.  Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK , 2000, The EMBO journal.

[41]  C. Der,et al.  Vav2 Is an Activator of Cdc42, Rac1, and RhoA* , 2000, The Journal of Biological Chemistry.

[42]  A. Debant,et al.  TrioGEF1 controls Rac- and Cdc42-dependent cell structures through the direct activation of rhoG. , 2000, Journal of cell science.

[43]  X. Bustelo,et al.  Biological and Regulatory Properties of Vav-3, a New Member of the Vav Family of Oncoproteins , 1999, Molecular and Cellular Biology.

[44]  Kenji Nakamura,et al.  Rac1 is required for the formation of three germ layers during gastrulation , 1998, Oncogene.

[45]  K. Schuebel,et al.  Phosphorylation‐dependent and constitutive activation of Rho proteins by wild‐type and oncogenic Vav‐2 , 1998, The EMBO journal.

[46]  M. Matsuda,et al.  Activation of Rac1 by a Crk SH3-binding protein, DOCK180. , 1998, Genes & development.

[47]  G M Bokoch,et al.  Activation of Rac and Cdc42 by integrins mediates cell spreading. , 1998, Molecular biology of the cell.

[48]  P. Roux,et al.  RhoG GTPase controls a pathway that independently activates Rac1 and Cdc42Hs. , 1998, Molecular biology of the cell.

[49]  E. Adamson,et al.  Vinculin knockout results in heart and brain defects during embryonic development. , 1998, Development.

[50]  S. Aizawa,et al.  Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice , 1995, Nature.

[51]  M. Katan,et al.  Role of phospholipase Cgamma1 in cell spreading requires association with a beta-Pix/GIT1-containing complex, leading to activation of Cdc42 and Rac1. , 2007, Molecular and cellular biology.

[52]  M. Schwartz,et al.  The Novel Cdc 42 Guanine Nucleotide Exchange Factor , Zizimin 1 , Dimerizes via the Cdc 42-binding CZH 2 Domain * , 2004 .

[53]  M. Matsuda,et al.  Activation of Rac 1 by a Crk SH 3-binding protein , DOCK 180 , 1998 .