XPLN, a Guanine Nucleotide Exchange Factor for RhoA and RhoB, But Not RhoC*

Rho proteins cycle between an inactive, GDP-bound state and an active, GTP-bound state. Activation of these GTPases is mediated by guanine nucleotide exchange factors (GEFs), which promote GDP to GTP exchange. In this study we have characterized XPLN, a Rho family GEF. Like other Rho GEFs, XPLN contains a tandem Dbl homology and pleckstrin homology domain topography, but lacks homology with other known functional domains or motifs. XPLN protein is expressed in the brain, skeletal muscle, heart, kidney, platelets, and macrophage and neuronal cell lines. In vitro, XPLN stimulates guanine nucleotide exchange on RhoA and RhoB, but not RhoC, RhoG, Rac1, or Cdc42. Consistent with these data, XPLN preferentially associates with RhoA and RhoB. The specificity of XPLN for RhoA and RhoB, but not RhoC, is surprising given that they share over 85% sequence identity. We determined that the inability of XPLN to exchange RhoC is mediated by isoleucine 43 in RhoC, a position occupied by valine in RhoA and RhoB. When expressed in cells, XPLN activates RhoA and RhoB, but not RhoC, and stimulates the assembly of stress fibers and focal adhesions in a Rho kinase-dependent manner. We also found that XPLN possesses transforming activity, as determined by focus formation assays. In conclusion, here we describe a Rho family GEF that can discriminate between the closely related RhoA, RhoB, and RhoC, possibly giving insight to the divergent functions of these three proteins.

[1]  S. Powers,et al.  Ras-15A protein shares highly similar dominant-negative biological properties with Ras-17N and forms a stable, guanine-nucleotide resistant complex with CDC25 exchange factor. , 1994, Oncogene.

[2]  David Michaelson,et al.  Differential Localization of Rho Gtpases in Live Cells , 2001, The Journal of cell biology.

[3]  A. Hall,et al.  Guanine nucleotide exchange factors for Rho GTPases: turning on the switch. , 2002, Genes & development.

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

[5]  W. Kiosses,et al.  Regulation of the small GTP‐binding protein Rho by cell adhesion and the cytoskeleton , 1999, The EMBO journal.

[6]  Takayuki Kato,et al.  Cooperation between mDia1 and ROCK in Rho-induced actin reorganization , 1999, Nature Cell Biology.

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

[8]  S. Merajver,et al.  RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype. , 2000, Cancer research.

[9]  A. Hall,et al.  Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. , 1998, Science.

[10]  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.

[11]  S. Antonarakis,et al.  Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP , 2001, Nature Cell Biology.

[12]  S. Smerdon,et al.  GTPase-activating proteins and their complexes. , 1998, Current opinion in structural biology.

[13]  F. McCormick,et al.  Regulation of Tiam1 Nucleotide Exchange Activity by Pleckstrin Domain Binding Ligands* , 2000, The Journal of Biological Chemistry.

[14]  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.

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

[16]  H. Ropers,et al.  Isolation of two novel human RhoGEFs, ARHGEF3 and ARHGEF4, in 3p13-21 and 2q22. , 2000, Biochemical and biophysical research communications.

[17]  C. Der,et al.  Structural basis for the selective activation of Rho GTPases by Dbl exchange factors , 2002, Nature Structural Biology.

[18]  M. Caligiuri,et al.  Leukemia-associated Rho Guanine Nucleotide Exchange Factor, a Dbl Family Protein Found Mutated in Leukemia, Causes Transformation by Activation of RhoA* , 2001, The Journal of Biological Chemistry.

[19]  Xing Shen,et al.  The Activity of Guanine Exchange Factor NET1 Is Essential for Transforming Growth Factor-β-mediated Stress Fiber Formation* , 2001, The Journal of Biological Chemistry.

[20]  John G. Collard,et al.  Invasion of T‐lymphoma cells: cooperation between Rho family GTPases and lysophospholipid receptor signaling , 1998, The EMBO journal.

[21]  Kozo Kaibuchi,et al.  Regulation of Myosin Phosphatase by Rho and Rho-Associated Kinase (Rho-Kinase) , 1996, Science.

[22]  K. Jakobs,et al.  Rho‐specific binding and guanine nucleotide exchange catalysis by KIAA0380, a Dbl family member , 1999, FEBS letters.

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

[24]  W. Arthur,et al.  RhoA inactivation by p190RhoGAP regulates cell spreading and migration by promoting membrane protrusion and polarity. , 2001, Molecular biology of the cell.

[25]  C. Der,et al.  Biological assays for Ras transformation. , 1995, Methods in enzymology.

[26]  S. Merajver,et al.  Characterization of RhoC expression in benign and malignant breast disease: a potential new marker for small breast carcinomas with metastatic ability. , 2002, The American journal of pathology.

[27]  C. Der,et al.  Ras and Rho regulation of the cell cycle and oncogenesis. , 2001, Cancer letters.

[28]  S. Sebti,et al.  Both Farnesylated and Geranylgeranylated RhoB Inhibit Malignant Transformation and Suppress Human Tumor Growth in Nude Mice* , 2000, The Journal of Biological Chemistry.

[29]  P. Thompson,et al.  RhoA Sustains Integrin αIIbβ3Adhesion Contacts under High Shear* , 2002, The Journal of Biological Chemistry.

[30]  A. Hall,et al.  The Rho Exchange Factor Net1 Is Regulated by Nuclear Sequestration* , 2002, The Journal of Biological Chemistry.

[31]  Shuh Narumiya,et al.  Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension , 1997, Nature.

[32]  A. Huttenlocher,et al.  Integrin-mediated adhesion regulates cell polarity and membrane protrusion through the Rho family of GTPases. , 2001, Molecular biology of the cell.

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

[34]  E. Sahai,et al.  ROCK and Dia have opposing effects on adherens junctions downstream of Rho , 2002, Nature Cell Biology.

[35]  C. Der,et al.  Molecular basis for Rac1 recognition by guanine nucleotide exchange factors , 2001, Nature Structural Biology.

[36]  S. Takai,et al.  Isolation of a novel oncogene, NET1, from neuroepithelioma cells by expression cDNA cloning. , 1996, Oncogene.

[37]  R. Treisman,et al.  Activation of RhoA and SAPK/JNK signalling pathways by the RhoA‐specific exchange factor mNET1 , 1998, The EMBO journal.

[38]  Yoshiharu Matsuura,et al.  Phosphorylation and Activation of Myosin by Rho-associated Kinase (Rho-kinase)* , 1996, The Journal of Biological Chemistry.

[39]  G. Prendergast,et al.  Actin' up: RhoB in cancer and apoptosis , 2001, Nature Reviews Cancer.

[40]  Daniel Zicha,et al.  A Role for Cdc42 in Macrophage Chemotaxis , 1998, The Journal of cell biology.

[41]  B. Zetter,et al.  Motility and invasion are differentially modulated by Rho family GTPases , 2000, Oncogene.

[42]  Eric S. Lander,et al.  Genomic analysis of metastasis reveals an essential role for RhoC , 2000, Nature.

[43]  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.

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

[45]  R. Cerione,et al.  Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using fluorescence spectroscopy. , 1994, Biochemistry.