Abstract
The Rho family of small GTPases plays a central role in intracellular signal transduction, particularly in reorganization of the actin cytoskeleton. Rho activity induces cell contractility, whereas Rac promotes cellular protrusion, which counteracts Rho signaling. In this regard, the reciprocal balance between these GTPases determines cell morphology and migratory behavior. Here we demonstrate that Tiam1/Rac1 signaling is able to antagonize Rho activity directly at the GTPase level in COS-7 cells. p190-RhoGAP plays a central regulatory role in this signaling pathway. Interfering with its activation by Src-kinase-dependent tyrosine phosphorylation or its recruitment to the membrane through interaction with the SH2 domains of p120-RasGAP blocks the Tiam1-mediated rapid downregulation of Rho. This process is mediated by Rac1, but not by Rac2 or Rac3 isoforms. Our data provide evidence for a biochemical pathway of the reciprocal regulation of two related small GTPases, which are key elements in cell migration.
References
Bishop, A.L. and Hall, A. (2000). Rho GTPases and their effector proteins. Biochem. J.348, 241–255.10.1042/bj3480241Search in Google Scholar
Bollag, G., Crompton, A.M., Peverly Mitchell, D., Habets, G.G., and Symons, M. (2000). Activation of Rac1 by human Tiam1. Methods Enzymol.325, 51–61.10.1016/S0076-6879(00)25430-XSearch in Google Scholar
Bouton, A.H., Kanner, S.B., Vines, R.R., Wang, H.C., Gibbs, J.B., and Parsons, J.T. (1991). Transformation by pp60src or stimulation of cells with epidermal growth factor induces the stable association of tyrosine phosphorylated cellular proteins with GTPase activating protein. Mol. Cell. Biol.11, 945–953.Search in Google Scholar
Bryant, S.S., Briggs, S., Smithgall, T.E., Martin, G.A., McCormick, F., Chang, J.H., Parsons, S.J., and Jove, R. (1995). Two SH2 domains of p120 Ras GTPase activating protein bind synergistically to tyrosine phosphorylated p190 Rho GTPase activating protein. J. Biol. Chem.270, 17947–17952.10.1074/jbc.270.30.17947Search in Google Scholar
Chang, J.H., Gill, S., Settleman, J., and Parsons, S.J. (1995). c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190 RhoGAP, and p120 RasGAP following epidermal growth factor stimulation. J. Cell Biol.130, 355–368.10.1083/jcb.130.2.355Search in Google Scholar
Chiarugi, P., Cirri, P., Taddei, L., Giannoni, E., Camici, G., Manao, G., Raugei, G., and Ramponi, G. (2000). The low Mr protein tyrosine phosphatase is involved in Rho mediated cytoskeleton rearrangement after integrin and platelet derived growth factor stimulation. J. Biol. Chem.275, 4640–4646.10.1074/jbc.275.7.4640Search in Google Scholar
Defilippi, P., Venturino, M., Gulino, D., Duperray, A., Boquet, P., Fiorentini, C., Volpe, G., Palmieri, M., Silengo, L., and Tarone, G. (1997). Dissection of pathways implicated in integrin-mediated actin cytoskeleton assembly. Involvement of protein kinase C, Rho GTPase, and tyrosine phosphorylation. J. Biol. Chem.272, 21726–21734.10.1074/jbc.272.35.21726Search in Google Scholar
Dovas, A. and Couchman, J.R. (2005). RhoGDI: multiple functions in the regulation of Rho family GTPase activities. Biochem. J.390, 1–9.10.1042/BJ20050104Search in Google Scholar
Driessens, M.H., Olivo, C., Nagata, K., Inagaki, M., and Collard, J.G. (2002). B plexins activate Rho through PDZ-RhoGEF. FEBS Lett.529,168–172.10.1016/S0014-5793(02)03323-9Search in Google Scholar
Eberth, A., Dvorsky, R., Becker, C.F., Beste, A., Goody, R.S., and Ahmadian, M.R. (2005). Monitoring the real-time kinetics of the hydrolysis reaction of guanine nucleotide-binding proteins. Biol. Chem.386, 1105–1114.10.1515/BC.2005.127Search in Google Scholar PubMed
Ellis, C., Moran, M., McCormick, F., and Pawson, T. (1990). Phosphorylation of GAP and GAP associated proteins by transforming and mitogenic tyrosine kinases. Nature343, 377–381.10.1038/343377a0Search in Google Scholar PubMed
Engers, R., Springer, E., Michiels, F., Collard, J.G., and Gabbert, H.E. (2001). Rac affects invasion of human renal cell carcinomas by upregulating tissue inhibitor of metalloproteinases TIMP 1 and TIMP 2 expression. J. Biol. Chem.276, 41889–41897.10.1074/jbc.M105049200Search in Google Scholar
Etienne Manneville, S. and Hall, A. (2002). Rho GTPases in cell biology. Nature420, 629–635.10.1038/nature01148Search in Google Scholar
Fiegen, D., Haeusler, L.C., Blumenstein, L., Herbrand, U., Dvorsky, R., Vetter, I.R., and Ahmadian, M.R. (2004). Alternative splicing of Rac1 creates a self-activating GTPase. J. Biol. Chem.279, 4743–4749.10.1074/jbc.M310281200Search in Google Scholar
Fukuhara, S., Murga, C., Zohar, M., Igishi, T., and Gutkind, J.S. (1999). A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho. J. Biol. Chem.274, 5868–5879.10.1074/jbc.274.9.5868Search in Google Scholar
Fukuhara, S., Chikumi, H., and Gutkind, J.S. (2001). RGS containing RhoGEFs: the missing link between transforming G proteins and Rho? Oncogene20, 1661–1668.10.1038/sj.onc.1204182Search in Google Scholar
Giannoni, E., Chiarugi, P., Cozzi, G., Magnelli, L., Taddei, M.L., Fiaschi, T., Buricchi, F., Raugei, G. and Ramponi, G. (2003). Lymphocyte function-associated antigen-1-mediated T cell adhesion is impaired by low molecular weight phosphotyrosine phosphatase-dependent inhibition of FAK activity. J. Biol. Chem.278, 36763–36776.10.1074/jbc.M302686200Search in Google Scholar
Habets, G.G., Scholtes, E.H., Zuydgeest, D., van der Kammen, R.A., Stam, J.C., Berns, A. and Collard, J.G. (1994). Identification of an invasion-inducing gene, Tiam-1, that encodes a protein with homology to GDP-GTP exchangers for Rho-like proteins. Cell77, 537–549.10.1016/0092-8674(94)90216-XSearch in Google Scholar
Haeusler, L.C., Blumenstein, L., Stege, P., Dvorsky, R., and Ahmadian, M.R. (2003). Comparative functional analysis of the Rac GTPases. FEBS Lett.555, 556–560.10.1016/S0014-5793(03)01351-6Search in Google Scholar
Hall, A. (1998). Rho GTPases and the actin cytoskeleton. Science279, 509–514.10.1126/science.279.5350.509Search in Google Scholar PubMed
Hart, M.J., Sharma, S., el Masry, N., Qiu, R.G., McCabe, P., Polakis, P., and Bollag, G. (1996). Identification of a novel guanine nucleotide exchange factor for the Rho GTPase. J. Biol. Chem.271, 25452–25458.10.1074/jbc.271.41.25452Search in Google Scholar PubMed
Hart, M.J., Jiang, X., Kozasa, T., Roscoe, W., Singer, W.D., Gilman, A.G., Sternweis, P.C., and Bollag, G. (1998). p115 RhoGEF, a GTPase activating protein for Gα12 and Gα13. Science280, 2112–2114.10.1126/science.280.5372.2112Search in Google Scholar
Horwitz, A.R. and Parsons, J.T. (1999). Cell migration – movin' on. Science286, 1102–1103.10.1126/science.286.5442.1102Search in Google Scholar
Hotchin, N.A. and Hall, A. (1995). The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular Rho/Rac GTPases. J. Cell Biol.131, 1857–1865.10.1083/jcb.131.6.1857Search in Google Scholar
Hu, K.Q. and Settleman, J. (1997). Tandem SH2 binding sites mediate the RasGAP RhoGAP interaction: a conformational mechanism for SH3 domain regulation. EMBO J.16, 473–483.10.1093/emboj/16.3.473Search in Google Scholar
Kozasa, T., Jiang, X., Hart, M.J., Sternweis, P.M., Singer, W.D., Gilman, A.G., Bollag, G. and Sternweis, P.C. (1998). RhoGEF, a GTPase activating protein for Gα12 and Gα13. Science280, 2109–2111.10.1126/science.280.5372.2109Search in Google Scholar
Kozma, R., Sarner, S., Ahmed, S. and Lim, L. (1997). Rho family GTPases and neuronal growth cone remodelling: relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid. Mol. Cell. Biol.17, 1201–1211.10.1128/MCB.17.3.1201Search in Google Scholar
Longenecker, K.L., Lewis, M.E., Chikumi, H., Gutkind, J.S., and Derewenda, Z.S. (2001). Structure of the RGS like domain from PDZ RhoGEF: linking heterotrimeric G protein coupled signaling to Rho GTPases. Structure9, 559–569.10.1016/S0969-2126(01)00620-7Search in Google Scholar
Mertens, A.E., Roovers, R.C., and Collard, J.G. (2003). Regulation of Tiam1/Rac signalling. FEBS Lett.546, 11–16.10.1016/S0014-5793(03)00435-6Search in Google Scholar
Mira, J.P., Benard, V., Groffen, J., Sanders, L.C., and Knaus, U.G. (2000). Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway. Proc. Natl. Acad. Sci. USA97, 185–189.10.1073/pnas.97.1.185Search in Google Scholar PubMed PubMed Central
McGlade, J., Brunkhorst, B., Anderson, D., Mbamalu, G., Settleman, J., Dedhar, G., Rozakis Adcock, M., Chen, L.B., and Pawson, T. (1993). The N-terminal region of GAP regulates cytoskeletal structure and cell adhesion. EMBO J.12, 3073–3081.10.1002/j.1460-2075.1993.tb05976.xSearch in Google Scholar PubMed PubMed Central
Michiels, F. and Collard, J.G. (1999). Rho-like GTPases: their role in cell adhesion and invasion. Biochem. Soc. Symp.65, 125–146.Search in Google Scholar
Michiels, F., Habets, G.G., Stam, J.C., van der Kammen, R.A., and Collard, J.G. (1995). A role for Rac in Tiam1-induced membrane ruffling and invasion. Nature375, 338–340.10.1038/375338a0Search in Google Scholar PubMed
Moran, M.F., Polakis, P., McCormick, F., Pawson, T., and Ellis, C. (1991). Protein tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase activating protein. Mol. Cell. Biol.11, 1804–1812.Search in Google Scholar
Nimnual, A.S., Taylor, L.J., and Bar Sagi, D. (2003). Redox dependent downregulation of Rho by Rac. Nat. Cell Biol.5, 236–241.10.1038/ncb938Search in Google Scholar PubMed
Nomanbhoy, T.K. and Cerione, R. (1996). Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy. J. Biol. Chem.271, 10004–10009.10.1074/jbc.271.17.10004Search in Google Scholar PubMed
Noren, N.K., Niessen, C.M., Gumbiner, B.M., and Burridge, K. (2001). Cadherin engagement regulates Rho family GTPases. J. Biol. Chem.276, 33305–33308.10.1074/jbc.C100306200Search in Google Scholar PubMed
Noren, N.K., Arthur, W.T. and Burridge, K. (2003). Cadherin engagement inhibits RhoA via p190RhoGAP. J. Biol. Chem.278, 13615–13618.10.1074/jbc.C200657200Search in Google Scholar PubMed
Perrot, V., Vazquez-Prado, J., and Gutkind, J.S. (2002). Plexin B regulates Rho through the guanine nucleotide exchange factors leukemia-associated Rho GEF (LARG) and PDZ-RhoGEF. J. Biol. Chem.277, 43115–43120.10.1074/jbc.M206005200Search in Google Scholar PubMed
Pop, M., Aktories, K., and Schmidt, G. (2004). Isotype specific degradation of Rac activated by the cytotoxic necrotizing factor 1. J. Biol. Chem.279, 35840–35848.10.1074/jbc.M404346200Search in Google Scholar PubMed
Raugei, G., Ramponi, G., and Chiarugi, P. (2002). Low molecular weight protein tyrosine phosphatases: small, but smart. Cell. Mol. Life Sci.59, 941–949.10.1007/s00018-002-8481-zSearch in Google Scholar PubMed
Reid, T., Bathoorn, A., Ahmadian, M.R., and Collard, J.G. (1999). Identification and characterization of hPEM 2, a guanine nucleotide exchange factor specific for Cdc42. J. Biol. Chem.274, 33587–33593.10.1074/jbc.274.47.33587Search in Google Scholar PubMed
Ridley, A.J. (2001). Rho GTPases and cell migration. J. Cell Sci.114, 2713–2722.10.1242/jcs.114.15.2713Search in Google Scholar PubMed
Ridley, A.J., Self, A.J., Kasmi, F., Paterson, H.F., Hall, A., Marshall, C.J., and Ellis, C. (1993). Rho family GTPase activating proteins p190, Bcr and RhoGAP show distinct specificities in vitro and in vivo. EMBO J.12, 5151–5160.10.1002/j.1460-2075.1993.tb06210.xSearch in Google Scholar
Roof, R.W., Haskell, M.D., Dukes, B.D., Sherman, N., Kinter, M., and Parsons, S.J. (1998). Phosphotyrosine (p-Tyr)-dependent and -independent mechanisms of p190 RhoGAP p120 RasGAP interaction: Tyr-1105 of p190, a substrate for c Src, is the sole p-Tyr mediator of complex formation. Mol. Cell. Biol.18, 7052–7063.10.1128/MCB.18.12.7052Search in Google Scholar
Rossman, K.L., Der, C.J., and Sondek, J. (2005). GEF means go: turning on Rho GTPases with guanine nucleotide-exchange factors. Nat. Rev. Mol. Cell Biol.6, 167–180.10.1038/nrm1587Search in Google Scholar
Ruemenapp, U., Blomquist, A., Schworer, G., Schablowski, H., Psoma, A., and Jakobs, K.H. (1999). Rho specific binding and guanine nucleotide exchange catalysis by KIAA0380, a dbl family member. FEBS Lett.459, 313–318.10.1016/S0014-5793(99)01270-3Search in Google Scholar
Sander, E.E., ten Klooster, J.P., van Delft, S., van der Kammen, R.A., and Collard, J.G. (1999). Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior. J. Cell Biol.147, 1009–1022.10.1083/jcb.147.5.1009Search in Google Scholar
Scheffzek, K. and Ahmadian, M.R. (2005). GTPase activating proteins: structural and functional insights 18 years after discovery. Cell. Mol. Life Sci.62, 3014–3038.10.1007/s00018-005-5136-xSearch in Google Scholar
Settleman, J., Narasimhan, V., Foster, L.C., and Weinberg, R.A. (1992). Molecular cloning of cDNAs encoding the GAP associated protein p190: implications for a signalling pathway from ras to the nucleus. Cell69, 539–549.10.1016/0092-8674(92)90454-KSearch in Google Scholar
Sussman, D.J. and Milman, G. (1984). Short-term, high-efficiency expression of transfected DNA. Mol. Cell. Biol.4, 1641–1643.Search in Google Scholar
Swiercz, J.M., Kuner, R., Behrens, J., and Offermanns, S. (2002). Plexin-B1 directly interacts with PDZ-RhoGEF/LARG to regulate RhoA and growth cone morphology. Neuron35, 51–63.10.1016/S0896-6273(02)00750-XSearch in Google Scholar
Taya, S., Inagaki, N., Sengiku, H., Makino, H., Iwamatsu, A., Urakawa, I., Nagao, K., Kataoka, S., and Kaibuchi, K. (2001). Direct interaction of insulin-like growth factor-1 receptor with leukemia-associated RhoGEF. J. Cell Biol.155, 809–820.10.1083/jcb.200106139Search in Google Scholar PubMed PubMed Central
Uhlenbrock, K., Eberth, A., Herbrand, U., Daryab, N., Stege, P., Meier, F., Friedl, P., Collard, J., and Ahmadian, M.R. (2004). The RacGEF Tiam1 inhibits migration and invasion of metastatic melanoma via a novel adhesive mechanism. J. Cell Sci.117, 4863–4871.10.1242/jcs.01367Search in Google Scholar PubMed
van Aelst, L. and D'Souza Schorey, C. (1997). Rho GTPases and signalling networks. Genes Dev.11, 2295–2322.10.1101/gad.11.18.2295Search in Google Scholar PubMed
van Leeuwen, F.N., van Delft, S., Kain, H.E., van der Kammen, R.A., and Collard, J.G. (1999). Rac regulates phosphorylation of the myosin II heavy chain, actinomyosin disassembly and cell spreading. Nat. Cell Biol.1, 242–248.10.1038/12068Search in Google Scholar PubMed
Vetter, I.R. and Wittinghofer, A. (2001). The guanine nucleotide binding switch in three dimensions. Science294, 1299–1304.10.1126/science.1062023Search in Google Scholar PubMed
Wells, C.D., Gutowski, S., Bollag, G., and Sternweis, P.C. (2001). Identification of potential mechanisms for regulation of p115 RhoGEF through analysis of endogenous and mutant forms of the exchange factor. J. Biol. Chem.276, 28897–28905.10.1074/jbc.M102913200Search in Google Scholar PubMed
Wells, C.D., Liu, M.Y., Jackson, M., Gutowski, S., Sternweis, P.M., Rothstein, J.D., Kozasa, T., and Sternweis, P.C. (2002). Mechanisms for reversible regulation between G13 and Rho exchange factors. J. Biol. Chem.277, 1174–1181.10.1074/jbc.M105274200Search in Google Scholar PubMed
Zondag, G.C., Evers, E.E., ten Klooster, J.P., Janssen, L., van der Kammen, R.A., and Collard, J.G. (2000). Oncogenic Ras downregulates Rac activity, which leads to increased Rho activity and epithelial mesenchymal transition. J. Cell Biol.149, 775–782.10.1083/jcb.149.4.775Search in Google Scholar PubMed PubMed Central
©2006 by Walter de Gruyter Berlin New York