Oncogenic Ras Leads to Rho Activation by Activating the Mitogen-activated Protein Kinase Pathway and Decreasing Rho-GTPase-activating Protein Activity

Abstract

Transformation by oncogenic Ras requires signaling through Rho family proteins including RhoA, but the mechanism(s) whereby oncogenic Ras regulates the activity of RhoA is (are) unknown. We examined the effect of Ras on RhoA activity in NIH 3T3 cells either stably transfected with H-Ras(V12) under control of an inducible promoter or transiently expressing the activated H-Ras. Using a novel method to quantitate enzymatically the GTP bound to Rho, we found that expression of the oncogenic Ras increased Rho activity ∼2-fold. Increased Rho activity was associated with increased plasma membrane binding of RhoA and decreased activity of the Rho/Ras-regulated p21WAF1/CIP1 promoter. RhoA activation by oncogenic Ras could be explained by a decrease in cytosolic p190 Rho-GAP activity and translocation of p190 Rho-GAP from the cytosol to a detergent-insoluble cytoskeletal fraction. Pharmacologic inhibition of the Ras/Raf/MEK/ERK pathway prevented Ras-induced activation of RhoA and translocation of p190 Rho-GAP; expression of constitutively active Raf-1 kinase or MEK was sufficient to induce p190 Rho-GAP translocation. We conclude that in NIH 3T3 cells oncogenic Ras activates RhoA through the Raf/MEK/ERK pathway by decreasing the cytosolic activity and changing the subcellular localization of p190 Rho-GAP. Transformation by oncogenic Ras requires signaling through Rho family proteins including RhoA, but the mechanism(s) whereby oncogenic Ras regulates the activity of RhoA is (are) unknown. We examined the effect of Ras on RhoA activity in NIH 3T3 cells either stably transfected with H-Ras(V12) under control of an inducible promoter or transiently expressing the activated H-Ras. Using a novel method to quantitate enzymatically the GTP bound to Rho, we found that expression of the oncogenic Ras increased Rho activity ∼2-fold. Increased Rho activity was associated with increased plasma membrane binding of RhoA and decreased activity of the Rho/Ras-regulated p21WAF1/CIP1 promoter. RhoA activation by oncogenic Ras could be explained by a decrease in cytosolic p190 Rho-GAP activity and translocation of p190 Rho-GAP from the cytosol to a detergent-insoluble cytoskeletal fraction. Pharmacologic inhibition of the Ras/Raf/MEK/ERK pathway prevented Ras-induced activation of RhoA and translocation of p190 Rho-GAP; expression of constitutively active Raf-1 kinase or MEK was sufficient to induce p190 Rho-GAP translocation. We conclude that in NIH 3T3 cells oncogenic Ras activates RhoA through the Raf/MEK/ERK pathway by decreasing the cytosolic activity and changing the subcellular localization of p190 Rho-GAP. mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid cytomegalovirus Dulbecco's modified Eagle's medium extracellular signal-regulated kinase fetal bovine serum GTPase-activating protein guanine dissociation inhibitor guanine nucleotide exchange factor glutathioneS-transferase guanosine 5′-O-(thiotriphosphate) long terminal repeat luciferase mitogen-activated protein Madin-Darby canine kidney Rho binding domain Rous sarcoma virus thymidine kinase catalytic domain of Raf-1 kinase Proteins of the Ras superfamily, including the Ras and Rho families, cycle between active GTP- and inactive GDP-bound forms and function as essential switches in signal transduction pathways that regulate cell growth, differentiation, and survival (1Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Google Scholar). Activating mutations in H-, K-, and N-Ras are found in up to 30% of all human cancers; in cancers with wild type Ras, overexpression of growth factor receptors frequently leads to activation of the Ras/Raf/MEK1/ERK pathway, suggesting an important contribution of Ras functions to the development of human cancers (1Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Google Scholar, 2von Lintig F.C. Dreilinger A.D. Varki N.M. Wallace A.M. Casteel D.E. Boss G.R. Br. Can. Res. Treat. 2000; 62: 51-62Google Scholar, 3Sivaraman V.S. Wang H. Nuovo G.J. Malbon C.C. J. Clin. Invest. 1997; 99: 1478-1483Google Scholar). Although there are no reports of activating mutations of Rho proteins in human tumors, several Rho proteins are overexpressed in tumors, and Rho family-activating guanine nucleotide exchange factors (GEFs) have been isolated in screens for transforming genes, suggesting a role of Rho proteins in tumorigenesis (4Sahai E. Marshall C.J. Nat. Rev. Cancer. 2002; 2: 133-142Google Scholar). Members of the Rho family regulate the actin cytoskeleton, thereby affecting cell morphology and motility; in addition, they modulate gene expression, cell cycle progression, and cell survival (1Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Google Scholar, 4Sahai E. Marshall C.J. Nat. Rev. Cancer. 2002; 2: 133-142Google Scholar, 5Kjoller L. Hall A. Exp. Cell Res. 1999; 253: 166-179Google Scholar). RhoA, B, and C and Rac1 play critical roles in cell transformation induced by activated, oncogenic Ras, with dominant negative Rho and Rac1 constructs inhibiting Ras-induced transformation and constitutively active constructs inducing anchorage-independent growth and other features of the transformed phenotype (4Sahai E. Marshall C.J. Nat. Rev. Cancer. 2002; 2: 133-142Google Scholar, 6Prendergast G.C. Khosravi-Far R. Solski P.A. Kurzawa H. Lebowitz P.F. Der C.J. Oncogene. 1995; 10: 2289-2296Google Scholar, 7Qiu R.-G. Chen J. McCormick F. Symons M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11781-11785Google Scholar, 8Sahai E. Olson M.F. Marshall C.J. EMBO J. 2001; 20: 755-766Google Scholar, 9Qiu R.G. Chen J. Kirn D. McCormick F. Symons M. Nature. 1995; 374: 457-459Google Scholar). The requirement of Rho for Ras-induced transformation exists in part because Ras and Rho play opposing roles in control of the cyclin-dependent kinase inhibitor p21WAF1/CIP1, with Ras inducing and Rho inhibiting p21WAF1/CIP1 transcription; thus increased Ras activity actually blocks cell cycle progression when Rho signaling is inhibited by C3 exoenzyme (8Sahai E. Olson M.F. Marshall C.J. EMBO J. 2001; 20: 755-766Google Scholar, 10Olson M.F. Paterson H.F. Marshall C.J. Nature. 1998; 394: 295-299Google Scholar, 11Danen E.H. Sonneveld P. Sonnenberg A. Yamada K.M. J. Cell Biol. 2000; 151: 1413-1422Google Scholar). Activated, oncogenic Ras may regulate RhoA and Rac1 activities, but the effects of Ras appear to be cell type-specific, vary with the Ras subtype, and depend on the kinetics and duration of Ras activation (8Sahai E. Olson M.F. Marshall C.J. EMBO J. 2001; 20: 755-766Google Scholar,12Nobes C.D. Hall A. Cell. 1995; 81: 53-62Google Scholar, 13Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Google Scholar, 14Zhong C. Kinch M.S. Burridge K. Mol. Biol. Cell. 1997; 8: 2329-2344Google Scholar, 15Khosravi-Far R. White M.A. Westwick J.K. Solski P.A. Chrzanowska-Wodnicka M. van Aelst L. Wigler M.H. Der C.J. Mol. Cell. Biol. 1996; 16: 3923-3933Google Scholar, 16Zondag G.C. Evers E.E. ten Klooster J.P. Janssen L. van der Kammen R.A. Collard J.G. J. Cell Biol. 2000; 149: 775-782Google Scholar, 17Karaguni I.M. Herter P. Debruyne P. Chtarbova S. Kasprzynski A. Herbrand U. Ahmadian M.R. Glusenkamp K.H. Winde G. Mareel M. Moroy T. Muller O. Cancer Res. 2002; 62: 1718-1723Google Scholar, 18Gupta S. Plattner R. Der C.J. Stanbridge E.J. Mol. Cell. Biol. 2000; 20: 9294-9306Google Scholar). Microinjection or transient transfection of oncogenic H-Ras(V12) into Swiss 3T3 cells leads to acute cytoskeletal changes, suggesting a hierarchal system with Ras activating Rac (causing membrane ruffling) and Rac in turn activating RhoA (causing induction of stress fibers); however, these studies were performed before direct measures of Rac and Rho·GTP levels were available (12Nobes C.D. Hall A. Cell. 1995; 81: 53-62Google Scholar, 13Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Google Scholar). Ras activation of Rac can occur through the Ras effector phosphatidylinositol 3-kinase, with increased phosphoinositides activating a multimolecular complex including a Rac-activating GEF (19Scita G. Tenca P. Frittoli E. Tocchetti A. Innocenti M. Giardina G. Di Fiore P.P. EMBO J. 2000; 19: 2393-2398Google Scholar, 20Nimnual A.S. Yatsula B.A. Bar-Sagi D. Science. 1998; 279: 560-563Google Scholar, 21Innocenti M. Tenca P. Frittoli E. Faretta M. Tocchetti A., Di Fiore P.P. Scita G. J. Cell Biol. 2002; 156: 125-136Google Scholar). How Rac activation can lead to activation of RhoA is less clear, but it may involve Rac activation of phospholipase A2 with subsequent arachidonic acid and leukotriene production, at least in Swiss 3T3 cells (22Peppelenbosch M.P. Qiu R.G. Vries-Smits A.M. Tertoolen L.G. de Laat S.W. McCormick F. Hall A. Symons M.H. Bos J.L. Cell. 1995; 81: 849-856Google Scholar). In contrast to the acute response to oncogenic Ras, studies in Ras-transformed cell lines have produced conflicting results, with some studies reporting decreased Rac and increased RhoA activation compared with nontransformed cells, and others reporting increased activation of both GTPases or no changes (8Sahai E. Olson M.F. Marshall C.J. EMBO J. 2001; 20: 755-766Google Scholar, 14Zhong C. Kinch M.S. Burridge K. Mol. Biol. Cell. 1997; 8: 2329-2344Google Scholar, 15Khosravi-Far R. White M.A. Westwick J.K. Solski P.A. Chrzanowska-Wodnicka M. van Aelst L. Wigler M.H. Der C.J. Mol. Cell. Biol. 1996; 16: 3923-3933Google Scholar, 16Zondag G.C. Evers E.E. ten Klooster J.P. Janssen L. van der Kammen R.A. Collard J.G. J. Cell Biol. 2000; 149: 775-782Google Scholar, 17Karaguni I.M. Herter P. Debruyne P. Chtarbova S. Kasprzynski A. Herbrand U. Ahmadian M.R. Glusenkamp K.H. Winde G. Mareel M. Moroy T. Muller O. Cancer Res. 2002; 62: 1718-1723Google Scholar, 18Gupta S. Plattner R. Der C.J. Stanbridge E.J. Mol. Cell. Biol. 2000; 20: 9294-9306Google Scholar). In v-H-Ras-transformed MDCK cells, decreased Rac activity appeared to be secondary to transcriptional down-regulation of the Rac-specific GEF Tiam1; the mechanism for increased RhoA activation was not elucidated, but the effect of oncogenic Ras on RhoA and Rac activity was mimicked by stable transfection of constitutively active Raf (16Zondag G.C. Evers E.E. ten Klooster J.P. Janssen L. van der Kammen R.A. Collard J.G. J. Cell Biol. 2000; 149: 775-782Google Scholar). H-Ras(V12)-transformed Swiss 3T3 cells also demonstrated decreased Rac and increased Rho activity compared with untransformed cells, but short term expression of a constitutively active Raf in Swiss 3T3 cells did not lead to elevation of RhoA activity; elevated RhoC·GTP levels were only seen after prolonged (>4 weeks) culture of the active Raf-overexpressing cells, suggesting that they were a consequence of selection rather than direct signaling (8Sahai E. Olson M.F. Marshall C.J. EMBO J. 2001; 20: 755-766Google Scholar). In HT1080 human fibrosarcoma cells containing oncogenic N-Ras, both Rac and RhoA activity were increased compared with cells lacking the mutant N-Ras; Rac and RhoA activities were also increased when cells lacking the mutant N-Ras were stably transfected with constitutively active Raf or MEK (18Gupta S. Plattner R. Der C.J. Stanbridge E.J. Mol. Cell. Biol. 2000; 20: 9294-9306Google Scholar). In K-Ras(V12)-transformed normal rat kidney cells, no significant change of RhoA activity was observed compared with untransformed cells (23Pawlak G. Helfman D.M. Mol. Biol. Cell. 2002; 13: 336-347Google Scholar). Several older studies reported loss of stress fibers in Ras-transformed Rat1 cells, with restoration of stress fibers upon transfection of constitutively active RhoA, suggesting loss of RhoA activity in the Ras-transformed cells (7Qiu R.-G. Chen J. McCormick F. Symons M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11781-11785Google Scholar, 24Izawa I. Amano M. Chihara K. Yamamoto T. Kaibuchi K. Oncogene. 1998; 17: 2863-2871Google Scholar); others reported increased stress fibers in Ras-transformed breast cancer cells and NIH 3T3 cells without direct measurement of Rho activity (14Zhong C. Kinch M.S. Burridge K. Mol. Biol. Cell. 1997; 8: 2329-2344Google Scholar, 15Khosravi-Far R. White M.A. Westwick J.K. Solski P.A. Chrzanowska-Wodnicka M. van Aelst L. Wigler M.H. Der C.J. Mol. Cell. Biol. 1996; 16: 3923-3933Google Scholar). Because the mechanism of Rho regulation by Ras is not clear, we decided to examine the effects of oncogenic Ras on the activation state of RhoA in NIH 3T3 cells stably expressing H-Ras(V12) under control of an inducible promoter (LTR-H-Ras(A) cells (25Schönthal A. Herrlich P. Rahmsdorf H.J. Ponta H. Cell. 1988; 54: 325-334Google Scholar)); these cells have low basal and high induced levels of H-Ras(V12) and allowed us to study short term effects of Ras activation avoiding complex genetic changes that may occur during long term culture of Ras-transformed cells. Using two different methods to assess Rho activation, we found that induction of H-Ras(V12) in LTR-H-Ras(A) cells or transient transfection of H-Ras(V12) into wild type NIH 3T3 cells caused an approximate 2-fold increase in Rho·GTP levels. Concomitant with Rho activation, we found increased RhoA translocation to membranes and decreased activity of a p21WAF1/CIP1 promoter construct. The mechanism for increased Rho activation appeared to be decreased p190 Rho-GAP activity because of translocation of p190 Rho-GAP from the cytosol to a detergent-insoluble cytoskeletal fraction. Wild type NIH 3T3 fibroblasts and LTR-H-Ras(A) NIH 3T3 cells stably expressing activated Ras(V12) under the inducible murine mammary tumor virus promoter were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) as described previously (26Scheele J.S. Rhee J.M. Boss G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1097-1100Google Scholar). To induce Ras(V12) expression, cells were treated with 1 μm dexamethasone for 24 h. For transient transfection experiments, cells were plated in six-well cluster dishes and 24 h later were transfected with a total of 1.5 μg of DNA/well using LipofectAMINE PlusTM or LipofectAMINE 2000TM (Invitrogen) as described previously (27Suhasini M., Li, H. Lohmann S.M. Boss G.R. Pilz R.B. Mol. Cell. Biol. 1998; 18: 6983-6994Google Scholar). The specific MEK inhibitor U0126 was from Calbiochem (28Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Google Scholar). The following plasmids were used: pcDNA3-EE-RhoA(wt), pcDNA3-EE-RhoA(V14), pcDNA3-EE-RhoA(63L), and pRC/CMV-BXB, described previously (27Suhasini M., Li, H. Lohmann S.M. Boss G.R. Pilz R.B. Mol. Cell. Biol. 1998; 18: 6983-6994Google Scholar, 29Gudi T. Chen J.C. Casteel D.E. Seasholtz T.M. Boss G.R. Pilz R.B. J. Biol. Chem. 2002; 277: 37382-37393Google Scholar); pDCR-H-Ras(V12) from M. Wigler (30White M.A. Nicolette C. Minden A. Polverino A. Van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 533-541Google Scholar); pΔN-p115Rho-GEF from M. Hart (31Hart M.J. Sharma S. elMasry N. Qiu R.G. McCabe P. Polakis P. Bollag G. J. Biol. Chem. 1996; 271: 25452-25458Google Scholar); p21-Luc from X.-F. Wang (32Datto M.B., Yu, Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Google Scholar); pEF-C3exo from R. Treisman (33Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Google Scholar); pMEK1(E218,D222) from S. Cowley (34Cowley S. Patterson H. Kemp P. Marshall C. Cell. 1994; 77: 841-852Google Scholar), courtesy of P. M. McDonough; and pHA-p190Rho-GAP from J. Settleman (35Settleman J. Narasimhan V. Foster L.C. Weinberg R.A. Cell. 1992; 69: 539-549Google Scholar). The activation state of endogenous Rho was measured by two different methods: (i) measurement of absolute amounts of GTP and GTP + GDP bound to Rho and (ii) assessment of Rho-bound GTP by Western blotting. In both methods, the Rho binding domain (RBD) of Rhotekin was used to isolate Rho·GTP as originally described by Ren et al. (36Ren X.-D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Google Scholar). Glutathione S-transferase (GST)-tagged Rhotekin RBD was purified from bacterial lysates; the bacterial expression vector was provided by M. A. Schwartz (36Ren X.-D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Google Scholar). In the first method, there is the potential to measure activation of RhoA, B, and C simultaneously; however, NIH 3T3 cells express mainly RhoA with low amounts of RhoC and negligible amounts of RhoB (37Fritz G. Kaina B. Aktories K. J. Biol. Chem. 1995; 270: 25172-25177Google Scholar). The first method was modified to allow quantitation of GTP and GTP + GDP bound to transfected RhoA constructs. This method is a modification of a procedure we have used previously to measure GTP, and GTP + GDP, bound to Ras, Rap1, and Rheb (2von Lintig F.C. Dreilinger A.D. Varki N.M. Wallace A.M. Casteel D.E. Boss G.R. Br. Can. Res. Treat. 2000; 62: 51-62Google Scholar, 26Scheele J.S. Rhee J.M. Boss G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1097-1100Google Scholar, 38Sharma P.M. Egawa K. Huang Y. Martin J.L. Huvar I. Boss G.R. Olefsky J.M. J. Biol. Chem. 1998; 273: 18528-18537Google Scholar, 39von Lintig F.C. Pilz R.B. Boss G.R. Oncogene. 2000; 19: 4029-4034Google Scholar, 40Im E. von Lintig F.C. Chen J. Zhuang S. Qui W. Chowdhury S. Worley P.F. Boss G.R. Pilz R.B. Oncogene. 2002; 21: 6356-6365Google Scholar). Cells grown on a 100-mm plate under the conditions indicated under "Results" and in the figure legends were extracted quickly in situ by washing once with ice-cold Tris-buffered saline, pH 7.4, and adding lysis buffer consisting of 50 mmTris-HCl, pH 7.4, 1% Nonidet P-40, 1% CHAPS, 200 mm NaCl, 1 mm MgCl2, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride. After a 1-min incubation on ice, the lysed cells were scraped with a rubber policeman, transferred to a microcentrifuge tube, and subjected to vortexing for 10 s. Cell extracts were centrifuged at 10,000 × g for 2 min, and a portion of the supernatant was added to tubes containing 10 mm MgSO4 and 30 μg of GST-tagged Rhotekin RBD bound to glutathione beads; these samples were used for measuring GTP bound to Rho ("unloaded" samples). The remaining supernatant was added to tubes containing 10 μmGTP, 10 mm EDTA, and 30 μg of GST-tagged RBD on glutathione beads, allowing the free GTP to exchange for GDP bound to Rho, thus converting all of the Rho to the GTP-bound state ("loaded" samples). After gentle shaking for 1 h at 4 °C, the beads with Rho·GTP bound to the Rhotekin RBD were washed four times with 50 mm Tris-HCl, pH 7.4, 2% Nonidet P-40, 500 mm NaCl, 10 mm MgSO4, and twice with 20 mm Tris-PO4, pH 7.4, 5 mmMgSO4. GTP was released from Rho by heating the beads for 3 min at 100 °C in 5 mm Tris-PO4, pH 7.4, 2 mm dithiothreitol, 2 mm EDTA (TDE buffer). We have shown previously >95% recovery of GTP under these conditions (26Scheele J.S. Rhee J.M. Boss G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1097-1100Google Scholar). GTP eluted from the unloaded and loaded samples was measured in a coupled enzymatic assay by conversion to ATP in the presence of ADP and nucleoside diphosphate kinase (26Scheele J.S. Rhee J.M. Boss G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1097-1100Google Scholar); the resulting ATP was measured by the firefly luciferase method in a photon-counting luminometer (MGM Instruments, Hamden, CT). This method is sensitive to 1 fmol of GTP and is quantitative because the second reaction is irreversible, from light generation, allowing both reactions to go to completion (26Scheele J.S. Rhee J.M. Boss G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1097-1100Google Scholar). Cells were extracted and processed as described above for the unloaded samples except the magnesium concentration in the initial lysis buffer was increased to 10 mm; Rho·GTP isolated by binding to the Rhotekin RBD-coated beads was quantitated by Western blotting using a RhoA-specific antibody (Santa Cruz Biotechnology), as described by Renet al. (36Ren X.-D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Google Scholar). Cells transfected with EE epitope-tagged RhoA constructs were extracted in situ in 50 mmTris-HCl, pH 7.4, 1% Nonidet P-40, 500 mm NaCl, 10 mm MgCl2, 0.5% deoxycholate, 0.05% SDS, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin (RIPA buffer). After centrifuging the extracts, were in and added to tubes containing protein beads with either a antibody or control The tubes were for 1 h at 4 °C, and the beads were washed four times with and twice with 20 pH 7.4, 5 mm GTP and GDP were released from the Rho as described above by heating the beads in In of the GTP was measured as described and in the of GDP GTP was measured as described previously P.M. Egawa K. Huang Y. Martin J.L. Huvar I. Boss G.R. Olefsky J.M. J. Biol. Chem. 1998; 273: 18528-18537Google by converting GDP to GTP using kinase and with the resulting GTP the of GDP measured as described Cells were plated at × on a culture and 24 h later the cells were transfected with of using (Invitrogen) to the cells of and as indicated some cells 50 pEF-C3exo or 100 of Cells were treated for 24 h with 1 μm and luciferase activity was measured in cell extracts as described previously T. Huvar I. M. Lohmann S.M. Boss G.R. Pilz R.B. J. Biol. Chem. 1996; 271: Scholar). We did not an control vector because all four that were and demonstrated some increase in when LTR-H-Ras(A) cells were treated with cells grown on two were extracted by for 2 min in and extracts were centrifuged at 10,000 × g for 2 The domain of human bound to beads was used to isolate and the of bound to the beads was quantitated by Western blotting with a antibody as described previously V. J. Biol. Chem. 1999; Scholar), using an assay from Cells grown on were extracted by in 10 mm pH 2 1 mm buffer). The resulting cell was centrifuged at 500 × g for 5 min to and subcellular and the supernatant was centrifuged at × g for 30 The supernatant and from the second are to as cytosol and with the membrane washed twice in buffer to were to Scholar), and amounts of protein from μg of 20 μg of and μg of were subjected to blotting using (Santa Cruz and or a antibody (Santa Cruz cytoskeletal were as described previously Oncogene. 1998; 17: Scholar). Cells were washed in and extracted in situ for at in 50 mm pH 3 5 mm MgCl2, 0.5% The supernatant was the detergent-insoluble was scraped with a rubber in the presence of and inhibitor and centrifuged for 10 min at × g at 4 were in buffer and by blotting using the antibody and an antibody Cruz extracts were as described above and subjected to using a antibody or control were on protein beads and by blotting using a antibody and the antibody from Cruz used at Cells grown on 100-mm were extracted by in 10 mm pH 7.4, 1 mm was purified on beads and was loaded with activity of by a incubation at °C in 50 mm pH 5 mm EDTA, as described previously A.J. Hall A. 1995; Scholar). The RhoA was with cell extracts in the presence of 1 mm GTP for 10 and 20 min at °C, and the reaction was by adding a of ice-cold After washing the beads times with they were on and was measured by The are as the increase in exchange compared with RhoA in Cells grown on were extracted by in buffer supplemented with 1 and a inhibitor and the cytosol was as described After of 1% or 500 μg of protein was subjected to using protein beads with either Rho-GAP antibody or control The beads were washed times with buffer and once with a reaction buffer containing 50 mmTris-HCl, pH 10 mm MgCl2, 1 1 bovine serum 1 mm The beads were in 100 of reaction and 50 of with activity was added to the reaction A.J. Hall A. 1995; Scholar). were in a at 20 °C, and at the indicated times the reaction was by 10 of the reaction to 1 of ice-cold buffer containing 50 mm Tris-HCl, pH 50 mm NaCl, 5 mm MgCl2, 1 mm were on were washed and was measured in a and were as a of the of bound to RhoA in the extracts were and subjected to with either the or as described were by using the The of Rho-GAP was by the with an antibody (Santa Cruz kinase activity was by Western blotting using a antibody that a to and of as described previously (27Suhasini M., Li, H. Lohmann S.M. Boss G.R. Pilz R.B. Mol. Cell. Biol. 1998; 18: 6983-6994Google Scholar). part of these we a quantitative method to assess Rho activation by measuring absolute amounts of GTP and of total the of GTP GDP, bound to To measure it was isolated from cell extracts to the method of Ren et al. (36Ren X.-D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Google using beads with a RBD however, of the Rho·GTP by Western we eluted GTP from Rho and measured it in a coupled enzymatic assay as described previously for measuring GTP bound to other proteins (26Scheele J.S. Rhee J.M. Boss G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1097-1100Google Scholar, 38Sharma P.M. Egawa K. Huang Y. Martin J.L. Huvar I. Boss G.R. Olefsky J.M. J. Biol. Chem. 1998; 273: 18528-18537Google Scholar, 39von Lintig F.C. Pilz R.B. Boss G.R. Oncogene. 2000; 19: 4029-4034Google Scholar, 40Im E. von Lintig F.C. Chen J. Zhuang S. Qui W. Chowdhury S. Worley P.F. Boss G.R. Pilz R.B. Oncogene. 2002; 21: 6356-6365Google Scholar). To measure total bound to Rho, we to Rho·GTP by a of in the of magnesium and in the presence of 10 μm under these is to Rho·GTP B. Y. Wang Y. J. Biol. Chem. 2000; Scholar), and the was measured as In extracts from NIH 3T3 cells, the assay a response a of protein for both unloaded in Rho·GTP was measured 1 and for loaded in total bound to Rho were measured after converting to Rho·GTP 1