Oncogenic Ras Leads to Rho Activation by Activating the Mitogen-activated Protein Kinase Pathway and Decreasing Rho-GTPase-activating Protein Activity
2003; Elsevier BV; Volume: 278; Issue: 5 Linguagem: Inglês
10.1074/jbc.m207943200
ISSN1083-351X
AutoresJeffrey C. Chen, Shunhui Zhuang, Tony H. Nguyen, Gerry R. Boss, Renate B. Pilz,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoTransformation 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, supernatants were split in half and added to tubes containing protein G-agarose beads coated with either a mouse monoclonal anti-EE antibody or control mouse IgG. The tubes were shaken gently for 1 h at 4 °C, and the beads were washed four times with RIPA buffer, and twice with 20 mmTris-PO4, pH 7.4, 5 mm MgSO4. GTP and GDP were released from the immunoprecipitated Rho as described above by heating the beads in TDE buffer. In one aliquot of the sample, GTP was measured as described above, and in another aliquot the sum of GDP plus GTP was measured as described previously (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) by converting GDP to GTP using pyruvate kinase and phosphoenolpyruvate with the resulting GTP (representing the sum of GDP plus GTP) measured as described above. Cells were plated at 8 × 104/well on a 24-well culture plate, and 24 h later the cells were transfected with 300 ng of DNA using PolyfectTM (Invitrogen) according to the manufacturer's recommendation. All cells received 25 ng of p21-Luc, and as indicated some cells additionally received 50 ng pEF-C3exo or 100 ng of pcDNA3-EE-Rho(63L). Cells were treated for 24 h with 1 μm dexamethasone, and luciferase activity was measured in cell extracts as described previously (41Gudi T. Huvar I. Meinecke M. Lohmann S.M. Boss G.R. Pilz R.B. J. Biol. Chem. 1996; 271: 4597-4600Google Scholar). We did not include an internal control vector because all four that were tested,i.e. pRSV-β-galactosidase, pCMV-β-galactosidase, pSV40-β-galactosidase, and pTK-β-galactosidase, demonstrated some increase in transcription when LTR-H-Ras(A) cells were treated with dexamethasone. Subconfluent cells grown on two 150-mm plates were extracted by incubating for 2 min in cold RIPA buffer, and extracts were centrifuged at 10,000 × g for 2 min. The p21(Rac/CDC42)-binding domain of human PAK-1 bound to glutathione-agarose beads was used to isolate Rac·GTP, and the amount of Rac·GTP bound to the beads was quantitated by Western blotting with a mouse monoclonal anti-Rac antibody as described previously (42Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Google Scholar), using an assay kit from Upstate Biotechnology. Cells grown on 150-mm plates were extracted by Dounce homogenization in 10 mm Hepes, pH 7.5, 2 mmEDTA, 1 mm MgCl2 (HEM buffer). The resulting cell homogenate was centrifuged at 500 × g for 5 min to remove nuclei and subcellular organelles, and the supernatant was centrifuged at 37,000 × g for 30 min. The supernatant and pellet from the second centrifugation are referred to as cytosol and membranes, respectively, with the membrane preparation washed twice in HEM buffer to remove contaminating cytosol. Protein concentrations were determined according to Bradford (43Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar), and equal amounts of protein from each preparation (30 μg of homogenate, 20 μg of cytosol, and 40 μg of membranes) were subjected to SDS-PAGE/Western blotting using mouse monoclonal anti-Rho-GAP (Santa Cruz Biotechnology, 1:1,000) and anti-Ras-GAP (Sigma, 1:500) antibodies, or a rabbit polyclonal anti-RhoA antibody (Santa Cruz Biotechnology, 1:1,000). Triton X-100-insoluble cytoskeletal fractions were prepared as described previously (44Sharma S.V. Oncogene. 1998; 17: 271-281Google Scholar). Cells were washed in phosphate-buffered saline and extracted in situ for 40 s at room temperature in 50 mm Na-Hepes, pH 6.4, 3 mmEGTA, 5 mm MgCl2, 0.5% Triton X-100. The detergent-soluble supernatant was removed; the detergent-insoluble material was scraped off with a rubber policeman in the presence of phosphate-buffered saline and protease inhibitor mixture, and centrifuged for 10 min at 300 × g at 4 °C. Pellets were resuspended in SDS-sample buffer and analyzed by SDS-PAGE/Western blotting using the monoclonal anti-Rho-GAP antibody and an actin-specific antibody (C2, Santa Cruz Biotechnology, 1:300 dilution). Cytosolic extracts were prepared as described above and subjected to immunoprecipitation using a rabbit polyclonal anti-Rho-GDI antibody or control rabbit IgG. Immunoprecipitates were collected on protein G-agarose beads and analyzed by SDS-PAGE/Western blotting using a mouse monoclonal anti-RhoA antibody and the rabbit polyclonal anti-Rho-GDI antibody (both from Santa Cruz Biotechnology, used at 1:1,000). Cells grown on 100-mm plates were extracted by sonication in 10 mm Hepes, pH 7.4, 1 mm EDTA. Bacterially expressed GST-RhoA was purified on glutathione-Sepharose beads and was loaded with [3H]GDP (specific activity of 11.7 Ci/mmol) by a 10-min incubation at 37 °C in 50 mm Hepes, pH 7.5, 5 mm EDTA, as described previously (45Self A.J. Hall A. Methods Enzymol. 1995; 256: 67-76Google Scholar). The [3H]GDP-loaded RhoA was incubated with cell extracts in the presence of 1 mm GTP for 10 and 20 min at 37 °C, and the reaction was stopped by adding a 40-fold excess of ice-cold RIPA buffer. After washing the beads three times with RIPA buffer, they were dried on filter paper, and radioactivity was measured by liquid scintillation counting. The data are expressed as the percent increase in GDP·GTP exchange compared with [3H]GDP-loaded RhoA incubated in extract buffer. Cells grown on 150-mm plates were extracted by Dounce homogenization in HEM buffer supplemented with 1 mmNa3VO4 and a protease inhibitor mixture, and the cytosol was prepared as described above. After addition of 1% Triton X-100, 250 or 500 μg of extract protein was subjected to immunoprecipitation using protein G-agarose beads precoupled with either mouse monoclonal anti-p190 Rho-GAP antibody or control mouse IgG. The beads were washed three times with Triton X-100-containing buffer and once with a reaction buffer containing 50 mmTris-HCl, pH 7.5, 10 mm MgCl2, 1 mmdithiothreitol, 1 mg/ml bovine serum albumin, 1 mm GTP. The beads were resuspended in 100 μl of reaction buffer, and 50 ng of GST-RhoA preloaded with [γ-32PO4]GTP (specific activity 6,000 Ci/mmol) was added to initiate the reaction (45Self A.J. Hall A. Methods Enzymol. 1995; 256: 67-76Google Scholar). Tubes were shaken vigorously in a water bath at 20 °C, and at the indicated times the reaction was stopped by transferring 10 μl of the reaction mixture to 1 ml of ice-cold stop buffer containing 50 mm Tris-HCl, pH 7.5, 50 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol. Samples were collected on nitrocellulose filters, which were washed and dried overnight. Cerenkov radiation was measured in a scintillation counter, and data were expressed as a percentage of the amount of [γ-32PO4]GTP bound to RhoA in the zero time samples. Cytosolic extracts were prepared and subjected to immunoprecipitation with either the anti-Rho-GAP or anti-Ras-GAP antibodies as described above. Immunoprecipitates were analyzed by SDS-PAGE/Western immunoblotting using the same antibodies. The amount of phosphorylated Rho-GAP was determined by probing the blots with an anti-phosphotyrosine-specific antibody (Santa Cruz Biotechnology, 1:500). MAP kinase activity was assessed by Western blotting using a phospho-ERK-specific antibody that recognizes a dually phosphorylated peptide sequence corresponding to Thr183 and Tyr185 of p42 MAP kinase, as described previously (27Suhasini M., Li, H. Lohmann S.M. Boss G.R. Pilz R.B. Mol. Cell. Biol. 1998; 18: 6983-6994Google Scholar). As part of these studies, we developed a new quantitative method to assess Rho activation by measuring absolute amounts of GTP and of total nucleotides i.e. the sum of GTP plus GDP, bound to Rho. To measure Rho·GTP, it was isolated from cell extracts according to the method of Ren et al. (36Ren X.-D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Google Scholar) using glutathione-agarose beads coated with a GST-Rhotekin RBD fusion protein; however, instead of assessing the Rho·GTP semiquantitatively by Western blotting, we eluted GTP from Rho and measured it in a coupled enzymatic assay as described previously for measuring GTP bound to other Ras-related 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 nucleotides bound to Rho, we converted Rho·GDP to Rho·GTP by incubating a separate aliquot of extract in the absence of magnesium and in the presence of 10 μm GTP; under these conditions, Rho·GDP is converted rapidly to Rho·GTP (46Zhang B. Zhang Y. Wang Z.-X. Zheng Y. J. Biol. Chem. 2000; 275: 25299-25307Google Scholar), and the latter was measured as just described. In extracts prepared from logarithmically growing NIH 3T3 cells, the assay yielded a linear response over a 5-fold range of cellular protein for both unloaded samples, i.e. those in which Rho·GTP was measured directly (Fig. 1 a), and for loaded samples, i.e. those in which total nucleotides bound to Rho were measured after converting Rho·GDP to Rho·GTP (Fig. 1
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