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• W2015493603 abstract "Dbl is a representative prototype of a growing family of oncogene products that contain the Dbl homology/pleckstrin homology elements in their primary structures and are associated with a variety of neoplastic pathologies. Members of the Dbl family have been shown to function as physiological activators (guanine nucleotide exchange factors) of the Rho-like small GTPases. Although the expression of GTPase-defective versions of Rho proteins has been shown to induce a transformed phenotype under different conditions, their transformation capacity has been typically weak and incomplete relative to that exhibited by dbl -like oncogenes. Moreover, in some cases (e.g. NIH3T3 fibroblasts), expression of GTPase-defective Cdc42 results in growth inhibition. Thus, in attempting to reconstitute dbl -induced transformation of NIH3T3 fibroblasts, we have generated spontaneously activated (“fast-cycling”) mutants of Cdc42, Rac1, and RhoA that mimic the functional effects of activation by the Dbl oncoprotein. When stably expressed in NIH3T3 cells, all three mutants caused the loss of serum dependence and showed increased saturation density. Furthermore, all three stable cell lines were tumorigenic when injected into nude mice. Our data demonstrate that all three Dbl targets need to be activated to promote the full complement of Dbl effects. More importantly, activation of each of these GTP-binding proteins contributes to a different and distinct facet of cellular transformation. Dbl is a representative prototype of a growing family of oncogene products that contain the Dbl homology/pleckstrin homology elements in their primary structures and are associated with a variety of neoplastic pathologies. Members of the Dbl family have been shown to function as physiological activators (guanine nucleotide exchange factors) of the Rho-like small GTPases. Although the expression of GTPase-defective versions of Rho proteins has been shown to induce a transformed phenotype under different conditions, their transformation capacity has been typically weak and incomplete relative to that exhibited by dbl -like oncogenes. Moreover, in some cases (e.g. NIH3T3 fibroblasts), expression of GTPase-defective Cdc42 results in growth inhibition. Thus, in attempting to reconstitute dbl -induced transformation of NIH3T3 fibroblasts, we have generated spontaneously activated (“fast-cycling”) mutants of Cdc42, Rac1, and RhoA that mimic the functional effects of activation by the Dbl oncoprotein. When stably expressed in NIH3T3 cells, all three mutants caused the loss of serum dependence and showed increased saturation density. Furthermore, all three stable cell lines were tumorigenic when injected into nude mice. Our data demonstrate that all three Dbl targets need to be activated to promote the full complement of Dbl effects. More importantly, activation of each of these GTP-binding proteins contributes to a different and distinct facet of cellular transformation. guanine nucleotide exchange factor hemagglutinin c-Jun NH2-terminal kinase Dulbecco's modified Eagle's medium GTPase activating protein glutathioneS -transferase guanosine 5′-3-O -(thio)triphosphate The dbl oncogene was first identified by transfection of fibroblasts with DNA from a human diffuse-B-cell lymphoma (1Eva A. Aaronson S.A. Nature. 1985; 316: 273-275Crossref PubMed Scopus (167) Google Scholar, 2Ron D. Tronick S.R. Aaronson S.A. Eva A. EMBO J. 1988; 7: 2465-2473Crossref PubMed Scopus (83) Google Scholar). Since then, over 15 different oncogene products have been described that bear strong sequence and functional homology to the original Dbl protein (3Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (466) Google Scholar, 4Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (334) Google Scholar). Operationally, Dbl family members have been defined as proteins that contain the tandem arrangement of a pleckstrin homology domain adjacent to a unique domain (approximately 180 amino acids) found only in members of this family, and hence termed the Dbl homology domain. Many of these proteins possess high oncogenic activity, and indeed, most of the Dbl family members were initially found in gene transfer experiments through their ability to potently transform fibroblasts. Oncogenic activation of these cellular proto-oncogenes often occurs by a specific mutation or a chromosomal rearrangement event, which results in continuous, unregulated activity of the mutated proteins. To date, most Dbl family members have been shown to serve as activators, or guanine nucleotide exchange factors (GEFs),1 for Rho-like proteins (i.e. Cdc42, Rac, and Rho) (3Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (466) Google Scholar). Like all GTP-binding proteins, members of the Rho subfamily function as binary molecular switches that are “on” in the GTP-bound state and “off” in the GDP-bound state (5Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1762) Google Scholar, 6Bourne H.R. Sanders D.A. McCormick F. Nature. 1990; 348: 125-131Crossref PubMed Scopus (1844) Google Scholar, 7Hall A. Science. 1990; 249: 635-640Crossref PubMed Scopus (676) Google Scholar). Deactivation (transition from the GTP to the GDP state) is achieved by their intrinsic GTP hydrolytic capability, which is further stimulated by GTPase activating proteins (GAPs). Activation of the GTP-binding proteins occurs in response to a variety of stimuli (such as cell cycle progression and growth factor/cytokine stimulation) and is mediated by GEFs, which stimulate the dissociation of bound GDP. GTP then rebinds, thus triggering the conformational change that leads to the activated state of the molecule. Because nucleotide exchange is the only biochemical activity demonstrated by Dbl proteins, and because transformation and exchange activities share common structure/function features (8Hart M.J. Eva A. Zangrilli D. Aaronson S.A. Evans T. Cerione R.A. Zheng Y. J. Biol. Chem. 1994; 269: 62-65Abstract Full Text PDF PubMed Google Scholar), it has been assumed that the activation of Rho proteins is the basis for the oncogenic activity demonstrated by Dbl proteins. A logical extension of this reasoning is that activated alleles of Rho proteins should be transforming when introduced into cells. Such dominant-positive reagents are typically generated by mutations of residues that are critical for GTP hydrolysis, thus rendering the protein GTPase-defective. When introduced into a cell, the GTPase-defective GTP-binding protein elicits a persistent stimulation of its signaling cascade, resulting in an exaggerated phenotype that directly demonstrates its involvement in a particular pathway. This is exemplified in the case of Ras, in which expression of either the Ras(G12V) or Ras(Q61L) GTPase-defective mutant is oncogenic (9Sigal I.S. Gibbs J.B. D'Alonzo J.S. Temeeles G.L. Wolanski B.S. Socher S.S. Scolnick E.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 952-956Crossref PubMed Scopus (187) Google Scholar), and indeed, such mutations are found in a significant fraction of human tumors (10Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3783) Google Scholar). For members of the Dbl family, elucidation of their transformation mechanism has not been straightforward. Some oncogenic activity has been observed upon expression of the GTPase-defective proteins RhoA(Q63L), Rac1(G12V), and Cdc42(G12V) in fibroblasts and in immunocompromised mice (11Avraham H. Weinberg R. Mol. Cell. Biol. 1989; 9: 2058-2066Crossref PubMed Scopus (95) Google Scholar, 12Qiu R.G. Chen J. Kirn D. McCormick F. Symons M. Nature. 1995; 374: 457-459Crossref PubMed Scopus (813) Google Scholar, 13Qiu R.G. Chen J. McCormick F. Symons M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11781-11785Crossref PubMed Scopus (488) Google Scholar, 14Qiu R.G. Abo A. McCormick F. Symons M. Mol. Cell. Biol. 1997; 17: 3449-3458Crossref PubMed Scopus (265) Google Scholar, 15Khosravi-Far R. Solski P.A. Clarck G.J. Kinch M.S. Der C.J. Mol. Cell. Biol. 1995; 15: 6443-6453Crossref PubMed Scopus (641) Google Scholar, 16Westwick J.K. Lambert Q.T. Clarck G.J. Symons M. Van Aelst L. Pestell R.G. Der C.J. Mol. Cell. Biol. 1997; 17: 1324-1335Crossref PubMed Scopus (385) Google Scholar, 17Whitehead I.P. Abe K. Gorski J.L. Der C.J. Mol. Cell. Biol. 1998; 18: 4689-4697Crossref PubMed Scopus (57) Google Scholar). Furthermore, dominant-negative mutants of these proteins were shown to block Ras-induced transformation, indicating their critical role in proliferative signaling pathways (12Qiu R.G. Chen J. Kirn D. McCormick F. Symons M. Nature. 1995; 374: 457-459Crossref PubMed Scopus (813) Google Scholar, 13Qiu R.G. Chen J. McCormick F. Symons M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11781-11785Crossref PubMed Scopus (488) Google Scholar, 14Qiu R.G. Abo A. McCormick F. Symons M. Mol. Cell. Biol. 1997; 17: 3449-3458Crossref PubMed Scopus (265) Google Scholar, 15Khosravi-Far R. Solski P.A. Clarck G.J. Kinch M.S. Der C.J. Mol. Cell. Biol. 1995; 15: 6443-6453Crossref PubMed Scopus (641) Google Scholar). However, the oncogenic capacity of these proteins has been typically incomplete and weak. Moreover, stable overexpression of GTPase-defective Rho proteins has tended to be difficult. In particular, we have consistently found that significant overexpression of the GTPase-defective alleles (i.e. G12V or Q61L) of Cdc42 in NIH3T3 cells actually has detrimental effects on cell growth. This has prompted us to consider the idea that for proper signaling, Cdc42 must undergo a complete cycle of GTP binding and hydrolysis. We have therefore used an alternative scheme for activation of ectopically expressed GTPases; rather than a mutation that blocks GTP hydrolysis, we have generated mutants that possess enhanced intrinsic GTP↔GDP exchange rate but maintain normal GTP hydrolytic activity. Thus, in vivo , these mutated (“fast-cycling”) GTP-binding proteins become activated spontaneously, and more closely reflect their in vivo activation by the Dbl oncoprotein. Indeed, we have previously shown that Cdc42(F28L) is activated in vivo , and that its stable overexpression in NIH3T3 cells is accompanied by a few hallmarks of malignant transformation (18Lin R. Bagrodia S. Cerione R. Manor D. Curr. Biol. 1997; 7: 794-797Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Here, we use the fast-cycling versions of Cdc42, Rac1, and RhoA (i.e. the primary GTP-binding protein targets of Dbl) to assess their relative contributions to the total phenotype exhibited by Dbl-transformed cells. Rac1(F28L) and RhoA(F30L) mutations were made using a polymerase chain reaction strategy identical to that used earlier for generating the Cdc42(F28L) mutant (18Lin R. Bagrodia S. Cerione R. Manor D. Curr. Biol. 1997; 7: 794-797Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). The reaction included two internal primers harboring the Phe → Leu mutation, two external pET15b primers, and a template of the wild-type gene in pET15b. Expression of recombinant proteins in Escherichia coli was performed exactly as described previously (18Lin R. Bagrodia S. Cerione R. Manor D. Curr. Biol. 1997; 7: 794-797Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 19Leonard D.A. Sartoskar R. Wu J.W. Cerione R.A. Manor D. Biochemistry. 1997; 36: 1173-1180Crossref PubMed Scopus (34) Google Scholar). For transient expression in COS cells, the cDNAs encoding the GTP-binding proteins were subcloned into the (HA-tagged) pKH3 vector or the (Myc-tagged) pCDNA3 vector, using theBam HI-Eco RI restriction sites. For stable expression in NIH3T3 cells, constructs were subcloned into the (HA-tagged) pJ4H vector using the same restriction sites. For focus formation assays, a 3′ Bam HI site was added to all constructs by polymerase chain reaction, and theBam HI-Bam HI fragments were subcloned into theBam HI-digested pZipNeo vector, where correct orientation was verified by restriction digestion. Stable cell lines were generated by co-transfection of NIH3T3 cells with the indicated genes in the pJ4H vector, together with pCDNA3-Neo using the LipofectAMINE method (Life Technologies, Inc.). Neomycin-resistant colonies were selected by two consecutive culturing steps in DMEM supplemented with 10% calf serum and neomycin (G418; 600 μg/ml; Life Technologies, Inc.). Resistant colonies were screened for expression of the desired protein by Western blotting the total lysates with anti-HA antibodies (HA.11; Berkely Antibody Co.). The Dbl-expressing cell line was generated by transformation of NIH3T3 cells with pZip-onco-Dbl (8Hart M.J. Eva A. Zangrilli D. Aaronson S.A. Evans T. Cerione R.A. Zheng Y. J. Biol. Chem. 1994; 269: 62-65Abstract Full Text PDF PubMed Google Scholar), followed by the isolation of a prominent focus and neomycin selection as described above. For primary focus formation assays, the indicated constructs in the pZipNeo vector were used to transfect subconfluent NIH3T3 cells in 6-well plates using the LipofectAMINE method. After 2 days, each well was split into two 100-mm plates and cultured in DMEM supplemented with 10% calf serum. Two weeks after transfection, cells were fixed with formaldehyde and stained with crystal violet, and foci were scored under a microscope. For secondary focus formation assays, 1000 cells stably expressing the indicated constructs were mixed with 2 × 105 NIH3T3 cells and cultured in DMEM supplemented with 10% calf serum. After 10 days, foci larger then 3 mm were scored from fixed and stained plates. Transfection protocols, cell culture and lysis, immunoprecipitation, kinase assays, and soft-agar growth assays were described in detail earlier (18Lin R. Bagrodia S. Cerione R. Manor D. Curr. Biol. 1997; 7: 794-797Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 20Bagrodia S. Taylor S. Creasy C. Chernoff J. Cerione R. J. Biol. Chem. 1995; 270: 22731-22737Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 21Bagrodia S. Derigard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). Nucleotide exchange was monitored using the mant-GDP fluorescence assay (22Leonard D.A. Evans T. Hart M. Cerione R.A. Manor D. Biochemistry. 1994; 33: 12323-12328Crossref PubMed Scopus (69) Google Scholar) or the binding of [35S]GTPγS as described (23Leonard D.A. Lin R. Cerione R.A. Manor D. J. Biol. Chem. 1998; 273: 16210-16215Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). For measurements of GTP hydrolysis, 1 μm purified protein was incubated with 20 mm Tris-HCl (pH 8.0), 100 mm NaCl, 1 mm dithiothreitol, 0.5 mg/ml bovine serum albumin, 1 μm GTP, 100 nm [γ-32P]GTP (30 Ci/mmol, NEN Life Science Products) in the presence of 15 mm EDTA for wild-type protein or 5 mm EDTA for fast-cycling proteins, at room temperature for 20 min. Hydrolysis was initiated by dilution with 20 mm Tris (pH 8.0), 100 mm NaCl, 1 mm dithiothreitol, 0.5 mg/ml bovine serum albumin, 20 mm MgCl2, with or without 0.01 μm Cdc42-GAP purified as described previously (23Leonard D.A. Lin R. Cerione R.A. Manor D. J. Biol. Chem. 1998; 273: 16210-16215Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). GTP hydrolysis was measured at room temperature for Cdc42 and Rac1 and at 37 °C for RhoA. This assay has been described in detail (24Taylor S.J. Shalloway D. Curr. Biol. 1996; 6: 1621-1627Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 25Bagrodia S. Taylor S.J. Jordon K.A. Van Aelst L. Cerione R.A. J. Biol. Chem. 1998; 273: 23633-23636Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Briefly, COS-7 cells were transiently transfected with the cDNA for the indicated GTP-binding protein in the pKH3 vector, with or without oncogenic Dbl in the pCMV vector. Twenty-four hours posttransfection, cells from 60-mm plates were lysed in 20 mm HEPES, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 20 mm NaF, 20 mm β-glycerol-phosphate, 20 μm GTP, 1 mm sodium vanadate, and 10 μg/ml each of leupeptin and aprotonin and incubated with 50 μg of recombinant glutathione S -transferase (GST)-PBD (20Bagrodia S. Taylor S. Creasy C. Chernoff J. Cerione R. J. Biol. Chem. 1995; 270: 22731-22737Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). GST-PBD was then precipitated with glutathione-agarose beads, washed three times with lysis buffer, and subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting using the indicated antibodies. Stable cell lines were cultured on dual-chamber microscope slides (Nunc) for 2 days in normal media, and then serum-starved for 12 h and fixed with 3.7% formaldehyde. Slides were then sequentially incubated with anti-vinculin antibodies (Sigma), Oregon Green-conjugated goat anti-mouse antibodies, Texas Red phalloidin, and Hoechst-33342 (all from Molecular Probes). The slides were visualized and photographed on a Nikon Eclipse 600 fluorescence microscope. The various stable cell lines were cultured in DMEM supplemented with 10% calf serum, trypsinized, and washed once with growth media and once with phosphate-buffered saline. 107 cells were injected subcutaneously into two dorsal sites of athymic nude mice (CD-1; Charles River Laboratories). Visible tumors (>0.5 cm) formed in the injection sites after the indicated latency and grew progressively for another week. A phenylalanine residue corresponding to position 28 in Ras is highly conserved in the Ras superfamily of small GTPases, in which it has been shown to interact with the guanine base of the nucleotide (26Pai E.F. Kabsch W. Krengel U. Holmes K.C. John J. Wittinghofer A. Nature. 1989; 341: 209-214Crossref PubMed Scopus (691) Google Scholar, 27Feltham J. Dotch V. Raza S. Manor D. Cerione R. Sutcliffe M. Wagner G. Oswald R. Biochemistry. 1997; 36: 8755-8766Crossref PubMed Scopus (79) Google Scholar, 28Hirshberg M. Stockley R.W. Dodson G. Webb M.R. Nature Struct. Biol. 1997; 4: 147-151Crossref PubMed Scopus (191) Google Scholar, 29Wei Y. Zhang Y. Derewenda U. Liu X. Minor W. Nakamoto R.K. Somlyo A.V. Somlyo A.P. Derewenda Z.S. Nat. Struct. Biol. 1997; 4: 699-703Crossref PubMed Scopus (156) Google Scholar). Conservative substitution of this residue to a leucine resulted in a reduced affinity of the protein to guanine nucleotides in Ras (30Reinstein J. Schlichting I. Frech M. Goody R. Wittinghofer A. J. Biol. Chem. 1991; 266: 17700-17706Abstract Full Text PDF PubMed Google Scholar) and Cdc42 (18Lin R. Bagrodia S. Cerione R. Manor D. Curr. Biol. 1997; 7: 794-797Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), leading to spontaneous activation (i.e. GTP binding) of the mutated protein when expressed in cultured cells. We have generated the corresponding mutations in Rac1 and RhoA (i.e. Rac1(F28L) and RhoA(F30L)) and expressed and purified these mutants to homogeneity from E. coli . The ability of these purified proteins to bind GTPγS was compared, and is shown in Fig. 1 A . As is typical for all GTP-binding proteins, the wild-type versions of Rac1, Cdc42, and RhoA show only negligible levels (<10%) of [35S]GTPγS binding activity in the presence of 15 mm MgCl2. The addition of EDTA, which chelates the tightly bound Mg2+ ion (31Hall A. Self A.J. J. Biol. Chem. 1986; 261: 10963-10965Abstract Full Text PDF PubMed Google Scholar), leads to complete exchange of the bound GDP for GTPγS (defined as 100% in Fig. 1 A ). The Cdc42, Rac1, and RhoA point mutants, on the other hand, exhibit significant [35S]GTPγS binding activity, even in the presence of high Mg2+ (i.e. 63, 76, and 53% of the maximal binding, respectively), indicating a significantly higher basal nucleotide exchange activity. To fully assess the biochemical properties of the mutated GTP-binding proteins, we have also measured their ability to hydrolyze GTP in the presence and absence of the Cdc42-GAP (32Barfod E.T. Zheng Y. Kuang W.J. Hart M.J. Evans T. Cerione R.A. Ashkenazi A. J. Biol. Chem. 1993; 268: 26059-26062Abstract Full Text PDF PubMed Google Scholar). The GTP-binding proteins were complexed with [γ-32P]GTP, and GTP hydrolysis was initiated by the addition of Mg2+ (33Hart M.J. Maru Y. Leonard D. Witte O.N. Evans T. Cerione R.A. Science. 1992; 258: 812-815Crossref PubMed Scopus (122) Google Scholar). As can be seen from Fig.1 B, both the point-mutated Cdc42 and Rac proteins showed intrinsic GTP hydrolytic rates that were comparable to their wild-type counterparts. The intrinsic GTP hydrolytic activity of RhoA has been consistently observed to be slower than the corresponding activities for Rac and Cdc42, and this activity is slightly reduced (by 30–50%) in the fast-cycling RhoA mutant. A similar affect has been seen when examining the analogous point mutation in Ras (30Reinstein J. Schlichting I. Frech M. Goody R. Wittinghofer A. J. Biol. Chem. 1991; 266: 17700-17706Abstract Full Text PDF PubMed Google Scholar). More importantly, each of the point mutants is fully responsive to GAP stimulation, yielding turnover numbers for GTP hydrolysis that are virtually indistinguishable from those for the wild-type proteins. Taken together, our in vitro results indicate that Cdc42(F28L), Rac1(F28L), and RhoA(F30L) all exhibit significantly higher GTP ↔ GDP exchange activity compared with their wild-type counterparts but maintain GTP hydrolytic capability. Because of their inherent ability to rapidly undergo GDP-GTP exchange and still hydrolyze GTP (thereby undergoing a fast GTP-binding/GTPase cycle), we have referred to these point mutants as fast-cycling mutants. It was our expectation that these mutants would be constitutively active in vivo (i.e. without the involvement of an extracellular signal or GEF activity), due to the high GTP:GDP ratio in cytosol (34Otero A.D. Biochem. Pharmacol. 1990; 39: 1399-1404Crossref PubMed Scopus (104) Google Scholar). In vivo activation of Cdc42, Rac, and Rho is accompanied by diverse biological phenotypes accomplished through interactions with different target proteins. To assess whether the Phe → Leu mutation indeed results in the spontaneous activation of the different GTP-binding proteins in vivo , we have utilized a modification of an assay developed to assess the activation level of Ras (24Taylor S.J. Shalloway D. Curr. Biol. 1996; 6: 1621-1627Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 25Bagrodia S. Taylor S.J. Jordon K.A. Van Aelst L. Cerione R.A. J. Biol. Chem. 1998; 273: 23633-23636Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). This assay is based on the GTP-specific high affinity interaction between the tested GTP-binding protein and the binding domain of its target, PAK-3 (20Bagrodia S. Taylor S. Creasy C. Chernoff J. Cerione R. J. Biol. Chem. 1995; 270: 22731-22737Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar), which is fused to GST to enable affinity precipitation. Thus, a recombinant GST fusion protein containing the p21-binding domain (PBD, also known as the CRIB (Cdc42/Rac-interaction binding) domain) (35Burbelo P.D. Drechsel D. Hall A. J. Biol. Chem. 1995; 270: 29071-29074Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar) of PAK-3 immobilized on glutathione-agarose was incubated with lysates from COS-7 cells transfected with the different forms of Cdc42 and Rac1. Following extensive washes, the lysates (Fig.2 A, middle panel ) and the precipitated GST-PBD beads (Fig. 2 A, top panel ) were electrophoresed and blotted for the (HA-tagged) GTP-binding proteins. Fig. 2 A, middle panel, shows that each of the GTP-binding proteins were expressed to significant and similar levels. As expected, the co-expression of Cdc42 and Dbl (compare lanes 1 and5 ), as well as Rac1 and Dbl (compare lanes 3 and6 ), resulted in the enhanced precipitation of the GTP-binding protein, indicating that Dbl activates each of these proteins in cells. More importantly, the amounts of the fast-cycling versions of Rac1 and Cdc42 precipitated with GST-PBD were markedly higher than those of the wild-type GTP-binding proteins (comparelanes 1 and 2 or lanes 3 and4 ). We have used this assay also to compare the activation level of the fast-cycling version (F28L) with that of the GTPase-defective version (Q61L) of Cdc42 and Rac1. Under essentially identical experimental conditions, 20% of the expressed Cdc42(Q61L) precipitated with GST-PBD, versus 18% of the fast-cycling, Cdc42(F28L) mutant (± 2%; data not shown). This verifies that a significantly larger fraction of each Phe → Leu mutant is in the GTP-bound state, compared with the corresponding wild-type protein, and is consistent with the idea that the fast-cycling versions of Cdc42 and Rac1 are spontaneously activated when ectopically expressed in cultured cells. Another well characterized signaling end point for Cdc42 and Rac1 is a nuclear transcriptional activator, the c-Jun kinase (JNK1) (21Bagrodia S. Derigard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 36Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1447) Google Scholar,37Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1570) Google Scholar). We have previously reported that the expression of the Cdc42(F28L) mutant in COS-7 cells leads to activation of JNK1 (18Lin R. Bagrodia S. Cerione R. Manor D. Curr. Biol. 1997; 7: 794-797Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). We show here that this is also true for the Rac1(F28L) mutant. Fig. 2 B shows the results of an experiment in which wild-type Rac1, the GTPase-defective Rac1(Q61L) mutant, and the fast-cycling Rac1(F28L) mutant were co-transfected into COS-7 cells together with flag-tagged JNK1, and then immunocomplex kinase assays were performed following anti-flag immunoprecipitation. The JNK1 precipitated from cells expressing the Rac1(F28L) mutant exhibited levels of protein kinase activity (measured by the phosphorylation of c-Jun) that were comparable to those measured in cells expressing the Rac1(Q61L) mutant and significantly higher than the activity precipitated from cells expressing wild-type Rac1 or vector alone. We have also established NIH3T3 cell lines that stably express HA-tagged forms of Cdc42(F28L), Rac1(F28L), and RhoA(F30L). Taking advantage of the high affinity interaction between JNK and c-Jun, we examined the endogenous JNK activity in these stable cell lines as well as from cells expressing the oncogenic Dbl protein. Lysates from the different cell lines were incubated with recombinant GST-c-Jun immobilized on glutathione beads. The precipitated GST-Jun·JNK1 complexes were washed, incubated with MgCl2 and [γ-32P]ATP, electrophoresed, and autoradiographed. JNK activity, visualized as 32P incorporation into the GST-Jun protein, is shown in Fig. 2 C . It is clear from these data that JNK activity is stimulated 3–5-fold in cell lines expressing fast-cycling Cdc42, Rac1, or oncogenic Dbl, relative to mock-transfected cells. Stimulation of JNK activity by fast-cycling RhoA, can be detected only in cells expressing relatively high levels of RhoA(F30L), in accordance with previous reports (36Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1447) Google Scholar, 37Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1570) Google Scholar). 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• W2015493603 date "1999-08-01" @default.
• W2015493603 modified "2023-10-01" @default.
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