The Dbl-related Protein, Lfc, Localizes to Microtubules and Mediates the Activation of Rac Signaling Pathways in Cells

The possibility that the Dbl family member Lfc can activate Rac1 in cells is investigated in this study. Previously, we demonstrated that both Lfc and Lsc, like their closest relative Lbc, can act catalytically in stimulating the guanine nucleotide exchange activity of RhoA in vitro. Neither Lfc nor Lsc stimulated the in vitro exchange activity of Cdc42 or Rac1; however, Lfc was capable of forming a tight complex with Rac1 in vitro. We show here that Lfc stimulates c-Jun kinase (JNK) activity in COS-7 cells. This stimulation was blocked by a dominant negative mutant of Rac1 and somewhat less effectively by dominant negative RhoA, but not by dominant negative Cdc42. Overexpression of Lfc in NIH 3T3 cells induced the formation of actin stress fibers and membrane ruffles, consistent with the activation of both RhoA and Rac1 signaling pathways, whereas overexpression of Lsc led exclusively to well developed stress fibers. Using a recently developed assay for measuring the cellular activation of Rac, we did not find that expression of Lfc increased the levels of GTP-bound Rac1. However, an examination of the cellular localization of Lfc showed that it was localized to microtubules, similar to what has been reported for activated Rac1, the mixed lineage kinase (MLK) and JNK. Moreover, we have found that the Pleckstrin homology (PH) domain of Lfc specifically associates with tubulin. Taken together, these findings suggest a model where the PH domain-mediated localization of Lfc to microtubules enables the recruitment of Rac to a site proximal to its signaling targets, resulting in JNK activation and actin cytoskeletal changes. The possibility that the Dbl family member Lfc can activate Rac1 in cells is investigated in this study. Previously, we demonstrated that both Lfc and Lsc, like their closest relative Lbc, can act catalytically in stimulating the guanine nucleotide exchange activity of RhoA in vitro. Neither Lfc nor Lsc stimulated the in vitro exchange activity of Cdc42 or Rac1; however, Lfc was capable of forming a tight complex with Rac1 in vitro. We show here that Lfc stimulates c-Jun kinase (JNK) activity in COS-7 cells. This stimulation was blocked by a dominant negative mutant of Rac1 and somewhat less effectively by dominant negative RhoA, but not by dominant negative Cdc42. Overexpression of Lfc in NIH 3T3 cells induced the formation of actin stress fibers and membrane ruffles, consistent with the activation of both RhoA and Rac1 signaling pathways, whereas overexpression of Lsc led exclusively to well developed stress fibers. Using a recently developed assay for measuring the cellular activation of Rac, we did not find that expression of Lfc increased the levels of GTP-bound Rac1. However, an examination of the cellular localization of Lfc showed that it was localized to microtubules, similar to what has been reported for activated Rac1, the mixed lineage kinase (MLK) and JNK. Moreover, we have found that the Pleckstrin homology (PH) domain of Lfc specifically associates with tubulin. Taken together, these findings suggest a model where the PH domain-mediated localization of Lfc to microtubules enables the recruitment of Rac to a site proximal to its signaling targets, resulting in JNK activation and actin cytoskeletal changes. guanine nucleotide exchange factor Dbl homology Pleckstrin homology p21-activated kinase mixed lineage kinase c-Jun kinase myelin basic protein p21 (Cdc42/Rac1) binding domain polymerase chain reaction polyacrylamide gel electrophoresis glutathione S- transferase. Lfc was initially identified based on its transforming activity when overexpressed in NIH 3T3 cells (1Whitehead I. Kirk H. Tognon C. Trigo-Gonzalez G. Kay R. J. Biol. Chem. 1995; 270: 18388-18395Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The Lfc oncoprotein is a member of the Dbl family of growth regulatory proteins. Many members of the Dbl family have been demonstrated to function upstream of the Rho-related GTP-binding proteins, acting as guanine nucleotide exchange factors (GEFs)1 by stimulating the exchange of GTP for GDP and thereby promoting G protein activation. This rapidly growing family of regulatory proteins includes greater than 20 members to date (reviewed in Ref. 2Whitehead I.P. Cambell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (334) Google Scholar). The fact that there exist so many GEFs for the Rho subfamily suggests that there will be multiple pathways leading to the activation of an individual GTP-binding protein. All members of the Dbl family possess a Dbl homology (DH) domain in tandem with a Pleckstrin homology (PH) domain, and it has been demonstrated that both domains are required for the transforming activities of oncogenic members of the family. The DH domain typically represents the limit motif for binding the G protein and stimulating nucleotide exchange (3Hart 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), whereas the PH domain appears to be essential for mediating the appropriate cellular localization of the protein (1Whitehead I. Kirk H. Tognon C. Trigo-Gonzalez G. Kay R. J. Biol. Chem. 1995; 270: 18388-18395Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 4Zheng Y. Zangrilli D. Cerione R.A. Eva A. J. Biol. Chem. 1996; 271: 19017-19020Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Additionally, members of the Dbl family contain a number of other structural motifs that indicate a role in signal transduction. These domains presumably function to mediate protein/protein and protein/lipid interactions and serve to link members of the Dbl family to upstream regulation (2Whitehead I.P. Cambell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (334) Google Scholar). This has been most carefully worked out for the Dbl family member, Vav, which is a Rac-GEF that is regulated by tyrosine phosphorylation and by the phospholipids, phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) (5Crespo P. Schubel K.E. Ostram A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (682) Google Scholar, 6Han J. Das B. Wei W. Van Aelst L. Mosteller R.D. Khosravi F.R. Westwick J.K. Der C.J. Broek D. Mol. Cell. Biol. 1997; 17: 1346-1353Crossref PubMed Scopus (277) Google Scholar, 7Han J. Luby-Phelps K. Das B. Shu X. Xia Y. Mostellar R.D. Krishna U.M. Falck J.R. White M.A. Broek D. Science. 1998; 279: 558-560Crossref PubMed Scopus (710) Google Scholar). Lfc contains a cysteine-rich domain similar to the diacylglycerol binding domain found in protein kinase C which may function to couple Lfc with upstream generation of lipids (1Whitehead I. Kirk H. Tognon C. Trigo-Gonzalez G. Kay R. J. Biol. Chem. 1995; 270: 18388-18395Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Determining the regulation and cellular localization of the growing family of Rho-GEFs will be important for understanding how Rho-related GTP-binding proteins mediate multiple cellular activities. Activated RhoA, Rac1, and Cdc42 regulate both gene transcription and the actin cytoskeleton, contributing to the control of cell morphology, motility, and growth (reviewed in Refs. 8Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2101) Google Scholar and 9Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar). Through their interactions with multiple targets, the Rho family of GTP-binding proteins is able to coordinate these diverse cellular functions. A number of targets have been identified including two related families of serine/threonine kinases, the p21-activated kinases (Paks) and the mixed lineage kinases (MLKs), which have been shown to play specific roles in regulating gene transcription and the actin cytoskeleton (10Bagrodia S. Taylor S.J. Creasy C.L. Chernoff J. Cerione R.A. J. Biol. Chem. 1995; 270: 22731-22737Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 11Brown J.L. Stowers L. Baer M. Trejo J. Coughlin S. Chant J. J. Curr. Biol. 1996; : 598-605Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 12Manser E. Chong C. Huang H.Y. Loo T.H. Chen X. Dong J.M. Leung T. Lim L. Mol. Cell. Biol. 1997; 17: 1129-1143Crossref PubMed Google Scholar, 13Sells M. Knaus U. Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1997; 7: 202-210Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 14Dharmawardhane S. Sanders L.C. Martin S.S. Daniels R.H. Bokoch G.M. J. Cell Biol. 1997; 138: 1265-1278Crossref PubMed Scopus (199) Google Scholar, 15Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). It has previously been reported that Rac1 and Cdc42 stimulate the enzymatic activity of the mitogen-activated protein kinases (MAPKs), JNK and p38 (16Coso O.A. Chiariello M. Yu J.C. Termoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1570) Google Scholar). Additionally, both the Paks and MLKs have also been shown to stimulate JNK activity (11Brown J.L. Stowers L. Baer M. Trejo J. Coughlin S. Chant J. J. Curr. Biol. 1996; : 598-605Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 15Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 17Bagrodia S. Derijard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). The JNKs in turn phosphorylate and regulate the activity of proteins that control the expression of specific gene products involved in regulating growth and morphology (18Davis R.J. Mol. Reprod. Dev. 1995; 42: 459-467Crossref PubMed Scopus (383) Google Scholar, 19Whitemarsh A.J. Yang S. Su M.S. Sharrocks A.D. Davis R.J. Mol. Cell. Biol. 1997; 17: 2360-2371Crossref PubMed Scopus (438) Google Scholar). The JNK family members have also been referred to as stress-activated protein kinases (SAPKs) because of the ability of ultraviolet radiation, osmotic shock, or inflammatory cytokines such as interleukin-1 and tumor necrosis factor-α to stimulate their activity (20Derijard B. Hibi M. Wu I.-H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2957) Google Scholar, 21Sluss H.K. Barrett T. Derijard B. Davis R.J. Mol. Cell. Biol. 1994; 14: 8376-8384Crossref PubMed Scopus (446) Google Scholar). We have previously shown that Lfc, which functions specifically as a GEF for RhoA in vitro, binds tightly to Rac1 in a nucleotide-independent manner (22Glaven J.A. Whitehead I.P. Nomanbhoy T. Kay R. Cerione R.A. J. Biol. Chem. 1996; 271: 27374-27381Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Additionally, Lfc strongly activates JNK in COS-7 cells. These findings suggest that Lfc may have a broader specificity in cells that would include promoting a Rac1-mediated pathway which leads to JNK activation. This led us to examine the cellular activity of Lfc. Thus far, Vav, Tiam-1, and SOS (7Han J. Luby-Phelps K. Das B. Shu X. Xia Y. Mostellar R.D. Krishna U.M. Falck J.R. White M.A. Broek D. Science. 1998; 279: 558-560Crossref PubMed Scopus (710) Google Scholar, 23Michiels F. Stam J.C. Hordijk P.L. van der Kammen R.A. Ruuls-Van Stalle L. Feltkamp C.A. Collard J.G. J. Cell Biol. 1997; 137: 387-398Crossref PubMed Scopus (211) Google Scholar, 24Nimnual A.S. Yatsula B.A. Bar-Sagi D. Science. 1998; 279: 560-563Crossref PubMed Scopus (389) Google Scholar) are Dbl family members that with the help of cellular co-factors (e.g. lipid second messengers) have been shown to stimulate Rac activation in cells. In the present studies, we show that Lfc is another Dbl family member that has a positive effect on Rac signaling. However, in this case, we do not detect a direct Lfc-stimulated activation of Rac1. Rather Lfc, through direct interactions with microtubules as mediated by its PH domain, may serve to mark the cellular site for a Rac-signaling complex that leads to JNK activation and/or actin cytoskeletal changes. Plasmids pAX142 HAD7-Lfc and HAD6-Lfc (1Whitehead I. Kirk H. Tognon C. Trigo-Gonzalez G. Kay R. J. Biol. Chem. 1995; 270: 18388-18395Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) and D7-Lsc (30Whitehead I.P. Khosravi-Far R. Kirk H. Trigo-Gonzalez G. Der C.J. Kay R. J. Biol. Chem. 1996; 271: 18643-18650Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) have been described previously. The GST-Lfc (PH) was generated by PCR from the D7-Lfc cDNA. Primers for PCR encoded the restriction sites HindIII and XbaI to subclone the PH domain fragment into the Escherichia coli expression vector pGXKG. The PCR generated fragment encompassed the entire PH domain plus a small amount of flanking sequence (amino acid sequence 450–573). Generation of all constructs encoding Rac1, Cdc42, and their dominant negative mutants have been described previously (10Bagrodia S. Taylor S.J. Creasy C.L. Chernoff J. Cerione R.A. J. Biol. Chem. 1995; 270: 22731-22737Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 22Glaven J.A. Whitehead I.P. Nomanbhoy T. Kay R. Cerione R.A. J. Biol. Chem. 1996; 271: 27374-27381Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 26Bagrodia S. Taylor S.J. Jordan A. Van Aelst L. Cerione R.A. J. Biol. Chem. 1998; 273: 23633-23636Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). These into the expression vector which has been to contain the or for expression in cells. The generation of has been described S. Derijard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google was by in the site to the PCR The Dbl was generated by a fragment encoding oncogenic Dbl from from into the site of encoding the and dominant negative mutants of Rho a from C. Der of and into which contains the for expression in cells. The has been described previously S. Derijard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). Plasmids and from J. S. Gutkind of All products to that of COS-7 cells and NIH 3T3 cells out to the the was with for COS-7 cells or for NIH 3T3 cells. complex kinase cells of studies, cells the of and for for to for kinase with in 20 and and for expression was by complex kinase for the amount of or JNK was with to protein was with to protein in and into two was to and the second was in the kinase JNK kinase in and and with of and 20 in 20 and for 20 kinase in and 20 and mixed with of myelin basic protein and 20 in a of for The kinase by the of and by The of was by The p21 (Cdc42/Rac1) binding domain of was in E. coli as a protein and by binding to The was to activated Rac1 from COS-7 cell in and in 20 and by for in and The expression of proteins was by of an to and for for out for in and by Rac1 was with The was with the studies, cells for with for and with for cytoskeleton was by with for by with studies, cells and as Lfc was for by for with from G. for by for The did not show or and a The expression and of Lfc from cells has been previously described (22Glaven J.A. Whitehead I.P. Nomanbhoy T. Kay R. Cerione R.A. J. Biol. Chem. 1996; 271: 27374-27381Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The protein was in E. coli and an that was from a was to a that was to of expression was induced by the of for by and The in 20 and on by by of and The by for by The proteins in from and out for and in cell and the to was with and the was with the It has been reported previously that both Rac1 and Cdc42 can activate the JNK mitogen-activated protein kinase in COS-7 whereas RhoA not (16Coso O.A. Chiariello M. Yu J.C. Termoto 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 that the overexpression of Lfc in COS-7 cells leads to JNK activation. In we show a of Lfc and Lsc, and the of the proteins in this study. Using an in vitro kinase assay to the activity of JNK from we find that Lfc and Dbl can activate JNK in COS-7 cells to an similar to the activation induced by cells to ultraviolet radiation, whereas Lsc stimulation The stimulation of JNK activity was not on the diacylglycerol binding domain the and D7-Lfc constructs both JNK activation The of stimulation by Lsc that RhoA can activate JNK in cell as reported by H. Crespo P. Coso O.A. Igishi T. Xu N. Gutkind J.S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), it not activate JNK in COS-7 cells. we out to Rac1 the Lfc-stimulated activation of JNK in COS-7 of JNK by Lfc in COS-7 cells with with an expression vector or with an expression vector encoding Lfc, Lsc, or Dbl and JNK activity was by complex kinase and c-Jun as by and by to show phosphorylation of and to show relative of in the kinase a of the ability of Lfc and Lfc the to stimulate JNK activity in and complex kinase described in We the activation of JNK in COS-7 cells be by dominant negative mutants of the Rho family of small GTP-binding proteins. on in vitro binding studies, which that Lfc with both RhoA and Rac1, we would that dominant negative of RhoA and Rac1 would be capable of binding Lfc and its activation of JNK. shown in blocked Lfc-stimulated JNK whereas levels of showed a levels of was able to an negative Cdc42 was not able to Lfc-stimulated JNK activity The which serve as targets for Cdc42 and Rac but not RhoA, have been shown to mediate the activation of JNK in cell (10Bagrodia S. Taylor S.J. Creasy C.L. Chernoff J. Cerione R.A. J. Biol. Chem. 1995; 270: 22731-22737Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 11Brown J.L. Stowers L. Baer M. Trejo J. Coughlin S. Chant J. J. Curr. Biol. 1996; : 598-605Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 17Bagrodia S. Derijard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). we a mutant of by with would JNK activation. A represents the that this was the However, it was that we did not find that Lfc stimulated activity in these cells. We this by with Lfc, Lsc, or Dbl in COS-7 cells. These showed that Lfc did not activate where Dbl stimulated activity This suggests that the direct stimulation of Rac activity by Lfc is not the by which Lfc JNK activation is another protein kinase for Cdc42 and Rac that has recently been shown to be a of JNK H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). that like the a of Lfc-stimulated JNK Thus far, we have not been able to Lfc stimulates activity because this protein kinase is when in and so it has not been to is an essential in the signaling pathway leading to JNK negative mutant of Lfc-stimulated JNK COS-7 cells with with an expression vector or with an expression vector encoding Lfc and of kinase or of and JNK activity was by complex kinase and c-Jun as by and by to show phosphorylation of and to show relative of in the kinase guanine nucleotide exchange in that Lfc stimulated exchange on RhoA but not on Rac1 (22Glaven J.A. Whitehead I.P. Nomanbhoy T. Kay R. Cerione R.A. J. Biol. Chem. 1996; 271: 27374-27381Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), that Lfc did not act as a GEF for Rac1. However, similar in vitro have been for Vav, Tiam-1, and but it was shown that cellular co-factors to of these to act as GEFs for Rac1 in cells (7Han J. Luby-Phelps K. Das B. Shu X. Xia Y. Mostellar R.D. Krishna U.M. Falck J.R. White M.A. Broek D. Science. 1998; 279: 558-560Crossref PubMed Scopus (710) Google Scholar, 23Michiels F. Stam J.C. Hordijk P.L. van der Kammen R.A. Ruuls-Van Stalle L. Feltkamp C.A. Collard J.G. J. Cell Biol. 1997; 137: 387-398Crossref PubMed Scopus (211) Google Scholar, 24Nimnual A.S. Yatsula B.A. Bar-Sagi D. Science. 1998; 279: 560-563Crossref PubMed Scopus (389) Google Scholar). we of a recently developed assay for Rac activation S. Taylor S.J. Jordan A. Van Aelst L. Cerione R.A. J. Biol. Chem. 1998; 273: 23633-23636Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar) to Lfc the levels of GTP-bound Rac1 in cells. the domain of was as a protein and on and was as an to GTP-bound from COS-7 cell shown in when this we to detect an in the levels of GTP-bound Rac1 in cells that with Lfc and However, we able to detect an in GTP-bound Rac1 when Dbl was with consistent with that Dbl can stimulate the activation of Rac in cells S. Taylor S.J. Jordan A. Van Aelst L. Cerione R.A. J. Biol. Chem. 1998; 273: 23633-23636Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). in the actin cytoskeleton have been shown to be regulated by members of the Rho subfamily of GTP-binding proteins (reviewed in Ref. 9Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar). Moreover, it has been shown that of the Rho family members of these we out to Lfc a with to the actin cytoskeleton in NIH 3T3 cells with an expression vector encoding Lfc or Lsc and expression vector of with an to cells Lfc or We also the actin induced by expression of RhoA and Rac1 shown in cells the RhoA mutant have actin stress fibers as with control NIH 3T3 cells NIH 3T3 cells Rac1 have an actin their resulting in membrane We show in that Lsc strongly actin stress fibers and a morphology similar to that when the activated RhoA Lfc, however, appears to not the formation of actin stress fibers but also and membrane of signaling through both activated RhoA and Rac1 We Lfc in to examine the cellular localization of Lfc. Using a as the and we a cellular for Lfc, as by a not This of the We cells Lfc to examine Lfc with Lfc was as described and microtubules an by The of this that Lfc with the A localization to microtubules has also been reported for Rac1 A. S. R. Lim L. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) and recently for and JNK K. A. C. P. E. T. N. A. J. 1998; 17: PubMed Scopus Google Scholar). The Lfc, which contains the domains of Lfc, was as an to binding that a protein with the GST-Lfc The which can be with not was identified as based on this we a the PH domain of Lfc the GST-Lfc (PH) protein was in E. coli and as an it was able to in an manner as we with the domain The (PH) domain was to the not Taken together, these findings as a binding for the PH domain of Lfc and suggest that it is the PH domain that is for the of Lfc with microtubules in cells. Lfc as a nucleotide exchange factor for RhoA in vitro and is able to actin stress fibers when overexpressed in NIH 3T3 cells. However, the fact that we have shown that Lfc a tight binding complex with Rac1 in vitro, with the that Lfc stimulates JNK in COS-7 led us to examine Lfc or the activated of Rac1 in cells. The activation of JNK by Vav, and Dbl have all been shown to be mediated by Rac1, such that the dominant negative mutant their ability to activate JNK (16Coso O.A. Chiariello M. Yu J.C. Termoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1570) Google P. Bustelo X.R. Aaronson Coso O.A. M. M. Gutkind J.S. 1996; Google Scholar). The here indicate that Rac1 also the activation of JNK by Lfc in COS-7 cells. Lfc-stimulated JNK activity was by expression of the dominant negative of Rac1, but not by dominant negative Cdc42. negative mutants of and two targets of Rac1, also Lfc-stimulated JNK