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• W1483753543 abstract "Dbl is a guanine nucleotide exchange factor that activates the Rho family GTPases Cdc42, Rac, and Rho. Dbl and all three GTPases are strong activators of transcription factor NFκB, which has been shown to have an important role in Dbl-induced oncogenic transformation. Here we show that although Dbl activation of NFκB requires Cdc42, Rac, and Rho, the different GTPases activate NFκB by different mechanisms. Whereas Rac stimulates the activity of the IκB kinase IKKβ, Cdc42 and Rho activate NFκB without activating either IKKα or IKKβ. Like Dbl, Rac activation of IKKβ is mediated by the serine/threonine kinases NIK but not MEKK. This differs from Rac activation of the JNK pathway, which was previously shown to be mediated by MEKK. The pathway leading from Rho and Cdc42 to NFκB is more elusive, but our results suggest that it involves an IKKα/IKKβ-independent mechanism. Finally, we show that the signaling enzymes that mediate NFκB activation by Dbl and the Rho GTPases are also necessary for malignant transformation induced by oncogenic Dbl. Dbl is a guanine nucleotide exchange factor that activates the Rho family GTPases Cdc42, Rac, and Rho. Dbl and all three GTPases are strong activators of transcription factor NFκB, which has been shown to have an important role in Dbl-induced oncogenic transformation. Here we show that although Dbl activation of NFκB requires Cdc42, Rac, and Rho, the different GTPases activate NFκB by different mechanisms. Whereas Rac stimulates the activity of the IκB kinase IKKβ, Cdc42 and Rho activate NFκB without activating either IKKα or IKKβ. Like Dbl, Rac activation of IKKβ is mediated by the serine/threonine kinases NIK but not MEKK. This differs from Rac activation of the JNK pathway, which was previously shown to be mediated by MEKK. The pathway leading from Rho and Cdc42 to NFκB is more elusive, but our results suggest that it involves an IKKα/IKKβ-independent mechanism. Finally, we show that the signaling enzymes that mediate NFκB activation by Dbl and the Rho GTPases are also necessary for malignant transformation induced by oncogenic Dbl. guanine nucleotide exchange factor IκB kinase NFκB-inducing kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase c-Jun NH2-terminal kinase JNK kinase nuclear transcription factor-κB tumor necrosis factor p21-activated kinase hemagglutinin glutathioneS-transferase luciferase The Rho family of GTPases, including members of the Cdc42, Rac, and Rho subfamilies, function as molecular switches cycling between an inactive GDP-bound state and an active GTP-bound state (1Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2101) Google Scholar). Guanine nucleotide exchange factors (GEFs)1 catalyze the activation of the GTPases by exchanging GDP for GTP. Dbl is a GEF that acts both in vivo and in vitro as an exchange factor for Cdc42, Rho, and Rac (2Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (466) Google Scholar, 3Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (334) Google Scholar). Dbl contains a Dbl homology domain that is required for GEF activity (4Hart 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) adjacent to a pleckstrin homology domain that is most likely responsible for proper localization at the membrane (5Zheng 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). Dbl is a representative prototype of a growing family of proto-oncogenes that contain Dbl homology/pleckstrin homology elements. Activated forms of the Dbl family members are associated with a variety of neoplastic pathologies (2Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (466) Google Scholar, 3Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (334) Google Scholar, 6Eva A. Aaronson S.A. Nature. 1985; 316: 273-275Crossref PubMed Scopus (167) Google Scholar). It is generally thought that the activation of Rho family GTPases may be responsible for their potent transformation capabilities. Indeed, Cdc42, Rac, and Rho were each shown to contribute to distinct aspects of Dbl-induced transformation (7Lin R. Cerione R.A. Manor D. J. Biol. Chem. 1999; 274: 23633-23641Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Rho proteins have also been shown to be necessary for transformation by other oncogenes including Ras (8Khosravi-Far R. Solski P.A. Clark G.J. Kinch M.S. Der C.J. Mol. Cell. Biol. 1995; 15: 6443-6453Crossref PubMed Scopus (641) Google Scholar, 9Lamarche N. Tapon N. Stowers L. Burbelo P.D. Aspenstrom P. Bridges T. Chant J. Hall A. Cell. 1996; 87: 519-529Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 10Qiu R.G. Abo A. McCormick F. Symons M. Mol. Cell. Biol. 1997; 17: 3449-3458Crossref PubMed Scopus (265) Google Scholar, 11Qiu R.G. Chen J. McCormick F. Symons M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11781-11785Crossref PubMed Scopus (488) Google Scholar, 12Qiu R.G. Chen J. Kirn D. McCormick F. Symons M. Nature. 1995; 374: 457-459Crossref PubMed Scopus (813) Google Scholar). The Rho family GTPases were originally identified as proteins that have important roles in regulating the organization of the actin cytoskeleton and the formation of focal adhesions (13Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (883) Google Scholar, 14Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3747) Google Scholar, 15Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3084) Google Scholar, 16Manser E. Huang H.Y. Loo T.H. Chen X.Q. Dong J.M. Leung T. Lim L. Mol. Cell. Biol. 1997; 17: 1129-1143Crossref PubMed Google Scholar, 17Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3843) Google Scholar, 18Dutartre H. Davoust J. Gorvel J.P. Chavrier P. J. Cell Sci. 1996; 109: 367-377Crossref PubMed Google Scholar). Later the GTPases were also found to activate signal transduction pathways that lead to the regulation of gene expression. Cytoskeletal organization and the regulation of gene expression are both likely to contribute to the cellular changes involved in cell growth and oncogenic transformation. Expression of constitutively active mutants of Rac and Cdc42 in many different cell types results in stimulation of the JNK (also known as stress-activated protein kinase) (19Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1710) Google Scholar, 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, 21Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2415) Google Scholar) and p38 pathways (22Coso 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, 23Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1447) Google Scholar), which in turn regulate expression of specific genes. All three GTPases also regulate other signaling pathways such as the pathway leading to activation of the serum response factor (24Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1207) Google Scholar). The signaling pathway by which Rac and Cdc42 activate JNK has been well characterized. JNK activation by Rac and Cdc42 was shown to be mediated by the mitogen-activated protein kinase kinase kinase MEKK, which phosphorylates the mitogen-activated protein kinase kinase JNKK (also known as SEK1 or MKK4 (25Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar, 26Sanchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Avruch J. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (917) Google Scholar, 27Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1415) Google Scholar)). JNKK in turn phosphorylates and activates JNK. Besides MEKK, other mitogen-activated protein kinase kinase kinases such as the mixed lineage kinases have also been shown to mediate JNK activation in response to the GTPases (28Fanger G.R. Gerwins P. Widmann C. Jarpe M.B. Johnson G.L. Curr. Opin. Genet. Dev. 1997; 7: 67-74Crossref PubMed Scopus (299) Google Scholar). More recently, the GTPases and some of their GEFs, including Dbl, have been shown to activate nuclear transcription factor-κB (NFκB) (29Perona R. Montaner S. Saniger L. Sanchez-Perez I. Bravo R. Lacal J.C. Genes Dev. 1997; 11: 463-475Crossref PubMed Scopus (537) Google Scholar,30Montaner S. Perona R. Saniger L. Lacal J.C. J. Biol. Chem. 1998; 273: 12779-12785Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). A major function of NFκB is the regulation of genes involved in immune and inflammatory responses (for review, see Ref. 31Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5592) Google Scholar). NFκB is also capable of protecting cells against apoptosis (32Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2940) Google Scholar, 33Mayo M.W. Wang C.Y. Cogswell P.C. Rogers-Graham K.S. Lowe S.W. Der C.J. Baldwin Jr., A.S. Science. 1997; 278: 1812-1815Crossref PubMed Scopus (506) Google Scholar, 34Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2452) Google Scholar, 35Wang J. Walsh K. 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Chem. 1996; 271: 7992-7998Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 45Yamaoka S. Inoue H. Sakurai M. Sugiyama T. Hazama M. Yamada T. Hatanaka M. EMBO J. 1996; 15: 873-887Crossref PubMed Scopus (179) Google Scholar, 46Whitehead I.P. Lambert Q.T. Glaven J.A. Abe K. Rossman K.L. Mahon G.M. Trzaskos J.M. Kay R. Campbell S.L. Der C.J. Mol. Cell. Biol. 1999; 19: 7759-7770Crossref PubMed Google Scholar). The signaling pathway by which NFκB is activated by cytokines such as TNFα or interleukin 1 is well characterized. In unstimulated cells, NFκB is usually found in the cytoplasm sequestered by a group of regulatory proteins known as IκBs (IκBα, -β, and -ε) (31Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5592) Google Scholar). Exposure of cells to TNFα or interleukin 1 results in phosphorylation of IκBα on two critical serines. This targets IκB for ubiquitination-dependent degradation by the proteosome complex and leads to the release and subsequent translocation of NFκB to the nucleus where it can regulate the expression of target genes (31Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5592) Google Scholar). A large multiprotein complex containing two catalytic subunits, IKKα and IKKβ, is rapidly stimulated by interleukin 1 and TNFα (47DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1917) Google Scholar, 48Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1595) Google Scholar, 49Zandi E. Chen Y. Karin M. Science. 1998; 281: 1360-1363Crossref PubMed Google Scholar, 50Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1855) Google Scholar). IKKα and IKKβ can form homodimers or heterodimersin vitro, and purified recombinant forms of each can directly phosphorylate IκBα and IκBβ at the proper sites (49Zandi E. Chen Y. Karin M. Science. 1998; 281: 1360-1363Crossref PubMed Google Scholar). In addition, the IKK complex contains a regulatory subunit, IKKγ, that appears to bind IKKα-IKKβ as a dimer (51Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar). The protein kinase NIK has been shown to phosphorylate and activate the IKKs and is thought to mediate IKK activation in response to stimuli such as TNFα (52Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1166) Google Scholar) and the expression of the Cot/Tpl-2 protein kinase (53Lin X. Cunningham Jr., E.T. Mu Y. Geleziunas R. Greene W.C. Immunity. 1999; 10: 271-280Abstract Full Text Full Text PDF PubMed Google Scholar). MEKK has also been shown to phosphorylate and activate IKK when overexpressed (54Yin M.J. Christerson L.B. Yamamoto Y. Kwak Y.T. Xu S. Mercurio F. Barbosa M. Cobb M.H. Gaynor R.B. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 55Nemoto S. DiDonato J.A. Lin A. Mol. Cell. Biol. 1998; 18: 7336-7343Crossref PubMed Google Scholar), and it has been proposed to mediate IKK and NFκB activation by the Tax transactivator protein of human T cell leukemia virus 1 (54Yin M.J. Christerson L.B. Yamamoto Y. Kwak Y.T. Xu S. Mercurio F. Barbosa M. Cobb M.H. Gaynor R.B. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Less is known about the signaling pathway by which Dbl and the Rho family GTPases activate NFκB. Here we show that the three GTPases, Cdc42, Rac, and Rho, activate NFκB by different pathways. Whereas Rac activates NFκB by a pathway that depends on IKKβ, Cdc42 and Rho activate NFκB in the absence of IKK stimulation. The Rac-dependent pathway requires NIK and the Rac effector PAK but does not require MEKK. Dbl requires both branches of the pathway for full activation of NFκB. pRK5-Myc-tagged Dbl (amino acids 495–826) was a gift from A. Hall and has been described previously (56Olson M.F. Pasteris N.G. Gorski J.L. Hall A. Curr. Biol. 1996; 6: 1628-1633Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). PAK1(T423E) and pEGFP-C1-hPAK1 (amino acids 83–149) (57Zhao Z.-S. Manser E. Chen X.-Z. Chong C. Leung T. Lim L. Mol. Cell. Biol. 1998; 18: 2153-2163Crossref PubMed Google Scholar) were gifts from J. Chernoff. pRC/β-actin HA-IKKβ, pEBG-IKKβ, pRC/β-actin HA-IKKα, GST-IKKβ, GST-IKKβ(S-A), and pCMV-IKKβ(SS-AA) were gifts from A. Lin and have been described elsewhere (55Nemoto S. DiDonato J.A. Lin A. Mol. Cell. Biol. 1998; 18: 7336-7343Crossref PubMed Google Scholar). pLPC-IKKα(S-A), pCMV4 IκBα(S-A) (S32A/S36A), and pBIIX-Luc, which contains two NFκB sites and a minimal fospromoter upstream of the luciferase gene, were gifts from A. Beg. pSRαRacL61, pSRαRacV12, pCMVCdc42V12, pEXVRhoV14, pEXVRacN17, and pCMVCdc42N17 have been described previously (23Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1447) Google Scholar). SRαMEKKΔ and dominant negative SRαMEKKΔ(K432M) have been described previously (58Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar). pCMVM2-JNK and GST-c-Jun have been described previously (23Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1447) Google Scholar). PAKR containing the N-terminal Rac-binding domain of human PAK65 (amino acids 1–225) was a gift from J. Chernoff and has been described previously (59Martin G.A. Bollag G. McCormick F. Abo A. EMBO J. 1995; 14: 1970-1978Crossref PubMed Scopus (305) Google Scholar). pCDNA3-NIK wild type and pCDNA3-NIK(KK-AA) were from R. Pestell. C3 transferase expression vector was a gift from R. Prywes. All cell lines were maintained at 37 °C in 5% CO2 and cultured in Dulbecco's modified Eagle's medium supplemented with 50 units/ml penicillin, 50 µg/ml streptomycin, and 4 mm glutamine. HeLa and 293 cells were cultured in 10% fetal bovine serum; NIH3T3 cells were cultured in 10% bovine calf serum. Transient transfections into HeLa and NIH3T3 cells were carried out using the LipofectAMINE method (Life Technologies, Inc.) according to the manufacturer's protocol. Cells were seeded at a density of 3.7 × 105/3.5-cm-diameter dish and were starved 24 h after transfection in 0.2% serum. 293 cells were transfected using a standard calcium phosphate precipitation method. Luciferase assays were carried out in both HeLa and NIH3T3 cells with similar results. However, because the basal levels of luciferase activity were lower in HeLa cells, only these results are shown in the figures. In both cases, cells were transfected as described above and harvested 48 h after transfection. Luciferase assays were carried out using the dual luciferase kit (Promega). Firefly luciferase reporter constructs (200 ng of the pBIIX-Luc) were transfected together with 50 ng of theRenilla luciferase reporter plasmid pRL-TK as an internal control. Cells were lysed in 150 µl of passive lysis buffer (Promega), and 7.5 µl of lysate was assayed for firefly andRenilla luciferase activity according to the manufacturer's instructions. Transfection efficiencies were corrected through normalization of the firefly luciferase activity to the activity obtained from the Renilla Luciferase. All experiments were performed at least three times, and the results averaged. Statistical analyses were performed using the Student t test with significant differences established as p < 0.05. GST-IκBα-(1–54), GST-IκBα-(1–54;TT), in which Ser-32 and -36 were replaced by threonines, and GST-c-Jun-(1–79) were purified on glutathione-agarose beads as described elsewhere (55Nemoto S. DiDonato J.A. Lin A. Mol. Cell. Biol. 1998; 18: 7336-7343Crossref PubMed Google Scholar). For IKKβ assays, NIH3T3 cells were transfected with either HA-tagged IKKβ or GST-tagged IKKβ (pEBG-IKKβ) expression vectors. Both vectors gave identical results. For IKKα assays, 293 cells were used instead of NIH3T3 cells because IKKα was poorly expressed in NIH3T3 cells, and we could not get sufficient expression for immune complex kinase assays in these cells. In both cases, cells from each transfection were lysed in M2 buffer (60Minden A. Lin A. Smeal T. Derijard B. Cobb M. Davis R. Karin M. Mol. Cell. Biol. 1994; 14: 6683-6688Crossref PubMed Scopus (436) Google Scholar) 48 h after transfection. Approximately 100 µg of cell extracts was incubated with either anti-HA monoclonal antibody and protein A-Sepharose (for isolation of HA-IKK) or glutathione-agarose beads (Sigma) (for isolation of GST-IKK) and incubated 2 h to overnight at 4 °C. The immune complexes were washed twice in M2 buffer (58Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar) and twice in kinase buffer (20 mm HEPES, pH 7.5, 10 mm MgCl2) and incubated at 30 °C in 30 µl of kinase buffer containing 20 mm β-glycerophosphate, 20 mm p-nitrophenyl phosphate, 1 mmdithiothreitol, 50 µmNa3V04, 20 µm ATP, and 5 µCi of [γ-32P]ATP. Approximately 2 µg of GST-IκBα wild type or S32T/S36T fusion protein was used as substrate in each reaction. Reactions were stopped after 30 min by denaturation in SDS loading buffer. Proteins were resolved by SDS-polyacrylamide gel electrophoresis, and substrate phosphorylation was visualized by autoradiography. For PAK1 autophosphorylation assays, Myc-tagged PAK was immunopurified from cell lysates using anti-Myc antibody. Immune complex kinase assays were carried out as described above for IKK in the absence of substrate, and the reaction was stopped after 20 min. PAK phosphorylation was then examined by SDS-polyacrylamide gel electrophoresis and autoradiography. JNK assays were performed as described previously (23Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1447) Google Scholar). Cells were harvested in M2 buffer (58Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar), and equal amounts of cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Immobilon P, Millipore Corp.). The membrane was immunoblotted with the appropriate antibody. The following antibodies were used: mouse monoclonal anti-HA 12CA5 (Roche Molecular Biochemicals), anti-GST mouse monoclonal antibody (Sigma), mouse monoclonal anti-Myc 9E10 (Santa Cruz Biochemicals), mouse monoclonal anti-FLAG (Eastman Kodak Co.), rabbit polyclonal anti-IKKα antibody (Santa Cruz Biochemicals), and rabbit polyclonal anti-MEKK antibody (Santa Cruz Biochemicals). Immunocomplexes were visualized by the enhanced chemiluminescence detection method (Amersham Pharmacia Biotech). Focus formation assays in NIH3T3 cells were carried out as described previously (23Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1447) Google Scholar). To examine activation of NFκB, HeLa cells were transfected with the pBIIX-Luc reporter (which contains two NFκB sites and a fos minimal promoter upstream of the luciferase gene) together with either empty vector or vector containing oncogenic Dbl. Luciferase activity was measured 48 h after transfection. As seen in Fig.1 A, oncogenic Dbl activation of pBIIX-Luc was completely blocked by the super-repressor IκBα(S-A) (61Traenckner E.B. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (934) Google Scholar), indicating that NFκB activation by Dbl is likely to be mediated by IκB phosphorylation (Fig. 1 A). To see whether Dbl activation of NFκB requires the Rho family GTPases, two inhibitors were used. The first was a PAKR expression vector. PAKR contains the regulatory domain of PAK2, which specifically binds to activated Rac and Cdc42 (59Martin G.A. Bollag G. McCormick F. Abo A. EMBO J. 1995; 14: 1970-1978Crossref PubMed Scopus (305) Google Scholar). PAKR serves as an inhibitor of Cdc42 and Rac by titrating out the activated forms of the GTPases therefore blocking their ability to activate downstream effectors. The other inhibitor was a C3 transferase expression vector. C3 transferase specifically inhibits Rho activity (15Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3084) Google Scholar, 17Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3843) Google Scholar). As seen in Fig.1 A, NFκB activity induced by Dbl is significantly blocked by expression of both PAKR and C3 transferase, suggesting that Cdc42 and/or Rac as well as Rho are necessary for its activation of NFκB. When both PAKR and C3 transferase were used together, the inhibition was even greater, suggesting that a pathway activated by Rac/Cdc42 cooperates with a Rho-activated pathway to activate NFκB. Although dominant negative Rac and Cdc42 also have an inhibitory effect on NFκB activation by Dbl (Fig. 1 A), PAKR is considered to be a more reliable inhibitor of endogenous Rac and Cdc42 in these assays because the N17 mutants are thought to function by binding to the GEFs and forming a rather stable complex that could titrate out the exchange factors (62Feig L.A. Nat. Cell Biol. 1999; 1: E25-E27Crossref PubMed Scopus (343) Google Scholar). An inhibitory effect could therefore be attributed to titration of the Dbl protein. PAKR in contrast should specifically inhibit the activities of endogenous Rac and Cdc42 rather than Dbl. Dominant negative Cdc42 and Rac have different effects on NFκB activation by Dbl. This may reflect a different binding affinity of the different dominant negative mutants to Dbl, or it may reflect the fact that both of these mutants were expressed at different levels as shown in Fig. 1 A. Dominant negative mutants of IKKα and IKKβ were analyzed for their abilities to block Dbl activation of NFκB. These constructs were transfected together with oncogenic Dbl expression vector and the pBIIX-Luc reporter construct. Although dominant negative IKKβ significantly blocked Dbl activation of NFκB, dominant negative IKKα had very little effect (Fig. 1 B). Furthermore, when expressed together with suboptimal doses of IKKβ, Dbl could synergize with IKKβ to stimulate NFκB activity (Fig.1 C). Because the IKKs form a large complex that binds many proteins, a dominant negative IKKβ might therefore have a rather global effect in that it may titrate other important signaling molecules. To examine the role of the IKKs in more detail, we looked at the induction of the IKK enzymatic activity in response to oncogenic Dbl. To determine whether Dbl activates IKKβ, an in vitro kinase assay was carried out. In this assay pEGB-IKKβ (a eukaryotic expression vector containing GST-tagged IKKβ) was transfected with either empty vector or oncogenic Dbl. MEKKΔ, an activated form of MEKK that has previously been shown to be a strong activator of IKKβ (55Nemoto S. DiDonato J.A. Lin A. Mol. Cell. Biol. 1998; 18: 7336-7343Crossref PubMed Google Scholar), was used as a positive control. After transient expression, GST-IKKβ expression levels were analyzed by Western blot and quantitated. Equal amounts of GST-IKKβ were then purified from cell lysates using glutathione-agarose-conjugated beads and assayed for the ability to phosphorylate bacterially expressed IκBα in the presence of [γ-32P]ATP. IκB phosphorylation was analyzed after SDS-polyacrylamide gel electrophoresis and autoradiography. Dbl stimulated IKKβ activity to levels comparable with MEKKΔ (Fig.2 A). As expected, IKKβ that was activated by Dbl or MEKKΔ was not able to phosphorylate IκBα(S32T/S36T). The GST-IκBα(S32T/S36T) mutant is a very poor IKK substrate because the phospho-acceptor sites (serine 32 and serine 36) are replaced by threonine residues (63DiDonato J. Mercurio F. Rosette C. Wu-Li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar) (Fig. 2 A,middle panel). Using a similar assay, we found that in contrast to IKKβ, Dbl could not activate IKKα (Fig. 2 B), whereas NIK, which was used as a positive control, activated IKKα, and MEKKΔ activated IKKα weakly. Because Dbl activation of NFκB appears to be mediated by IKKβ and the Rho family GTPases, we were interested in determining whether the Rho GTPases Cdc42, Rac, and Rho activate NFκB by an IKK-dependent pathway. All three GTPases activated the pBIIX-Luc promoter ∼6–10-fold (see Fig.3 A). Immune complex kinase assays were carried out to see whether the GTPases could also activate IKK. Surprisingly, we found that only activated Rac stimulated IKKβ activity, whereas activated RhoA only activated the kinase minimally, and activated Cdc42 did not activate the kinase at all (Fig.3 B). None of the GTPases activated IKKα (data not shown). This suggests that although Rac can activate NFκB via activation of IKK, Cdc42 and Rho may activate NFκB by an IKK-independent pathway. A well known target for Rac is the serine/threonine kinase PAK. PAK1 was recently shown to activate NFκB but not IKK (64Frost J.A. Swantek J.L. Stippec S. Yin M.J. Gaynor R. Cobb M.H. J. Biol. Chem. 2000; 275: 19693-19699Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). To determine whether PAK is required for Rac activation of IKK, IKKβ and activated Rac vectors were transfected along with either empty vector or the PAK1 autoinhibitory domain (PAK1-(83–149)), which is known to block endogenous PAK activity (57Zhao Z.-S. Manser E. Chen X.-Z. Chong C. Leung T. Lim L. Mol. Cell. Biol. 1998; 18: 2153-2163Crossref P" @default.
• W1483753543 created "2016-06-24" @default.
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• W1483753543 creator A5054787277 @default.
• W1483753543 date "2001-07-01" @default.
• W1483753543 modified "2023-10-17" @default.
• W1483753543 title "Dbl and the Rho GTPases Activate NFκB by IκB Kinase (IKK)-dependent and IKK-independent Pathways" @default.
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• W1483753543 doi "https://doi.org/10.1074/jbc.m011345200" @default.
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