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Originally published In Press as doi:10.1074/jbc.M011345200 on May 3, 2001

J. Biol. Chem., Vol. 276, Issue 28, 25876-25882, July 13, 2001
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Dbl and the Rho GTPases Activate NFkappa B by Ikappa B Kinase (IKK)-dependent and IKK-independent Pathways*

Marta S. Cammarano and Audrey MindenDagger

From the Department of Biological Sciences, Columbia University, New York, New York 10027

Received for publication, December 15, 2000, and in revised form, May 3, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 NFkappa B, which has been shown to have an important role in Dbl-induced oncogenic transformation. Here we show that although Dbl activation of NFkappa B requires Cdc42, Rac, and Rho, the different GTPases activate NFkappa B by different mechanisms. Whereas Rac stimulates the activity of the Ikappa B kinase IKKbeta , Cdc42 and Rho activate NFkappa B without activating either IKKalpha or IKKbeta . Like Dbl, Rac activation of IKKbeta 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 NFkappa B is more elusive, but our results suggest that it involves an IKKalpha /IKKbeta -independent mechanism. Finally, we show that the signaling enzymes that mediate NFkappa B activation by Dbl and the Rho GTPases are also necessary for malignant transformation induced by oncogenic Dbl.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1). 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 (2, 3). Dbl contains a Dbl homology domain that is required for GEF activity (4) adjacent to a pleckstrin homology domain that is most likely responsible for proper localization at the membrane (5). 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 (2, 3, 6). 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 (7). Rho proteins have also been shown to be necessary for transformation by other oncogenes including Ras (8-12).

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 (13-18). 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) (19-21) and p38 pathways (22, 23), 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 (24). 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 (25-27)). 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 (28).

More recently, the GTPases and some of their GEFs, including Dbl, have been shown to activate nuclear transcription factor-kappa B (NFkappa B) (29, 30). A major function of NFkappa B is the regulation of genes involved in immune and inflammatory responses (for review, see Ref. 31). NFkappa B is also capable of protecting cells against apoptosis (32-37) most likely by activating antiapoptotic genes (38). NFkappa B may also control cell cycle regulatory genes such as cyclin D1 (39-41) and has been found to be required for oncogenic transformation by a number of oncogenes (33, 42-46).

The signaling pathway by which NFkappa B is activated by cytokines such as TNFalpha or interleukin 1 is well characterized. In unstimulated cells, NFkappa B is usually found in the cytoplasm sequestered by a group of regulatory proteins known as Ikappa Bs (Ikappa Balpha , -beta , and -epsilon ) (31). Exposure of cells to TNFalpha or interleukin 1 results in phosphorylation of Ikappa Balpha on two critical serines. This targets Ikappa B for ubiquitination-dependent degradation by the proteosome complex and leads to the release and subsequent translocation of NFkappa B to the nucleus where it can regulate the expression of target genes (31). A large multiprotein complex containing two catalytic subunits, IKKalpha and IKKbeta , is rapidly stimulated by interleukin 1 and TNFalpha (47-50). IKKalpha and IKKbeta can form homodimers or heterodimers in vitro, and purified recombinant forms of each can directly phosphorylate Ikappa Balpha and Ikappa Bbeta at the proper sites (49). In addition, the IKK complex contains a regulatory subunit, IKKgamma , that appears to bind IKKalpha -IKKbeta as a dimer (51). 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 TNFalpha (52) and the expression of the Cot/Tpl-2 protein kinase (53). MEKK has also been shown to phosphorylate and activate IKK when overexpressed (54, 55), and it has been proposed to mediate IKK and NFkappa B activation by the Tax transactivator protein of human T cell leukemia virus 1 (54).

Less is known about the signaling pathway by which Dbl and the Rho family GTPases activate NFkappa B. Here we show that the three GTPases, Cdc42, Rac, and Rho, activate NFkappa B by different pathways. Whereas Rac activates NFkappa B by a pathway that depends on IKKbeta , Cdc42 and Rho activate NFkappa B 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 NFkappa B.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- pRK5-Myc-tagged Dbl (amino acids 495-826) was a gift from A. Hall and has been described previously (56). PAK1(T423E) and pEGFP-C1-hPAK1 (amino acids 83-149) (57) were gifts from J. Chernoff. pRC/beta -actin HA-IKKbeta , pEBG-IKKbeta , pRC/beta -actin HA-IKKalpha , GST-IKKbeta , GST-IKKbeta (S-A), and pCMV-IKKbeta (SS-AA) were gifts from A. Lin and have been described elsewhere (55). pLPC-IKKalpha (S-A), pCMV4 Ikappa Balpha (S-A) (S32A/S36A), and pBIIX-Luc, which contains two NFkappa B sites and a minimal fos promoter upstream of the luciferase gene, were gifts from A. Beg. pSRalpha RacL61, pSRalpha RacV12, pCMVCdc42V12, pEXVRhoV14, pEXVRacN17, and pCMVCdc42N17 have been described previously (23). SRalpha MEKKDelta and dominant negative SRalpha MEKKDelta (K432M) have been described previously (58). pCMVM2-JNK and GST-c-Jun have been described previously (23). 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 (59). pCDNA3-NIK wild type and pCDNA3-NIK(KK-AA) were from R. Pestell. C3 transferase expression vector was a gift from R. Prywes.

Cell Lines and Transfections-- 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.

Dual Luciferase Assays-- 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 the Renilla 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 and Renilla 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.

Purification of Recombinant GST Fusion Proteins-- GST-Ikappa Balpha -(1-54), GST-Ikappa Balpha -(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 (55).

Immunoprecipitations and Kinase Assays-- For IKKbeta assays, NIH3T3 cells were transfected with either HA-tagged IKKbeta or GST-tagged IKKbeta (pEBG-IKKbeta ) expression vectors. Both vectors gave identical results. For IKKalpha assays, 293 cells were used instead of NIH3T3 cells because IKKalpha 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 (60) 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 (58) 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 beta -glycerophosphate, 20 mM p-nitrophenyl phosphate, 1 mM dithiothreitol, 50 µM Na3V04, 20 µM ATP, and 5 µCi of [gamma -32P]ATP. Approximately 2 µg of GST-Ikappa Balpha 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 (23).

Western Blots-- Cells were harvested in M2 buffer (58), 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-IKKalpha 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-- Focus formation assays in NIH3T3 cells were carried out as described previously (23).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Dbl Activation of NFkappa B Is Mediated by the Rho Family GTPases-- To examine activation of NFkappa B, HeLa cells were transfected with the pBIIX-Luc reporter (which contains two NFkappa B 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. 1A, oncogenic Dbl activation of pBIIX-Luc was completely blocked by the super-repressor Ikappa Balpha (S-A) (61), indicating that NFkappa B activation by Dbl is likely to be mediated by Ikappa B phosphorylation (Fig. 1A). To see whether Dbl activation of NFkappa B 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 (59). 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 (15, 17). As seen in Fig. 1A, NFkappa B 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 NFkappa B. 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 NFkappa B. Although dominant negative Rac and Cdc42 also have an inhibitory effect on NFkappa B activation by Dbl (Fig. 1A), 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 (62). 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 NFkappa B 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. 1A.


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  		Fig. 1.   Activation of the NFkappa B pathway by Dbl is mediated by IKKbeta and the Rho GTPases. A, cells were transfected with a pBIIX-Luc plasmid (200 ng) together with empty vector or 0.1 µg of oncogenic Dbl expression vector along with Ikappa B(S-A) (0.1 µg), 0.1 µg of PAKR and/or 0.1 µg of C3 transferase expression vector, or 1 µg of RacN17 or Cdc42N17 expression vectors. 50 ng of an internal control plasmid, pRL-TK, expressing Renilla luciferase (Promega) was added to each transfection. Firefly luciferase activity was measured and normalized to the internal control Renilla luciferase activity. The relative luciferase activity of lysates from cells transfected with Dbl as compared with the lysate from cells transfected with the reporter alone is taken as the -fold increase. Data are shown as the mean ± S.E. *, a significant difference from Dbl alone (p < 0.05). Expression levels of RacN17 and Cdc42N17 were visualized by Western blot analysis using an anti-Myc antibody. B, cells were transfected with 200 ng of the pBIIX-Luc reporter alone or in the presence of Dbl expression vector (0.1 µg) along with either empty vector or expression vectors containing dominant negative mutants of either IKKalpha or IKKbeta (500 ng or 1 µg). 50 ng of the pRL-TK plasmid was included in each transfection. Luciferase activity was measured and normalized to the Renilla luciferase activity. The -fold activation refers to the increase in luciferase activity in cells transfected with Dbl over that found in cells transfected with the reporter and the empty vector. Western blots assessing the expression level of the dominant negative IKKbeta and IKKalpha using an anti-FLAG and an anti-IKKalpha antibody, respectively, are shown. NS, a nonspecific band present in all the lanes of the Western blot. Data are shown as the mean ± S.E. *, a significant difference from Dbl alone (p < 0.05). C, cells were cotransfected with a pBIIX-Luc reporter and the indicated combinations of expression vectors encoding Dbl (0.1 µg) and wild type IKKbeta (0.1, 0.2, or 0.5 µg). Luciferase activity was determined 24 h post-transfection. Data are shown as the mean ± S.E. *, a significant difference from Dbl alone (p < 0.05).

Dbl Activation of NFkappa B Is Blocked by Dominant Negative IKKbeta -- Dominant negative mutants of IKKalpha and IKKbeta were analyzed for their abilities to block Dbl activation of NFkappa B. These constructs were transfected together with oncogenic Dbl expression vector and the pBIIX-Luc reporter construct. Although dominant negative IKKbeta significantly blocked Dbl activation of NFkappa B, dominant negative IKKalpha had very little effect (Fig. 1B). Furthermore, when expressed together with suboptimal doses of IKKbeta , Dbl could synergize with IKKbeta to stimulate NFkappa B activity (Fig. 1C).

Dbl Stimulates IKKbeta Activity-- Because the IKKs form a large complex that binds many proteins, a dominant negative IKKbeta 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 IKKbeta , an in vitro kinase assay was carried out. In this assay pEGB-IKKbeta (a eukaryotic expression vector containing GST-tagged IKKbeta ) was transfected with either empty vector or oncogenic Dbl. MEKKDelta , an activated form of MEKK that has previously been shown to be a strong activator of IKKbeta (55), was used as a positive control. After transient expression, GST-IKKbeta expression levels were analyzed by Western blot and quantitated. Equal amounts of GST-IKKbeta were then purified from cell lysates using glutathione-agarose-conjugated beads and assayed for the ability to phosphorylate bacterially expressed Ikappa Balpha in the presence of [gamma -32P]ATP. Ikappa B phosphorylation was analyzed after SDS-polyacrylamide gel electrophoresis and autoradiography. Dbl stimulated IKKbeta activity to levels comparable with MEKKDelta (Fig. 2A). As expected, IKKbeta that was activated by Dbl or MEKKDelta was not able to phosphorylate Ikappa Balpha (S32T/S36T). The GST-Ikappa Balpha (S32T/S36T) mutant is a very poor IKK substrate because the phospho-acceptor sites (serine 32 and serine 36) are replaced by threonine residues (63) (Fig. 2A, middle panel). Using a similar assay, we found that in contrast to IKKbeta , Dbl could not activate IKKalpha (Fig. 2B), whereas NIK, which was used as a positive control, activated IKKalpha , and MEKKDelta activated IKKalpha weakly.


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  		Fig. 2.   Dbl regulates IKKbeta kinase activity. A, cells were transfected with 0.5 µg of GST-IKKbeta expression vector together with empty vector, Dbl (0.5 µg), or MEKKDelta (0.5 µg) expression vectors. IKK activity was assayed after normalizing for IKK expression by Western blot. Either GST-Ikappa Balpha -(1-54) (top panel) or GST-Ikappa Balpha (S32T/S36T) (middle panel) were used as substrates. The segments of the autoradiograms that contain the phosphorylated substrates are shown on the top and middle panels. The level of IKKbeta present in the extracts used for the kinase assay was assessed by Western blot using anti-HA antibody and is shown in the bottom panel. B, cells were transfected with 4 µg of HA-IKKalpha expression vector together with either empty vector, NIK, MEKKDelta , or Dbl expression vectors (8 µg). IKKalpha activity was measured as described above for IKKbeta . The level of IKKalpha present in the extracts used for the kinase assay was assessed by Western blot using an anti-HA antibody and is shown in the bottom panel.

Rac, but Not Cdc42 and Rho, Activates IKKbeta -- Because Dbl activation of NFkappa B appears to be mediated by IKKbeta and the Rho family GTPases, we were interested in determining whether the Rho GTPases Cdc42, Rac, and Rho activate NFkappa B by an IKK-dependent pathway. All three GTPases activated the pBIIX-Luc promoter ~6-10-fold (see Fig. 3A). Immune complex kinase assays were carried out to see whether the GTPases could also activate IKK. Surprisingly, we found that only activated Rac stimulated IKKbeta activity, whereas activated RhoA only activated the kinase minimally, and activated Cdc42 did not activate the kinase at all (Fig. 3B). None of the GTPases activated IKKalpha (data not shown). This suggests that although Rac can activate NFkappa B via activation of IKK, Cdc42 and Rho may activate NFkappa B by an IKK-independent pathway. A well known target for Rac is the serine/threonine kinase PAK. PAK1 was recently shown to activate NFkappa B but not IKK (64). To determine whether PAK is required for Rac activation of IKK, IKKbeta 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 (57). As shown in Fig. 3C, the PAK1 autoinhibitory domain completely blocked Rac activation of IKKbeta , indicating that PAK is necessary for IKKbeta activation by Rac. The PAK autoinhibitory domain also blocked oncogenic Dbl activation of IKKbeta . These results suggest that PAK1 is necessary for NFkappa B activation by Dbl and Rac. However, an activated PAK1 mutant, PAK1(T423E), was not sufficient to activate IKK on its own (see Fig. 3D), suggesting that although PAK is necessary for IKK activation, it is not sufficient. Likewise, we were not able to observe NFkappa B activation in response to activated PAK1 using luciferase reporter assays (data not shown), although the activated PAK1 had considerable kinase activity when assayed for autophosphorylation (Fig. 3D) and myelin basic protein phosphorylation (data not shown).


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  		Fig. 3.   The three Rho GTPases Rac, Rho, and Cdc42 activate NFkappa B by different mechanisms. A, cells were transfected with 200 ng of pBIIX-Luc plasmid together with 0.1 µg of Myc-Cdc42V12, M2-RacV12, or Myc-RhoV14 expression vector along with either empty vector or 0.1 µg of an expression vector containing dominant negative Ikappa B and 50 ng of the internal control pRL-TK vector. Luciferase activity was measured and normalized to the Renilla luciferase activity. The -fold activation refers to the value of luciferase activity obtained in the presence of the activators compared with the activity obtained in their absence. Data are shown as the mean ± S.E. B, cells were transfected with 0.5 µg of GST-IKKbeta vector together with 0.25 µg of M2-RacL61, Myc-RhoV14, or Myc-Cdc42V12. IKKbeta activity was assayed as described in Fig. 2, and Ikappa B phosphorylation is shown in the top panel. The IKKbeta expression level in the extracts used for the kinase assay is shown in the middle panel as detected by Western blots probed with anti-GST antibody. Western blots of the Rho GTPases probed with antibodies directed against the epitope tags are shown in the bottom panel. C, cells were transfected with 0.25 µg of HA-IKKbeta vector together with 0.5 µg of Dbl or RacL61 in the absence or presence of 1 µg of the PAK autoinhibitory domain (PAK1-(83-149)) or with 0.5 µg of wild type NIK. IKKbeta activity was assayed as described above. Shown in the bottom panel are IKKbeta expression levels in a Western blot that was performed by probing the proteins immunoprecipitated from the same amount of extracts used in the kinase assay with an anti-HA antibody. D, cells were transfected with 0.5 µg of GST-IKKbeta expression vector along with empty vector or 0.5 µg of Dbl or constitutively active Myc-PAK1 (PAK1(T423E)). IKKbeta activity was assessed as described above, and Ikappa B phosphorylation is shown in the top panel. A Western blot showing IKKbeta expression levels in the extracts used for the kinase assay as detected with an anti-GST antibody is shown in the bottom panel. As a positive control, PAK1(T423E) (PAK1TE) autophosphorylation was examined by immune complex kinase assay using anti-Myc antibody, and PAK1 expression levels were examined by probing Western blots with anti-Myc antibody. WT, wild type.

Dbl and Rac Activation of IKK Is Mediated by NIK-- Because Dbl and Rac activate IKK, we were interested in identifying the signaling enzymes that mediate this activation. Dbl and Rac both activate JNK by a pathway that requires MEKK (23). Surprisingly however, dominant negative MEKK did not block activation of NFkappa B activity by Dbl or Rac (Fig. 4A). In fact, although it did not activate NFkappa B on its own (data not shown), dominant negative MEKK actually slightly enhanced Dbl and Rac activation of NFkappa B. NIK is a well known activator of NFkappa B and has been shown to mediate NFkappa B activation in response to TNFalpha (52). Interestingly, dominant negative NIK had an inhibitory effect on NFkappa B activation by both Dbl and Rac (see Fig. 4A). Likewise, dominant negative NIK blocked IKKbeta activation by both Dbl and Rac, whereas dominant negative MEKK had no effect (Fig. 4B). Dominant negative MEKK did, however, inhibit JNK activation by Rac, indicating that it functioned normally as a dominant negative mutant (Fig. 4C). These data suggest that Dbl and Rac activate IKK by a pathway that requires NIK but not MEKK.


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  		Fig. 4.   Dbl and Rac activation of IKKbeta requires NIK but not MEKK. A, cells were transfected with 200 ng of pBIIX-Luc reporter together with either 0.1 µg of Dbl (open bars) or RacV12 (filled bars) along with either empty vector or increasing amounts (0.5 or 1 µg) of a dominant negative forms of NIK (NIKAA) or MEKK (MEKKDelta (K432M)). Total luciferase activity in the lysates from cells transfected with Dbl or RacV12 and empty vector is taken as 100%. Data are shown as the mean ± S.E. *, a significant decrease relative to Dbl or Rac alone (p < 0.05). B, cells were transfected with 0.25 µg of HA-IKKbeta vector together with either empty vector, 0.5 µg of Dbl, or 0.5 µg of RacL61 vectors and 0.5 or 1 µg of dominant negative NIK or MEKKDelta expression vectors. IKKbeta activity was detected as described in Fig. 2. Levels of expression of IKKbeta were visualized by Western blot using an anti-HA antibody after immunoprecipitation as previously described and are show in the middle panel. The bottom panel shows expression of the dominant negative MEKKDelta protein in whole cell extracts as detected using an anti-MEKK antibody. C, to test the activity of dominant negative MEKKDelta , cells were transfected with 0.25 µg of M2-JNK vector together with either empty vector or 0.5 µg of RacL61 vector and 0.5 µg of dominant negative MEKK expression vector. After transient expression, JNK activity was assessed by immune complex kinase assays using GST-c-Jun as a substrate. Phosphorylated c-Jun is shown in the top panel. The total level of JNK in the cell lysates as assessed by probing with anti-M2 antibody is shown in the bottom panel. MEKKDelta K-M, MEKKDelta (K432M); WT, wild type; NIKAA, NIK(KK-AA).

Dbl is a potent oncogene, and NFkappa B has been shown to be necessary for Dbl-induced oncogenesis (46). We have shown that NFkappa B activation by Dbl requires NIK, PAK, IKK, and Ikappa B. We therefore used focus formation assays to determine whether these signaling enzymes are important for Dbl-induced oncogenic transformation. NIH3T3 fibroblasts were transfected with oncogenic Dbl together with either empty vector, dominant negative IKK, Ikappa B super-repressor, dominant negative PAK, or dominant negative NIK, and foci were scored after 2 weeks. All of the dominant negative mutants in the NFkappa B pathway and dominant negative PAK inhibited focus formation by Dbl (see Fig. 5).


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  		Fig. 5.   Focus formation by oncogenic Dbl requires enzymes in the NFkappa B signaling pathway. A, NIH3T3 cells were transfected with oncogenic Dbl (200 ng) together with 1 µg of either empty vector, Ikappa B(S-A), IKKbeta (SS-AA), NIK(KK-AA) (NIKAA), or dominant negative PAK1. Cells were grown for 2 weeks following transfection. The plates were then stained with crystal violet and photographed. Representative plates are shown. B, foci were counted, and the number of foci are indicated as the percentage of foci induced by Dbl. Dbl produced ~500 foci/µg of transfected Dbl DNA. The results are an average of three independent experiments and are presented as ± S.E.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Dbl activation of NFkappa B requires the Rho family GTPases Cdc42, Rac, and Rho. However, the different Rho proteins activate NFkappa B by different mechanisms. Of the three GTPases, only Rac activated IKKbeta in an in vitro kinase assay and is therefore likely to activate NFkappa B by an IKKbeta -dependent pathway. Although Cdc42 and Rac have distinct effects in cells (1), this study is one of the first demonstrations that these two GTPases can operate through different signaling pathways. Like Rac, Dbl also stimulated IKKbeta kinase activity. Our results suggest a mechanism by which Dbl and Rac may activate IKKbeta leading to NFkappa B activation. First, we found that Dbl and Rac activation of IKKbeta and NFkappa B requires the serine/threonine kinase NIK but not MEKK. Second, we analyzed a well known effector molecule for Rac, the serine/threonine kinase PAK. Our results suggest that PAK1 is necessary for IKKbeta activation by Rac, although it is not sufficient to activate IKK on its own. Taken together, our results suggest a signaling pathway where Dbl activates Rac and PAK, which in turn activates NIK, either directly or indirectly, and most likely in cooperation with other signaling enzymes. NIK in turn phosphorylates IKKbeta , leading to Ikappa B phosphorylation and degradation followed by nuclear translocation of NFkappa B. It should be noted that our results differ somewhat from those of Frost et al. (64), who showed that Rac does not activate IKKbeta and that activated PAK can stimulate NFkappa beta activity, albeit in the absence of IKKbeta activation. These results may be explained by the fact that different cell types and a different constitutively active PAK1 mutant were used in the two studies.

Our results suggest that PAK is an important mediator of IKKbeta activation by Dbl and Rac. The PAK1 autoinhibitory domain, which is quite specific for PAK (57), completely inhibited IKKbeta activation by Rac and Dbl. However, we have found that activated PAK1 is not sufficient on its own to activate IKKbeta or the NFkappa B luciferase reporter strongly. Likewise, we have not seen NFkappa B activation in response to other members of the PAK family (data not shown). The results from our study suggest that although PAK is necessary for IKKbeta activation by Rac, it is not sufficient. This suggests that other factors may cooperate with PAK to activate IKKbeta . One possibility is that reactive oxygen may be involved in this pathway. Reactive oxygen was previously shown to have an important role in the signaling pathway leading from Rac to NFkappa B activation (65). Alternatively, other Rac/Cdc42 effectors such as mixed lineage kinase, which was shown to activate NFkappa B, may have a role in IKK activation by Rac (66).

It is interesting that Rac activates NFkappa B via NIK rather than MEKK. Although dominant negative MEKK was shown to block NFkappa B activation by Rac in COS cells (30), we did not see any inhibition by dominant negative MEKK in HeLa or NIH3T3 cells. Consistent with this finding, by using MEKK-null cells, Xia et al. (67) have recently reported that although MEKK is necessary for JNK activation by proinflammatory stimuli, it is dispensable for NFkappa B activation by the same signals. Our results suggest a model in which Rac activates two diverging signaling pathways. One pathway is mediated by MEKK and leads to JNK activation (23). Although MEKK is required for the activation of JNK by Rac, Rac has not been shown to stimulate MEKK activity on its own. Thus, although when overexpressed MEKK can stimulate NFkappa B activity (54, 55), Rac most likely does not stimulate MEKK activity sufficiently to allow it to activate NFkappa B. Instead, we propose that a second pathway exists in which Rac activates PAK, which in turn activates NIK either directly or indirectly. Activation of NIK in turn leads to NFkappa B activation most likely by phosphorylating IKKbeta .

In contrast to Rac, we have found that Cdc42 and Rho activate NFkappa B without activating either IKKbeta or IKKalpha . Although we cannot completely rule out the possibility that IKKalpha and IKKbeta activation by Rho and Cdc42 is too weak to be detected in our assays, even expression of high levels of Cdc42 and Rho did not produce noticeable IKK activity. Thus, our results strongly suggest that these two GTPases can trigger an IKK-independent pathway leading to NFkappa B activation. Recently, UV irradiation was also shown to activate NFkappa B by an IKK-independent mechanism (68, 69). In the case of Rho and Cdc42, however, this result is quite surprising because activation of NFkappa B by Rho and Cdc42 does appear to require Ikappa B phosphorylation as assessed by experiments with the Ikappa B super-repressor. Our results suggest that a kinase other than IKKalpha or IKKbeta may phosphorylate Ikappa B and thereby activate NFkappa B in response to Rho and Cdc42. It should be noted to this regard that dominant negative IKKbeta did partially inhibit Cdc42 and Rho activation of the NFkappa B luciferase reporter (data not shown). The most likely explanation for this is that dominant negative IKKbeta binds to and titrates a Rho and Cdc42 activated kinase and thereby indirectly inhibits NFkappa B activation by the GTPases. Alternatively, it could act by titrating IKKgamma or an IKKgamma -related protein, which could potentially be part of an IKKalpha /beta -independent kinase complex that phosphorylates Ikappa B. Interestingly, Cdc42 but not Rho activation of NFkappa B was partially blocked by dominant negative NIK (data not shown). This suggests that although it does not activate IKKbeta , Cdc42 still requires NIK activity to activate NFkappa B. These data are consistent with recent work done with NIK knockout mice that suggests that NIK may have a role in NFkappa B activation that is independent of IKKalpha /beta activity in response to some extracellular stimuli (70). The exact role for NIK in the NFkappa B pathway thus still remains to be fully clarified.

Understanding the signaling pathways activated by Dbl is especially important because Dbl is a potent oncogene. All three Rho family GTPases, Cdc42, Rac, and Rho, have been shown to contribute to different aspects of oncogenic transformation by Dbl (7). The NFkappa B pathway is particularly relevant to studying Dbl-induced oncogenesis because NFkappa B was recently shown to be one of the factors that is important in this process (46). Here we show that all the enzymes that we found to be involved in NFkappa B activation by Dbl, PAK1, NIK, IKKbeta , and Ikappa B are all necessary for focus formation induced by oncogenic Dbl. The mechanism by which NFkappa B regulates transformation in response to Dbl is not known, although NFkappa B was shown to regulate transformation by Ras by promoting cell survival (42). Elucidating how the NFkappa B pathway contributes to oncogenesis by other oncogenes such as Dbl will be critical for understanding the signaling pathways that control cell growth and proliferation.

    ACKNOWLEDGEMENTS

We thank A. Lin, A. Beg, J. Didonato, R. Pestell, and M. Cobb for reagents and plasmids used in this study and O. Karni for technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant R01 CA76342 and an American Scientist Development Grant Award from the American Heart Association (to A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biological Sciences MC 2460, Columbia University, Sherman Fairchild Center, Rm. 813, 1212 Amsterdam Ave., New York, NY 10027. Tel.: 212-854-5632; Fax: 212-865-8246; E-mail: agm24@columbia.edu.

Published, JBC Papers in Press, May 3, 2001, DOI 10.1074/jbc.M011345200

    ABBREVIATIONS

The abbreviations used are: GEF, guanine nucleotide exchange factor; IKK, Ikappa B kinase; NIK, NFkappa B-inducing kinase; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase; JNK, c-Jun NH2-terminal kinase; JNKK, JNK kinase; NFkappa B, nuclear transcription factor-kappa B; TNF, tumor necrosis factor; PAK, p21-activated kinase; HA, hemagglutinin; GST, glutathione S-transferase; Luc, luciferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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