Dbl and the Rho GTPases Activate NF k B by I k B Kinase (IKK)-dependent and IKK-independent Pathways*

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 NF k B, which has been shown to have an important role in Dbl-induced oncogenic transformation. Here we show that although Dbl activation of NF k B requires Cdc42, Rac, and Rho, the different GTPases activate NF k B by different mechanisms. Whereas Rac stimulates the activity of the I k B kinase IKK b , Cdc42 and Rho activate NF k B without activating either IKK a or IKK b . Like Dbl, Rac activation of IKK b 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 NF k B is more elusive, but our results suggest that it involves an IKK a / IKK b -independent mechanism. Finally, we show that the signaling enzymes that mediate NF k B activation by Dbl and the Rho GTPases are also necessary for malig-nant transformation induced by oncogenic Dbl. The Rho family of GTPases, including members of the Cdc42, Rac, and Rho subfamilies, function as molecular switches cy-cling 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 incubate toovernight The immune complexes twice M2 buffer (58) and twice in kinase m M HEPES, m M MgCl and incubated at 30 in 30 m l of kinase buffer containing 20 m M b -glycerophosphate, 20 m M p -nitrophenyl phosphate, 1 m M dithiothreitol, 50 m M Na 3 V0 4, 20 m M ATP, and 5 m Ci of [ g - 32 P]ATP. Approximately 2 m g of GST-I k B a wild type or S32T/S36T fusion protein was used as substrate in each reac- tion. Reactions were stopped after 30 min by denaturation in SDS loading buffer. Proteins were resolved by SDS-polyacrylamide gel elec- trophoresis, and substrate phosphorylation was visualized by autora-diography. 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 mem- brane (Immobilon P, Millipore Corp.). The membrane was immuno-blotted with the appropriate antibody. The following antibodies were used: mouse monoclonal anti-HA 12CA5 (Roche Molecular Biochemi-cals), anti-GST mouse monoclonal antibody (Sigma), mouse monoclonal anti-Myc 9E10 (Santa Cruz Biochemicals), mouse monoclonal anti-FLAG (Eastman Kodak Co.), rabbit polyclonal anti-IKK a 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).

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 GTPbound 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)(14)(15)(16)(17)(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)(26)(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-B (NFB) (29,30). A major function of NFB is the regulation of genes involved in immune and inflammatory responses (for review, see Ref. 31). NFB is also capable of protecting cells against apoptosis (32-37) most likely by activating antiapoptotic genes (38). NFB 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)(43)(44)(45)(46).
The signaling pathway by which NFB is activated by cytokines such as TNF␣ or interleukin 1 is well characterized. In unstimulated cells, NFB is usually found in the cytoplasm sequestered by a group of regulatory proteins known as IBs (IB␣, -␤, and -⑀) (31). Exposure of cells to TNF␣ or interleukin 1 results in phosphorylation of IB␣ on two critical serines. This targets IB for ubiquitination-dependent degradation by the proteosome complex and leads to the release and subsequent translocation of NFB to the nucleus where it can regulate the expression of target genes (31). A large multiprotein complex containing two catalytic subunits, IKK␣ and IKK␤, is rapidly stimulated by interleukin 1 and TNF␣ (47)(48)(49)(50). IKK␣ and IKK␤ can form homodimers or heterodimers in vitro, and purified recombinant forms of each can directly phosphorylate IB␣ and IB␤ at the proper sites (49). In addition, the IKK complex contains a regulatory subunit, IKK␥, that appears to bind IKK␣-IKK␤ 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 TNF␣ (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 NFB 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 NFB. Here we show that the three GTPases, Cdc42, Rac, and Rho, activate NFB by different pathways. Whereas Rac activates NFB by a pathway that depends on IKK␤, Cdc42 and Rho activate NFB 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 NFB.
Cell Lines and Transfections-All cell lines were maintained at 37°C in 5% CO 2 and cultured in Dulbecco's modified Eagle's medium supplemented with 50 units/ml penicillin, 50 g/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 ϫ 10 5 /3.5-cmdiameter 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 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 Ϯ S.E. *, a significant difference from Dbl alone (p Ͻ 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 g) along with either empty vector or expression vectors containing dominant negative mutants of either IKK␣ or IKK␤ (500 ng or 1 g). 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 IKK␤ and IKK␣ using an anti-FLAG and an anti-IKK␣ antibody, respectively, are shown. NS, a nonspecific band present in all the lanes of the Western blot. Data are shown as the mean Ϯ S.E. *, a significant difference from Dbl alone (p Ͻ 0.05). C, cells were cotransfected with a pBIIX-Luc reporter and the indicated combinations of expression vectors encoding Dbl (0.1 g) and wild type IKK␤ (0.1, 0.2, or 0.5 g). Luciferase activity was determined 24 h post-transfection. Data are shown as the mean Ϯ S.E. *, a significant difference from Dbl alone (p Ͻ 0.05).
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 l of passive lysis buffer (Promega), and 7.5 l 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 Ͻ 0.05.
Immunoprecipitations and Kinase Assays-For IKK␤ assays, NIH3T3 cells were transfected with either HA-tagged IKK␤ or GSTtagged IKK␤ (pEBG-IKK␤) expression vectors. Both vectors gave identical results. For IKK␣ assays, 293 cells were used instead of NIH3T3 cells because IKK␣ 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 g 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 MgCl 2 ) and incubated at 30°C in 30 l of kinase buffer containing 20 mM ␤-glycerophosphate, 20 mM p-nitrophenyl phosphate, 1 mM dithiothreitol, 50 M Na 3 V0 4, 20 M ATP, and 5 Ci of [␥-32 P]ATP. Approximately 2 g of GST-IB␣ 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-IKK␣ 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
Dbl Activation of NFB Is Mediated by the Rho Family GTPases-To examine activation of NFB, HeLa cells were transfected with the pBIIX-Luc reporter (which contains two NFB 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 IB␣(S-A) (61), indicating that NFB activation by Dbl is likely to be mediated by IB phosphorylation (Fig. 1A). To see whether Dbl activation of NFB 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, NFB 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 NFB. 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 NFB. Although dominant negative Rac and Cdc42 also have an inhibitory effect on NFB 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 NFB 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.
Dbl Activation of NFB Is Blocked by Dominant Negative IKK␤-Dominant negative mutants of IKK␣ and IKK␤ were analyzed for their abilities to block Dbl activation of NFB. These constructs were transfected together with oncogenic Dbl expression vector and the pBIIX-Luc reporter construct. Although dominant negative IKK␤ significantly blocked Dbl activation of NFB, dominant negative IKK␣ had very little effect (Fig. 1B). Furthermore, when expressed together with suboptimal doses of IKK␤, Dbl could synergize with IKK␤ to stimulate NFB activity (Fig. 1C). Dbl Stimulates IKK␤ Activity-Because the IKKs form a large complex that binds many proteins, a dominant negative IKK␤ 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 IKK␤, an in vitro kinase assay was carried out. In this assay pEGB-IKK␤ (a eukaryotic expression vector containing GST-tagged IKK␤) was transfected with either empty vector or oncogenic Dbl. MEKK⌬, an activated form of MEKK that has previously been shown to be a strong activator of IKK␤ (55), was used as a positive control. After transient expression, GST-IKK␤ expression levels were analyzed by Western blot and quantitated. Equal amounts of GST-IKK␤ were then purified from cell lysates using glutathione-agarose-conjugated beads and assayed for the ability to phosphorylate bacterially expressed IB␣ in the presence of [␥-32 P]ATP. IB phosphorylation was analyzed after SDS-polyacrylamide gel electrophoresis and autoradiography. Dbl stimulated IKK␤ activity to levels comparable with MEKK⌬ ( Fig.  2A). As expected, IKK␤ that was activated by Dbl or MEKK⌬ was not able to phosphorylate IB␣(S32T/S36T). The GST-IB␣(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 IKK␤, Dbl could not activate IKK␣ (Fig. 2B), whereas NIK, which was used as a positive control, activated IKK␣, and MEKK⌬ activated IKK␣ weakly.
Rac, but Not Cdc42 and Rho, Activates IKK␤-Because Dbl activation of NFB appears to be mediated by IKK␤ and the Rho family GTPases, we were interested in determining whether the Rho GTPases Cdc42, Rac, and Rho activate NFB by an IKK-dependent pathway. All three GTPases activated the pBIIX-Luc promoter ϳ6 -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 IKK␤ 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 IKK␣ (data not shown). This suggests that although Rac can activate NFB via activation of IKK, Cdc42 and Rho may activate NFB by an IKK-independent pathway. A well known target for Rac is the serine/threonine kinase PAK. PAK1 was recently shown to activate NFB but not IKK (64).
To determine whether PAK is required for Rac activation of IKK, IKK␤ 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 IKK␤, indicating that PAK is necessary for IKK␤ activation by Rac. The PAK autoinhibitory domain also blocked oncogenic Dbl activation of IKK␤. These results suggest that PAK1 is necessary for NFB 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 NFB 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).
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 NFB activity by Dbl or Rac (Fig. 4A). In fact, although it did not activate NFB on its own (data not shown), dominant negative MEKK actually slightly enhanced Dbl and Rac activation of NFB. NIK is a well known activator of NFB and has been shown to mediate NFB activation in response to TNF␣ (52). Interestingly, dominant negative NIK had an inhibitory effect on NFB activation by both Dbl and Rac (see Fig.  4A). Likewise, dominant negative NIK blocked IKK␤ 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.
Dbl is a potent oncogene, and NFB has been shown to be necessary for Dbl-induced oncogenesis (46). We have shown that NFB activation by Dbl requires NIK, PAK, IKK, and IB. 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, IB super-repressor, dominant negative PAK, or dominant negative NIK, and foci were scored after 2 weeks. All of the dominant negative mutants in the NFB pathway and dominant negative PAK inhibited focus formation by Dbl (see Fig. 5).

DISCUSSION
Dbl activation of NFB requires the Rho family GTPases Cdc42, Rac, and Rho. However, the different Rho proteins activate NFB by different mechanisms. Of the three GTPases, only Rac activated IKK␤ in an in vitro kinase assay and is therefore likely to activate NFB by an IKK␤-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 IKK␤ kinase activity. Our results suggest a mechanism by which Dbl and Rac may activate IKK␤ leading to NFB activation. First, we found that Dbl and Rac activation of IKK␤ and NFB 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 IKK␤ 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 IKK␤, leading to IB phosphorylation and degradation followed by nuclear translocation of NFB. It should be noted that our results differ somewhat from those of Frost et al. (64), who showed that Rac does not activate IKK␤ and that activated PAK can stimulate NF␤ activity, albeit in the absence of IKK␤ 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 IKK␤ activation by Dbl and Rac. The PAK1 autoinhibitory domain, which is quite specific for PAK (57), completely inhibited IKK␤ activation by Rac and Dbl. However, we have found that activated PAK1 is not sufficient on its own to activate IKK␤ or the NFB luciferase reporter strongly. Likewise, we have not seen NFB 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 IKK␤ activation by Rac, it is not sufficient. This suggests that other factors may cooperate with PAK to activate IKK␤. 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 NFB activation (65). Alternatively, other Rac/Cdc42 effectors such as mixed lineage kinase, which was shown to activate NFB, may have a role in IKK activation by Rac (66).
It is interesting that Rac activates NFB via NIK rather than MEKK. Although dominant negative MEKK was shown to block NFB 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 MEKKnull cells, Xia et al. (67) have recently reported that although MEKK is necessary for JNK activation by proinflammatory stimuli, it is dispensable for NFB 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 NFB activity (54,55), Rac most likely does not stimulate MEKK activity sufficiently to allow it to activate NFB. 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 NFB activation most likely by phosphorylating IKK␤.
In contrast to Rac, we have found that Cdc42 and Rho activate NFB without activating either IKK␤ or IKK␣. Although we cannot completely rule out the possibility that IKK␣ and IKK␤ 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 NFB activation. Recently, UV irradiation was also shown to activate NFB by an IKK-independent mechanism (68,69). In the case of Rho and Cdc42, however, this result is quite surprising because activation of NFB by Rho and Cdc42 does appear to require IB phosphorylation as assessed by experiments with the IB super-repressor. Our results suggest that a kinase other than IKK␣ or IKK␤ may phosphorylate IB and thereby activate NFB in response to Rho and Cdc42. It should be noted to this regard that dominant negative IKK␤ did partially inhibit Cdc42 and Rho activation of the NFB luciferase reporter (data not shown). The most likely explanation for this is that dominant negative IKK␤ binds to and titrates a Rho and Cdc42 activated kinase and thereby indirectly inhibits NFB activation by the GTPases. Alternatively, it could act by titrating IKK␥ or an IKK␥-related protein, which could potentially be part of an IKK␣/␤-independent kinase complex that phosphorylates IB. Interestingly, Cdc42 but not Rho activation of NFB was partially blocked by dominant negative NIK (data not shown). This suggests that al-though it does not activate IKK␤, Cdc42 still requires NIK activity to activate NFB. These data are consistent with recent work done with NIK knockout mice that suggests that NIK may have a role in NFB activation that is independent of IKK␣/␤ activity in response to some extracellular stimuli (70). The exact role for NIK in the NFB 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 NFB pathway is particularly relevant to studying Dbl-induced oncogenesis because NFB 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 NFB activation by Dbl, PAK1, NIK, IKK␤, and IB are all necessary for focus formation induced by oncogenic Dbl. The mechanism by which NFB regulates transformation in response to Dbl is not known, although NFB was shown to regulate transformation by Ras by promoting cell survival (42). Elucidating how the NFB pathway contributes to oncogenesis by other oncogenes such as Dbl will be critical for understanding the signaling pathways that control cell growth and proliferation.