IκB Kinase γ/Nuclear Factor-κB-Essential Modulator (IKKγ/NEMO) Facilitates RhoA GTPase Activation, which, in Turn, Activates Rho-associated Kinase (ROCK) to Phosphorylate IKKβ in Response to Transforming Growth Factor (TGF)-β1*

Background: TGF-β1 activates RhoA and nuclear factor-κB (NF-κB), but the activation mechanism was not clearly elucidated. Results: IKKγ disrupts RhoA-Rho guanine nucleotide dissociation inhibitor (RhoGDI) complex, facilitating GTP binding to RhoA, resulting in IKKβ phosphorylation by ROCK. Conclusion: IKKγ facilitates RhoA activation, which in turn activates NF-κB. Significance: We found the new mechanism of IKKγ to activate RhoA and NF-κB by TGF-β1. Transforming growth factor (TGF)-β1 plays several roles in a variety of cellular functions. TGF-β1 transmits its signal through Smad transcription factor-dependent and -independent pathways. It was reported that TGF-β1 activates NF-κB and RhoA, and RhoA activates NF-κB in several kinds of cells in a Smad-independent pathway. However, the activation molecular mechanism of NF-κB by RhoA upon TGF-β1 has not been clearly elucidated. We observed that RhoA-GTP level was increased by TGF-β1 in RAW264.7 cells. RhoA-GDP and RhoGDI were bound to N- and C-terminal domains of IKKγ, respectively. Purified IKKγ facilitated GTP binding to RhoA complexed with RhoGDI. Furthermore, Dbs, a guanine nucletotide exchange factor of RhoA much more enhanced GTP binding to RhoA complexed with RhoGDI in the presence of IKKγ. Indeed, si-IKKγ abolished RhoA activation in response to TGF-β1 in cells. However, TGF-β1 stimulated the release of RhoA-GTP from IKKγ and Rho-associated kinase (ROCK), an active RhoA effector protein, directly phosphorylated IKKβ in vitro, whereas TGF-β1-activated kinase 1 activated RhoA upon TGF-β1 stimulation. Taken together, our data indicate that IKKγ facilitates RhoA activation via a guanine nucletotide exchange factor, which in turn activates ROCK to phosphorylate IKKβ, leading to NF-κB activation that induced the chemokine expression and cell migration upon TGF-β1.

factor (GDF) to disrupt RhoA-RhoGDI complex, is required for the activation of RhoA by GEFs. Signals from active Rho GTPases transmit to a variety of effector proteins, including Rho-associated coiled-coil forming serine/threonine kinase (ROCK), which is activated by RhoA, and p21-activated kinase, which is activated by Cdc42/Rac (7).
Nuclear factor-B (NF-B) is a transcription factor that controls the expression of specific target genes, including cytokines, chemokines, cell adhesion molecules, and inducible enzymes to regulate inflammation, cancer, apoptosis, and several other physiological phenomena (8). The NF-B family consists of p65 (RelA), RelB, c-Rel, NF-B1 (p105, precursor of p50), and NF-B2 (p100, precursor of p52) forming hetero-or homodimers. Two principal pathways of NF-B activation have been elucidated: the classical pathway and the alternative pathway. In the classical pathway, the IB kinase (IKK) complex consists of two catalytic subunits, IKK␣ (IKK1) and IKK␤ (IKK2), and a regulatory subunit, IKK␥ (also referred to as NEMO (NF-B essential modifier). When IKK␤ is activated by phosphorylation, it can then phosphorylate inhibitor of NF-B (IB), which is bound to NF-B. When the phosphorylated IB is ubiquitinated and degraded, NF-B dimer such as p65/p50, which is released from IB, is translocated into the nucleus, where it binds to and activates the transcription of specific target genes. In the alternative pathway, the activation of IKK␣ homodimer by phosphorylation induces p100 processing and the nuclear translocation of the RelB/p52 dimer (9). Intriguingly, ubiquitination, as well as phosphorylation, is involved in the activation of IKK (10).
It is noteworthy that Rho subfamily small GTPases are implicated in NF-B activation (11)(12)(13)(14)(15). In addition, TGF-␤1 can rapidly activate Rho subfamily GTPases, including RhoA, Cdc42, and Rac1, depending on the cell lines (3, 16 -18). Furthermore, TGF-␤1 induces the activation of NF-B signaling (3,19,20) leading to cell motility (16,21). Although the relevance of TGF-␤1, Rho GTPases, and NF-B has been reported in a variety of cells, the molecular mechanism of the activation of NF-B by TGF-␤1 via RhoA GTPase activation in macrophages has not been well elucidated (17). Therefore, we attempted to discover the underlying molecular mechanism how RhoA regulates NF-B or vice versa upon TGF-␤1. We found that IKK activates RhoA; IKK␥ binds to the RhoA-RhoGDI complex, facilitating the activation of RhoA, likely by disrupting the RhoA-RhoGDI complex. Thereafter, active RhoA-GTP and its downstream component ROCK phosphorylates IKK␤, which in turn phosphorylates IB and p65, thereby leading to NF-B activation.
Cell Culture, Fluorescence Microscopy, and Confocal Microscopy-The RAW264.7 (mouse macrophage) cell line was cultured (22); if necessary, TGF-␤1 (5 ng/ml) was treated. HeLa cells were cultured in DMEM containing 10% FBS, 100 units/ml streptomycin, and 100 units/ml penicillin at 37°C in 5% CO 2 . The cells were fixed with 4% paraformaldehyde for 10 min, neutralized with 20 mM glycine for 10 min, and then washed three times with PBS containing 0.1% Triton X-100. The samples were incubated with primary antibody (1:100) overnight at 4°C, washed, and then incubated with the appropriate fluorescent dye-conjugated secondary antibody for 2 h at 24°C. DAPI (1 g/ml) was added 10 min before washing. Fluorescence was observed by fluorescence microscopy (Axiovert 200; Carl Zeiss; Göttingen, Germany) and confocal microscopy (LSM 780NLO; Carl Zeiss). RhoA was identified using an anti-RhoA antibody, which is recognized by an Alexa Fluor 488-conjugated secondary antibody (green), and IKK␥ was identified by an anti-IKK␥ antibody, which is recognized by an Alexa Flour 568-conjugated secondary antibody (red). Nuclei were identified with DAPI staining (blue).
Assay of Cell Migration-Migration of RAW264.7 cells was determined using a Transwell permeable support kit with polycarbonate filter (22).
Luciferase Reporter Assays-RAW264.7 cells were grown to 80% confluence in six-well plates and then transiently transfected with the pNF-B-Luc cis reporter plasmid (Stratagene; Santa Clara, CA) by incubating with Lipofectamine 2000 (Invitrogen) or Attractene (Qiagen; Hilden, Germany) for 3 h according to the manufacturer's instructions. To calibrate the variation in transfection efficiency, the cells were co-transfected with 1 g of pCS2ϩ-␤-galactosidase plasmids, an expression plasmid for the E. coli galactosidase gene. The transfected cells were incubated in serum-free medium for 24 h, rinsed with PBS, lysed in 1ϫ reporter lysis buffer (Promega; Madison, WI), and the cell debris was removed by centrifugation. The relative luciferase activity of the supernatant was measured using a luminometer according to the manufacturer's instructions (Lumat LB 9057; EG & G Bertold).
Loading of GDP and GTP␥S onto GTP-binding Proteins in Vitro-Cell lysates (1 g/l protein in 500 l) were incubated with 10 mM EDTA, pH 8.0. Next, GTP␥S or GDP was added to the cell lysates to a final concentration of 0.1 or 1 mM, respectively, and incubated at 30°C for 30 min with constant agitation. The reaction was terminated by thoroughly mixing with MgCl 2 at a final concentration of 60 mM on ice. To determine the level of RhoA-GTP, GST-rhotekin-RBD beads (23) and an EZ-Detect Rho activation kit containing GST-RBD (Pierce) were used (24).
Assay of GTP Binding to RhoA-RhoA in the absence or presence of RhoGDI or the RhoA-RhoGDI complex in buffer (10 mM HEPES, pH 7.4, 50 mM NaCl, 1 or 5 mM MgCl 2 , 2 or 1 mM EDTA, respectively, 1 mM DTT, 0.1% CHAPS) was incubated with Construction and Transient Transfection of a Small Hairpin RNA Targeting RhoA-A shRNA-expressing sequence for targeting RhoA mRNA was cloned into the pSUPER RNAi system (Oligoengine; Seattle, WA) (22,26).
Immunoprecipitation-Immunoprecipitation was performed according to the previous report (17). IKK␤ and IKK␥ were immunoprecipitated with an anti-IKK␤ antibody and an anti-IKK␥ antibody, respectively.
To determine protein-protein binding, GST-IKK␥ beads and the proteins were incubated in a buffer (50 mM Tris-HCl, pH 7.5, 1ϫ PBS, 10% glycerol, and 1 g/ml each aprotinin, leupeptin, and pepstatin A and 1 mM PMSF) for 2 h at 4°C. After washing the beads, bound proteins were identified with Western blotting.
Proximity Ligation Assay-To detect the interaction between RhoA and IKK, we utilized the DuoLink in situ proximity ligation assay (Olink Bioscience; Uppsala, Sweden) according to the manufacturer's protocol.
Measurement of MIP-1␣-Macrophage inflammatory protein (MIP)-1␣ secreted from the RAW264.7 cells in response to TGF-␤1 was quantitatively determined using ELISA (R&D Systems) according to the manufacturer's instructions. RAW264.7 cells were incubated with TGF-␤1 for various time periods. Reverse transcription PCR and real-time PCR for MIP-1␣ mRNA expression was performed according to the previous report (22).
Statistical Analysis-Data are presented as the means Ϯ S.E. of at least three independent experiments. The Student's t test was used to compare groups using the GraphPad Prism program (San Diego, CA).

RhoA Interacts with the IKK Complex-TGF-␤1 increased
RhoA-GTP levels in a 30 -60-min treatment and then decreased (Fig. 1A), which is accordance with the previous results (17). Considering the relevance between RhoA and NF-B, we found that the amino acid sequence of IKK␥ is partially identical to RhoA effector proteins, including ROCK1, protein kinase N, rhophilin, and rhotekin although the homologous regions of IKK␥ are not identical with the known Rhobinding domains of the effector proteins (supplemental Fig. S1). Therefore, we examined the possible binding of RhoA to IKK␥. Immunoprecipitation of IKK␥ resulted in the co-precipitation of RhoA in a resting state, whereas TGF-␤1 reduced the co-precipitation of RhoA and IKK␥ (Fig. 1B). In addition, RhoA was co-immunoprecipitated with IKK␣/␤ without TGF-␤1, but TGF-␤1 also reduced the amount of RhoA that was co-immunoprecipitated with IKK␣/␤ (Fig. 1B). Whereas co-immunoprecipitation of RhoA with IKK␥ was reduced in 1 h treatment of TGF-␤1, their interaction was recovered after 24 h (Fig. 1C). This suggests that RhoA-GDP instead of RhoA-GTP is preferentially bound to IKK␥.
In addition, the proximity ligation assay, which evaluates the interaction of two proteins in situ showed an interaction between RhoA and IKK␥ in a resting state, but TGF-␤1 markedly reduced the interaction between RhoA and IKK␥ (Fig. 1D). Consistently, confocal microscopy showed the co-localization of RhoA and IKK␥ in HeLa cells. However, TGF-␤1 reduced the co-localization of RhoA and IKK␥; RhoA was instead translocated to the plasma membrane (Fig. 1E). Notably, RhoA was observed in both the cytosol and nucleus of HeLa cells (Fig. 1E). Furthermore, purified RhoA was bound with purified recombinant GST-IKK␥ in a concentration-dependent manner (Fig.  1F), suggesting that RhoA directly interacts with IKK␥. RhoA interacts with the IKK complex. A, RAW264.7 cells were incubated with 5 ng/ml TGF-␤1 at 37°C, and GTP-RhoA levels were determined. B, the cells were treated TGF-␤1 for 3 h. IKK␣/IKK␤ and IKK␥ were immunoprecipitated with an anti-IKK␥ antibody for 2 h at 4°C, and then their co-precipitation with RhoA was determined by Western blot. C, control; T, TGF-␤1 treatment. C, RAW264.7 cells were incubated with TGF-␤1, and IKK␥ was immunoprecipitated. The bound RhoA were analyzed by Western blot. Intensity of RhoA was quantified by densitometry (lower panel). D, RAW264.7 cells were treated with TGF-␤1 for 1 h, and the interaction between RhoA and IKK was visualized in situ using a proximity ligation assay (PLA). The results are given as the means Ϯ S.E. of three independent experiments (*, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001). E, HeLa cells were treated with TGF-␤1 for 1 h, and RhoA (green), IKK␥ (red), and nuclei (blue) were visualized. Arrows indicate RhoA localization in the membrane. F, GST-IKK␥ (1 g of protein)-Sepharose 4B beads were incubated with 0.5-4 g of purified M-RhoA for 2 h at 4°C, and RhoA bound to GST-IKK␥ was detected by Western blot. The amount of RhoA bound to IKK␥ was calculated from the control standard curve. GST-IKK␥ was detected with Ponceau S staining. CON, control; IB, immunoblot.

RhoA-GDP and RhoGDI
Interact with IKK␥-To investigate whether the GDP-or GTP-bound state of RhoA affects its interaction with IKK␥, cell lysates were preincubated with GDP or GTP␥S, and immunoprecipitation was performed. RhoA underwent co-immunoprecipitation with IKK␥ in the presence of GDP, but the co-immunoprecipitation was markedly reduced in the presence of GTP␥S ( Fig. 2A). Consistently, RhoA purified from the membranous (M-RhoA) and cytosolic (C-RhoA) fractions of Sf9 insect cells directly bound to GST-IKK␥ in its GDP-bound state, whereas RhoA-GTP␥S rarely interacted with GST-IKK␥ (Fig. 2B). The recombinant RhoA-GDP purified from E. coli was able to bind to GST-IKK␥, instead RhoA-GTP␥S rarely bound to IKK␥ (Fig. 2C), suggesting that prenyl group of RhoA is not essential for the binding to IKK␥.
Because cytosolic RhoA-GDP in a resting state was considered to form a complex with RhoGDI, we examined whether RhoGDI is also capable of binding to IKK␥. Indeed, cytosolic RhoGDI was bound to IKK␥ irrespective of the presence of either GDP or GTP␥S (Fig. 2D). The purified RhoGDI directly bound to GST-IKK␥/glutathione-Sepharose beads in a concentration-dependent manner (Fig. 2E). In addition, the RhoA-RhoGDI complex, as well as RhoA or RhoGDI alone, was bound to IKK␥, suggesting that a trimeric complex of RhoA-RhoGDI-IKK␥ is formed in vitro (Fig. 2F).
IB and RhoGDI for the binding toward IKK␥ was explored. High concentration of IB prevented RhoGDI from binding to IKK␥ in vitro (Fig. 3C). Consistently, co-immunoprecipitation of IKK␥ with IB increased upon TGF-␤1 in the presence of MG132, an inhibitor of proteasomal degradation, and the coimmunoprecipitation of IKK␥ with RhoGDI was slightly reduced in cells (Fig. 3D), suggesting that TGF-␤1 stimulates IB binding to IKK␥.
Similarly, because the N-terminal domain (aa 44 -111) was known to be a binding site of IKK␤ (29), the competition between RhoA-GDP and IKK␤ was examined; high concentration of IKK␤ did not interfere with the binding of RhoA-GDP to IKK␥ (Fig. 3E), suggesting that IKK␤ and RhoA-GDP do not compete each other for their binding to IKK␥. The proposed diagram of the interaction of the proteins was shown in Fig. 3F.
IKK␥ Facilitates the Activation of RhoA Complexed with RhoGDI in Vitro-Herein, we inferred that IKK␥ could disrupt the interaction between RhoA and RhoGDI because RhoA-GDP bound to the N-terminal domain, and RhoGDI bound to the C-terminal domain of IKK␥, respectively. To demonstrate this hypothesis, we prepared purified proteins, including RhoA, RhoGDI, a complex of RhoA-RhoGDI, and IKK␥ (Fig. 4A).
Next, we examined whether IKK␥ is able to facilitate the incorporation of GTP into RhoA in the complex of RhoA-RhoGDI. The preliminary experiment showed that the fluorescence intensity of 2Ј(3Ј)-O-(N-methylanthraniloyl) (Mant)-GTP (30,31) was linearly correlated with RhoA concentration, suggesting that the extent of fluorescence presents the binding of Mant-GTP to RhoA. The fluorescence intensity of Mant-GTP bound to M-RhoA (Fig. 4B) and C-RhoA (data not shown) increased in a time-dependent manner, but RhoGDI interfered with the increase in Mant-GTP binding. However, IKK␥ significantly augmented the fluorescence of Mant-GTP by binding to RhoA complexed with RhoGDI (Fig. 4B). However, IKK␥ could not augment the fluorescence of Mant-GTP-bound to RhoA alone (Fig. 4F), suggesting that IKK␥ does not play a role as a GEF.
To confirm this result again, we measured the binding of [ 35 S]GTP␥ to RhoA. As expected, RhoGDI suppressed the binding of [ 35 S]GTP␥ to RhoA. However, IKK␥ significantly increased the binding of [ 35 S]GTP␥ to RhoA complexed with RhoGDI (Fig. 4, C and D), which was in a concentration-dependent manner (Fig. 4E). At a low MgCl 2 concentration, much more [ 35 S]GTP␥ was incorporated into RhoA than at a high MgCl 2 concentration (Fig. 4E). Furthermore, Dbs, a GEF for RhoA (13) much more enhanced the binding of [ 35 S]GTP␥ to RhoA complexed with RhoGDI in the presence of IKK␥ than in the absence of it (Fig. 4, C and D). Here, the detergent CHAPS (1%) was used to disrupt the RhoA-RhoGDI complex. CHAPS seemed to impair Dbs activity, likely by preventing Dbs from acessing RhoA. Consistently, knockdown of IKK␥ by si-IKK␥ blocked the induction of RhoA-GTP in cells upon TGF-␤1 stimulation (Fig. 4G), suggesting that IKK␥ is indispensable for RhoA activation by TGF-␤1.
RhoA Is Involved in NF-B Activation in Response to TGF-␤1-We tried to ascertain the involvement of RhoA in NF-B acti-vation due to TGF-␤1. TGF-␤1 increased the NF-B reporter gene activity in 1 h, but its activity then decreased after 12-24 h (Fig. 5A). However, both treatment with an IKK␤ inhibitor (BMS34551) and transfection of an IB super suppressor (SR, IB S32A/S36A) abolished NF-B activation by TGF-␤1 (Fig.  5B). TGF-␤1 induced IB degradation, but this degradation was blocked by the proteasomal inhibitor MG132 (Fig. 5C), suggesting that TGF-␤1 activates NF-B by degrading IB in the proteasome.
ROCK Is Involved in NF-B Activation-Furthermore, Y27632 and HA1077 (Fasudil), inhibitors of ROCK reduced the NF-B reporter gene luciferase activities by TGF-␤1 (Fig. 6A). However, Y27632 did not alter the level of phosphorylated Smad3 in response to TGF-␤1 (data not shown), indicating that although TGF-␤1 can stimulates the Smad pathway, RhoA/ ROCK activation is not implicated in Smad activation. Moreover, Y27632 and 5z-7-oxozeaenol, a TAK1 inhibitor (32), prevented the degradation of IB, as well as the phosphorylation of IB, IKK␣/␤, and p65 by TGF-␤1 (Fig. 6B), suggesting that ROCK and TAK1 are essential for NF-B activity in response to TGF-␤1. Thus, we clarified whether ROCK directly phosphorylates IKK␤. When the constitutively active form of ROCK (aa 17-535) lacking the C-terminal region of the Rho-binding domain (33) and recombinant GST-IKK␤ were incubated in the presence (Fig. 6D) or in the absence of cytosol (Fig. 6C), the phosphorylated IKK␤ was detected with anti-phospho-IKK␤ antibodies (Ser-177 and Ser-181), suggesting that ROCK FIGURE 5. TGF-␤1 induces RhoA-mediated NF-B activation. A, RAW 264.7 cells were co-transfected with a NF-B luciferase cis-reporter construct and a pCS2ϩ-␤-galactosidase plasmid, incubated for 24 h, and then stimulated with TGF-␤1. Luciferase activity was measured with a luminometer. B, cells were co-transfected with the NF-B luciferase reporter construct and pCS2ϩ-␤-galactosidase plasmids, or each 1 g/ml mock (M) vector and the IB␣ super repressor (S32A/S36A) DNA construct (SR) and incubated for 24 h. Cells transfected with the NF-B luciferase construct were also preincubated with DMSO or 10 M IKK␤ inhibitor (BMS345541, BMS) dissolved in DMSO for 1 h. Then, the cells were incubated with TGF-␤1 for 1 h, and the NF-B activity was determined. C, cells were pretreated with DMSO or 10 M MG132 dissolved in DMSO for 1 h and incubated with TGF-␤1 at 37°C. IB␣ and ␤-actin were analyzed by Western blotting. D, RAW 264.7 cells were co-transfected with the NF-B-luciferase reporter construct, pCS2ϩ-␤-galactosidase plasmids, and 1 g/ml mock vector (M) or an HA-tagged RhoA construct (WT, constitutively active (G14V), or dominant-negative (T19N)), incubated for 24 h, and then incubated with TGF-␤1 for 1 h. Luciferase activity was determined, and RhoA expression was assessed by Western blotting using an anti-HA antibody. E, cells were co-transfected with the NF-B-luciferase construct and pCS2ϩ-␤-galactosidase plasmids. After 24 h, the cells were incubated with 10 g/ml Tat-C3 for 1 h and then with TGF-␤1 for 1 h. The resulting luciferase activity was measured. F, cells were co-transfected with the NF-B-luciferase reporter construct, pCS2ϩ-␤-galactosidase plasmids, and either scrambled RNA (SCR) or 2 g/ml sh-RhoA plasmid (Sh). After 72 h, the cells were incubated with or without TGF-␤1 for 1 h, and then the luciferase activity was measured. G, cells were transfected with scrambled RNA or sh-RhoA and then stimulated with TGF-␤1. The phosphorylations of IB, IKK␣/␤, and p65 were analyzed by Western blotting. When the control value was set to 1, the amount of RhoA expressed in the presence of sh-RhoA was 0.33 Ϯ 0.058. The values represent the means Ϯ S.E. of three independent experiments (*, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001).
directly phosphorylates Ser-177 and Ser-181 of IKK␤. Here, ROCK activity was demonstrated by measurement of the phosphorylation of myosin light chain phosphatase using anti-phospho-myosin light chain phosphatase antibody (Fig. 6D). However, because TAK1 is required for NF-B activation (34), we examined the involvement of TAK1 in the regulation of the activation of RhoA. A TAK1 inhibitor, 5z-7-oxozeaenol, markedly abolished a TGF-␤1-dependent increase of RhoA-GTP levels (Fig. 6E), suggesting that TAK1 is involved in RhoA activation upon TGF-␤1 stimulation.
NF-B Regulates Chemokine Expression-Finally, we observed that TGF-␤1 induced the transcription and protein expression of the chemokine such as MIP-1␣ (Fig. 7, A and B) and cell migration (Fig. 7C). However, BMS-345541 (an inhibitor of IKK␤) inhibited the MIP-1␣ and cell migration, suggesting that NF-B activation is involved in the cell migration upon TGF-␤1 stimulation. Consistently, we recently reported that RhoA is required for the expression of MIP-1␣ upon TGF-␤1 stimulation (17,22).

DISCUSSION
Regulation of Transcription by RhoA-Rho proteins such as RhoA, Cdc42, and Rac1 regulate transcription via several tran-scription factors, including serum response factor (SRF), NF-B, and others regulated by the kinases such as JNK1 and p38 MAPK. The substrates of these kinases include Stat3, Stat5a, ELK, PEA3, ATF2, Max, and CHOP/GADD153 (35). Among them, it has been well known that RhoA activates SRF (36). The mechanism by which RhoA activates SRF has been proposed as follows; MAL/MKL1, a member of the myocardinrelated transcription factor (MRTF) is a cofactor of SRF that binds to monomeric G-actin in the cytosol but translocates to the nucleus after MAL is released from polymerized F-actin upon serum stimulation (37). However, in this study, we disclosed that the activation of NF-B by RhoA is utterly different from SRF activation by RhoA.
RhoA-RhoGDI Complex Binds to IKK␥ Allowing RhoA Activation-Generally, IKK␥ provides binding sites for many proteins; 16 proteins have been reported as directly binding to IKK␥ and promoting the activation of NF-B (38). Here, we presented that RhoA-GDP and RhoGDI are also bound to IKK␥; RhoA-GDP was bound to the N-terminal domain (aa  and RhoGDI was bound to the large C-terminal domain (estimated aa 112-419) of IKK␥. The Rho-binding domain of IKK␥ is not identical with the known Rho-binding domains of effector proteins, including ROCK1, protein kinase N, rhophi- lin, and rhotekin (supplemental Fig. S1). It is noteworthy that RhoA-GDP was also bound to small C-terminal domain (aa 351-419) of IKK␥, but not to the large C-terminal domain, suggesting that a part of C-terminal region (aa 101-350) interfere with RhoA-GDP binding to IKK␥. However, CC2 (coiled coil 2) and leucine zipper domains of IKK␥ (Fig. 3A) directly interact in an anti-parallel orientation through the connecting loop containing proline (39), suggesting that N and C termini may be localized in the same direction (Fig. 3F). Interestingly, IB binds to the C-terminal zinc finger domain (aa 389 -419) (28), and IKK␤ binds to the N-terminal domain of IKK␥ (aa 44 -111) (29). These results indicate that IKK␤ and IB bound to IKK␥ might be spatially close each other (Fig. 3F). Indeed, IKK␤ itself interacts with the C terminus of IB forming a stable ternary complex with IKK␥ (40). In addition, IB competes with RhoGDI for the binding to IKK␥ in vitro and in vivo upon TGF-␤1 (Fig. 3, C and D, respectively). It remains unclear how the binding of IB to IKK␥ increases upon TGF-␤1 with the decrease of RhoGDI. However, RhoA-binding domain may be a relatively short length of N terminus (aa 1-43), which is not overlapped with the IKK␤-binding site of IKK␥ (aa 44 -111); therefore, RhoA-GDP did not compete with IKK␤ for the binding to IKK␥ (Fig. 3E).
In conclusion, we here propose that IKK␥ likely functions to activate RhoA by serving as a scaffold protein to recruit the RhoA-RhoGDI complex. It was indeed known that IKK␥ recruits IB to IKK␤ as a scaffold protein, allowing IKK␤ to phosphorylate IB (28,41). Because RhoA-GDP complexed with RhoGDI is not directly activated by GEFs (5, 6), a specific GDF to disrupt RhoA-RhoGDI complex has been accepted to be required for the activation of RhoA. Herein, we demonstrated that IKK␥ could allow RhoA of RhoA-RhoGDI complex to be readily incorporated with GTP (Fig. 3). Moreover, the Dbs, a GEF of RhoA, much enhanced GTP binding to the RhoA-RhoGDI complex in the presence of IKK␥ (Fig. 3), suggesting that IKK␥ allows the GEF to recognize, access, or act on RhoA complexed with RhoGDI; IKK␥ may play a role as a GDF.
Depending on the signaling pathway, there may be diverse GDFs that activate RhoA by a variety of specific stimuli. Indeed, several GDFs, including ezrin/radixin/moesin, Etk/Bmx, and neurotrophin receptor p75 NTR , have been reported (6,42,43). Given that there are several GDFs, it is likely that each GDF activates RhoA in a different particular signal pathway.
In addition to GDFs, modification of RhoGDI itself releases Rho GTPases from the RhoA-RhoGDI complex; phosphorylation of RhoGDI at Tyr-156 by Src alleviates its affinity for RhoA, Rac1, and Cdc42 (44), and phosphorylation RhoGDI by PKC␣ leads to dissociation from RhoA (45). Because IKK␥ can be also modified with proteins similar to ubiquitin (46), it is possible that a variety modification of RhoA, RhoGDI, or IKK␥ actually regulates to disrupt the RhoA-RhoGDI complex in response to several stimuli in vivo. Nonetheless, it is evident that in vitro system unmodified recombinant IKK␥ facilitates GTP-binding to RhoA complexed with recombinant RhoGDI without modification of these proteins by any other stimuli.
Although RhoA was reported to be activated by TGF-␤1 via a GEF, including Vav2 or Net1 (18,47), Lbc, which is also referred to as AKAP13, AKAP-Lbc, and ARHGEF13 (48) was recently reported to be associated with ␣-catulin, which is bound to IKK-␤ (49). It is likely that different types of GEFs in different cells may be involved in the activation of RhoA in response to TGF-␤1.
Active RhoA/ROCK Activates NF-B-Eventually, RhoA-GTP released from IKK␥ by TGF-␤1 was thought to activate ROCK. Indeed, active ROCK directly phosphorylated the recombinant GST-IKK␤ at Ser-177 and Ser-181 residues in vitro (Fig. 6C). Although TGF-␤1 activates TAK1 (50) and only TAK1 is generally accepted as an IKK kinase in a canonical pathway (51,52), it remains unclear whether IKK is activated by an upstream kinase or autophosphorylation (53,54). Here, we demonstrated that TAK1 is involved in RhoA activation upon TGF-␤1 (Fig. 6E). Because ROCK and TAK1 are involved in NF-B activation (Fig. 6B), we examined whether ROCK regulates TAK1 activity in a positive feedback manner. However, Y27632, an inhibitor of ROCK, did not influence the phosphorylation of TAK1. Consistent with this observation, neither si-ROCK1 nor si-ROCK2 had an influence on the phosphorylation of TAK1 (data not shown). These results suggest that RhoA/ROCK does not promote TAK1 activity. Although it is still ambiguous that TAK1 and/or ROCK directly activate(s) IKK␤, we propose that ROCK is able to directly phosphorylate IKK␤ and TAK1 is instead involved in the activation of RhoA (Fig. 6E). However, it remains to be studied in the future how FIGURE 7. NF-B mediates TGF-␤1-induced cell migration. A, BMS345541 (BMS; IKK␤ inhibitor, 10 M) was pretreated for 1 h, TGF-␤1 was treated for 1 h, and then secreted MIP-1␣ was determined by reverse transcription PCR. B, cells were pretreated with DMSO or 10 M BMS345541 dissolved in DMSO for 1 h. Then, cells were treated with TGF-␤1 for 6 h, and secreted MIP-1␣ was measured using ELISA. C, cells were preincubated with DMSO and 10 M IKK␤ inhibitor (BMS345541) for 1 h and then incubated with or without TGF-␤1 for 1 h in the lower chamber, allowing cells of the upper chamber to migrate for 6 h. D, proposed scheme of the molecular mechanism to induce NF-B activation through RhoA activation by IKK␥ and in turn IKK␤ phosphorylation by ROCK. The data represent the means Ϯ S.E. of three independent experiments (*, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001). ave., average. TAK1 is involved in RhoA regulation in TGF-␤1 signaling. On the other hand, a long term treatment of TGF-␤1 inactivates RhoA (Fig. 1A) through p190RhoGAP and cAMP/Epac/ ARAP3 activation (17,22). Subsequently, NF-B activation was also abolished by a long term treatment of TGF-␤1 (Fig. 5A).
Thus, it is a very intriguing novel finding that IKK␥ facilitates RhoA activation, which in turn activates ROCK, leading to direct phosphorylation of IKK␤ and subsequent activation of NF-B. Conclusively, the RhoA and IKK complexes may regulate each other and form a positive feedback loop to activate NF-B. Thus, we proposed the scheme of the new regulatory mechanism of NF-B activation through RhoA activation in response to TGF-␤ (Fig. 7D).