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Inhibition of NF-B Activity by IBß in Association with B-Ras

Yi Chen,^1, Sebastien Vallee,^1, Joann Wu,¹ Don Vu,¹ John Sondek,² and Gourisankar Ghosh^1*

Department of Chemistry and Biochemistry, University of California—San Diego, La Jolla, California 92093,¹ Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599²

Received 3 December 2003/ Accepted 6 January 2004

	ABSTRACT

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IBß, one of the major IB proteins, is only partially degraded in response to most extracellular signals. However, the molecular mechanism of this event is unknown. We show here that IBß exists in at least two different forms: one that is bound to the NF-B dimer and the other bound to both NF-B and B-Ras, a Ras-like small G protein. Removal of cellular B-Ras enhances whereas excess B-Ras blocks induced IBß degradation. Remarkably, B-Ras functions in both GDP- and GTP-bound states, and mutations of the conserved guanine-binding residues of B-Ras abrogate its ability to block degradation of IBß. B-Ras also directly blocks the in vitro phosphorylation of IBß by IKKß. These observations suggest that IBß in the ternary complex is resistant to degradation by most signals. We suggest that specific signals, in addition to those that activate only IKK, are essential for the complete degradation of IBß.

	INTRODUCTION

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The dimeric NF-B transcription factors are inhibited in quiescent cells through stable association with inhibitor IBs. A large number of extracellular stimuli transmit signals to relieve this inhibition (2, 17, 38). Almost all of these signals lead to the activation of a specific kinase known as IB kinase (IKK), which phosphorylates IB. Phosphorylated IB proteins are degraded by the sequential actions of ubiquitin ligase and the 26S proteasome releasing free NF-B (23).

The major IB proteins, IB and IBß, resemble each other in both primary sequence and tertiary structure, with the exception of a 40-residue-long insert present within the ankyrin repeat 3 in IBß. However, these two proteins exhibit one major functional difference (17, 35, 36). While signal-induced degradation of IB is responsible for rapid NF-B activation, prolonged activation of NF-B, which is essential for certain biological functions such as T-cell activation, requires IBß degradation (1). Several pathological conditions, such as asthma, cystic fibrosis, and viral and bacterial infection, also require prolonged NF-B activation (3, 4, 10, 19, 22, 29, 33, 35). We do not know why prolonged NF-B activation requires IBß degradation. Two other functional properties of IBß distinguish it from IB. Unlike IB, IBß does not fully degrade in response to most stimuli, and IBß/NF-B complexes are exclusively cytoplasmic in resting cells (16, 17, 25, 34). How these two properties contribute to persistent NF-B activation through IBß degradation is not known. A recent report shows that different MEKK kinases recruit IKK to IB/NF-B and IBß/NF-B complexes in tumor necrosis factor alpha (TNF-)-activated cells (31). This suggests that the compositions of IB and IBß complexes are different, which may lead to their differential functional properties.

Yeast two-hybrid screening has identified two Ras-like small GTPases, B-Ras1 and -2, as inhibitors of NF-B transcriptional activity (14). B-Ras proteins belong to the Ras family due to their high sequence similarity (5, 6, 39). However, there are some critical differences in the sequences of B-Ras. In addition to the fact that these two proteins lack lipid modification sites, B-Ras proteins contain two Ras oncogenic mutations that drastically reduce GTP hydrolysis. The mutations maintain Ras in the constitutively active GTP-bound conformation, converting it to its oncogenic form (5, 6). These differences in sequence suggest that B-Ras might not function as Ras and other small GTPases.

Although in vivo studies showed that B-Ras proteins were associated specifically with the IBß/p65 complex, in vitro pull-down and transfection experiments suggested that B-Ras was also able to bind and stabilize IB (14). We have shown that B-Ras1 binds directly to the IBß/p65 complex and masks the exposed p65 nuclear localization signal (NLS) (26). We also identified that along with the p65 NLS, the insert of IBß is the other primary site of B-Ras binding.

The focus of this study was to test whether B-Ras plays a role in the incomplete degradation of IBß bound to NF-B dimers. Our results demonstrate that in quiescent cells, a pool of IBß is primarily present in a form that cannot be phosphorylated by active IKK. In vitro experiments reveal that active IKK is unable to phosphorylate IBß or IBß/NF-B complexes in the presence of B-Ras. We further show that IBß/NF-B complexes represent subcomplexes within larger protein complexes, and at least some of these complexes contain B-Ras. In addition to the insert, B-Ras also requires the N-terminal signal response region (SRR) of IBß for binding. B-Ras therefore directly masks the signal induced phosphorylation sites (Ser19 and Ser23) located within the SRR of IBß. Overexpression of B-Ras blocks stimulus-dependent degradation of IBß, and removal of B-Ras enhances it. Interestingly, either GTP or GDP is sufficient to function as a cofactor for its inhibitory function in vitro. We suggest that a pool of IBß/NF-B complexes are associated with B-Ras and respond to extracellular signals differently.

	MATERIALS AND METHODS

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Materials. p65 NLS monoclonal antibody was a generous gift from Roche Diagnostics. c-Rel antibody was a gift from Nancy Rice (National Institutes of Health). p65 and IBß polyclonal antibodies were purchased from Santa Cruz. B-Ras antibody was a gift from Sankar Ghosh (Yale University). GTP and GDP were purchased from Sigma Chemicals. Duplex short interfering RNA (siRNA) was purchased from Dharmacon. Single-stranded deoxy oligonucleotides were purchased form Allele Biotechnology, San Diego, Calif.

Protein purification. The cloning and purification methods used for NF-B and IB proteins have been described elsewhere (20). The coding sequence for B-Ras1 was amplified by using two terminal primers with NdeI and BamHI sites at the 5' and 3' ends, respectively. The restricted fragment was inserted into the NdeI- and BamHI-cleaved pET15b vector. Escherichia coli BL21(DE3) cells harboring the expression plasmid were grown and induced with 0.1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) at room temperature. The cell pellet was suspended in a buffer containing 20 mM Tris (pH 7.5), 1 M NaCl, and 2 mM ß-mercaptoethanol followed by lysis of cells by sonication. Clear, soluble crude extracts were loaded onto an Ni²⁺ affinity column (Novagen) and eluted with 200 mM imidazole after extensive washing. Peak fractions containing B-Ras1 were pooled and further purified by size exclusion chromatography (Superdex 75; Amersham Biosciences). B-Ras1 was also purified under a denatured condition and subsequently refolded. E. coli cells expressing B-Ras1 were solubilized in a buffer containing 7 M urea, 1 M NaCl, 20 mM Tris (pH 7.5), and 2 mM ß-mercaptoethanol. Cells were lysed by sonication followed by centrifugation to remove cell debris. The clear denatured extract was loaded onto an Ni²⁺ affinity column equilibrated with lysis buffer. After extensive washing, B-Ras1 was eluted with 200 mM imidazole. B-Ras1 was refolded first by diluting the protein to 0.4 mg/ml in the lysis buffer and then subsequently removing urea by dialysis in three steps. Refolded protein was concentrated and subjected to size exclusion chromatography. The elution profile of the protein was same as that of the B-Ras1 purified with Ni-nitrilotriacetic acid (NTA) and size exclusion chromatography (Superdex 75) under native conditions. The other Ras and Ras-related proteins were expressed in E. coli. Proteins were purified by Ni-NTA and size exclusion chromatography.

Cells, transfection, and extract preparation. HeLa, Cos-7, and 293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1 mM glutamine, 100-U/ml penicillin, and 100-µg/ml streptomycin. Transfections were performed with Lipofectamine Plus (Invitrogen). Cytosolic extract of HeLa cells was concentrated by ammonium sulfate precipitation to 75% saturation. The pellet was suspended in chromatography buffer (150 mM KCl, 20 mM Tris [pH 7.5], 1 mM dithiothreitol [DTT]) and loaded onto a Superose 6 column.

Plasmids and site-directed mutagenesis. All plasmids were constructed by standard recombinant DNA procedures. Site-directed mutagenesis and deletions were performed with the Stratagene Quickchange kit.

In vitro kinase assays. Baculovirus-expressed, His-tagged IKKß was purified by nickel affinity chromatography. Ten nanograms of pure IKKß was used in each kinase reaction. Kinase assays were performed at 30°C for 30 min in buffer containing 20 mM Tris (pH 7.6), 10 mM MgCl₂, 2 mM DTT, 20 µM ATP, and 15 µCi of [-³²P]ATP. For kinase assays, 2 µg of IB or IBß was mixed with or without 4 µg of p65 homodimer or p50/p65 heterodimer on ice for 30 min. Two or 4 µg of B-Ras1 was added to the IB/NF-B or IBß/NF-B complexes wherever necessary. Mixtures were incubated on ice for 60 min before kinase assays were performed.

Immunoprecipitation and Western analysis. Cells were washed three times in phosphate-buffered saline (PBS) buffer. Cytoplasmic extracts were made by lysing cells in buffer containing 1% Triton X-100, 20 mM Tris-HCl (pH 7.6), 200 mM NaCl, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride (lysis buffer). One to 2 mg of the extract was mixed with protein A-agarose and primary antibodies and incubated at 4°C overnight. The immunoprecipitates were washed three times in lysis buffer and eluted with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) buffer by heating at 100°C for 5 min. The supernatant was separated by SDS-PAGE (12.5% polyacrylamide). The separated proteins in the gel were transferred to Hybond nitrocellulose membrane (Amersham-Pharmacia Biotech). The membrane was blocked with 5% milk in PBS-Tween buffer and incubated with anti-p65 polyclonal antibody (Santa Cruz Biotechnology) for 1 h at room temperature. The membrane was washed and incubated with horseradish peroxidase-conjugated antirabbit immunoglobulin (Ig) (Santa Cruz Biotechnology). Blots were visualized by use of the ECL enhanced chemiluminescence reagent kit (Amersham-Pharmacia Biotech).

Immunoprecipitations and kinase assays. 293 cell lysates (1.5 mg) were immunoprecipitated with anti-p65 goat polyclonal antibody. Immunoprecipitates were washed four times with lysis buffer and were used as substrates for kinase assays with purified baculovirus-expressed IKKß.

RNAi experiments. RNA interference (RNAi) experiments were performed as follows. The sequence of siRNA for the B-Ras1 was 5'-AAGAUUGCGAAACAAUGGAGG-3'. siRNA (1.0 µg) was transfected into 293 cells in six-well plates with Lipofectamine Plus (Invitrogen). Three days posttransfection, cells were treated with 10 ng of TNF- for the indicated times. Whole-cell lysates were subjected to SDS-PAGE and Western blot analysis. Six pSUPER siRNA expression vectors, three each for B-Ras1 and -2, were prepared by inserting duplex oligonucleotides based on the sequence of B-Ras1 and B-Ras2 cDNAs between the BglII and HindIII sites of the vector. The sequences of the top strands of these oligonucleotides are listed below. The sequence underlined represents the loop.

The following oligonucleotides were used for pSuper-B-Ras1: 1 (nucleotides 103 to 121), 5'-GATTGCGAAACAATGGAAGTTCAAGAGACTTCCATTGTTTCGCAATC-3'; 2 (nucleotides 202 to 220), 5'-GGCGTGGAGCTGCCAAAGCTTCAAGAGAGCTTTGGCAGCTCCACGCC-3'; and 3 (nucleotides 388 to 406), 5'-GTGGACGCTGAAGTGGCACTTCAAGAGAGTGCCACTTCAGCGTCCAC-3'. The oligonucleotides for pSuper-B-Ras2 were as follows: 1 (nucleotides 84 to 102), 5'-CCATGTAGTGGGTTCGGAGTTCAAGAGACTCCGAACCCACTACATGG-3'; 2 (nucleotides 333 to 351), 5'-GGAGGTCACCATCGTGGTCTTCAAGAGAGACCACGATGGTGACCTCC-3'; and 3 (nucleotides 435 to 453), 5'-GCTGTGGGAGGTGTCAGTGTTCAAGAGACACTGACACCTCCCACAGC-3'.

All six of these expression vectors independently worked well as siRNA to knock down B-Ras1 and -2. The control pSUPER expression vector was prepared by inserting duplex DNA based on the sequence of NEIL-2, a protein involved in DNA repair pathway. The sequence of the top strand of the duplex is 5'-GCTGGCGGGCTGTAGCTTC-3', located at position +88 of NEIL-2 mRNA. This vector has tested positive for knockdown NEIL-2 expression.

GTP-GDP exchange assays. GTP-GDP exchange was performed with 2 µg of purified B-Ras protein incubated for 1 h and 30 min with 1 mM GTP or GDP (Sigma) and 1 mM MgCl₂ at 30°C, and this reaction mixture was directly used for the kinase assay.

	RESULTS

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In vivo association of B-Ras with IBß. To investigate if cellular IBß/NF-B complexes are associated with B-Ras, we first size fractionated the cytosolic extracts of HeLa cells by Superose 6 (Amersham-Biosciences) size exclusion chromatography. The proteins in each fraction were separated by SDS-PAGE followed by immunoblotting with IBß, IB, p65, c-Rel, and B-Ras antibodies. As expected, IB and NF-B proteins are in general present in similar fractions and elute as broad peaks over large volumes (Fig. 1A). A comparison of the elution profiles of IBß and IB reveals that the IBß is shifted toward a larger molecular weight than the IB profile (Fig. 1B). It is important to note that each peak represents a convolution of multiple IB/NF-B complexes of similar molecular weights, because each of the inhibitors in each fraction is associated with several NF-B dimers. We observe that B-Ras elutes in two peaks: one as a high-molecular-weight complex (fractions 32 to 34) and the other as a low-molecular-weight, possibly uncomplexed, free molecule. Although B-Ras elution does not coincide with the peak fraction of IBß, fractions containing B-Ras also contain substantial amounts of IBß. These observations, along with earlier results that B-Ras associates with IBß, suggest that a fraction of IBß may specifically bind B-Ras (8, 14).

View larger version (39K): [in this window] [in a new window]   FIG. 1. (A) Fractionation of IBß/NF-B complexes. The presence of c-Rel, p65, IBß, IB, and B-Ras in the fractions was confirmed by separation of proteins by SDS-PAGE and immunoblotting. Anti-c-Rel, anti-IBß(C-20), anti-IB, and anti-p65(C-20) antibodies (Ab), respectively, were used for immunoblotting (IB). (B) Graphic presentation of IB and IBß amounts across the chromatographic fractions shown in panel A. (C) B-Ras associates with IBß in vivo. Cytosolic extract from Jurkat T cells expressing Flag-IBß or IB were immunoprecipitated (IP), followed by immunoblotting with anti-B-Ras, anti-p65, or anti-c-Rel antibodies. These results show that Flag-IBß associates with p65, c-Rel, and B-Ras. Flag-IB associates with p65 and c-Rel but not with B-Ras.

B-Ras1 blocks phosphorylation of IBß by IKKß. It is known that in contrast to complete degradation of IB, IBß only partially degrades in response to most stimuli. We wanted to investigate whether inefficient IBß degradation is due to its inability to be phosphorylated by IKK. To test this, we immunoprecipitated IB/p65 complexes by using anti-p65 activation domain antibody and tested the ability of IB and IBß in the immunoprecipitation complex for phosphorylation by IKKß (Fig. 2A, panel 1, lane 1). In parallel, we have performed phosphorylation reaction on an equimolar mixture of purified free IB and IBß and analyzed the products. We observed that both of these pure E. coli-derived proteins are efficiently phosphorylated by IKKß (lane 3). The immunoprecipitation complex is a mixture of all IB/p65 complexes, including the IB/p65 and IBß/p65 complexes. The presence of IB and IBß in the immunoprecipitation complex is revealed by immunoblotting (Fig. 2A, panels 2 and 3, lanes 1 and 2). Because E. coli-derived pure IBß is a poly-histidine fusion protein, it migrates as a slightly-higher-molecular-weight protein compared to native IBß (Fig. 2A, panel 3, compare lanes 1 to 2 versus lane 3). The addition of active baculovirus-derived IKKß to the immunoprecipitation complex under appropriate phosphorylation reaction conditions and subsequent autoradiography clearly reveal a band at a position corresponding to IB (Fig. 2A, panel 1, lane 1). In contrast, we did not observe a clear radiolabeled band from the immunoprecipitation complex that corresponds to IBß (panel 1, lane 1). In the absence of IKKß, no specific phosphorylation was observed, suggesting that the phosphorylation is IKKß specific (panel 1, lane 2). These results suggest that the IKK phosphorylation sites on IB in the IB/p65 complexes are free, but the homologous sites on IBß might not be free in the IBß/NF-B complexes. However, we cannot rule out the possibility that the differential phosphorylation efficiency might be due to a smaller amount of IBß in the pull-down reaction.

View larger version (49K): [in this window] [in a new window]   FIG. 2. (A) Phosphorylation sites of IBß are blocked in vivo. IB/p65 complexes were immunoprecipitated (IP) from the extract of 293 cells. Immunoprecipitated complexes were subjected to phosphorylation by pure (panel 1, lane 1) or mock (panels 1, lane 2) IKKß. In parallel, a mixture of equimolar amounts of IB and IBß was subjected to phosphorylation by pure IKKß (panel 1, lane 3). The reaction products were identified by separation of proteins by SDS-PAGE followed by autoradiography. The presence of IB and IBß in the immunoprecipitate or pure IB and IBß is shown by the Western blot (panels 2 and 3). Ab, antibody; mAb, monoclonal antibody; IB, immunoblotting. (B) Inhibition of IBß phosphorylation by B-Ras in vitro. Pure IBß (1.5 µg) was subjected to phosphorylation by IKKß, and the products were resolved by SDS-PAGE, transferred to membrane, and exposed to phosphorimaging. Phosphorylated IBß is shown in the absence (top panel, lane 1) or presence of 2 or 4 µg of pure B-Ras1 (lanes 2 and 3). The effects of p65 homodimer (top panel, lanes 4 to 6) and p50/p65 heterodimer (top panel, lanes 7 to 9) on phosphorylation inhibition are shown in the absence (lanes 4 and 7) or the presence of B-Ras1 (lanes 5 and 6 and 8 and 9). The bottom panel shows Coomassie staining of B-Ras1 of the same blot used for the autoradiography. (C) Phosphorylation of IB by IKKß. IB (top panel, lanes 1 to 3), IB/p65 complex (lanes 4 to 6), and IB/p50/p65 (lanes 7 to 9) were subjected to phosphorylation in the absence (top panel, lanes 1, 4, and 7) or presence (lanes 2 and 3, 5 and 6, and 8 and 9) of increasing amounts of B-Ras. The bottom panel shows Coomassie staining of the same blot. Only the B-Ras portion of the blot is shown. (D) Phosphorylations of IBß in the presence or absence of B-Ras1, H-Ras, cdc42, Rac1, and RhoA (top panel). The bottom panel shows the Coomassie staining of the Ras proteins used in the reaction.

There is ample evidence in the literature suggesting that other small GTPases, such as Ras, Rho, and cdc42, are also capable of modulating NF-B activation (27, 30). It is possible that some of these Ras and Ras-related proteins alter NF-B activation by directly inhibiting IBß phosphorylation. To test this, we used H-Ras, Rac1, RhoA, and cdc42 to see if they could inhibit phosphorylation of IBß by IKK (Fig. 2D). We observed that the inhibitory activity is specific to B-Ras; other small G proteins failed to inhibit phosphorylation of IBß. It is likely that these GTPases are unable to bind IBß. These results suggest that Ras and other GTPases modulate NF-B activation pathways differently.

Removal of B-Ras from cells enhances the rate of signal-induced degradation of IBß. The above experiments suggest that blockade of the IBß SRR by B-Ras might be responsible, at least in part, for the incomplete degradation of IBß in induced cells. To test this, we selectively removed B-Ras1 from cells by RNAi and observed the effect of inducers on IBß degradation (11, 12). We transfected siRNA directed against B-Ras1, and the transfected cells were induced with TNF- (Fig. 3). We observed that selective removal of B-Ras1 renders IBß more susceptible to degradation by TNF-. After 30 min of TNF treatment, more than 50% of IBß is degraded in B-Ras-deficient cells compared to a <10% reduction in wild-type cells (Fig. 3A, middle panel, compare lanes 2 and 4). In contrast, we found that removal of B-Ras1 has little effect on IB degradation, because the degradation pattern of IB remained unchanged (Fig. 3A, top panel). We further tested TNF--induced degradation of IBß in cells expressing siRNA for both B-Ras1 and -2 in an expression vector. HeLa cells were transfected with pSUPER expressing a control siRNA and combined B-Ras1/B-Ras 2 vectors, and after 24 h, cells were treated with TNF- (7). As expected, no change in IB degradation was observed, but significant amounts of IBß were degraded after 30 min of TNF- treatment in cells expressing siRNA for B-Ras1 or -2 (middle panel, lanes 3 and 6). Whereas RNAi experiments conducted with an RNA duplex against only B-Ras1 did not generate complete degradation of IBß, pSUPER vector-transfected cells expressing siRNA against both B-Ras1 and -2 showed complete degradation of IBß upon stimulation. These experiments clearly demonstrate the importance of B-Ras in regulating induced degradation of IBß.

View larger version (32K): [in this window] [in a new window]   FIG. 3. siRNA-treated 293 cells are susceptible to IBß degradation in response to inducers. (A) 293 cells transfected with siRNA directed against B-Ras1 were induced with TNF- after 48 h of growth for the indicated period of time. (B) HeLa cells were transfected with pSUPER vector expressing siRNA for B-Ras1/2 or siRNA for NEIL-2 as a control. Transfected cells were treated with TNF- after 24 h of growth for the indicated period of time.

B-Ras uses the unique insert and the N-terminal SRR of IBß for complex formation.

View larger version (28K): [in this window] [in a new window]   FIG. 4. B-Ras1 uses the SRR and insert of IBß for binding. (A) Schematic representation of IB, IBß, and various mutants used in these experiments. The domain boundaries of wild-type IB and IBß are indicated. (B) The top panel shows inhibition of phosphorylation of full-length and truncated IBß proteins. Lanes 2, 4, 5, 7, 8, and 10 show the efficiency of phosphorylation of full-length IBß and mutants in the presence of increasing amounts of pure B-Ras. Lanes 1, 3, 6, and 9 indicate phosphorylation in the absence of B-Ras1. The bottom panel shows Coomassie staining of the same blot used for phosphorimaging. (C) Co-IP of IBß and B-Ras from COS cells transfected with HA-B-Ras1 and wild-type or mutant IBß. Immunoprecipitation was done with anti-B-Ras antibody immunoblotted (IB) with antihemagglutinin (HA) antibody. The top panel shows that wild-type IBß forms complex with B-Ras (lane 1). A hydrid IB protein containing the SRR of IB and the rest of IBß binds poorly with B-Ras (lane 3). Both wild-type IBß and hybrid IBß with the insert deleted do not bind B-Ras (lanes 2 and 4). The bottom panels show the expression of IBß and B-Ras.

Overexpression of wild-type B-Ras1 blocks IBß degradation. The above results prompted us to examine the effect of overexpression of B-Ras on IBß degradation. We tested the effect of B-Ras1 on inducer-dependent IBß degradation in HeLa cells. HeLa cells transfected with mammalian vectors expressing IBß and p65, in the presence or absence of B-Ras1, were treated with TNF-. We observed that B-Ras1 blocked TNF--induced degradation of wild-type IBß (Fig. 5A).

View larger version (45K): [in this window] [in a new window]   FIG. 5. B-Ras 1 protects IBß degradation. (A) HeLa cells cotransfected with hemagglutinin (HA)-tagged IBß and B-Ras1 or empty vector were grown for 2 days followed by treatment with TNF- for the indicated period of time. Similar experiments were done with three different IBß mutants: HA-IBß152-192 (B), HA-IB(1-65)/IBß(50-359) (C), and HA-IB (1-65)/IBß(50-359)(152-192). ns, nonspecific binding.

Both GDP- and GTP-bound forms of B-Ras1 inhibit IBß phosphorylation. We wanted to examine the relationship between B-Ras activity and guanine (G)-nucleotide binding states. B-Ras1 preparations used in all previous in vitro assays are proteins expressed in E. coli in soluble forms. To convincingly prove if both GDP and GTP or only one form of B-Ras acts as an inhibitor of IBß phosphorylation, it is essential to prepare homogeneous B-Ras loaded with either GDP or GTP. In Ras and most related small GTPases, E. coli-expressed proteins are loaded with GDP (37). In vitro nucleotide exchange reactions are required to prepare homogeneous preparations of GDP- or GTP-bound G proteins. Interestingly, the conventional in vitro GTP/GDP exchange reaction does not work for B-Ras1 (data not shown) (21). We prepared B-Ras1 by purifying denatured protein and subsequently refolding it under native conditions. The refolded protein was further purified by size exclusion chromatography. We observed that although the refolded protein behaved similarly to the soluble form of B-Ras1 chromatographically, it failed to block IBß phosphorylation by IKK (Fig. 6A). This observation suggested that lack of the nucleotide in refolded B-Ras may have rendered it inactive. Indeed, we observed that GTP was present in B-Ras1 purified under native conditions, whereas refolded B-Ras1 did not contain any GTP or GDP (data not shown). The refolded protein can be loaded with either GDP or GTP in the presence of MgCl₂. Nucleotide-loaded B-Ras1 was tested for phosphorylation of IBß by IKK. Figure 6B shows that both the GDP- and GTP-bound forms of B-Ras1 efficiently block phosphorylation of IBß by IKK.

View larger version (26K): [in this window] [in a new window]   FIG. 6. GDP or GTP binding is essential for B-Ras1 function. (A) Autoradiogram of phosphorylation of IBß by IKKß in the presence of B-Ras 1 purified as a soluble or refolded protein (top panel). Coomassie staining of the same membrane shows the quality of B-Ras 1 preparations (bottom panel). (B) Refolded B-Ras 1 was used for nucleotide loading with GDP and GTP. IBß was subjected to phosphorylation in the presence of nucleotide-loaded B-Ras1. (C) Effect of a mutant B-Ras1 in protecting IBß degradation in HeLa cells. HeLa cells were transfected with vectors expressing wild-type (WT) IBß and wild-type or mutant B-Ras followed by TNF treatment for indicated times. Degradation of IBß was monitored by Western blotting with antihemagglutinin (HA) antibody.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

 
One of the fundamental questions about cellular signaling is how different members of a protein family impart specificity in signal transduction pathways. With respect to NF-B-mediated regulation of gene expression, it is now known from knockout experiments that all five members of the NF-B family play distinct, albeit overlapping roles. Whereas promoter accessibility, combinational dimer formation, DNA binding affinity, and posttranslational modification contribute to achieving such specificity, inhibition of NF-B dimers by IB family proteins is perhaps the most important mechanism of NF-B regulation. How different IB proteins regulate each NF-B dimer is not known.

A pool of IBß/NF-B complexes associate with B-Ras. In most cells, almost equal amounts of NF-B dimers are associated with IB and IBß. However, IBß is less sensitive to several extracellular signals (16, 17). The biochemical mechanism for this lack of stimulus-dependent complete IBß degradation has not been addressed. Whereas incomplete degradation of IBß can result from defects at any one of the multiple steps required for degradation, we suspected that defects in IBß phosphorylation by IKK were principally responsible. Using a series of biochemical experiments, we have shown that there are two pools of IBß in quiescent cells: one present as a binary complex with NF-B and the other as a ternary complex with both NF-B and B-Ras. Our in vitro experiments reveal that IB/NF-B and IBß/NF-B binary complexes are fully competent substrates for IKK phosphorylation. When HeLa cells are induced with TNF-, the kinetics of IB and IBß degradations are different. We believe that IBß of the ternary complex is more resistant to stimulus-dependent degradation. Reduction or elimination of B-Ras by RNAi showed enhanced degradation of IBß. We suggest that IBß of the IBß/NF-B complex is degraded quickly in wild-type cells, while IBß in the ternary complex remains resistant to degradation. The latter pool becomes susceptible only after the removal of B-Ras by siRNA.

The question that remains unanswered is whether a specific NF-B dimer dictates the formation of IBß binary and ternary complexes. Although NF-B subunit-dependent ternary complex formation may appear unlikely based on our current results, which show that B-Ras binds primarily through IBß, we cannot entirely rule out this possibility. Under physiological conditions, B-Ras may have a higher affinity for a specific IBß/NF-B complex compared to other binary complexes. Depending on the cellular concentrations of these proteins and in the presence of competing binding partners, a high-affinity ternary complex may predominate. In vitro binding experiments reveal that IB binds NF-B p50/p65 heterodimer more strongly than the homodimers of p65 and c-Rel or the p50/c-Rel heterodimer (C. Phelps and G. Ghosh, unpublished results). IBß, on the other hand, binds all of these dimers more weakly than IB (25). It is possible that IBß displays increased variations in binding affinity and specificity in the presence of B-Ras (i.e., IBß may preferentially bind to a specific dimer in the presence of B-Ras). Future experiments will be needed to resolve whether the B-Ras/IBß/NF-B ternary complex requires specific NF-B dimers.

A second important question is how the sequestration of IBß/NF-B complexes are regulated. It has been shown by us and others that all IBß/NF-B complexes are cytoplasmic. However, in this study, we have shown that B-Ras binds to only a fraction of these IBß/NF-B complexes and masks their apparently exposed NF-B NLS. What regulates the cytoplasmic sequestration of the rest of the IBß/NF-B complexes that do not bind to B-Ras? At this point, we do not know the answer to this question. It is possible that IBß in the binary complex might be modified, which causes the masking of the second NLS. Yet another possibility is that this second pool of IBß/NF-B complex is not actually binary but interacts with another protein that blocks only the second NLS, but unlike B-Ras does not prevent phosphorylation of IBß.

Unique sequence and structural features of IBß explain its functional differences. Although the presence of highly homologous ankyrin repeats and the PEST sequence of IB and IBß masks the clear differences in their sequences, it is the nonhomologous segments that are the key to the formation of a larger complex nucleated by IBß. Results presented in this paper indicate that B-Ras1 interacts with IBß by using two different segments of IBß: the insert and the SRR. The insert is absent in IB, and the SRR and PEST are not completely homologous in IB and IBß. The most critical of these segments for B-Ras1 binding is the insert. The insert, which is not a part of the relatively rigid ARD structure, is also present in Cactus, the Drosophila homolog of IB proteins (15, 24). It is possible that Cactus interacts with Drosophila B-Ras in a manner similar to IBß.

Our results also reveal that the SRR of IBß, but not IB, is able to make direct contact with B-Ras1. The SRR of IB is different in sequence from the SRR of IBß. The SRR is important for IKK phosphorylation and recognition by the E3RS/ß-TRCP ubiquitination machinery. However, a stringent sequence requirement for these two functions is restricted only to a small, highly homologous segment of 16 amino acids. Outside of this small homologous region, the SRR sequences of the two IB proteins are different. We do not, however, know if the IKK-phosphorylatable serines (Ser19 and Ser23) are directly involved in B-Ras binding or are occluded from phosphorylation because of indirect blockade of IKKß recognition.

B-Ras1 is an unusual small G protein. B-Ras proteins are somewhat unusual members of the Ras family of GTPases in that they contain the constitutively active oncogenic mutations G12L and Q61L in Ras (5). Because of the similarity to oncogenic Ras mutations within B-Ras sequence, it was originally thought that native B-Ras exists in cells in a constitutively active form. While it still might be true that B-Ras binds GTP in vivo and acts as a constitutively active form of GTPase, several observations indicate that B-Ras proteins are clearly different from other GTPases. First, we observed that both GDP- and GTP-bound forms of B-Ras are equally potent in blocking phosphorylation of IBß by IKKß in vitro (Fig. 6B). This suggests that even if other accessory proteins induce GTP hydrolysis, the hydrolyzed product GDP would remain bound and maintain its activity in IBß binding. Second, B-Ras functions as an inhibitor of signal transduction (i.e., signals are terminated through B-Ras). In contrast, oncogenic Ras proteins sustain signals by continuously binding to effector molecules. It is therefore likely that B-Ras proteins represent a different paradigm in cell signaling.

The fact that B-Ras1 binds to IBß in both its GDP- and GTP-bound states suggests that this protein may not assume two different conformations. We suggest that B-Ras1 is not a molecular switch like other small G proteins. We have not been able to show if the B-Ras/GDP complex is a physiologic entity and functions as an inhibitor of IBß phosphorylation by IKK in vivo. Based on its simple role as an inhibitory adapter molecule, there is no reason for B-Ras to not use both GDP and GTP as cofactors to carry out its function in vivo. G nucleotides contacting residues in all four G-boxes in B-Ras proteins (both 1 and 2), however, are invariant, and alteration of conserved G-nucleotide binding residues or the removal of G nucleotide by a denaturation-renaturation process makes B-Ras inactive (Fig. 6C). These results indicate that G-nucleotide binding is essential for B-Ras function.

At least some small G protein families contain members that are GTP hydrolysis-deficient (13, 28). Exactly how they function is unclear. It is possible that like B-Ras, these non-GTPase small G proteins are functionally distinct from the prototypical members. These proteins simply bind G nucleotides as a cofactor for their cellular activities and do not undergo GTP-GDP exchange cycles.

Conclusions. We have demonstrated the biochemical mechanism of the origin of differential degradation of IB and IBß in response to inducers. We suggest that a pool of IBß/NF-B complex is bound to B-Ras as a ternary complex. IBß of this ternary complex is not degradable by most inducers (Fig. 7). It is likely that a specific NF-B dimer is a part of this ternary complex. Further biochemical purification of these complexes is necessary to determine the composition of these complexes.

View larger version (31K): [in this window] [in a new window]   FIG. 7. Model of NF-B activation from the IB- and IBß-bound states. IB and a pool of IBß complexes are present as binary complexes with NF-B. These IBs are rapidly degraded by inducers such as TNF-. A second pool of IBß remains as a ternary complex with NF-B and B-Ras. Release of an NF-B dimer from IBß from the second pool requires compound signals.

	ACKNOWLEDGMENTS

 
We thank Tom Huxford, Rasmi Talwar, and Anu K. Moorthy for critically reading the manuscript; Sankar Ghosh and Tapas Hazra for reagents and helpful discussion; and Chun Wu for helpful suggestions. We also thank Anu K. Moorthy for help with HeLa cytoplasmic extracts, Michael Karin and Mireille Delhase for discussion and reagents, and Ju Chen for support.

This research was supported by grants from the National Cancer Institute, the Cystic Fibrosis Foundation, and the Human Frontier Science Program.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0359. Phone: (858) 822-0469. Fax: (858) 534-7042. E-mail: gghosh{at}chem.ucsd.edu.

Y.C. and S.V. contributed equally to this work.

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