KappaB-Ras is a nuclear-cytoplasmic small GTPase that inhibits NF-kappaB activation through the suppression of transcriptional activation of p65/RelA - PubMed (original) (raw)

KappaB-Ras is a nuclear-cytoplasmic small GTPase that inhibits NF-kappaB activation through the suppression of transcriptional activation of p65/RelA

Kenji Tago et al. J Biol Chem. 2010.

Abstract

NF-κB is an important transcription factor involved in various biological responses, including inflammation, cell differentiation, and tumorigenesis. κB-Ras was identified as an IκB-interacting small GTPase and is reported to disturb cytokine-induced NF-κB activation. In this study, we established that κB-Ras is a novel type of nuclear-cytoplasmic small GTPase that mainly binds to GTP, and its localization seemed to be regulated by its GTP/GDP-binding state. Unexpectedly, the GDP-binding form of the κB-Ras mutant exhibited a more potent inhibitory effect on NF-κB activation, and this inhibitory effect seemed to be due to suppression of the transactivation of a p65/RelA NF-κB subunit. κB-Ras suppressed phosphorylation at serine 276 on the p65/RelA subunit, resulting in decreased interaction between p65/RelA and the transcriptional coactivator p300. Interestingly, the GDP-bound κB-Ras mutant exhibited higher interactive affinity with p65/RelA and inhibited the phosphorylation of p65/RelA more potently than wild-type κB-Ras. Taken together, these findings suggest that the GDP-bound form of κB-Ras in cytoplasm suppresses NF-κB activation by inhibiting its transcriptional activation.

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Figures

FIGURE 1.

FIGURE 1.

κB-Ras is a nuclear-cytoplasm shuttling small GTPase. A, immunoblotting analysis of κB-Ras1 and κB-Ras2 in adult mouse tissues. Upper and lower photographs show the expression of κB-Ras1 and κB-Ras2. B, immunoblotting analysis of κB-Ras1 and κB-Ras2 in lysates of various human and mouse cell lines. C, immunostaining analysis of κB-Ras2 in HeLa cells. Endogenous κB-Ras2 was detected by anti-κB-Ras2 polyclonal antibody and Alexa594-conjugated secondary antibody. Nucleus was visualized by Hoechst 33258. D, confocal microscopy analysis of κB-Ras2 expressed in NIH-3T3 cells. NIH-3T3 cells expressing FLAG-κB-Ras2 were treated with LMB for 6 h. κB-Ras2 localization was detected by anti-FLAG (M2) antibody and anti-mouse IgG conjugated with Alexa594. Nucleus was visualized by Hoechst 33258. E, confocal microscopy analysis of endogenous κB-Ras2 in HeLa cells. Cells were treated with LMB as in D. κB-Ras2 localization was detected by anti-κB-Ras2 antibody and anti-mouse IgG conjugated with Alexa594. Nucleus was visualized by Hoechst 33258.

FIGURE 2.

FIGURE 2.

T18N mutant of κB-Ras exhibits low affinity to GTP. A, molecular scheme of wild-type κB-Ras2 and T18N mutant. B, purified recombinant GST, GST-κB-Ras2, and GST-T18N mutant were analyzed by Coomassie Brilliant Blue (CBB) staining. C, GTPγS binding activity of κB-Ras2 and T18N mutant in vitro. Recombinant proteins of κB-Ras2 and T18N mutant were incubated with [35S]GTPγS during the indicated periods, and then protein-GTPγS complexes were collected on a nitrocellulose membrane. D, binding ability of κB-Ras2 to GTPγS and GDP was tested. Recombinant protein of κB-Ras2 was incubated with the indicated concentration of [35S]GTPγS or [3H]GDP for 30 min. Protein-GTPγS or -GDP complexes were collected on a nitrocellulose membrane, and radioactivity was measured. E, expressions of κB-Ras2 and T18N mutant in HEK293T cells. F, cells expressing FLAG-κB-Ras2 or T18N were metabolically labeled with [32P]phosphorus. Cell lysates were utilized for immunoprecipitation using anti-FLAG (M2) antibody. Bound guanine nucleotides were developed by TLC. G, percentage of GTP-bound form is shown on the graph. Error bars, S.D. (n = 3). IP, immunoprecipitation; IB, immunoblot.

FIGURE 3.

FIGURE 3.

κB-Ras changes its localization in a GTP/GDP-dependent manner. A, immunostaining analysis of κB-Ras2 and T18N mutant in NIH-3T3 cells. Experiments were performed as in Fig. 1, C and D. κB-Ras2 localization was detected by anti-FLAG (M2) antibody and anti-mouse IgG conjugated with Alexa594 (green). Nucleus was visualized by Hoechst 33258 (blue). To confirm the localization of κB-Ras2, merged photographs are shown. B, percentage of positive cells with nuclear localization of κB-Ras2 and T18N mutant in NIH-3T3 cells were calculated and are shown in the graph. In the graph, error bars indicate S.D. (n = 3). C, to analyze the subcellular localization of the GDP- or GTP-bound form of κB-Ras, cytosolic and nuclear fractions were prepared from HEK293T cells expressing FLAG-κB-Ras2 or T18N. Each fraction was analyzed by immunoblot analysis with anti-FLAG (M2) antibody. WCL, whole cell lysate.

FIGURE 4.

FIGURE 4.

Effect of κB-Ras on cytokine-induced NF-κB activation. A, HEK293T cells transfected with pNF-κB-luciferase plus FLAG-κB-Ras2 or T18N were stimulated with TNFα, and luciferase activity was measured. To verify the protein expression, immunoblot analysis was performed for κB-Ras2 and T18N mutant. B, HEK293T cells seeded on a 60-mm dish were transfected with κB-Ras2 (0.1 or 0.3 μg) or T18N (1 μg). Cells expressing κB-Ras2 (0.1 or 0.3 mg) or T18N were stimulated with TNFα. RT-PCR analysis for IL-8 and GAPDH was performed. The expression level of κB-Ras2 and T18N mutant was evaluated by immunoblot analysis. In the experiments in C and D, NIH-3T3 cells that stably harbor κB-responsive luciferase gene (KF-8 cells) were utilized. C, KF-8 cells were infected with retrovirus harboring shRNA for luciferase (sh-Luc) or murine κB-Ras2 (sh_-κ_B-Ras2). Six days later, cells were harvested for immunoblot analysis to test the expressions of κB-Ras2, IκBα, IκBβ, and β-actin. D, KF-8 cells stably harboring the κB-luciferase gene were infected with control virus (sh-luciferase) or sh-κB-Ras2 virus. Cells were stimulated with TNFα (10 ng/ml) for 12 h. Cells were harvested, and luciferase activity was measured. In each experiment, error bars = S.D. (n = 3; *, p < 0.005).

FIGURE 5.

FIGURE 5.

Effect of κB-Ras on TNFα-induced IκB degradation. A, FLAG-κB-Ras2 and T18N were ectopically expressed in HEK293T cells. Forty eight hours later, cells were stimulated with/without TNFα (10 ng/ml) for the indicated periods, and cells were then lysed with lysis buffer. Obtained lysates were analyzed by immunoblot analysis using anti-IκBα, IκBβ, and β-actin antibodies. CTL, control. To detect overexpressed κB-Ras2, anti-FLAG (M2) antibody was utilized. The degradation of IκBα and IκBβ was normalized with the protein of β-actin, and the quantified ratios of IκBα and IκBβ are shown in B and C, respectively. In each experiment, error bars = S.D. (n = 3; *, p < 0.005). D, NIH-3T3 cells were infected with control virus (sh-Luc) or sh-κB-Ras2 virus. Six days later, cells were stimulated with 10 ng/ml TNFα for the indicated periods and then harvested for immunoblot analysis to test the expressions of κB-Ras2, IκBα, IκBβ, and β-actin. E, relative protein of IκBα and IκBβ was normalized with the protein of β-actin, and the quantified ratios of IκBα and IκBβ are shown. Error bars, S.D. (n = 3; *, p < 0.005).

FIGURE 6.

FIGURE 6.

κB-Ras inhibits p65/RelA activation in an IκBβ stabilization-independent manner. A, HEK293T cells expressing κB-Ras2 or T18N were stimulated with TNFα for the indicated periods, and nuclear extracts were then prepared and analyzed by EMSA using an NF-κB-specific probe. CTL indicates control cells transfected with empty vector. B, schemes of wild-type p65/RelA and p65/RelA (S276A) mutant were drawn. RHD, CBD, and TAD indicate Rel homology domain, CBP/p300-binding domain, and transcriptional activating domain, respectively. Numbers mean the position of amino acids in p65/RelA. C, HEK293T cells were transfected with pNF-κB-luciferase plus the indicated combination of plasmids encoding wild-type p65/RelA, p65/RelA (S276A), FLAG-κB-Ras2, and/or T18N mutant. Cells were harvested, and the luciferase activity in each transfected cell was measured. CTL indicates cells transfected without p65/RelA (WT) or p65/RelA (S276A) mutant. Error bars, S.D. (n = 3).

FIGURE 7.

FIGURE 7.

GDP-bound form of κB-Ras directly interacts with p65/RelA. A, direct interaction between κB-Ras2/T18N and p65/RelA was analyzed by in vitro pulldown assay. Various amounts (10, 30, 100, and 300 pmol) of GST-κB-Ras2 (wild type) or T18N were incubated with 10 μ

m

GTPγS or GDP for 30 min. Then 20 pmol of recombinant p65/RelA was added. Binding reaction was performed for 30 min at 25 °C, and then protein complexes were collected using glutathione-Sepharose. Protein complexes were analyzed by immunoblot (IB) analysis using anti-p65/RelA or anti-GST antibody. B, protein of myc-p65/RelA bound to GST-κB-Ras2 or T18N is shown on the graph. The amount of bound p65/RelA is shown as a percentage of the total amount of input p65/RelA. Error bars, S.D. (n = 3; *, p < 0.005).

FIGURE 8.

FIGURE 8.

κB-Ras specifically inhibits the transcriptional activation of p65/RelA. A, schemes of GAL4DBD-p65/RelA and GAL4DBD-p65/RelA (S276A) were drawn. RHD, CBD, and TAD indicate the Rel-homology domain, CBP/p300-binding domain, and transcriptional activating domain, respectively. Numbers in parentheses mean the original position of amino acids in p65/RelA. B, HEK293T cells were transfected with pFR-luciferase plus the indicated combination of plasmids encoding GAL4DBD-p65/RelA, GAL4DBD-p65/RelA (S276A), FLAG-κB-Ras2, and/or T18N mutant. Cells were stimulated with TNFα. Cells were harvested and their luciferase activity was measured. CTL indicates unstimulated control. Error bars, S.D. (n = 3; *, p < 0.005).

FIGURE 9.

FIGURE 9.

κB-Ras interrupts the interaction between p65/RelA and p300. A, effect of κB-Ras on PKA-catalyzed phosphorylation of p65/RelA in vitro. Recombinant proteins of p65/RelA or p65/RelA (S276A) were incubated with recombinant PKA in kinase reaction buffer including [γ-32P]ATP for 2 h at 37 °C. Phosphorylated samples were resolved by SDS-PAGE and analyzed by autoradiography. B, HEK293T cells were transfected with the indicated combination of plasmids encoding myc-p65/RelA, FLAG-κB-Ras2, and T18N mutant. Cells were stimulated with TNFα for 30 min, and their cell lysates were then used for immunoprecipitation (IP) with anti-Myc (9E10) antibody. Immunoprecipitates and whole cell lysates (WCL) were analyzed by immunoblotting with anti-p65/RelA, anti-p300, and anti-κB-Ras2 antibodies. CTL indicates transfection without κB-Ras2 or T18N. C, HEK293T cells expressing myc-p65/RelA or myc-p65/RelA (S276A) mutant were stimulated with 10 ng/ml TNFα for the indicated periods, and then their cell lysates were used for immunoprecipitation with anti-Myc (9E10) antibody. Immunoprecipitated samples and WCL were analyzed by immunoblotting with anti-p65/RelA and anti-p300 antibodies.

FIGURE 10.

FIGURE 10.

Working hypothesis of κB-Ras-induced NF-κB inhibition. According to our current study, κB-Ras seems to inhibit NF-κB activation by interrupting the transcriptional activation of p65/RelA. The GDP-bound form of κB-Ras localized in cytosol mainly contributes to this suppressive mechanism.

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