AMAP1 as a negative-feedback regulator of nuclear factor-κB under inflammatory conditions

Abstract

NF-κB is a major transcriptional factor regulating many cellular functions including inflammation; therefore, its appropriate control is of high importance. The detailed mechanism of its activation has been well characterized, but that of negative regulation is poorly understood. In this study, we showed AMAP1, an Arf-GTPase activating protein, as a negative feedback regulator for NF-κB by binding with IKKβ, an essential kinase in NF-κB signaling. Proteomics analysis identified AMAP1 as a binding protein with IKKβ. Overexpression of AMAP1 suppressed NF-κB activity by interfering the binding of IKKβ and NEMO, and deletion of AMAP1 augmented NF-κB activity. The activation of NF-κB induced translocation of AMAP1 to cytoplasm from cell membrane and nucleus, which resulted in augmented interaction of AMAP1 and IKKβ. These results demonstrated a novel role of AMAP1 as a negative feedback regulator of NF-κB, and presented it as a possible target for anti-inflammatory treatments.

Figure 1. AMAP1 interacts with IKKβ in vivo.

(a) The binding of AMAP1 and IKKβ. FLAG-tagged IKKβ and HA-tagged AMAP1 were expressed in HEK293T cells and immunoprecipitated with anti-FLAG and anti-HA antibodies. AMAP1 and IKKβ were detected in the precipitate by immunoblotting with anti-FLAG and anti-HA antibodies. (b) Schematic representation of IKKβ and its mutants. (c) FLAG-tagged IKKβ/mutants and HA-tagged AMAP1 were expressed in HEK293T cells and immunoprecipitated with anti-FLAG antibody. AMAP1 was detected in the precipitate by immunoblotting with anti-HA antibody. (d) Schematic representation of AMAP1 and its mutants. (e) FLAG-tagged IKKβ and GST-tagged AMAP1/mutants were expressed in HEK293T cells and immunoprecipitated with anti-FLAG antibody. AMAP1 and its mutants were detected in the precipitate by immunoblotting with anti-GST antibody. The data shown are from one representative experiment of the three that were performed. Full-length blots are presented in Supplementary Figure 4.

Figure 2. AMAP1 negatively regulates NF-κB.

(a) AMAP1 interfered with the association of IKKβ and NEMO. FLAG-tagged IKKβ and NEMO were expressed in HEK293T cells along with control vector or HA-tagged AMAP1 and immunoprecipitated with anti-FLAG antibody. AMAP1, IKKβ and NEMO were detected in the precipitates by immunoblotting with anti-FLAG, anti-HA and anti-NEMO antibodies. (b) The induction of NEMO reduced the interaction of IKKβ and AMAP1. FLAG-tagged IKKβ and HA-tagged AMAP1 were expressed in HEK293T cells along with control or NEMO vector and immunoprecipitated with anti-FLAG antibody. AMAP1, IKKβ and NEMO were detected in the precipitates by immunoblotting with anti-FLAG, anti-HA and anti-NEMO antibodies. (c) NF-κB activity from nuclear extracts of overexpressed AMAP1 and control cells with or without IL-1β (2.5 ng/mL) treatment for 30 min. (d) Immunoblots of AMAP1 (whole cell) and p65 (nuclear extract) of HEK293T cells transfected with pcDNA3.1-HA-AMAP1 (AMAP1 overexpression) or pcDNA3.1 (control) and the cumulative, quantitative densitometry data. (e) NF-κB activity from nuclear extracts of shAMAP1 and shControl cells with or without IL-1β (2.5 ng/mL) treatment for 30 min. (f) Immunoblots of AMAP1 (whole cell) and p65 (nuclear extract) of HEK293T cells transfected with AMAP1 shRNA (shAMAP1) or Control shRNA (shControl) with or without IL-1β (2.5 ng/mL) and the cumulative, quantitative densitometry data. The data from three independent experiments are shown. Error bar: ±SD. * P < 0.05, ** P < 0.01 in a two-sided, Student's t-test. Full-length blots are presented in Supplementary Figure 4.

Figure 3. IL-1β induces AMAP1 translocation and the AMAP1 and IKKβ interaction.

(a) Representative AMAP1 immunoblots of fractions from sucrose gradient centrifugation of HEK293T cells treated with or without IL-1β (2.5 ng/mL) for 30 min and the cumulative, quantitative densitometry data from three independent experiments are shown. (b) HUVECs were treated with or without IL-1β (2.5 ng/mL) for 30 min and then stained with anti-AMAP1 antibody, WGA and DAPI (n = 3). Scale bar = 50 μm. (c) Representative AMAP1 immunoblots of fractions from sucrose gradient centrifugation of HUVECs treated with or without IL-1β (2.5 ng/mL) for 30 min and the cumulative, quantitative densitometry data from three independent experiments are shown. Representative blots from three immunoprecipitation experiments using anti-AMAP1 (d) and anti-IKKβ (e) antibodies in HUVECs treated with or without IL-1β (2.5 ng/mL) for 30 min. The graph represents the cumulative, quantitative densitometry data of the IKKβ blot in anti-AMAP1 antibody precipitates (d), of the AMAP1 blot in anti-IKKβ antibody precipitates (e). Error bar: ±SD. * P < 0.05, *** P < 0.005 in a two-sided, Student's t-test. Full-length blots are presented in Supplementary Figure 4.

Figure 4. Proposed model for AMAP1 as a binding protein of IKKβ and negative regulator of NF-κB.

Dashed lines indicate the feedback mechanism.