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

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.

reported the inhibitory effect of AMAP1 (ASAP1) on the production of proinflammatory mediators 23 , but the detailed mechanism remains unknown.
In this report, we carefully examined the involvement of AMAP1 in the regulation of NF-kB, and found that it has an unanticipated role to interfere the IKKb2NEMO binding, which results in negative-feedback regulation of NF-kB activity. This report describes the detailed role of AMAP1 in the inflammatory responses, and AMAP1 can be a new target for developing anti-inflammatory treatments.

Results
AMAP1 interacts with IKKb. To discover novel roles of IKKb, we started with identifying proteins that bind to IKKb. Three kinds of FLAG-tagged IKKb were overexpressed in HEK293T cells, and binding proteins were immunoprecipitated using anti-FLAG antibody. The samples were subjected to proteomic analyses using the nano-LC/MALDI-TOF system. Of the proteins identified repeatedly, we focused on AMAP1 since it is clinically important due to its augmented expression in cancer 15,16,19,22,24 , which is one of the major inflammatory diseases 7,9 . To confirm the physical association of IKKb and AMAP1, we performed co-immunoprecipitation (Co-IP) assays using the lysate of HEK293T cells co-overexpressed with HA-tagged AMAP1 and FLAG-tagged IKKb. IKKb and AMAP1 were detected in the precipitates with either the anti-HA antibody or the anti-FLAG antibody, respectively (Fig. 1a).
Next, we determined the binding sites of these proteins. Based on the functional modules of IKKb, including an amino-terminal kinase domain (KD), a ubiquitin-like domain (ULD), an elongated a-helical scaffold/dimerization domain (SDD) and a carboxyl-terminal NEMO Binding Domain (NBD) 25 , we divided IKKb into 4 overlapping FLAG-tagged fragments. The constructs are IKKb-D1 comprising the KD, ULD and SDD, IKKb-D2 comprising the KD and ULD, IKKb-D3 comprising only the KD, and IKKb-D4 comprising the ULD, SDD and NBD (Fig. 1b), and each construct was expressed in HEK293T cells along with HA-tagged AMAP1. We found that AMAP1 was co-precipitated only with IKKb-D4, but not with any other construct of IKKb (Fig. 1c), indicating that AMAP1 binds to IKKb at the C-terminal region containing the NBD. Further, we examined which region of AMAP1 is essential for its binding to IKKb. Each of 6 GST-tagged fragments of AMAP1, which are Bar (Bin-Amphiphysin-Rvs), PH (Pleckstrin Homology), Arf-GAP, ANK (Ankyrin repeat), PRD (Proline Rich Domain) and SH3 (Src Homology3) domains (Fig. 1d), was expressed in HEK293T cells along with FLAG-tagged IKKb. We found only SH3 domain construct was co-precipitated with IKKb as shown in Fig. 1e. To confirm it further, we made construct of SH3-domain deleted AMAP1 (AMAP1DSH3). Cells were expressed with full-length AMAP1 or AMAP1DSH3 along with FLAG-tagged IKKb, and we found only full-length AMAP1, not AMAP1DSH3, was co-precipitated with IKKb ( Supplementary Fig. S1 online). Taken together, these results indicated that the SH3 domain of AMAP1 is responsible for the binding with IKKb, which implicates that the GTPase activity of AMAP1 is not involved in the interaction with IKKb.
AMAP1 negatively regulates NF-kB. The C-terminal region of IKKb identified as the binding site for AMAP1 contains the binding domain for NEMO/IKKc, which is the regulatory subunit of the IKK complex 3 . It prompted us to verify the effect of AMAP1 on the association of IKKb and NEMO because it is required for NF-kB activation 26 . As shown in Fig. 2a, overexpression of AMAP1 inhibited the binding of IKKb and NEMO, and it was in a dosedependent manner ( Supplementary Fig. S2 online). In addition, the increased level of NEMO could mitigate the interaction between AMAP1 and IKKb (Fig. 2b). Taken together, there is competition between AMAP1 and NEMO in IKKb interaction. As expected from these results, overexpression of AMAP1 suppressed NF-kB activity (Fig. 2c), and it was confirmed by less translocation of p65 into nucleus after IL-1b stimulation in AMAP1-overexpressed cells (Fig. 2d). Additionally, suppression of AMAP1 by shRNA enhanced NF-kB activity (Fig. 2e) and augmented p65 translocation into the nucleus after IL-1b stimulation (Fig. 2f). Thus, AMAP1 interferes the binding of IKKb and NEMO, and negatively regulates NF-kB.
NF-kB activation induces AMAP1 translocation to cytoplasm and augments the AMAP1-IKKb interaction. In the experiments mentioned above, we sometimes observed that more amount of AMAP1 was detected after treatment with IL-1b ( Supplementary  Fig. S3a online). This observation coincides with previous report showing LPS significantly boosted AMAP1 protein levels within 15 min 23 . However, considering that 15 min is too short for synthesis or degradation of proteins, it was highly possible that AMAP1 might not be completely dissolved in cell lysis buffer because AMAP1 can be localized in lipid rafts [11][12][13][14] and nucleus 27 , both of which might not be fully dissolved by 1% Triton X-100 of cell lysis buffer used in this study. Indeed, we also observed similar change of AMAP1 by LPS stimulation, but it disappeared when cells were lysed in 1% SDS cell lysis buffer ( Supplementary Fig. S3b online). Therefore, we hypothesized that NF-kB activation induces AMAP1 translocation to the cytoplasm from cell membrane or nucleus, which could result in detection of more AMAP1 in cell lysate by 1% Triton X-100. As shown in Fig. 3a, fractionation by sucrose gradient indicated that IL-1b stimulation induced the translocation of AMAP1 from lipid rafts to the cytoplasm. We also performed confocal immunocytochemistry with using human umbilical vein endothelial cells (HUVECs) since HEK293T cells were not appropriate for observation of intracellular localization due to their large nuclei and thin cytoplasm. It was hard to see AMAP1 staining on cellular membrane, but we observed that IL-1b stimulation induced translocation of AMAP1 in nuclei to the cytoplasm (Fig. 3b). Finally, we repeated the sucrose gradient fractionation with HUVECs, and observed the translocation of AMAP1 from the membrane in response to IL-1b stimulation (Fig. 3c), similarly with HEK293T cells.
Considering that IKKb is nearly entirely cytoplasmic protein 28,29 , the translocation of AMAP1 to cytoplasm in response to IL-1b stimulation raised the question as to whether the activation of NF-kB would strengthen the interaction between AMAP1 and IKKb. As shown in Fig. 3d and e, more IKKb was co-precipitated with anti-AMAP1 antibody after IL-1b stimulation, and conversely, more AMAP1 was co-precipitated with anti-IKKb antibody after IL-1b stimulation, revealing that NF-kB activation enhances the interaction between AMAP1 and IKKb.

Discussion
Most biological processes require positive and negative regulatory mechanisms to maintain equilibrium. The results from this study suggest a novel mechanism for NF-kB termination and contribute new knowledge to the mechanisms of NF-kB regulation. So far, the re-synthesis of IkB proteins induced by the activation of NF-kB is a widely accepted mechanism for terminating the NF-kB response. These proteins enter the nucleus, remove NF-kB from the DNA and re-localize it to the cytosol [30][31][32][33] . Here, we show that NF-kB activation induces AMAP1 translocation to cytoplasm from nucleus or lipid rafts and accelerates binding of IKKb and AMAP1, which inhibits the association of IKKb and NEMO, eventually leading to inactivation of NF-kB. Taken together, AMAP1 works as an inhibitory feedback mechanism for regulating the NF-kB pathway (Fig. 4).
Recent studies have established strong support for the critical role of NF-kB in cancer. Activation of NF-kB controls multiple cellular processes in cancer, including inflammation, transformation, proliferation, angiogenesis, invasion, metastasis, chemoresistance and      radioresistance 7,9 . Considering the role of AMAP1 explored in this study, augmented expression of AMAP1 observed in cancer might indicate overwhelming NF-kB activation and self-repairing against cancer.
In summary, this study rediscovered AMAP1 as a negative regulator of NF-kB activity in a feedback mechanism, and AMAP1 could be a novel target for treatment or prevention against inflammatory diseases including cancer.
Proteomics analysis. A Nano-LC/MALDI-TOF system was used with three types of IKKb (wild-type IKKb, a dominant-positive mutant of IKKb and a dominantnegative mutant of IKKb) that were overexpressed in HEK293T cells and immunoprecipitated by a specific antibody. Each sample was reduced with 45 mM of DTT (Wako), alkylated with 100 mM of iodoacetamide (Sigma-Aldrich) and digested with 2000 ng of trypsin (Promega, Madison, WI, USA). One-dimensional peptide fractionation was performed with a DiNa Direct Nano-Flow LC/MALDI system (KYA Tech., Tokyo, Japan) using a reverse-phase (RP) trap column (HiQ Sil C18-3, 0.8 mm i.d. 3 3 mm) and an RP analytical column (HiQ Sil C18-3 Gradient, 0.15 mm i.d. 3 50 mm). The peptides was subjected to the trap column and then to the analytical column using a gradient of 0-50% solvent B in solvent A over 65 minutes [solvent A: 0.1% trifluoroacetic Acid (TFA), 2% acetonitrile; solvent B: 0.1%TFA, 70% acetonitrile] followed by 50-100% solvent B for 15 minutes at a flow rate 200 nL/minute. The RP column eluent was spotted onto a MALDI sample plate using a DiNa Direct Nano-flow LC/MALDI system (KYA Tech.) and analyzed using a 4800 mass spectrometer (Applied Biosystems Inc., Foster City, CA, USA). The peptides were fragmented under collision-induced dissociation conditions to give fragment ions that produced sequence information for the peptide. The software packages used for data acquisition and analysis included GPS explorer (Applied Biosystems Inc.) and Mascot (Matrix Science, Boston, MA, USA), respectively. The parameters of tolerance for the searches were set to 100 ppm for the MS and 0.2 Da for the MS/MS analyses.
Co-immunoprecipitation (Co-IP). HEK293T cells (8 3 10 5 ) were cotransfected with 0.5 mg of AMAP1 plasmids and 0.5 mg of IKKb plasmids using Effectene reagent (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. After 48 h, the cells were washed with PBS and lysed with Cell Lysis Buffer (CST). One milligram of the lysate was incubated with 1 mg of control IgG, anti-HA-tag or anti-FLAG antibody overnight at 4uC with agitation. Twenty-five microliters of protein G Sepharose (GE Healthcare, Uppsala, Sweden) was added, and the samples were rotated at 4uC for 1 h. The beads were washed three times with Cell Lysis Buffer and one time with Tris buffer pH 7.5. Proteins that were bound to the beads were eluted into SDS sample buffer, and the eluted material was analyzed by immunoblotting using anti-HA, anti-FLAG and anti-GST antibodies. Co-precipitation of endogenous AMAP1 with IKKb was performed using anti-AMAP1 and anti-IKKb antibodies.
Measurement of p65 activity. HEK293T cells were lysed with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) for nuclear and cytoplasmic extractions. Transcription factor kits were used to detect the active NF-kB in the nuclear extract for NF-kB p65 according to the manufacturer's protocol. Briefly, the biotinylated NF-kB consensus sequence was bound to streptavidin-coated ELISA wells. Because only the active form of NF-kB (p65) binds to the DNA sequence, nonspecific binding was minimized. The p65 that was bound to the consensus sequence was incubated with anti-p65 antibody and then with a secondary, HRPconjugated antibody. A chemiluminescent substrate was added to the wells, and the resulting signal was detected using a luminometer.
Sucrose gradient. Cells were treated with 5 ng/mL IL-1b or PBS for 30 min and lysed in cell lysis buffer (CST) containing 1 mM PMSF. An identical amount of protein from each sample was mixed with 90% sucrose in MES buffer (6 mL final volume, sucrose concentration 51.7-58.7%) and transferred to a Beckman ultracentrifuge tube. Four milliliters of 35% sucrose followed by 3 mL of 5% sucrose were overlaid, the samples were spun in a Beckman Coulter ultracentrifuge (39,000 rpm; approximately 180,000 3 g in a SW40Ti rotor, 20 h), and 26 fractions were collected from the top of the gradient. For the detection of the lipid raft fractions, all fractions were dot-blotted with HRP-labeled cholera toxin B (Sigma-Aldrich) to detect Ganglioside GM1.
Immunofluorescence. HUVECs were grown on a 4-well chamber slide (Thermo Scientific) and treated with or without IL-1b 2.5 ng/mL for 30 min. The cells were washed with PBS, fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. The cells were then incubated with blocking solution (5% goat normal serum in PBS) and labeled with anti-AMAP1 and anti-vimentin antibodies that were coupled to Alexa Fluor 488-and 584-conjugated secondary antibodies. The stained cells were mounted using Mounting Medium with DAPI (Vector Laboratories Inc., Burlingame, CA, USA). Images were captured and exported using a confocal microscope (Olympus FV1000; 603 oil-immersion lens, FLUOVIEW 3.0 software).
Statistical analysis. The data are presented as the mean of 6SD. Groups were compared using a two-tailed, Student's t-test. P , 0.05 was considered significant.  www.nature.com/scientificreports