Fig 1: AP2M1 and its phosphorylation are important for RABV infection. (A) HEK293 cells were transfected with siRNAs AP2M1 or NT. AP2M1 expression level was determined by Western blotting. ß-actin was used as a loading control. (B) mRNA levels of AP2M1 were determined by qRT-PCR in cells followed by siRNA transfection. A t-test was used for the statistical analysis. ****, p < 0.0001. Representative images of ERA-mCherry-infected (MOI = 0.01) HEK-293 cells (C) and SK cells (E) transfected with siRNAs targeting either AP2M1 or RABV-L, or the NT sequence. A high-content quantitative image-based analysis was used to measure the relative infection ratio (normalized to NT siRNA-treated cells) of RABV in either HEK-293 cells (D) or SK cells (F) transfected with the indicated siRNAs. Cell viability was measured at 48 h post-transfection by using the CellTiter-Glo reagent. Data are relative fluorescence values normalized to the level of the NT control. Values represent the mean ± SD. A one-way ANOVA was used for the statistical analysis. **, p < 0.01, ***, p < 0.001, ****, p < 0.0001; n.s., not significant. (G) HEK-293 cells were treated with various concentrations of sunitinib or erlotinib for 1 h at 37 °C before infection of ERA-mCherry at an MOI of 10, and then incubated for 1 h on ice to allow attachment of viral particles to cell surface receptors. Then the residual viruses were removed, and cell plasma membrane proteins were isolated to detect the phosphorylation of AP2M1 by Western blotting. (H) HEK-293 cells were transfected with pCAGGS-AP2M1(T156A) or pCAGGS. At 48 h post-transfection, the cells were infected with ERA-mCherry (MOI = 0.05), and virus titers were determined as focus forming units (FFU) in BSR-T7/5 cells. Multiple t-tests were used for the statistical analysis, *, p < 0.05. AP2M1-T156 overexpression was confirmed by Western blotting (right panel).
Fig 2: WNT Treatment Induces AAK1-, PIP2-Dependent Phosphorylation of AP2M1(A) Western blot analysis of HT1080 cells treated with WNT3A CM for indicated times.(B) Western blot analysis of HT1080 cells transfected with either AAK1 or control siRNA for 72 hr. Cells were then treated with rhWNT3A (200 ng/mL) for 15 min, then the medium was changed for fresh, complete DMEM, and cells were incubated for 9 hr.(C) Western blot analysis of pAP2M1 levels in HEK293T cells treated with either rhWNT3A (3A) or rhWNT5A (5A) (200 ng/mL) for 9 hr. The box-and-whisker plot represents pAP2M1/AP2M1 expression from four biological replicates quantified by LiCOR software.(D) Western blot analysis of HT1080 cells treated with either CHIR99021 compound (1 µM) or rhWNT3A (200 ng/mL) for the indicated time.(E) HEK293T cells were transfected with FLAG-dnTCF7L2 for 24 hr. Cells were then treated with WNT3A for 15 min, then the medium was changed for fresh, complete DMEM, incubated for 9 hr, and analyzed using western blot.(F) Western blot analysis of HEK293T WT, DVL TKO clone #4, DVL TKO clone #6, and LRP5/6 DKO cells treated with WNT3A CM for 9 hr.(G) Western blot analysis of HT1080 cells treated with WNT3A CM for 15 mins, then the medium was changed for complete DMEM containing either carbachol (50 µM) or neomycin (100 µM). Cells were incubated for 9 hr prior to cell harvest.(H) Western blot analysis of HT1080 cells transfected with siRNA against PI4K2a for 72 hr and pulsed with WNT3A CM for 15 mins and then incubated for 9 hr.All panels are representative of biological triplicates, unless otherwise noted. For complete statistics, see STAR Methods.See also Figure S4.
Fig 3: Proposed Model for AAK1-Dependent and CME of LRP6(A) Within minutes of WNT ligand exposure, FZD and LRP5/6 co-complex, allowing formation of the LRP6 signalosome in nascent clathrin-coated pits. Signalosome formation requires DVL domain swapping and polymerization, as well as PIP2 and AP2 recruitment. This promotes accumulation of ß-catenin and activation of the WNT signaling pathway.(B) Prolonged exposure to WNT ligand drives AAK1-dependent AP2M1 phosphorylation, CME, and ultimately removal of the WNT receptors from the plasma membrane.
Fig 4: SGC-AAK1-1 Is a Potent and Specific Inhibitor of AAK1 and BMP2K(A) Chemical structures of the AAK1/BMP2K chemical probe SGC-AAK-1, the related AAK1/BMP2K inhibitor 25A, and the negative control compound 34A that has a similar chemical structure but does not inhibit AAK1 or BMP2K.(B) SGC-AAK-1 selectively bound AAK1 and BMP2K with a more than 30-fold difference in Ki in a TR-FRET binding displacement assay over the related kinases GAK and STK16. Sixteen-point dose-response curves were measured in duplicate.(C) SGC-AAK1-1 has good selectivity over the human kinome. SGC-AAK1-1 was used at 1 µM concentration and tested against over 400 wild-type human kinases. KD determination was completed for all kinases that exhibited >85% inhibition and/or were observed as common off-targets for this structural class. Kinases for which SGC-AAK1-1 has a KD < 100 nM are marked with a large red circle and 100 nM < KD < 1.0 µM with a smaller red circle.(D) ITC confirmed a KD of 120 nM for binding of SGC-AAK1-1 to AAK1.(E) A co-crystal structure of BMP2K (2.41 Å) bound to 25A revealed the binding mode of the inhibitor in the ATP site. This structure revealed five key hydrogen bonds and revealed the orientation of the central hydrophobic portion of the inhibitor with respect to the binding pocket.(F and G) NanoBRET cellular target engagement assays in HEK293T cells showed that 25A (F) and SGC-AAK1-1 (G) both entered cells and directly bound to AAK1 and displaced a fluorescent tracer molecule from the ATP site. Cells were treated with serially diluted inhibitors in the presence of four different concentrations of a NanoBRET tracer molecule, and IC50 values were calculated from a linear interpolation of the IC50 values at each tracer concentration to obtain the predicted IC50 in the absence of tracer. Measurements were made with three technical replicates.(H) Binding of SGC-AAK1-1 to BMP2K in cells was weaker than to AAK1 with IC50 > 1 µM. Displacement of the same fluorescent tracer molecule required increased concentrations of SGC-AAK1-1, revealing an elevated IC50 value for binding to BMP2K versus AAK1 in the cellular target engagement assay.(I) SGC-AAK1-1 inhibited the phosphorylation of the AP2M1 subunit at Thr156 in HEK293T cells in a concentration-dependent manner or with 10 µM 34A. HEK293T cells were treated with inhibitors for 2 hr before western blot analysis.Error bars represent SE. See also Figure S1 and Tables S2, S3, S4, and S5.
Supplier Page from Abcam for Anti-AP2M1 (phospho T156) antibody [EPR4700]