Wnt5a signaling induced phosphorylation increases APT1 activity and promotes melanoma metastatic behavior
- University of Pennsylvania, United States
- University of Chicago, United States
Figures
Figure 1
Wnt5a signaling induces APT1 phosphorylation on serines 209 and 210.
(A) Annotated MS/MS spectrum of identified S209 phosphopeptide of protein APT1. The image represents the observed fragment ions collected using MS/MS (HCD). Colored lines represent matches between …
Figure 2 with 1 supplement
Wnt5a stimulation increases APT1 depalmitoylating activity through phosphorylation.
(A) In vitro fluorescence assays using 5 μM of depalmitoylation probe DPP-3 in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1WT, APT1S209D or catalytically …
Figure 2—figure supplement 1
Activity of APT1 in response to Wnt5a stimulation or the phosphomimetic mutation.
(A) Representative images of WM239A cells loaded with 10 μM DPP-3, treated with control buffer or 150 ng/ml Wnt5a, and then analyzed by live-cell fluorescence microscopy at 15 min, 30 min, and 45 …
Figure 3
APT1 phosphorylation increases APT1 depalmitoylating activity in cells and increases melanoma invasion.
(A) HEK 293T cells transfected with constitutively transcribed MCAM-GFP and doxycycline inducible APT1WT-FLAG (WT), APT1SA-FLAG (SA), or APT1S209D-FLAG (SD). After 15 hr of induction with 1 μg/ml …
Figure 4
APT1 phosphorylation impedes APT1 dimer formation.
(A) Differential scanning fluorimetry (DSF) of purified APT1WT, APT1SA and APT1S209D. (B) Melting curves of purified APT1WT, APT1SA and APT1S209D as determined by DSF. Results shown from DSF …
Figure 5 with 1 supplement
Increased phospho-APT1 staining correlates with increased melanoma tumor grade and metastasis.
(A) Schematic of APT1 mutations from human tumor samples. Adapted from results generated using cBioPortal (Gao et al., 2013 and Cerami et al., 2012). (B) In vitro fluorescence assays of 5 μM of …
Figure 5—figure supplement 1
Response of the APT1 S210L mutant to the APT1 inhibitor ML348.
(A) Quantification of fluorescence response of 5 μM DPP-3 treated with either 50 nM purified APT1 (APT1WT, APT1S209D, APT1S210L, or catalytically inactive APT1S119A) for 30 min each in the presence …
Author response image 1
Author response image 2
Tables
Table 1
Enzyme kinetics of APT1WT and APT1S209D.
Table of initial velocities (V0) of APT1WT and APT1S209D. Values were determined by incubating purified APT1WT and APT1S209D with increasing concentrations of DPP-3 substrates and measuring …
| Fluoresence/Time (V0) | ||
|---|---|---|
| Substrate | APT1WT | APT1SD |
| 0.25μM | 0.2012 ± 0.008627 | 0.4704 ± 0.01765 |
| 0.5 µM | 0.2316 ± 0.02656 | 0.8083 ± 0.03173 |
| 1.0 µM | 0.5496 ± 0.01478 | 1.612 ± 0.04416 |
| 5.0 µM | 0.6573 ± 0.03547 | 4.627 ± 0.2237 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.34362.010
