Wnt5a signaling induced phosphorylation increases APT1 activity and promotes melanoma metastatic behavior

Abstract

Wnt5a has been implicated in melanoma progression and metastasis, although the exact downstream signaling events that contribute to melanoma metastasis are poorly understood. Wnt5a signaling results in acyl protein thioesterase 1 (APT1) mediated depalmitoylation of pro-metastatic cell adhesion molecules CD44 and MCAM, resulting in increased melanoma invasion. The mechanistic details that underlie Wnt5a-mediated regulation of APT1 activity and cellular function remain unknown. Here, we show Wnt5a signaling regulates APT1 activity through induction of APT1 phosphorylation and we further investigate the functional role of APT1 phosphorylation on its depalmitoylating activity. We found phosphorylation increased APT1 depalmitoylating activity and reduced APT1 dimerization. We further determined APT1 phosphorylation increases melanoma invasion in vitro, and also correlated with increased tumor grade and metastasis. Our results further establish APT1 as an important regulator of melanoma invasion and metastatic behavior. Inhibition of APT1 may represent a novel way to treat Wnt5a driven cancers.

Keywords: Wnt signaling; Wnt5a; cancer biology; cell biology; cell lines; human; melanoma; palmitoylation.

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 observed and expected fragment ions of the given peptides. Specifically, blue lines represent matches with y-type fragments, red lines with b-type fragments, and purple lines show immonium ions. (B) Annotated MS/MS spectrum of identified S210 phosphopeptide of protein APT1, as described in A. (C) APT1^WT-CFP-FLAG was immunoprecipitated from lysates prepared from WM239A melanoma cells expressing APT1^WT-CFP-FLAG treated with control buffer or 150 ng/ml of purified Wnt5a for 15 min and analyzed by SDS-PAGE followed by immunoblotting to detect phosphorylated APT1 using antibodies to pS209-APT1 and pS210-APT1. (D) Western blot of whole cell lysate (WCL) from WM239A cells expressing APT1^WT-CFP-FLAG treated with control buffer or 150 ng/ml of Wnt5a for 15 min. (E) WM239A cells expressing APT1^WT-CFP-FLAG were treated with control buffer or 150 ng/ml of Wnt5a for increasing lengths of time and then APT1^WT-CFP-FLAG was immunoprecipitated and analyzed by SDS-PAGE and immunoblotting to detect phosphorylated APT1 with anti-p210-APT1 antibody. (F) WM239A cells expressing APT1^WT-CFP-FLAG (WT) or APT1^SA -CFP-FLAG (SA) were treated with control buffer or 150 ng/ml of Wnt5a for 15 min. APT1 was immunoprecipitated and analyzed by SDS-PAGE and immunoblotting to detect phosphorylated APT1 with anti-pS209-APT1 antibodies. (G) Three-dimensional model of APT1, showing the positions of Ser209 and Ser210 (magenta) adjacent to the catalytic residues Ser119, Asp174 and His208 (cyan).

Figure 2.. 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 APT1^WT, APT1^S209D or catalytically inactive APT1^S119A and fluorescence emission was measured over time (λ_ex490/9 nm; λ_em545/20 nm). Error bars indicate s.e.m., n = 3 replicates per condition. Results shown are from a representative trial from three independent experiments. (B) Linear regression of APT1^WT (WT) enzymatic activity measured by fluorescence emission of increasing DPP-3 substrate concentrations over time. Results are averaged from six independent experiments. (C) Linear regression of APT1^S209D (SD) enzymatic activity measured by fluorescence emission of increasing DPP-3 substrate concentrations over time. Results are averaged from six independent runs. (D) Quantification of relative fluorescence of WM239A cells treated with control buffer or 150 ng/ml of Wnt5a, loaded with 10 μM DPP-3, and then analyzed by live-cell fluorescence microscopy over time. Error bars indicate s.d., n = 25 cells per condition, ****p<0.0001 by unpaired t-test analysis. Results shown are from three experiments. (E) Quantification of relative fluorescence of WM239A cells expressing APT1^WT treated with control buffer, 150 ng/ml of Wnt5a, or 150 ng/ml of Wnt3a, loaded with 5 μM DPP-3, and then analyzed by live-cell fluorescence microscopy after 30 min. Error bars indicate s.d., n = 25 cells per condition, ****p<0.0001 by unpaired t-test analysis. Results shown are from three experiments. (F) Quantification of relative fluorescence generated by WM239A cells expressing either APT1^WT (WT), APT1^SA (SA), or APT1^S209D (SD) treated with control buffer or 150 ng/ml Wnt5a, loaded with 10 μM DPP-3, and then analyzed by live-cell fluorescence microscopy after 30 min. Error bars indicate s.d., n = 17–32 cells per condition, ****p<0.0001 by unpaired t-test analysis. Results shown are from three experiments. (G) Quantification of relative fluorescence of WM239A APT1^WT cells treated with kinases inhibitors 10 μM BI-D1870, or 0.2 μM staurosporine or DMSO control for 1 hr, loaded with 10 μM DPP-3, treated with Wnt5a and analyzed by live-cell fluorescence microscopy after 30 min. Error bars indicate s.d., n = 25 cells per condition, ****p<0.0001 by unpaired t-test analysis. Results shown are from three experiments.Quantification for all live-cell microscopy was determined by measuring the mean intensity of relative fluorescence for region of interests (background fluorescence was subtracted from mean intensity).

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 min, quantified in Figure 2D. (B) APT1^WT was immunoprecipitated from WM239A whole cell lysate pre-treated with control buffer or 150 ng/ml Wnt5a and analyzed by in vitro fluorescence assay. *p<0.05 by unpaired t-test analysis of – Wnt5a and +Wnt5 a groups. (C) WM239A APT1^WT cells were treated with control buffer, 75 nl/ml, or 150 ng/ml of recombinant Wnt3a for 1 hr, lysate was harvested and analyzed by SDS-PAGE to determine β-catenin stabilization. (D) Top panel: Representative images of APT1^WT, APT1^SA and APT1^S209D WM239A cells treated with 10 μM of DPP-3 for 15 min, and then analyzed by live-cell microscopy. Bottom panel: Quantification of relative fluorescence of WM239A APT1^WT (WT), APT1^SA (SA), and APT1^S209D (SD) cells treated with control buffer or 150 ng/ml Wnt5a, loaded with 10 μM DPP-3, and then analyzed by live-cell fluorescence microscopy after 30 min. Error bars indicate s.d., n = 17–32 cells per condition, ****p<0.0001 by unpaired t-test analysis.

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 APT1^WT-FLAG (WT), APT1^SA-FLAG (SA), or APT1^S209D-FLAG (SD). After 15 hr of induction with 1 μg/ml doxycycline, APT1 protein expression was determined by immunoblotting with anti-GFP (MCAM-GFP) and anti-FLAG (APT1-FLAG) antibodies. (B) An acyl biotin exchange (ABE) assay was used to measure MCAM palmitoylation in cell lysates from cell lines described in (A). In the ABE assay addition of hydroxylamine (+HAM) removes palmitate from cysteine residues that are then conjugated to biotin-HPDP. Biotinylated proteins are then isolated on streptavidin beads and palmitoylated proteins are analyzed by SDS-PAGE followed by immunoblotting. Hydroxylamine is withheld as a negative control (-HAM) MCAM-GFP is detected with anti-GFP antibodies and APT1-FLAG is detected with anti-FLAG antibodies. Results shown are a representative trial from three independent experiments. (C) WM239A melanoma cells expressing APT1^WT (WT), APT1^SA (SA), or APT1^S209D (SD) were grown on agarose to form spheroids that were embedded in collagen and images were taken on day seven and the distance invaded was measured. Representative images from day seven are shown. (D) Quantification of WM239A spheroid invasion assay in (C). Error bars indicate s.e.m., n = 14–34 spheroids counted per condition, ****p<0.0001 by unpaired t-test analysis. Results shown are from four experiments. (E) Quantification of WM239A spheroid invasion assay of APT1^S209D (SD) treated with DSMO control or 10 μM ML348, every other day for 7 days and distance invaded measured at day 7. Error bars indicate s.e.m., n = 6 spheroids counted per condition, **p=0.0017 by unpaired t-test analysis. Results shown are a representative trial from three independent experiments.

Figure 4.. APT1 phosphorylation impedes APT1 dimer formation.

(A) Differential scanning fluorimetry (DSF) of purified APT1^WT, APT1^SA and APT1^S209D. (B) Melting curves of purified APT1^WT, APT1^SA and APT1^S209D as determined by DSF. Results shown from DSF analysis are a representative trial from three independent experiments. (C) Left panel: Three-dimensional model of crystal structure of APT1 dimer interface. Ser209 and Ser210 (pink) are located 4.3 Å away from Met65 (blue) of the adjacent APT1 monomer. Catalytic triad Ser119, Asp174 and His208 are highlighted (cyan). Right panel: Zoom in of APT1 dimer interface. (D) Top panels: Chromatogram of purified APT1^WT (cyan), APT1^S209D (magenta), and molecular weight standards Ovalbumin (44kD) and Carbonic anhydrase (29kD) (black) from size-exclusion chromatography at three protein concentrations (Y-axis indicates the mAU of the APT1^SD). Bottom panel: SDS-PAGE followed by coomassie staining of eluted fractions after separation by size-exclusion chromatography of APT1^WT and APT1^S209D.

Figure 5.. 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 DPP-3 with either 50 nM purified APT1^WT, APT1^S210L APT1^S209D, or catalytically inactive APT1^S119A and fluorescence emission was measured (λ_ex490/9 nm; λ_em545/20 nm). Error bars indicate s.e.m., n = 3 replicates per condition. Results shown are a representative trial from three independent experiments. (C) Three-dimensional model of crystal structure of APT1 dimer interface with Ser210 to Leu mutation. Ser210Leu (pink) is located 5.5 Å away from Met65 (blue) of the adjacent APT1 monomer. Catalytic triad Ser119, Asp174 and His208 are shown in cyan. (D) Quantification of WM239A CFP-FLAG (CFP), APT1^WT-CFP-FLAG (WT), and APT1^S210L-CFP-FLAG (S210L) spheroids embedded in collagen and distance invaded measured at day 8. Error bars indicate s.e.m., n = 8 spheroids per condition, *p=0.0150 by unpaired t-test analysis. Results shown are from three independent experiments. (E) Immunohistochemistry staining of melanoma tumors in a human melanoma tumor array using pS209-APT1 and APT1 antibodies. (F) Immunohistochemistry staining of metastatic tumor samples in a human melanoma tumor array using anti-pS209-APT1.

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 (APT1^WT, APT1^S209D, APT1^S210L, or catalytically inactive APT1^S119A) for 30 min each in the presence or absence of 50 µM ML348 (λ_ex490/9 nm; λ_em545/20 nm). Error bars indicate s.d., n = 3 replicates per condition. Results shown are a representative trial from three experiments. (B) Representative images of APT1^WT and APT1^S210L collagen invasion assay in Figure 5D. Images are of spheroids on day 8 (D8). Scale bar = 500 µm. (C) Quantification of WM239A spheroid invasion assay of APT1^S210L (SL) treated with DSMO control or 10 μM ML348 every other day for 7 days and distance invaded measured on day 7. Error bars indicate s.e.m., n = 5–6 spheroids per condition, ****p<0.0001 by unpaired t-test analysis. Results are from three independent experiments. (D) Immunohistochemistry staining of metastatic tumors in a human melanoma tumor array using anti-APT1 antibodies.

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