Identification and functional analysis of SOX10 phosphorylation sites in melanoma

<div><p>The transcription factor SOX10 plays an important role in vertebrate neural crest development, including the establishment and maintenance of the melanocyte lineage. SOX10 is also highly expressed in melanoma tumors, and SOX10 expression increases with tumor progression. The suppression of SOX10 in melanoma cells activates TGF-β signaling and can promote resistance to BRAF and MEK inhibitors. Since resistance to BRAF/MEK inhibitors is seen in the majority of melanoma patients, there is an immediate need to assess the underlying biology that mediates resistance and to identify new targets for combinatorial therapeutic approaches. Previously, we demonstrated that SOX10 protein is required for tumor initiation, maintenance and survival. Here, we present data that support phosphorylation as a mechanism employed by melanoma cells to tightly regulate SOX10 expression. Mass spectrometry identified eight phosphorylation sites contained within SOX10, three of which (S24, S45 and T240) were selected for further analysis based on their location within predicted MAPK/CDK binding motifs. SOX10 mutations were generated at these phosphorylation sites to assess their impact on SOX10 protein function in melanoma cells, including transcriptional activation on target promoters, subcellular localization, and stability. These data further our understanding of SOX10 protein regulation and provide critical information for identification of molecular pathways that modulate SOX10 protein levels in melanoma, with the ultimate goal of discovering novel targets for more effective combinatorial therapeutic approaches for melanoma patients.</p></div

SOX10 post-translational modifications identified in MG132-treated 501mel cells.

<p>This schematic representing the SOX10 protein indicates known domains, SOXE conserved regions, and phosphorylated residues, as follows: black bars show known phosphorylation sites, green bars show known sites that were confirmed in this study, and red bars show novel sites from this study. The phosphorylated residues S224, S232, T240 and T244 were observed on numerous peptide fragments, and one or all four are plausible; their close proximity and the limited fragmentation capability in the digest restrict more precise determination among these residues. The nuclear localization and nuclear export signal regions are unaffected by the phosphorylation sites.</p

Mutation of SOX10 phosphorylation sites causes distinct changes in protein stability.

<p>Cycloheximide pulse-chase assays in 501mel (A-C) and MeWo (D-F) cells revealed altered stability of SOX10 phospho-mutants compared to WT SOX10 protein. A. WT SOX10 showed a half-life of 8.3 hours in 501mel cells. B,C. Stability of SOX10 phospho-mutants S24A and T240A is not significantly different from WT SOX10 in 501mel cells (two-way ANOVA, p = 0.25). D. WT SOX10 stabilty in MeWo cells exhibited a half-life of 19.5 hours. E. S24A SOX10 mutant protein showed reduced stability in MeWo cells with a half-life of 4.7 hours. F. T240A SOX10 mutant protein showed reduced stability in MeWo cells with a half-life of 11.7 hours. Both the S24A and the T240A mutant proteins exhibited significant differences relative to WT SOX10 protein in MeWo cells (two-way ANOVA, p = 0.0057 for protein type, p<0.0001 for time and interaction); by Bonferroni’s multiple comparisons post-test, these differences were significant for SOX10 S24A from 4 hours through 10 hours, and were significant for SOX10 T240A from 4 hours through 16 hours (P-values: *≤0.05, **≤0.01, ***≤0.001, ***≤0.0001). Data are compiled from 3 independent assays, with standard deviations plotted.</p

Mass spectrometry analysis identifies SOX10 phosphorylation sites.

<p>A. Workflow schematic of SOX10 protein analysis by mass spectrometry. 501mel cells were treated with MG132 proteasomal inhibitor before scraping cells and performing immunoprecipitation (IP) using SOX10 antibody to isolate protein. The eluted proteins were separated by SDS-page, followed by staining and removal of bands corresponding to 55kD, 75kD and 100kD. All three gel bands were subjected to destaining, in-gel digestion and extraction before running LC-MS/MS. B. Portions of IP samples were separated on SDS-page gel, followed by transfer onto PVDF membrane and Western blotting to confirm SOX10 isolation in the eluted samples being used for mass spectrometry. C. The three phosphorylation sites selected for mutation and characterization are shown in the context of full length SOX10 (Genbank ID NM_006941).</p

SOX10 phospho-mutants exhibit cell-specific differences in activation of the MITF promoter.

<p>A. Over-expression of WT and phospho-mutant SOX10 yields similar protein levels in HeLa cells at 48 hours on Western blot. SOX10 phospho-mutants show bands running at slightly different sizes; the SOX10 Sumo3x mutant is included as a control for protein band shifting that results from altering amino acid residues at sites of post-translational modifications. B,C. Representative luciferase data showing activation of p<i>MITF</i> from WT and SOX10 phospho-mutants in HeLa (B) and NIH3T3 cells (C); the S24A and T240A constructs showed significantly greater promoter activation in HeLa cells, while the S24A, S45A construct showed significantly greater promoter activation in NIH3T3 cells. Replicate data sets for p<i>MITF</i> can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190834#pone.0190834.s002" target="_blank">S2 Fig</a>. D. p<i>TYR</i> promoter luciferase data showed no significant differences between phospho-mutants and WT SOX10 in HeLa cells; representative dataset is shown. E. p<i>DCT</i> promoter luciferase data showed no significant differences between phospho-mutants and WT SOX10 in HeLa cells; representative dataset is shown. Statistics were calculated using one-way ANOVA with Bonferroni’s multiple comparison test, three independent assays per promoter construct.</p

SOX10 phosphorylation mutants retain nuclear localization.

<p>A,B. HeLa cells (A) and 501mel melanoma cells (B) were transfected with WT and phospho-mutant SOX10 constructs, and after 48 hours were fixed and stained to visualize subcellular localization of WT SOX10 and SOX10 phosphoryation mutant proteins. The Sumo3x SOX10 mutant was used as a post-translational modification control, as it is known to express in the nucleus despite mutations in all 3 sumoylation sites. No differences in localization are seen in the SOX10 phosphorylation mutants relative to WT SOX10. The V5 antibody (V5-488) stains exogenous SOX10 in both cell lines, while the SOX10 antibody (SOX10-568) stains both exogenous and endogenous SOX10 in 501mel cells (HeLa cells do not express endogenous SOX10).</p

LPP melanocytes are reduced in <i>Tg(DctSox10)</i> homozygotes during hair morphogenesis.

<p>(A) Brightfield images of hairs in <i>Tg(DctSox10)</i> and <i>+/+</i> littermates at P2. (B) Number of DCT⁺ melanocytes within the LPP of hairs at P2 (stage 6 hairs) and P7/8. At both time points, LPP melanocytes per hair are reduced in <i>Tg(DctSox10)/Tg(DctSox10)</i> compared to <i>Tg(DctSox10)/+</i> and <i>+/+</i> mice (*p<0.017). (C, D) Quantitative immunohistochemical analysis of stage 6 hairs from P2 skins for DCT and TRP1, or DCT and KIT. The population of DCT⁺/TRP1⁺ cells is significantly reduced in <i>Tg(DctSox10)/Tg(DctSox10)</i> in comparison to <i>Tg(DctSox10)/+</i> and +/+ mice (*p<0.008). <i>Tg(DctSox10)</i> also causes a switch in KIT intensity from KIT^hi in wild type to KIT^low in <i>Tg(DctSox10)</i> animals (*KIT^lo and **KIT^hi comparisons made between +/+ and <i>Tg(DctSox10)/+</i> or <i>+/+</i> and <i>Tg(DctSox10)/Tg(DctSox10)</i>; p<0.005).</p

<i>Sox10</i> is required by bulb melanocytes postnatally.

<p>(A–B) <i>Sox10^fl/fl</i> (<i>fl/fl; +/+</i>) and <i>Sox10^fl/fl; Tyr::CreERT2</i> (<i>fl/fl; Cre/+</i>) pups treated with TAM by IP injection to the lactating mother on P0–3 display variegated hypopigmentation on the belly and back and exhibit a white head spot upon the emergence of the morphogenetic coat (P10 shown here, n>5). (C) Number of PAX3⁺ melanocytes per hair bulb in skins harvested from these mice at P10 are significantly decreased in <i>Sox10^fl/fl; Tyr::CreERT2</i> animals compared to similarly-treated <i>Sox10^fl/fl</i> animals (*p = 0.002). (D–E) Adult <i>Sox10^fl/fl; Tyr::CreERT2</i> mice treated with TAM by IP injection on 0–3dpp exhibit white hairs within the plucked region upon hair regrowth that is not visible in similarly treated <i>Sox10^fl/fl</i> mice (brackets indicate plucked region, lower image is a magnification of plucked region). (F) Number of PAX3⁺ melanocytes per hair bulb in skins harvested from similarly-treated mice at 7dpp are significantly decreased in <i>Sox10^fl/fl; Tyr::CreERT2</i> animals compared to <i>Sox10^fl/fl</i> animals (*p = 0.001). (G–H) Fluorescent and corresponding brightfield images of hair bulbs from mice described in D–E. Arrows and arrowheads indicate PAX3⁺/SOX10⁺ and PAX3⁺/SOX10⁻ melanocytes, respectively. (I) Distribution of melanocytes double-labeled for PAX3 and SOX10 within pigmented (gray) and non-pigmented (white) hair bulbs in skins from <i>Sox10^fl/fl</i> (n = 3) and <i>Sox10^fl/fl; Tyr::CreERT2</i> (n = 4) harvested on 7dpp from mice treated with TAM on 0–3dpp (*p<0.006).</p

Overexpression of <i>Sox10</i> results in premature differentiation of LPP melanocytes in anagen hairs.

<p>(A) Number of DCT⁺ LPP melanocytes per anagen III/IV hair follicle (independent of the presence or absence of hair pigmentation) is significantly reduced in <i>Tg(DctSox10)/Tg(DctSox10)</i> mice when compared to wild type and <i>Tg(DctSox10)/+</i> mice (*p<0.0003). The ages of mice analyzed ranged between 9–22 weeks at harvest. (B) Eosin-stained skin sections of these hairs demonstrate the presence of ectopic pigmentation in the LPP of <i>Tg(DctSox10)/+</i> and <i>Tg(DctSox10)/Tg(DctSox10)</i> hairs (arrows) that is not see in wild type hairs. In <i>Tg(DctSox10)/+</i> LPP regions, this pigmentation often appeared in cells that were highly dendritic. (C, D) Brightfield and corresponding fluorescent images of anagen III/IV hair follicles double labeled for DCT and TRP1 (C) or KIT (D) in wild type and <i>Tg(DctSox10)/+</i> animals. The intensity of KIT fluorescence expression was variable, and categorized as KIT^lo (arrows) or KIT^hi (arrowheads), and did not appear to correlate with the presence or absence of pigmentation. (E,F) Comparison of the number LPP melanocytes per anagen III/IV hair follicle in <i>+/+</i> and <i>Tg(DctSox10)/+</i> animals that express DCT, and TRP1 or KIT, and produce ectopic pigmentation (*p<0.008).</p

<i>Tg(DctSox10)</i> results in congenital white spotting and premature hair graying.

<p>(A, B) Ventral and dorsal views demonstrating variable hypopigmentation in <i>Tg(DctSox10)/+</i> and <i>Tg(DctSox10)/Tg(DctSox10)</i> mice during hair morphogenesis and adult hair cycling. (C) Frequency of pigmented (pig+) and non-pigmented (pig−) anagen III/IV (7dpp) hairs that contain (DCT+ LPP cells) or do not contain (no LPP cells) LPP melanocytes within <i>Tg(DctSox10)</i> or <i>+/+</i> mice. The ages of mice analyzed ranged between 9–22 weeks at harvest. Significance determined by chi-square analysis (p<<0.0001) and evaluation of standardized residuals (*, z = −8.84; **, z = 12.24).</p