13 research outputs found
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
Heterozygous mutation of <i>Rps7</i> results in visible white spotting, small body size, and tail kinking.
<p>(A) Montu (<i>Mtu</i>) heterozygous mice exhibiting a white belly spot and kinked tail were identified in an ENU mutagenesis screen. An independent ENU screen identified zuma (<i>Zma</i>) mice with similar phenotypes (data not shown). (B) Heterozygote <i>Mtu/+</i> male (blue) and female (green) mice have a significantly reduced body weight compared to wild-type male (gray) and female (red) littermates. (C) Sequencing revealed novel <i>Rps7</i> point mutations in <i>Mtu/+</i> (c.574T>G, encoding p.V156G) and <i>Zma/+</i> (c.637A>C, encoding p.Y177S) mice. Sequence traces shown are for the antisense strand. Detailed <i>Rps7</i> genomic structure information can be found at <a href="http://www.ncbi.nlm.nih.gov/gene/20115" target="_blank">http://www.ncbi.nlm.nih.gov/gene/20115</a>. (D) The predicted structural locations of mutated amino acids in <i>Rps7<sup>Mtu</sup></i> and <i>Rps7<sup>Zma</sup></i> alleles. The three-dimensional structures of RPS7 orthologs from <i>S. cerevisiae</i> (PDB ID 3U5C_H, red) and <i>T. thermophila</i> (PDB ID 2XZM_3, gray) are superimposed. The locations of the residues homologous to mouse p.V156 (*) and p.Y177 (arrow) are shown in green (<i>S. cerevisiae</i>) and in yellow (<i>T. thermophila</i>). Image generated with UCSF Chimera.</p
Peripheral-blood parameters appear normal with slight developmental delay in <i>Rps7<sup>Zma</sup>/+</i> fetal liver red cell precursors.
<p>(A) <i>Rps7<sup>Mtu</sup>/+</i> mice displayed similar complete blood count (CBC) values to their wild-type littermates with the exception of a very slightly elevated mean corpuscular volume (MCV) (* indicates p<0.05; N = 7). (B) <i>Rps7<sup>Zma</sup>/+</i> mice did not differ significantly from their wild-type littermates in any CBC measurements (N = 5). (C) A typical example of FACS analysis of fetal liver cells. The 5 characterized erythroid cell populations, R1–R5, are boxed in pink. (D) The average values and standard deviation for each fetal liver erythroid cell population are given as a percentage of total viable cells in Rps7+/+ and <i>Rps7<sup>Zma</sup>/+</i> samples (* indicates p<0.001; N = 6).</p
Brain size and behavioral abnormalities in <i>Rps7</i> mutants.
<p>(A) A Nissl stained coronal section of a 5 month old <i>Rps7<sup>Mtu</sup>/+</i>brain shows a thinner cortex and larger ventricles when compared to an <i>Rps7</i>+/+ littermate. (B, C) Dissected whole brains show that the cortex is reduced in size in adult <i>Rps7<sup>Mtu</sup>/+</i> (B) and postnatal day 0 <i>Rps7<sup>Zma</sup>/+</i>mice (C) when compared to <i>Rps7</i>+/+ littermates. (D–I) Magnetic Resonance Microscopy (MRM) was used to visualize brain development in late gestation (E18.5) <i>Rps7</i>+/+(D–F) and <i>Rps7<sup>Zma</sup>/+</i>(G–I) embryos. Enlarged ventricles (v in panel H) were apparent in all <i>Rps7<sup>Zma</sup>/+</i> samples (N = 3). Representative slices are shown in sagittal (D,G), coronal (E,H), and axial (F,I) views. Scale bars = 1 mm. (J) The volume of each brain region was quantitated as a percentage of total brain volume in <i>Rps7</i>+/+(black columns) and <i>Rps7<sup>Zma</sup>/+</i>(gray columns) (N = 3). Abbreviations: Olfactory bulbs (OB), lateral ventricles (LV), cortex (Ct), septum (Sp), striatum (St), 4<sup>th</sup> ventricle (4V), hippocampus (Hp), thalamus (Th), colliculi (Co), cerebellum (Ce). ** indicates p<0.005. (K) Assessment of working memory by measuring spontaneous alternation in a T-maze showed a significant difference between <i>Rps7<sup>Mtu</sup>/+</i>mice and <i>Rps7</i>+/+ littermate controls (* indicates P = 0.01, N = 9). In all panels +/+ = <i>Rps7</i>+/+; Mtu/+ = <i>Rps7<sup>Mtu</sup>/+</i>; Zma/+ = <i>Rps7<sup>Zma</sup>/+</i>.</p
Increased apoptosis occurs in the developing CNS of <i>Rps7<sup>Zma</sup>/+</i> mutant embryos; however, this apoptosis is reduced in <i>Rps7<sup>Zma</sup>/+</i>; <i>Trp53<sup>KO</sup>/+</i> embryos.
<p>(A–C) Increased apoptosis was observed in E11.5 <i>Rps7<sup>Zma</sup>/+</i> (Z/+) coronal sections through the neocortex compared to <i>Rps7</i>+/+ (+/+) and <i>Rps7<sup>Zma</sup>/+</i>; <i>Trp53<sup>KO</sup>/+</i> (Z/+;p53/+), as measured by cleaved caspase-3 (CC3) staining shown in green. (D–F) Apoptosis was also relatively increased in neural tube cross-sections of <i>Rps7<sup>Zma</sup>/+</i> embryos at E11.5 (E). (G–I) Cellular disorganization was apparent in mitotic, phospho-histone H3-positive (PH3+) cells surrounding the lumen of the neural tube in E11.5 <i>Rps7<sup>Zma</sup>/+</i> embryos (H). (J) E11.5 <i>Rps7<sup>Zma</sup>/+</i> (z) embryos showed no difference from <i>Rps7</i>+/+(+) or <i>Rps7<sup>Zma</sup>/+</i>; <i>Trp53<sup>KO</sup>/+</i>(p) in total counts of PH3+ cells surrounding the lumen of the neural tube. (K) CC3+ cell counts confirmed significantly increased apoptosis in E11.5 <i>Rps7<sup>Zma</sup>/+</i> (z) neural tube as compared to <i>Rps7</i>+/+ (+) or <i>Rps7<sup>Zma</sup>/+</i>; <i>Trp53<sup>KO</sup>/+</i> (p) (* indicates p<0.001). Scale bars: in A, D, G = 100 µM with equivalent magnification across all genotypes.</p