Phosphorylation Modulates the Subcellular Localization of SOX11

Abstract

SOX11 is a key Transcription Factor (TF) in the regulation of embryonic and adult neurogenesis, whose mutation has recently been linked to an intellectual disability syndrome in humans. SOX11's transient activity during neurogenesis is critical to ensure the precise execution of the neurogenic program. Here, we report that SOX11 displays differential subcellular localizations during the course of neurogenesis. Western-Blot analysis of embryonic mouse brain lysates indicated that SOX11 is post-translationally modified by phosphorylation. Using Mass Spectrometry, we found 10 serine residues in the SOX11 protein that are putatively phosphorylated. Systematic analysis of phospho-mutant SOX11 resulted in the identification of the S30 residue, whose phosphorylation promotes nuclear over cytoplasmic localization of SOX11. Collectively, these findings uncover phosphorylation as a novel layer of regulation of the intellectual disability gene Sox11.

Keywords: SOX11; cancer; intellectual disability; neurogenesis; subcellular localization; transcription factor phosphorylation.

Figure 1

SOX11’s subcellular localization in embryonic neurogenesis. Representative line intensity plots of cells from E11.5 (A), E13.5 (B), E15.5 (C), E18.5 (D) and P0 (E). The blue line represents intensity of DAPI, the red line represents intensity of SOX11. Coronal sections of brains derived from E11.5 (A′), E13.5 (B′), E15.5 (C′), E18.5 (D′) and P0 (E′) mice were stained for SOX11 (RED) and DAPI (blue). Arrows: cells with nuclear and cytoplasmic localization of SOX11; arrow heads: cells with exclusively nuclear localization of SOX11. Note the almost exclusive nuclear localization of SOX11 at E15.5. Scale bars: 20 μm. Graphs represent the percentage of cells with nuclear localization or nuclear and cytoplasmic localization of SOX11 over all the SOX11 positive cells counted from E11.5 (A″), E13.5 (B″), E15.5 (C″), E18.5 (D″) and P0 (E″). The data are presented as mean ± SEM.

Figure 2

SOX11’s subcellular localization in adult neurogenesis. Immunofluorescent analysis of SOX11’s subcellular localization in the dentate gyrus (DG; A), the subventricular zone (SVZ; B), the rostral migratory stream (RMS; C) and the olfactory bulb (OB; D). SOX11 (red), DCX (green), DAPI (blue). Scale bars: 20 μm.

Figure 3

SOX11 is phosphorylated in vivo and has at least 10 phospho-serines in vitro. (A) Western Blot analysis of nuclear (N) and cytoplasmic (C) extracts treated with Phospho Stop (PS) or lambda phosphatase (λPP) from E15.5 and E18.5 WT mice (wt) and Sox11 knockout mice (ko). First row: blotting with an anti-SOX11 antibody. Second row: blotting with an anti-pCREB (S133) antibody verifies the functionality of λPP treatment and the enrichment of nuclear proteins. Third row: blotting with an anti-GAPDH validates the enrichment of cytoplasmic proteins. Fourth row: blotting with an anti-pRNA polymerase II validates enrichment of nuclear proteins and functionality of λPP. E18.5, knock out for Sox11, brain extracts (E18.5ko) validates that the bands in the WT brains are specific for SOX11. Note the different band pattern of the SOX11 signal between E15.5 nuclear extracts treated with PS and λPP. The SOX11 band pattern also appears to be changed by phosphatase treatment in E15.5 and E18.5 cytoplasmic extracts. (B) Identification of SOX11 phosphorylation sites by mass spectrometry. The table reports the SOX11 site, the peptide sequence, ion score and the phosphoRS metanalysis to identify the exact sites within the peptide sequence based on the MS/MS spectra. Peptides with highest site probabilities/ion scores have been selected from Supplementary Table S2 and contain peptides from both cell lines (N2A and HEK293T). All phosphopeptides were identified in both lines: (1) peptide with the highest peptide score contained a deubitquitination on Q(11)/Q(2) indicated by a small q; (2) peptides contain one missed cleavage. (C) Schematic representation of the putative phosphorylated serines on SOX11 protein. (D) Schematic representation of SOX11 mutants in which the serine residues that have been replaced by Alanine (NON-phosphorylatable amino acid) are marked with blue while the residues replaced by Aspartate (amino acid that MIMICS phosphorylation) are marked with red.

Figure 4

Conservation of the phosphorylatable serines. Alignment of SOX11 amino acid sequence from different species shows the conservation of the phosphorylatable serines (A). Alignment of murine amino acid sequences of SOX11 (row1), SOX4 (row2) and SOX12 (row3) shows the conservation of the phosphorylatable serines among the SoxC family (B). Black boxes indicate the regions of interest. Red letters indicate the occurrence of conservation. Note that the phosphorylatable serines surrounding the HMG-box (S30, S133, S137) are unique to SOX11.

Figure 5

SOX11’s non-phosphorylatable form leads to nuclear localization. (A–G) Representative line intensity plots of HEK293T cells transfected with SOX11 wildtype (WT) and mutants. The blue line represents DAPI intensity, the red line represents SOX11 intensity. (A′–G′) Immunostaining for SOX11 (red) and DAPI (blue) of cells transfected with SOX11 WT and mutants were to determine the subcellular localization of SOX11 phospho-mutants. Note that the C3-Sox11p^NON (B′) and C3-Sox11p^N3W7 (D′) are almost exclusively localized in the nucleus. Arrow: cell with nuclear and cytoplasmic localization of SOX11. Arrow head: cell with only nuclear localization of SOX11. Scale bars: 20 μm. (A″–G″) Percentage of cells with nuclear localization (N) or nuclear and cytoplasmic (N + C) localization of the different SOX11 mutants.

Figure 6

SOX11’s subcellular localization depends on the phosphorylation of S30. (A–E) Representative line intensity plots of HEK293T cells transfected with SOX11 WT and mutants. The blue line represents DAPI intensity, the red line represents SOX11 intensity. (A’–E’) Immunostaining for SOX11 (red) and DAPI (blue) of cells transfected with SOX11 WT and mutants were to determine the subcellular localization of SOX11 phospho-mutants. Note that the C3-Sox11p^N1W9 is almost exclusively localized in the nucleus (B’). Arrow: cell with nuclear and cytoplasmic localization of SOX11. Arrow head: cell with only nuclear localization of SOX11. Scale bars: 20 μm. (A″–E″) Percentage of cells with nuclear localization (N) or nuclear and cytoplasmic (N + C) localization of the different SOX11 mutants. (F) Western Blot analysis of nuclear (N) and cytoplasmic (C) extracts of Neuro2a cells overexpressing C3-Sox11p^Wt (WT), C3-Sox11p^N1W9 (N1W9), C3-Sox11p^M1W9 (M1W9), C3-Sox11p^W1N2W7 (W1N2W7) and C3-Sox11p^W1M2W7 (W1M2W7). First row: blotting with an anti-SOX11 antibody; Second row: Blotting with an anti-pCREB (S133) antibody validates enrichment of nuclear proteins; third row: Blotting with an anti-αTUBULIN validates enrichment of cytoplasmic proteins. Note that the N1W9 mutant is exclusively detected in the nuclear fraction. (G) Activity of a luciferase reporter driven by a minimal SOX11 responsive promoter in HEK293T cells overexpressing C3 (empty backbone), C3-Sox11p^Wt, C3-Sox11p^N1W9 and C3-Sox11p^M1W9, n = 3 (***p < 0.001).