Fig. 7: Phosphorylation of SOX4 by PKA facilitates its nuclear translocation and enhances the transcription of EBF2.

A BAT SVF cells were treated with or without induction medium for 1 h. Then cells were fixed and subjected into immunostaining by using anti-SOX4 antibody. Scale bar, 20 μm. B Control and 3X HA-SOX4 expressing BAT SVF cells were treated with or without induction medium for 1 h. Cells were harvested and subjected into chromatin immunoprecipitation by using anti-HA antibody. ChIP-Seq analysis showed binding profiles of SOX4 on the promoter of Ebf2 and de novo motif analysis of SOX4 binding sites. C BAT SVF cells were treated with DMSO, forskolin (20 μM) or H89 (30 μM) prior to stimulation with forskolin (20 μM) for 60 min. After 1 hour of stimulation, we performed immunofluorescence assay to detect the cytoplasmic-nuclear distribution of SOX4 protein. Scale bar, 30 μm. D ChIP-qPCR showing Sox4 occupancy at the promoter of Ebf2 in BAT SVF treated without or with forskolin (20 μM) for 1 h (n = 3). E BAT SVF cells isolated from 3-week-old Sox4-MKO and control littermates were cultured to confluence. Cells were treated with or without forskolin (20 μM) for 1 h and then harvested for RT-qPCR analysis (n = 3). 18S was used as an invariant control. The mRNA levels in control cells were normalized to 1.0. F Sox4^f/f and Sox4-MKO mice (8-week-old male, n = 4) were housed at RT or 4 °C for 4 h. Subsequently, mice were dissected, and BAT was collected. BAT SVFs were isolated from BAT for qPCR analysis of Ebf2. G BAT SVF reaching indicated confluence were subjected to different supplement (PBS, forskolin or H89) as described in (C). Transcriptional activity of the Ebf2 enhancer in NIH3T3 were analyzed by luciferase reporter assay (n = 3). H Mass spectrometry analysis of phosphorylation sites on SOX4. HEK293T cells were transfected with WT-PRKACA (catalytic subunit of PKA) or a kinase-dead mutant of PKA (KD-PRKACA) together with HA-SOX4 for 48 h. The cells were lysed and subjected to immunoprecipitation (IP) against HA, then the pellet was separated on SDS-PAGE and subjected to mass spectrometry analysis. I Alignment of SOX4 residues S325, along with the flanking amino acid residues from different species. HEK293T cells were transfected with WT or S235A mutant of SOX4. After 48 hours, cells were harvested and analyzed by western blot (n = 3). J, K NIH3T3 were transfected with Flag-SOX4 (WT) or S235A mutant. 36 h later, cells were treated with DMSO or forskolin (20 μM) for 1 h. And then cells were fixed for immunofluorescence assay (J) (Scale bar, 30 μm) or collected for qPCR analysis (K) (n = 3). L The illustration showed that forskolin induces PKA activation, leading to the phosphorylation of SOX4 at S235 and its subsequent translocation into the nucleus. Once in the nucleus, SOX4 cooperates with EBF2 to activate transcription of EBF2. Asterisks (*) denote the level of statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. Data are presented as mean ± SEM. Statistical analyses were determined by unpaired two-tailed Student’s t-test (D, F), one-way ANOVA followed by Tukey’s test (G) and two-way ANOVA followed by Tukey’s test (E, K).