Fig. 3
ERK2 directly phosphorylates SOX10 at T240 and T244. a Sequence alignment of the putative ERK phosphorylation motifs in SOX10 from different species (top) and among SOXE family proteins (bottom). The putative phosphorylation motifs were highlighted in box and phosphorylation sites (T) are shown in bold. The consensus ERK phosphorylation motif is shown below the sequences. b In vivo detection of SOX10 phosphorylation at T240 and T244. HA-SOX10 proteins were immunoprecipitated from A375-TR HA-SOX10 cell lysates, digested by trypsin and analyzed by multiple reactions monitoring (MRM) mass spectrometry. MRM spectra of non-phosphorylated and T240/T244-phosphorylated SOX10 tryptic fragments are shown. c Quantitation of SOX10 phosphorylation in A375 cells treated with or without 2 μM Vemurafenib for 6 h. The areas of the peptide peaks in MRM chromatograms were measured to estimate the relative quantities of corresponding peptides. A SOX10 tryptic fragment (183 AAQGEAECPGGEAEQGGTAAIQAHYK 208, 848.7188+++) was used as the internal control. d In vitro kinase assays were performed using recombinant ERK2 (activated) and synthetic SOX10 peptides (236-HGPPTPPTTPKTELQ-250) and the reaction products were analyzed by LC–MS. The HPLC results for SOX10 peptide variants including WT, T240A, T244A, and AA are shown. Different peptide species detected in the reaction products were designated by arrows with their molecular weights and identities shown beside. Mass spectrometry results were included in the supplemental information (Supplementary Fig. 3). e WT, T240A, T244A, AA HA-SOX10 proteins were precipitated from lysates of lentivirus-transduced A375 cells treated with or without 1 μM SCH772984 and probed with anti-HA or anti-phospho-PXTP antibodies. Uncropped images are shown in Supplementary Fig. 10. f 293T cells were transduced with WT or AA HA-SOX10 constructs along with empty vector or V600E BRAF constructs, ±AZD6244 treatment. Cells were lysed and HA-SOX10 were immunoprecipiated and probed with indicated antibodies. Uncropped images are shown in Supplementary Fig. 11
