Figure 8. Phosphorylation of PAX3 at Ser205 is critical for stimulation of muscle cell migration.
(A) Exchange of serine by alanine in PAX3 at Ser205 reduces C2C12 myoblast migration after transfection with CA Braf (V600E). Cultures were either transfected with a control vector (-), with wildtype Pax3 (WT) or with mutated Pax3 constructs. Mutated serine residues are indicated (S201A, S205A, S209A, S222A). Cell numbers were determined by counting the number of DAPI-stained nuclei. n = 10; Mann-Whitney-U test, (p*<0.05). (B) Phosphorylated PAX3 proteins were isolated from Pax3 and mutant Braf (V600E) transfected HEK293T or C2C12 cells by phospho-column affinity purification and detected by immunoblotting. Phosphorylated ERK1/2 (P-ERK1/2), ERK2 and EZRIN served as controls. Two sets of independent experiments are shown for HEK293T cells. Molecular sizes (in kDa) are indicated by the overlay of the colored marker with the PAX3 and BRAF bands visualized by chemiluminescence. Note that PAX3 shows a shift in the molecular weight due to the HA-tag. Stained membranes with marker are shown. (C) In vitro kinase assay of GST, GST-PAX3, PAX3 and different PAX3 mutants demonstrating phosphorylation of PAX3 by BRAF. Bacterially produced GST-PAX3 (WT and mutants) was purified by GST-affinity chromatography. Column eluates and inputs were incubated in vitro together with BRAF. n = 2. Phosphorylated PAX3 was detected using an antibody specific for p-Ser205 PAX3. Specificity of the p-Ser205 PAX3 antibody was assessed by the failure to detect the S205A PAX3 mutant (bottom panel).