![Figure 5. p38-γ directly phosphorylates Ser199 and Ser200 within the C terminus of MyoD. (A) IP-kinase assays from 10T1/2 fibroblasts expressing MKK6EE and p38-γ, or empty vector. Extracts were immunoprecipitated with antibody reactive to the Myc tag on p38-γ, and incubated with the recombinant substrates GST–MyoD–N terminal (GST-MyoD-NT) and GST–MyoD–C terminal (GST-MyoD-CT), along with GST (negative) and GST-ATF2 (positive) as controls. (B) MALDI-TOF MS spectra of IP-kinase assays from 10T1/2 fibroblasts expressing empty vector or MKK6EE and p38-γ. The peak with an m/z ratio of 1,255.69 (indicated by the asterisks) represents the phosphorylated MyoD peptide. (C) IP-kinase assay from 10T1/2 fibroblasts expressing MKK6EE and p38-γ, or empty vector. Myc–p38-γ immunoprecipitates were incubated with the recombinant substrates GST-MyoD-CT or GST-MyoD-CT [S199A/S200A] containing mutated p38-γ phosphorylation sites. GST and GST-ATF2 were used as controls. (D) Immunoblot analysis of 293T extracts expressing MKK6EE and p38-γ, or empty vector. Wild type (wt) and mutant (mt) MyoD S199A/S200A were expressed as indicated. (E) LC-MS/MS analysis of 293T nuclear extracts expressing MyoD alone or together with MKK6EE and p38-γ. For relative abundance calculations, indicated MyoD peptides were normalized against additional MyoD peptides and α-actin peptides. Representative data are shown. (F) Immunoblot analysis of S200-phosphorylated MyoD and total MyoD in proliferating p38-γ+/+ and p38-γ−/− satellite cell–derived myoblasts. Tubulin was used as a loading control. Fold change is shown relative to p38-γ+/+ satellite cells after quantification and normalization to tubulin. Error bars represent ±SEM for n = 3. Asterisk denotes significance (P < 0.004). CT, C terminus; NT, N terminus.](https://cdn.rupress.org/rup/content_public/journal/jcb/187/7/10.1083_jcb.200907037/6/jcb_200907037r_gs_fig5.jpeg?Expires=1767688221&Signature=gDCT12csakm5UZnANeJSYYFmCtN~IyPxgyL0cPFHBqydNsMo2ulfH7d316-qeMi9JP9Hd5aI0vhMEIC839sh-W~vDvSawrFSP7~xm3i2CbvnXeHSuR86TXECV~KBiWpsqa4rze-urtMGMHTgRqHFd8Djh5y43nVCnAYg4bWFcsLJ8NtPil464T9XuCM9~eBTLkxabGejHNXW5~tL8AYq3HgGPcdG8hYucKmPm6FG-2VlMCyzBldSaB9pYy1uk4chmeh2LA9zwPXMrmKrom3JwX5BBrWMfA093jTVt4zXYpOux312UftqVMocIKsYmLS0iElAJvtXD-J4ZYXPq1~1-Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
p38-γ directly phosphorylates Ser199 and Ser200 within the C terminus of MyoD. (A) IP-kinase assays from 10T1/2 fibroblasts expressing MKK6EE and p38-γ, or empty vector. Extracts were immunoprecipitated with antibody reactive to the Myc tag on p38-γ, and incubated with the recombinant substrates GST–MyoD–N terminal (GST-MyoD-NT) and GST–MyoD–C terminal (GST-MyoD-CT), along with GST (negative) and GST-ATF2 (positive) as controls. (B) MALDI-TOF MS spectra of IP-kinase assays from 10T1/2 fibroblasts expressing empty vector or MKK6EE and p38-γ. The peak with an m/z ratio of 1,255.69 (indicated by the asterisks) represents the phosphorylated MyoD peptide. (C) IP-kinase assay from 10T1/2 fibroblasts expressing MKK6EE and p38-γ, or empty vector. Myc–p38-γ immunoprecipitates were incubated with the recombinant substrates GST-MyoD-CT or GST-MyoD-CT [S199A/S200A] containing mutated p38-γ phosphorylation sites. GST and GST-ATF2 were used as controls. (D) Immunoblot analysis of 293T extracts expressing MKK6EE and p38-γ, or empty vector. Wild type (wt) and mutant (mt) MyoD S199A/S200A were expressed as indicated. (E) LC-MS/MS analysis of 293T nuclear extracts expressing MyoD alone or together with MKK6EE and p38-γ. For relative abundance calculations, indicated MyoD peptides were normalized against additional MyoD peptides and α-actin peptides. Representative data are shown. (F) Immunoblot analysis of S200-phosphorylated MyoD and total MyoD in proliferating p38-γ+/+ and p38-γ−/− satellite cell–derived myoblasts. Tubulin was used as a loading control. Fold change is shown relative to p38-γ+/+ satellite cells after quantification and normalization to tubulin. Error bars represent ±SEM for n = 3. Asterisk denotes significance (P < 0.004). CT, C terminus; NT, N terminus.
