FIG. 4.

p38 phosphorylates MEF2C and increases its transcriptional activity during muscle differentiation. (a) 10T1/2 cells were cotransfected with a Gal4-Luc reporter and expression vectors for wild type (wt) and mutant (mt) Gal4-MEF2 fusion proteins [MEF2A(A312/A319) and MEF2C(A293)] along with either an empty vector or an MKK6EE expression vector. Fold activation is the ratio of the luciferase activity in cells transfected with the activator (Gal4-MEF2) to that of cells transfected with the reporter but no activators. The data are representative of three independent experiments. SB, SB202190. (b) C2C12 cells were transfected with either wild-type or mutant Gal4-MEF2C vectors along with either an empty vector or an MKK6EE expression vector. After in vivo labeling with ³²P, Gal4-MEF2C was immunoprecipitated, separated by SDS-PAGE, and visualized by autoradiography. The level of Gal4-MEF2C expression was determined by immunoblotting (IB) with anti-Gal4 antibody (α-gal4). (c, upper panels) C2C12 cells kept in either GM or DM (2 days) were metabolically labeled with ³²P in the absence or presence of SB202190 as indicated. Endogenous MEF2C was immunoprecipitated, the immune complexes were separated by SDS-PAGE, and the MEF2C band was excised and analyzed by two-dimensional tryptic phosphopeptide mapping. (lower panels), M2-tagged wild-type MEF2C or mutant MEF2C(A293) were transfected into C2C12 cells and were kept in DM for 2 days before being subjected to metabolic labeling as described for panel a. Tagged MEF2C proteins were immunoprecipitated and subjected to two-dimensional mapping. o, the origin of the chromatogram. The phosphopeptide containing T293 is indicated by an arrow.