PMC

Sumoylation of MEF2C inhibits its transcriptional activity. (A) Gal4, Gal4-MEF2C-WT, or Gal4-MEF2C-K391R construct was co-transfected with GAL4×5-luciferase reporter and pRL-tk reporter into HeLa cells. Firefly luciferase activities were measured and normalized for transfection efficiency by using Renilla luciferase activities. (B) Gal4-MEF2C-WT or Gal4-MEF2C-K391R construct was co-transfected with GAL4×5-luciferase reporter, pRL-tk reporter, and SENP2, DN-UBC9-Flag, or vector construct into HeLa cells. Firefly luciferase activities were measured and normalized for transfection efficiency by using Renilla luciferase activities and then divided by the luciferase activities of Gal4-MEF2C-WT or Gal4-MEF2C-K391R, respectively, to show the fold differences. (C) Gal4, Gal4-MEF2C-WT, or Gal4-MEF2C-K391R construct was co-transfected with LexA×8-GAL4×5-luciferase reporter, pRL-tk reporter, and LexA-VP16 construct into HeLa cells. Firefly luciferase activities were measured and normalized for transfection efficiency by using Renilla luciferase activities.

Phosphorylation of MEF2C at S396 and sumoylation of MEF2C at K391 repress transcription through the same pathway. (A) Three possible models with respect to the relationship between phosphorylation and sumoylation of MEF2C. (B) GAL4×5-luciferase reporter and pRL-tk reporter constructs were co-transfected with various Gal4-MEF2C constructs into HeLa cells. Firefly luciferase activities were measured and normalized for transfection efficiency by using Renilla luciferase activities. (C) LexA×8-GAL4×5-luciferase reporter, pRL-tk reporter, and LexA-VP16 constructs were cotransfected with various Gal4-MEF2C constructs into HeLa cells. Firefly luciferase activities were measured and normalized for transfection efficiency by using Renilla luciferase activities.

MEF2 proteins are sumoylated. (A) The ³⁵S-labeled MEF2C protein obtained through in vitro transcription and translation was incubated with SUMO reaction mixtures and analyzed by SDS-PAGE followed by autoradiography. (B) Sequence alignment of the MEF2 family of proteins. The lysine residue of a sumoylation consensus motif and a serine residue that is phosphorylated are shown in bold. (C) The ³⁵S-labeled wild-type (WT) and K391R mutant of MEF2C were incubated with SUMO reaction mixtures and analyzed by SDS-PAGE followed by autoradiography. (D) HeLa cells were transfected with the indicated plasmids. Myc-MEF2C was immunoprecipitated with anti-Myc. Cell lysates and Myc IP were resolved by SDS-PAGE and blotted with anti-Myc or anti-GFP. (E) HeLa cells were transfected with the indicated plasmids. Cell lysates were resolved by SDS-PAGE and blotted with anti-Myc. (F) HeLa cells were transfected with the indicated plasmids. Cell lysates were resolved by SDS-PAGE and blotted with anti-Myc.

A small C-terminal fragment of MEF2C is sufficient to repress transcription in a sumoylation-dependent manner. (A) Schematic drawing of the functional domains and two C-terminal fragments of MEF2C. (B) Gal4-MEF2C-WT, Gal4-MEF2C-K391R, Gal4-MEF2C-ΔN1, or Gal4-MEF2C-ΔN1-K391R construct was co-transfected with GAL4×5-luciferase reporter and pRL-tk reporter constructs into HeLa cells. Firefly luciferase activities were measured and normalized for transfection efficiency by using Renilla luciferase activities. (C) Gal4, Gal4-MEF2C-ΔN2, or Gal4-MEF2C-ΔN2-K391R construct was cotransfected with LexA×8-GAL4×5-luciferase reporter, pRL-tk reporter, and LexA-VP16 constructs into HeLa cells. Firefly luciferase activities were measured and normalized for transfection efficiency by using Renilla luciferase activities. (D) HeLa cells were transfected with the indicated plasmids. The total cell lysates were resolved by SDS-PAGE and blotted with anti-MEF2C. The asterisk indicates the endogenous MEF2 proteins.

MEF2C-S396A is less efficiently sumoylated in vitro and in vivo. (A) The recombinant GST-MEF2C-ΔN2-WT or GST-MEF2C-ΔN2-S396A proteins were subjected to CDK1 kinase assays and analyzed by SDS-PAGE followed by autoradiography. (B) 0.75 μg of GST-MEF2C-ΔN2-WT, S396A, or S396E proteins were incubated with 1 μg of E1, 0.5 μg of E2, and 1.25 μg of His₆-SUMO1 for the indicated time and stopped by SDS-PAGE sample buffer. The samples were resolved by SDS-PAGE and blotted with anti-MEF2C. (C) 0.3 μg of GST-MEF2C-ΔN2-WT, S396A, or S396E proteins were subjected to CDK1 kinase assays and then incubated with 0.2 μg of E1, 0.1 μg of E2, and 0.3 μg of His₆-SUMO1 for 15 min and stopped by SDS-PAGE sample buffer. The samples were resolved by SDS-PAGE and blotted with anti-MEF2C. (D) 10T1/2 cells were transfected with the indicated plasmids. Cell lysates were resolved by SDS-PAGE and blotted with anti-MEF2C. (E) Sequence alignment of the sumoylation sites of several SUMO substrates. Conserved amino acids were shown in bold.

Sumoylation-deficient mutant of MEF2C promotes myogenic conversion more efficiently. (A) MEF2C-WT- or MEF2C-K391R-expressing plasmids were co-transfected with MEF2×3-luciferase reporter, pRL-tk reporter, and GFP-SUMO1 or empty vector plasmids into HeLa cells. Firefly luciferase activities were measured and normalized for transfection efficiency by using Renilla luciferase activities. (B) MEF2×3-luciferase reporter, pRL-tkreporter, and MKK6-DD plasmids were co-transfected with GFP-SUMO1, SENP2, or empty vector plasmids into C2C12 cells. The cells were cultured in differentiation medium for 2 days. Firefly luciferase activities were measured and normalized for transfection efficiency by using Renilla luciferase activities. (C) Vector, MEF2C-WT-, MEF2C-K391R-, or GFP-SUMO1-expressing constructs were co-transfected with Myc-MyoD into 10T1/2 cells. The cells were cultured in differentiation medium for 5 days, fixed, and stained with DAPI (blue) and an anti-myosin heavy chain (MHC) monoclonal antibody (red). (D) MHC-positive cells were scored by random selection of 20 optical fields of cells in (C). The results of two independent experiments were averaged with the standard deviation indicated. P value was calculated using student t test. Cell lysates were resolved by SDS-PAGE and blotted with anti-MEF2C or Myc.