Figure 2: Folate decreases K81 acetylation and stabilizes MAT IIα.
From: Acetylation of MAT IIα represses tumour cell growth and is decreased in human hepatocellular cancer
(a) Folate deprivation increases K81 acetylation of MAT IIα and decreases its protein level. HEK293T cells were cultured in medium containing folate of different concentrations as indicated for 48 h. Western blotting was used to determine MAT IIα protein and K81 acetylation levels. (b) Folate-deprivation has no effect on MAT2A mRNA level. MAT2A mRNA was determined by qPCR and normalized against β-actin. Error bars represent ±s.d. of triplicate experiments. The two-tailed Student’s t-test was used. NS denotes no significance. (c) MG132 rescues the decrease in MAT IIα protein induced by folate deprivation. HEK293T cells were cultured as indicated and treated with either dimethylsulphoxide (DMSO; solvent) or 10 μM MG132 for 6 h. Cell lysates were analysed by western blotting. (d) Folate deprivation exclusively shortens the half-life of wild-type MAT IIα, but not K81R or K81Q mutant. HEK293 stable cell lines expressing wild-type MAT IIα, or K81R/K81Q mutants were cultured in folate-deprived condition for 48 h, CHX (10 μg ml-1) treatment was applied for different time course before harvest. MAT IIα protein levels were determined by western blotting (left panel). The right panel showcases relative protein amounts of different groups. Error bars represent ±s.d. of triplicate experiments. The two-tailed Student’s t-test was used. **P<0.01; ***P<0.001. (e) K81R mutation blocks MAT IIα ubiquitylation induced by TSA. HEK293T cells were transfected with the indicated plasmids. After TSA and MG132 treatment as indicated, ubiquitylation of purified flag-MAT IIα proteins were determined. (f) Folate deprivation promotes ubiquitylation of wild-type MAT IIα but not K81R mutant. HEK293T cells were transfected as indicated. Ubiquitylation of flag-MAT IIα protein was determined by western blotting. (g) MG132 stabilizes MAT IIα upon folate deprivation. HEK293T cells were cultured under normal or folate-deprived condition, followed by CHX and MG132 treatment. Cell lysates were directly subjected to western blotting (left panel). The right panel showcases relative protein amounts of different groups. Error bars represent ±s.d. of triplicate experiments. The two-tailed Student’s t-test was used. **P<0.01; ***P<0.001.
