Figure 2. E318K prevents MITF sumoylation and results in differential expression of MITF target genes.
a, His-tagged wild-type MITF or the indicated single or double point mutants were co-transfected with HA–SUMO1 in COS-7 cells or b, HA–SUMO was transfected alone into homozygous mutant E318K MITF melanoma cells (NAE). Single- and double-sumoylated forms of MITF are indicated by a dagger and double dagger, respectively. The doublet bands are caused by MAPK-mediated phosphorylation at serine 73 (ref. 30). c, UACC62 human melanoma cells were transfected with TRPM1-promoter constructs with indicated amounts of expression vector encoding wild-type or mutant forms of MITF. Fold induction is shown as the ratio to the average of no MITF transfection (0 ng). Data are mean ± s.d. of at least four independent experiments. d, Expression of MITF in two melanoma cell lines (HT144 and C32) engineered to inducibly express wild-type (WT) or mutant (E318K) MITF after treatment with tetracycline for 48 h (48), as determined by qRT–PCR. Performed in triplicate, error bars depict s.d. e, Expression of MITF target genes DCT (top left), MLANA (top right) and THBS1 (bottom left) determined by qRT–PCR in melanoma cell lines 48 h after induction of wild-type or E318K MITF. Gene expression is normalized to GAPDH and shown as fold change compared to uninduced cells. Performed in triplicate, error bars denote s.d. f, qRT–PCR analysis of total RNA isolated from UACC62 human melanoma cells, which were transfected with expression vector encoding wild-type or mutant forms of MITF. The expression level of each target gene was normalized to MITF mRNA. Fold induction is shown as the ratio to each mRNA expression with wild-type MITF. Data are mean ± s.d. of at least three independent experiments. *P < 0.05, **P < 0.01.