Lysine 182 and lysine 316 are the sites of SUMO-conjugation in MITF in vitro and in vivo. (A) Schematic representation of MITF. Two possible sumoylation sites are present at position 182 and 316. The putative acceptor sites and the alignment of sumoylation motifs in MITF and reference proteins are shown on the right. (B) Protein extracts from cells co-transfected with constructs encoding wild type MITF or MITF with the indicated mutations [K182R, K316R, both K182/316R (2KR) or K201R], and flag-tagged SUMO-1 were immunoprecipitated with anti-V5 antibody. The precipitates were analyzed by immunoblotting with horseradish peroxidase-coupled anti-V5 antibody. The V5-His tagged Mitf is indicated by an arrowhead and its corresponding conjugated forms by arrows. (C) In vitro sumoylation of MITF. In vitro translated wild type and mutant MITF were subjected to in vitro sumoylation using purified recombinant E1 (SAE I/SAE II) enzyme, E2 (Ubc9) enzyme, and recombinant SUMO-1 or SUMO-3 in the presence of ATP. The reaction mixes were subjected to SDS-PAGE and immunoblotted with anti-V5 antibody. Note that in vitro reactions can result in additional MITF forms not detected in transfected cells which may reflect conformational isomers. (D) MITF with mutations in phosphorylation sites are normally sumoylated. The indicated plasmids encoding wild type and mutant MITF were transfected into HEK293 cells along with the SUMO-1 expression plasmid, and cellular extracts subjected to electrophoresis and immunoblotting. Note that only 2KR mutant MITF shows no SUMO-modified forms. Also note that the S73A/409A double mutant, modified or not, shows a faster migration than the other mutants, consistent with previous results (Wu et al., 2000) and independent unpublished observations (Arnheiter et al., in press). Longer exposures (not shown) clearly revealed both mono- and doubly sumoylated forms in wild type MITF and all mutants except 2KR-MITF.

Sumoylation does not affect DNA binding of MITF. (A) The purified GST-wild type MITF or mutant MITF (K182R/K316R) were subjected to SDS-PAGE and the proteins were detected by Coomassie stain (left panel). GST-MITF fusion protein was subjected to in vitro sumoylation in the presence or absence of Ubc9. The reaction mixtures were subjected to SDS-PAGE and immunoblotted with anti-MITF antibody (right panel). (B) The reaction mixtures were subsequently analyzed by EMSA. For competition assays, all DNA-binding reactions contained 4 fmol Biotin-labeled double-stranded oligonucleotide and various amounts of unlabeled double-stranded oligonucleotide.

Synergistic transcriptional activities are enhanced by sumoylation site mutations of MITF. (A) Schematic illustration of GAL4-responsive reporter genes used in this study. (B) Expression plasmids for GAL4 DNA binding domain fused with the wild type and 2KR mutant of MITF were transfected into NIH3T3 cells with a luciferase reporter gene containing one (1X GAL), three (3X GAL) or five (5X GAL) GAL4 binding sites upstream of the TATA box and phRL. (C) Ratio of mutant over WT activity measured in Figure 5B. (D) Schematic representation of promoter fragment of the human Dct gene. Effects of the 2KR mutation on the synergistic transcription between MITF and SOX10. Expression plasmids for wild type or 2KR mutant MITF, wild type or SOX10, a luciferase reporter gene containing the DCT promoter, and phRL-TK were cotransfected into NIH3T3 cells. Luciferase activity was measured as described for Figure 5.

MITF is modified by SUMO in mammalian cells. (A) Schematic representation of V5-His tagged MITF and Flag-tagged ubiquitin and ubiquitin-like proteins used in this study. (B) HEK293 cells were transfected with the plasmid expressing V5-tagged Mitf together with the plasmids expressing Flag-tagged SUMO-1, SUMO-2, Nedd8, ubiquitin and GFP-tagged SUMO-1. Thirty-six hours after transfection, cell lysates were prepared and immunoprecipitated with anti-V5 antibody. The immunoprecipitates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-V5 antibody (left panel). Whole cell lysates were subjected to SDS-PAGE and analyzed by immunoblot with anti-Flag antibody (right panel). The V5-His tagged MITF (several isoforms representing different degrees of phosphorylation as previously described (Wu et al., 2000) is indicated by an arrowhead and its corresponding conjugated forms by arrows. (C) Flag-tagged SUMO-1 or Nedd8 were co-expressed with V5-tagged MITF with or without exon 6a (+6 MITF or)6 MITF) in HEK293 cells. The cell lysates were immunoprecipitated with anti-V5 or anti-Flag antibodies and the immunoprecipitates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-V5 or anti-Flag antibodies. (D) Endogenous MITF is modified by SUMO-1. Whole cell lysates from B16 melanoma cells were prepared either in the presence or absence of isopeptidase inhibitors and then immunoprecipitated with anti-MITF. The precipitates were analyzed by immunoblotting with horseradish peroxidase coupled anti-MITF or anti-SUMO-1 antibodies. Note that in all figures, MITF proteins are indicated by an arrowhead, and their corresponding conjugated forms by arrows.

PIAS family members do not alter in vitro MITF sumoylation. (A) Ubc9 and PIAS interact with MITF in vitro. In vitro translated V5-tagged MITF was incubated with GST or GST-Ubc9 fusion protein immobilized on glutathione-agarose beads. Bound proteins were separated on 10% SDS-PAGE and subjected to immunoblotting with anti-V5 antibody (left panel). The lysates from HEK293 cells expressing Flag-tagged PIAS-1, -3, or -Xα were incubated with GST-MITF immobilized on glutathione-agarose beads. Bound proteins were detected with anti-Flag antibody. (B) Schematic representation of GST-MITF fusion proteins used in this study (left panel). In vitro translated myc-tagged Ubc9 or lysates of HEK293 cells expressing Flag-tagged PIAS Xα protein were incubated with the designated GST-MITF fusion proteins that were immobilized on glutathione agarose beads. Bound proteins were detected with anti-Flag or anti-myc antibody respectively. (C) Schematic representation of GAL4 DNA binding domain (GAL DB)-MITF fusion proteins used in this study. In vitro translated GAL DB fusion proteins were incubated with GST-PIAS Xα or GST-Ubc9. Bound proteins were detected by anti-V5 antibody. (D) PIAS does not enhance the sumoylation of MITF in vitro. In vitro translated V5-tagged MITF or V5-tagged c-JUN were subjected to in vitro sumoylation in the presence of GST-PIAS-1, Xα, or GST. The reaction mixtures contain the indicated proteins. Anti-V5 western blotting was used to detect free and SUMO-1 conjugated MITF. (E) PIAS proteins do not alter sumoylation in transfected cells. HEK293 cells were cotransfected with the indicated plasmids and subjected electrophoresis and western blotting as indicated above. Note that neither PIAS-1 nor PIAS-3 altered the extent of sumoylation after co-expression of SUMO-1. Also note that under the conditions employed, the doubly sumoylated MITF form was not detected.

Effect of SUMO modification on transcriptional activity of MITF. (A) B16 melanoma cells; or (B) NIH3T3 cells were transiently transfected with the 4X M-box-luciferase reporter construct and phRL-TK carrying Renilla luciferase gene together with the expression plasmids carrying wild type MITF, MITF-K182R, MITF-K201R, MITF-K316R, MITF-K182R/K316R or control (empty) plasmid. Gel electrophoresis and MITF immunoblottting indicated that similar amounts of MITF were expressed in NIH3T3 cells [upper panel in (B), arrow head marking MITF protein]. Luciferase activity was measured 24 h after transfection and normalized using the activities of the Renilla luciferase. Results represent averages from at least three independent experiments. (C) NIH3T3 cells were transfected with pFR-luc and phRL-TK together with pcDNAGAL4 DB-wild type MITF, mutants, or pcDNAGAL4DB and luciferase activities were analyzed as in (A). The upper panel shows the expression levels of GAL4 DNA binding domain and fusion proteins, as revealed by immunoblotting. (D) NIH3T3 cells were cotransfected with the 4X M-box-luciferase reporter construct, phRL-TK, and the plasmids expressing wild-type MITF or mutant together with the plasmids expressing SUMO-1, SUMO-2, Nedd8, ubiquitin, PIAS family members, or with the empty plasmid (pcDNA), and luciferase activity was measured. (D) NIH3T3 cells were co-transfected with pGL-tyrosinase-, tyrp-1, Dct, or cathepsin K promoter as well as phRL-Tk and the plasmids expressing wild-type or mutant MITF, or empty plasmid (pcDNA).

Sumoylation sites and neighboring proline residues suggesting their function as ‘synergy control’ motifs are conserved throughout evolution. Note that D. melanogaster (Hallsson et al., 2004) and C. elegans (Rehli et al., 1999) each have only one gene most closely related to MITF while D. rerio and X. laevis each have two and G. gallus, M. musculus and H. sapiens each have one MITF gene. In addition, vertebrates have several more distantly MITF-related genes (Tfe genes) that were not analyzed in this study. For each species, only one exemplary MITF (or closely MITF-related) sequence is shown.