The Microphthalmia associated transcription factor MITF and the Mitotic Activated Protein Kinase signaling pathway (MAPK) are crucial elements for the differentiation, proliferation and survival of melanocytes. The alteration of one of these two elements results in the impairment of this cell type. In contrast, in most cases, melanoma is associated with a constitutive activation of the MAPK pathway and often with a gain-of-function of MITF. In order to study interactions between the MAPK pathway and MITF activity, we focused our insterest on post-translational modifications that give rise to multiple isoforms of different activities. In fact, MITF contains a phosphorylation site, Serine-73 (S73), which has been implicated both in the increase of activity and the degradation of MITF protein in vitro. To assess the role of this S73 in vivo, our group previously generated targeted mice harboring a S73A mutation in the Mitf gene. However, the S73 codon is part of an Exon Splicing Enhancer sequence, whose mutation leads to a decreased binding affinity of the splice regulatory protein SRp40 and exclusion of exon 2B coding for that serine. In order to dissociate potential effects of residue-73 mutation from the exclusion of exon 2B, we retargeted Mitf in a way that renders the 5' splice site of exon 2B non-functional and force the incorportation of exon 2B. The phenotypic analysis of 3 new alleles Mitf S-S73A, SS73D and SS73-S demonstrates a gain-of-function of the non-phosphorylatable MITF 2B+ isoform.
