title: In vivo role of alternative splicing and serine phosphorylation of the microphthalmia-associated transcription factor.
abstract: The microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper protein that plays major roles in the development and physiology of vertebrate melanocytes and melanoma cells. It is regulated by post-translational modifications, including phosphorylation at serine 73, which based on in vitro experiments imparts on MITF an increased transcriptional activity paired with a decreased stability. Serine 73 is encoded by the alternatively spliced exon 2B, which is preferentially skipped in mice carrying a targeted serine-73-to-alanine mutation. Here, we measured the relative abundance of exon 2B+ and exon 2B- RNAs in freshly isolated and FACS-sorted wild-type melanoblasts and melanocytes and generated a series of knock-in mice allowing forced incorporation of either alanine, aspartate, or wild-type serine at position 73. None of these knock-in alleles, however, creates a striking pigmentation phenotype on its own, but differences between them can be revealed either by a general reduction of Mitf transcript levels or in heteroallelic combinations with extant Mitf mutations. In fact, compared with straight serine-73 knock-in mice with their relative reduction of 2B+ Mitf, forced incorporation of alanine 73 leads to greater increases in MITF protein levels, melanoblast and melanocyte numbers, and extent of pigmentation in particular allelic combinations. These results underscore, in vivo, the importance of the link between alternative splicing and post-translational modifications and may bear on the recent observation that exon 2B skipping can be found in metastatic melanoma.
authors.affiliation: Mammalian Development Section, National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Julien
authors.initials: J
authors.lastname: Debbache
authors.forename: M Raza
authors.initials: MR
authors.lastname: Zaidi
authors.forename: Sean
authors.initials: S
authors.lastname: Davis
authors.forename: Theresa
authors.initials: T
authors.lastname: Guo
authors.forename: Keren
authors.initials: K
authors.lastname: Bismuth
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.forename: Susan
authors.initials: S
authors.lastname: Skuntz
authors.forename: Dragan
authors.initials: D
authors.lastname: Maric
authors.forename: James
authors.initials: J
authors.lastname: Pickel
authors.forename: Paul
authors.initials: P
authors.lastname: Meltzer
authors.forename: Glenn
authors.initials: G
authors.lastname: Merlino
authors.forename: Heinz
authors.initials: H
authors.lastname: Arnheiter
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D005091:Exons / Q000235:genetics; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008544:Melanocytes / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D051739:Microphthalmia-Associated Transcription Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D010641:Phenotype; D010766:Phosphorylation; D010858:Pigmentation / Q000235:genetics; D012694:Serine / Q000378:metabolism*
chemical_list: D051739:Microphthalmia-Associated Transcription Factor; D012694:Serine