The Role of MITF Phosphorylation Sites During Coat Color and Eye Development in Mice Analyzed by BAC Transgene Rescue

The microphthalmia-associated transcription factor (Mitf) has emerged as an important model for gene regulation in eukaryotic organisms. In vertebrates, it regulates the development of several cell types including melanocytes and has also been shown to play an important role in melanoma. In vitro, the activity of MITF is regulated by multiple signaling pathways, including the KITL/KIT/B-Raf pathway which results in phosphorylation of MITF on serine residues 73 and 409. However, the precise role of signaling to MITF in vivo remains largely unknown. Here, we use a BAC transgene rescue approach to introduce specific mutations in MITF to study the importance of specific phosphoacceptor-sites and protein domains. We show that mice which carry a BAC transgene where single-amino acid substitutions have been made in the Mitf gene rescue the phenotype of loss-of-function mutations in Mitf. This may indicate that signaling from KIT to MITF affects other phosphoacceptor sites in MITF, or that alternative sites can be phosphorylated when Ser73 and Ser409 have been mutated. Our results have implications for understanding signaling to transcription factors. Furthermore, as MITF and signaling mechanisms have been shown to play an important role in melanomas, our findings may lead to novel insights into this resilient disease.

[1]  W. Pavan,et al.  Transcriptional and signaling regulation in neural crest stem cell-derived melanocyte development: do all roads lead to Mitf? , 2008, Cell Research.

[2]  A. Dutra,et al.  An Unstable Targeted Allele of the Mouse Mitf Gene With a High Somatic and Germline Reversion Rate , 2008, Genetics.

[3]  E. Steingrímsson,et al.  Evolutionary sequence comparison of the Mitf gene reveals novel conserved domains. , 2007, Pigment Cell Research.

[4]  H. Arnheiter Mammalian paramutation: a tail's tale? , 2007, Pigment cell research.

[5]  D. Fisher,et al.  MITF: master regulator of melanocyte development and melanoma oncogene. , 2006, Trends in molecular medicine.

[6]  Tania Nolan,et al.  SPUD: a quantitative PCR assay for the detection of inhibitors in nucleic acid preparations. , 2006, Analytical biochemistry.

[7]  Nancy A. Jenkins,et al.  Simple and highly efficient BAC recombineering using galK selection , 2005, Nucleic acids research.

[8]  D. Fisher,et al.  Mechanisms of Hair Graying: Incomplete Melanocyte Stem Cell Maintenance in the Niche , 2005, Science.

[9]  N. Copeland,et al.  Melanocytes and the microphthalmia transcription factor network. , 2004, Annual review of genetics.

[10]  Janice P. Evans,et al.  BRCA2 deficiency in mice leads to meiotic impairment and infertility , 2004, Development.

[11]  S. Sharan,et al.  A simple two-step, 'hit and fix' method to generate subtle mutations in BACs using short denatured PCR fragments. , 2003, Nucleic acids research.

[12]  F. Speleman,et al.  Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes , 2002, Genome Biology.

[13]  N. Copeland,et al.  Mitf and Tfe3, two members of the Mitf-Tfe family of bHLH-Zip transcription factors, have important but functionally redundant roles in osteoclast development , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Michael C. Ostrowski,et al.  Microphthalmia Transcription Factor Is a Target of the p38 MAPK Pathway in Response to Receptor Activator of NF-κB Ligand Signaling* , 2002, The Journal of Biological Chemistry.

[15]  P. Cohen,et al.  GSK3 takes centre stage more than 20 years after its discovery. , 2001, The Biochemical journal.

[16]  N. Copeland,et al.  Genomic, transcriptional and mutational analysis of the mouse microphthalmia locus. , 2000, Genetics.

[17]  E. Price,et al.  c-Kit triggers dual phosphorylations, which couple activation and degradation of the essential melanocyte factor Mi. , 2000, Genes & development.

[18]  S Shibahara,et al.  An L1 element intronic insertion in the black-eyed white (Mitf[mi-bw]) gene: the loss of a single Mitf isoform responsible for the pigmentary defect and inner ear deafness. , 1999, Human molecular genetics.

[19]  E. Price,et al.  MAP kinase links the transcription factor Microphthalmia to c-Kit signalling in melanocytes , 1998, Nature.

[20]  James A. Vaught,et al.  microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family. , 1994, Genes & development.

[21]  A. Ferré-D’Amaré,et al.  Molecular basis of mouse microphthalmia (mi) mutations helps explain their developmental and phenotypic consequences , 1994, Nature Genetics.

[22]  N. Jenkins,et al.  Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein , 1993, Cell.

[23]  A. Bernstein,et al.  The c-fms gene complements the mitogenic defect in mast cells derived from mutant W mice but not mi (microphthalmia) mice. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[24]  N. Copeland,et al.  Organization, distribution, and stability of endogenous ecotropic murine leukemia virus DNA sequences in chromosomes of Mus musculus , 1982, Journal of virology.

[25]  X. Wang,et al.  The basic-helix-loop-helix-leucine zipper gene Mitf: analysis of alternative promoter choice and splicing. , 2010, Methods in molecular biology.

[26]  D. Fisher,et al.  Ser298 of MITF, a mutation site in Waardenburg syndrome type 2, is a phosphorylation site with functional significance. , 2000, Human molecular genetics.

[27]  S Rozen,et al.  Primer3 on the WWW for general users and for biologist programmers. , 2000, Methods in molecular biology.