Originally published as Genetics Published Articles Ahead of Print on July 27, 2009.

Genetics, Vol. 183, 581-594, October 2009, Copyright © 2009
doi:10.1534/genetics.109.103945

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

Georg L. Bauer^*, Christian Praetorius^*, Kristín Bergsteinsdóttir^*, Jón H. Hallsson^*,1, Bryndís K. Gísladóttir^*, Alexander Schepsky^*, Deborah A. Swing, T. Norene O'Sullivan, Heinz Arnheiter, Keren Bismuth^,2, Julien Debbache, Colin Fletcher, Søren Warming^,3, Neal G. Copeland^**, Nancy A. Jenkins^** and Eiríkur Steingrímsson^*,4

^* Department of Biochemistry and Molecular Biology and Biomedical Center, Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland, Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, Laboratory of Developmental Neurogenetics, National Institute of Neurological Disorders and Stroke, Porter Neuroscience Research Center, Bethesda, Maryland 20892-3706, Knock Out Mouse Program, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, 20892-9305 and ^** Institute of Molecular and Cell Biology, Proteos, Singapore 138673

⁴ Corresponding author: Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, Vatnsmyrarvegur 16, 101 Reykjavik, Iceland.
E-mail: eirikurs{at}hi.is

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 phospho-acceptor sites and protein domains. We show that mice that carry a BAC transgene where single-amino-acid substitutions have been made in the Mitf gene rescue the phenotype of the loss-of-function mutations in Mitf. This may indicate that signaling from KIT to MITF affects other phospho-acceptor 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.

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