title: Phosphorylation of the leukemic oncoprotein EVI1 on serine 196 modulates DNA binding, transcriptional repression and transforming ability.
abstract: The EVI1 (ecotropic viral integration site 1) gene at 3q26 codes for a transcriptional regulator with an essential role in haematopoiesis. Overexpression of EVI1 in acute myeloid leukaemia (AML) is frequently associated with 3q26 rearrangements and confers extremely poor prognosis. EVI1 mediates transcriptional regulation, signalling, and epigenetic modifications by interacting with DNA, proteins and protein complexes. To explore to what extent protein phosphorylation impacts on EVI1 functions, we analysed endogenous EVI1 protein from a high EVI1 expressing Fanconi anaemia (FA) derived AML cell line. Mass spectrometric analysis of immunoprecipitated EVI1 revealed phosphorylation at serine 196 (S196) in the sixth zinc finger of the N-terminal zinc finger domain. Mutated EVI1 with an aspartate substitution at serine 196 (S196D), which mimics serine phosphorylation of this site, exhibited reduced DNA-binding and transcriptional repression from a gene promotor selectively targeted by the N-terminal zinc finger domain. Forced expression of the S196D mutant significantly reduced EVI1 mediated transformation of Rat1 fibroblasts. While EVI1-mediated serial replating of murine haematopoietic progenitors was maintained by EVI1-S196D, this was associated with significantly higher Evi1-trancript levels compared with WT-EVI1 or EVI1-S196A, mimicking S196 non-phosphorylated EVI1. These data suggest that EVI1 function is modulated by phosphorylation of the first zinc finger domain.
authors.affiliation: Stem Cell and Leukaemia Proteomics Laboratory, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: White
authors.forename: Richard D
authors.initials: RD
authors.lastname: Unwin
authors.forename: Eric
authors.initials: E
authors.lastname: Bindels
authors.forename: Andrew
authors.initials: A
authors.lastname: Pierce
authors.forename: Hsiang-Ying
authors.initials: HY
authors.lastname: Teng
authors.forename: Joanne
authors.initials: J
authors.lastname: Muter
authors.forename: Brigit
authors.initials: B
authors.lastname: Greystoke
authors.forename: Tim D
authors.initials: TD
authors.lastname: Somerville
authors.forename: John
authors.initials: J
authors.lastname: Griffiths
authors.forename: Simon
authors.initials: S
authors.lastname: Lovell
authors.forename: Tim C P
authors.initials: TC
authors.lastname: Somervaille
authors.forename: Ruud
authors.initials: R
authors.lastname: Delwel
authors.forename: Anthony D
authors.initials: AD
authors.lastname: Whetton
authors.forename: Stefan
authors.initials: S
authors.lastname: Meyer
mesh_terms: D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005347:Fibroblasts; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D000074008:MDS1 and EVI1 Complex Locus Protein; D013058:Mass Spectrometry; D010766:Phosphorylation; D011519:Proto-Oncogenes; D012694:Serine / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D004268:DNA-Binding Proteins; D000074008:MDS1 and EVI1 Complex Locus Protein; C063575:MECOM protein, human; D014157:Transcription Factors; D012694:Serine