Phosphorylation of the Leukemic Oncoprotein EVI1 on Serine 196 Modulates DNA Binding, Transcriptional Repression and Transforming Ability

Daniel J White,Ruud Delwel,Joanne Muter,Tim C P Somervaille,Simon Lovell,Brigit Greystoke,Eric Bindels,Anthony D Whetton,Andrew Pierce,Stefan Meyer,Tim D Somerville,Richard D Unwin,John Griffiths,Hsiang-Ying Teng,Pierre-Antoine Defossez

doi:10.1371/journal.pone.0066510

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.

Highlights

The Ecotropic viral integration site-1 gene (EVI1), at the MECOM (MDS-EVI1 complex) locus at 3q26.2 encodes a nuclear protein with an essential role in haematopoiesis [1]
EVI1 is phosphorylated on Serine 196 We detected high EVI1 protein expression in the Fanconi Anaemia (FA)-derived
acute myeloid leukaemia (AML) cell line SB1690CB, in which FA-associated 3q gains result in high EVI1 transcript levels [5] (Figure 1A)

Summary

Introduction

The Ecotropic viral integration site-1 gene (EVI1), at the MECOM (MDS-EVI1 complex) locus at 3q26.2 encodes a nuclear protein with an essential role in haematopoiesis [1]. Overexpression of EVI1 in acute myeloid leukaemia (AML) is commonly a result of chromosomal rearrangements involving the 3q26.2 region and is associated with an extremely poor clinical outcome [2,3]. EVI1 overexpression is in particular linked to leukemic transformation in individuals with Fanconi Anaemia (FA), which is an inherited chromosomal fragility disorder with predisposition to AML [4,5]. EVI1 functions as a transcriptional regulator with two zinc finger domains that recognize specific genomic DNA target sequences [6,7] and mediates interaction of DNA with chromatin modifying proteins and protein complexes to regulate gene expression [8,9,10]. EVI1 regulated genes have been reported to include Pbx and Fos [11,12].

Methods

Results

Conclusion