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

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

doi:10.1371/journal.pone.0066510

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

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

Cancer Research UK Manchester Institute

Research output: Contribution to journal › Article › peer-review

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. © 2013 White et al.

Original language	English
Article number	e66510
Journal	PLoS ONE
Volume	8
Issue number	6
DOIs	https://doi.org/10.1371/journal.pone.0066510
Publication status	Published - 12 Jun 2013

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White, D. J., Unwin, R. D., Bindels, E., Pierce, A., Teng, H. Y., Muter, J., Greystoke, B., Somerville, T. D., Griffiths, J., Lovell, S., Somervaille, T. C. P., Delwel, R., Whetton, A. D., & Meyer, S. (2013). Phosphorylation of the Leukemic Oncoprotein EVI1 on Serine 196 Modulates DNA Binding, Transcriptional Repression and Transforming Ability. PLoS ONE, 8(6), Article e66510. https://doi.org/10.1371/journal.pone.0066510

@article{5cd1e4d9b867410f9626325469e59bd5,

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. {\textcopyright} 2013 White et al.",

author = "White, {Daniel J.} and Unwin, {Richard D.} and Eric Bindels and Andrew Pierce and Teng, {Hsiang Ying} and Joanne Muter and Brigit Greystoke and Somerville, {Tim D.} and John Griffiths and Simon Lovell and Somervaille, {Tim C P} and Ruud Delwel and Whetton, {Anthony D.} and Stefan Meyer",

note = "C1860/A5901, Cancer Research UK, United Kingdom",

year = "2013",

month = jun,

day = "12",

doi = "10.1371/journal.pone.0066510",

language = "English",

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White, DJ, Unwin, RD, Bindels, E, Pierce, A, Teng, HY, Muter, J, Greystoke, B, Somerville, TD, Griffiths, J, Lovell, S , Somervaille, TCP, Delwel, R, Whetton, AD & Meyer, S 2013, 'Phosphorylation of the Leukemic Oncoprotein EVI1 on Serine 196 Modulates DNA Binding, Transcriptional Repression and Transforming Ability', PLoS ONE, vol. 8, no. 6, e66510. https://doi.org/10.1371/journal.pone.0066510

TY - JOUR

T1 - Phosphorylation of the Leukemic Oncoprotein EVI1 on Serine 196 Modulates DNA Binding, Transcriptional Repression and Transforming Ability

AU - White, Daniel J.

AU - Unwin, Richard D.

AU - Bindels, Eric

AU - Pierce, Andrew

AU - Teng, Hsiang Ying

AU - Muter, Joanne

AU - Greystoke, Brigit

AU - Somerville, Tim D.

AU - Griffiths, John

AU - Lovell, Simon

AU - Somervaille, Tim C P

AU - Delwel, Ruud

AU - Whetton, Anthony D.

AU - Meyer, Stefan

N1 - C1860/A5901, Cancer Research UK, United Kingdom

PY - 2013/6/12

Y1 - 2013/6/12

N2 - 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. © 2013 White et al.

AB - 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. © 2013 White et al.

U2 - 10.1371/journal.pone.0066510

DO - 10.1371/journal.pone.0066510

M3 - Article

C2 - 23776681

VL - 8

JO - PLoS ONE

JF - PLoS ONE

IS - 6

M1 - e66510

ER -

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

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