Emilie A. Bard-Chapeaua, Jayantha Gunaratneb, Pankaj Kumarc, Belinda Q. Chuaa, Julius Mullera, Frederic A. Bardc, Walter Blackstockb, Neal G. Copelanda,1, and Nancy A. Jenkinsa,1,2
a - Cancer Genetics Group,
b - Quantitative Proteomics Group,
c - Cell Structure and Function Group, Institute of Molecular and Cell Biology, Singapore 138673
1 - Present address: Methodist Hospital Research Institute, Houston, TX 77030.
2 - To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
Published in PNAS on 15 July 2013.
Ecotropic viral integration site 1 (EVI1) is an oncogenic zinc finger transcription factor whose expression is frequently up-regulated
in myeloid leukemia and epithelial cancers. To better understand the mechanisms underlying EVI1-associated disease, we sought to define the EVI1 interactome in cancer cells. By using stable isotope labeling by amino acids in cell culture-based quantitative proteomics, we could confidently assign 78 proteins as EVI1-interacting partners for FLAG-tagged EVI1. Subsequently, we showed that 22 of 27 tested interacting proteins could coimmunoprecipitate with endogenous EVI1 protein, which represented an 81.5% validation rate. Additionally, by comparing the stable isotope labeling by amino acids in cell culture data with high-throughput yeast twohybrid results, we showed that five of these proteins interacted directly with EVI1. Functional classification of EVI1-interacting proteins
revealed associations with the cellular transcription machinery; modulators of transcription; components of WNT, TGF-β, and RAS pathways; and proteins regulating DNA repair, recombination, and mitosis. We also identified several EVI1 phosphorylation sites by MS analysis and showed that two sites, Ser538 and Ser858, can be phosphorylated and dephosphorylated by two EVI1 interactome proteins, casein kinase II and protein phosphatase- 1α. Finally, mutations that impair EVI1 phosphorylation at these sites reduced the ability of EVI1 to bind DNA through its C-terminal zinc finger domain and to induce cancer cell proliferation.
Collectively, these combinatorial proteomic approaches demonstrate that EVI1 interacts with large and complex networks of proteins, which integrate signals from various different signaling pathways important for oncogenesis. Comprehensive analysis of the EVI1 interactome has thus provided an important resource for dissecting the molecular mechanisms of EVI1-associated disease.
Figure Legend: (A) EVI1 interactome. Networks were drawn using Cytoscape from “experimental knowledge-based” interactions (protein-protein interactions only) contained in the String database. The proteins examined were the ones identified in our proteomics experiments or those described in the literature as EVI1 binding partners. (B,C) Identification of EVI1 phosphorylation sites. Mass spectra showing phosphorylation of EVI1 on Ser538 (B) and Ser858 or Ser860 (C). (D) CK2 kinase and PP1α phosphatase assays after immunoprecipitation of EVI1 endogenous protein from SKOV3 nuclear lysates. CK2 and PP1 α phosphorylate and dephosphorylate EVI1 respectively. (E) CK2 kinase assay after immunoprecipitation of the FLAG tag only, FLAG-EVI1 wild type or FLAG-EVI1 mutant proteins from transfected HeLa nuclear lysates (top panel). A control Western blot (bottom panel) confirmed that equal amounts of FLAG-EVI1 protein were expressed in HeLa nuclear lysates. Serines 538 and 858 account for EVI1 phosphorylation by CK2.