Ecotropic viral integration site-1 (EVI1), an oncogenic zinc finger transcription factor, has been recognized since 1988. EVI1 is upregulated in myeloid leukemia, epithelial and other cancers, but its mechanism of action and regulation are unknown.
In a series of elegant experiments, Bard-Chapeau et al. (2013) have described the oncogene’s interactome.1 Using stable isotope labeling by amino acids in cell culture (SILAC) and employing quantitative mass spectrometric proteomic analysis, they have revealed the proteins with which this transcription factor interacts. Moreover, they have determined post-translational modification sites on the EVI1 protein. Using cell culture assays, the researchers demonstrated how other proteins affect EVI1’s oncogenic activity through upregulation or downregulation of serine phosphorylation at these sites.
The researchers first demonstrated that cellular EVI1 is commonly found within high molecular weight complexes. This was accomplished via gel filtration chromatography of lysates from an ovarian carcinoma cell line, SKOV3, that naturally expresses the oncogene. EVI1, a 1,015-amino-acid protein that is thought to play a role in the progression of ovarian carcinoma, was found only at molecular weights above 2,000 kDa.
By transiently expressing FLAG-tagged EVI1 in the SKOV3 cell line, the researchers discovered which proteins the oncogene interacts with. In-gel trypsin digests of cell lysates were analyzed by LTQ Orbitrap mass spectrometry (Thermo Scientific). Analysis of the data identified 78 proteins interacting with EVI1. Twenty-seven of these proteins, including eight known associates, were examined further. Of these, more than 81.5% co-immunoprecipitated with EVI1 using specific antibodies. These results were repeated and confirmed using a myeloid leukemia cell line, K562, which expresses EVI1 but at a lower level.
Following this, the researchers also used a yeast two-hybrid system to investigate the binding sites for the protein interactome. Mapping showed binding sites predominantly between the two zinc finger domains, including the EVI1 repressor domain.
Biological processes involved in EVI1 protein interactions were determined by functional clustering analysis of proteomic data. Pathways identified included those associated with DNA damage repair and recombination, and transcription — all important pathways in oncogenic transformation.
Mass spectrometry data also showed interaction between EVI1 and proteins commonly involved in post-translational modifications, especially phosphorylation. The mass spectrometry data identified these phosphorylation sites as serine residues: Ser436, Ser538, and Ser858 or 860. Sequence analysis of these sites showed potential binding motifs for major phosphorylation factors, casein kinase 2 (CK2) and protein Ser/Thr phosphatase-1α (PP1α).
In subsequent studies, CK2 assays showed increased EVI1 uptake of labeled phosphorus by immunoprecipitated SKOV3 cell eluates. Phosphorylation was reduced following treatment with the PP1α phosphatase. Using a mutated FLAG-labelled EVI1 to transfect HeLa cells, phosphorylation in response to CK2 was reduced when the serine residues at positions 538 and 858 were replaced.
The importance of phosphorylation on these two serine residues to EVI1 oncogenic activity was confirmed by cell culture assay. Proliferation of and the ability to form colonies by HeLa cells transfected with the FLAG-EVI1 were reduced when the two serine residues were substituted to reduce phosphorylation.
By combining different cell biology and proteomic assay techniques, the research group has been able to further clarify pathways and possible regulation steps in the transformation of normal cells towards oncogenesis by EVI1. These findings show potential in the therapy of cancers involving EVI1.
1. Bard-Chapeau, E. A., et al. (2013) “EVI1 oncoprotein interacts with a large and complex network of proteins and integrates signals through protein phosphorylation,” Proceedings of the National Academy of Science, 110(31) (pp. E2885–94), doi: 10.1073/pnas.1309310110.
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