Proteasomal Degradation of the EWS-FLI1 Fusion Protein Is Regulated by a Single Lysine Residue

E-26 transformation-specific (ETS) proteins are transcription factors directing gene expression through their conserved DNA binding domain. They are implicated as truncated forms or interchromosomal rearrangements in a variety of tumors including Ewing sarcoma, a pediatric tumor of the bone. Tumor c...

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Bibliographic Details
Published in:The Journal of biological chemistry 2016-12, Vol.291 (52), p.26922-26933
Main Authors: Gierisch, Maria E., Pfistner, Franziska, Lopez-Garcia, Laura A., Harder, Lena, Schäfer, Beat W., Niggli, Felix K.
Format: Article
Language:English
Subjects:
Bone Neoplasms - genetics
Bone Neoplasms - metabolism
Bone Neoplasms - pathology
cancer biology
ETS transcription factor family
Gene Expression Regulation, Neoplastic
HEK293 Cells
Humans
Lysine - genetics
Lysine - metabolism
Molecular Bases of Disease
Mutant Proteins - genetics
Mutant Proteins - metabolism
Mutation - genetics
Oncogene Proteins, Fusion - genetics
Oncogene Proteins, Fusion - metabolism
Promoter Regions, Genetic
proteasome
Proteasome Endopeptidase Complex - metabolism
Proteolysis
Proto-Oncogene Protein c-fli-1 - genetics
Proto-Oncogene Protein c-fli-1 - metabolism
RNA-Binding Protein EWS - genetics
RNA-Binding Protein EWS - metabolism
Sarcoma, Ewing - genetics
Sarcoma, Ewing - metabolism
Sarcoma, Ewing - pathology
transcription target gene
ubiquitin
Ubiquitination
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Summary:E-26 transformation-specific (ETS) proteins are transcription factors directing gene expression through their conserved DNA binding domain. They are implicated as truncated forms or interchromosomal rearrangements in a variety of tumors including Ewing sarcoma, a pediatric tumor of the bone. Tumor cells express the chimeric oncoprotein EWS-FLI1 from a specific t(22;11)(q24;12) translocation. EWS-FLI1 harbors a strong transactivation domain from EWSR1 and the DNA-binding ETS domain of FLI1 in the C-terminal part of the protein. Although Ewing cells are crucially dependent on continuous expression of EWS-FLI1, its regulation of turnover has not been characterized in detail. Here, we identify the EWS-FLI1 protein as a substrate of the ubiquitin-proteasome system with a characteristic polyubiquitination pattern. Using a global protein stability approach, we determined the half-life of EWS-FLI1 to lie between 2 and 4 h, whereas full-length EWSR1 and FLI1 were more stable. By mass spectrometry, we identified two ubiquitin acceptor lysine residues of which only mutation of Lys-380 in the ETS domain of the FLI1 part abolished EWS-FLI1 ubiquitination and stabilized the protein posttranslationally. Expression of this highly stable mutant protein in Ewing cells while simultaneously depleting the endogenous wild type protein differentially modulates two subgroups of target genes to be either EWS-FLI1 protein-dependent or turnover-dependent. The majority of target genes are in an unaltered state and cannot be further activated. Our study provides novel insights into EWS-FLI1 turnover, a critical pathway in Ewing sarcoma pathogenesis, and lays new ground to develop novel therapeutic strategies in Ewing sarcoma.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M116.752063