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.