Elucidating the molecular mechanism of TRIB2-mediated degradation of C/EBPa in AML

[摘要] The pseudokinase TRIB2 is a potent AML oncogene, capable of inducing transplantable AML with a relatively short latency in murine models. Functionally, the oncogenicity of TRIB2 has been linked to its degradation of C/EBPα, a transcription factor necessary for regulation of HSCs, myeloid differentiation and is identified mutated in ~10-15 % of cytogenetically normal AMLs. Previously, we have demonstrated that elevated TRIB2 mRNA expression is associated with a subset of C/EBPα dysregulated AML patients. Here, using in vivo ubiquitination assays I determined TRIB2 exerts its effect through K48 specific ubiquitin-dependent proteasomal degradation of C/EBPαp42. Peptide array analysis identified the specific amino acids involved in the direct binding of these two proteins. Site-directed mutagenesis of these amino acids demonstrated that the direct binding of TRIB2 and C/EBPα was required for TRIB2-mediated C/EBPαp42 ubiquitination. C/EBPαp42 may exist as a dimer or a monomer; however differential dimerisation perturbs terminal myeloid differentiation. TRIB2 binds both monomeric and dimeric C/EBPαp42 but ubiquitination assays revealed it preferentially mediates ubiquitin-dependent degradation of dimeric C/EBPαp42, suggesting dimeric C/EBPαp42 provides a structurally preferential confirmation for TRIB2-mediated ubiquitination of C/EBPαp42. In order to determine if a post-translational modification of C/EBPαp42 was a trigger for TRIB2-mediated binding and degradation, I assessed the phosphorylation status of C/EBPα, often a modification involved in C/EBPα ubiquitination. I determined TRIB2 decreased the levels of phosphorylated Serine 21 (S21) C/EBPα through preferential binding to the phosphorylated S21 form of C/EBPαp42 and mediating increased ubiquitination. Further molecular elucidation of the TRIB2:C/EBPαp42 proteolytic relationship identified C/EBPαp42-K313 as the site of ubiquitin conjugation on C/EBPα and subsequent annotation of this region revealed it is mutated in ~10 % of C/EBPα mutAMLs.Using the clinically available proteasome inhibitor Bortezomib I investigated the targeted inhibition of the TRIB2 degradation function to induce cell death in AML cells. In TRIB2 overexpressing AML cell lines, and in AML patient samples identified to have elevated levels of TRIB2, I have demonstrated that high TRIB2 expressing samples are more sensitive than low TRIB2 expressing samples to cell death induced by proteasomal inhibition.I propose TRIB2 mediates is leukaemogenic effects through functioning as an adaptor protein, facilitating the formation of a multiprotein complex COP1:TRIB2:C/EBPα resulting in ubiquitin-dependent C/EBPαp42 degradation. Dimeric P-Ser21-C/EBPαp42 provides a structurally favourable confirmation for enhanced TRIB2-mediated ubiquitination. As C/EBPαp42 plays a key role in both stem cell function and myeloid differentiation in AML, the targeted inhibition of TRIB2-mediated C/EBPαp42 degradation may provide therapeutic avenues in AML.

[发布机构] University:University of Glasgow;Department:Institute of Cancer Sciences

[关键词] TRIB2, C/EBPA, AML, ubiquitin