E6AP, an E3 ubiquitin ligase negatively regulates granulopoiesis by targeting transcription factor C/EBPα for ubiquitin mediated proteasome degradation

Abstract

CCAAT/Enhancer Binding Protein Alpha (C/EBPα) is an important transcription factor involved in granulocytic differentiation. Here for the first time we demonstrate that E6AP, an E3 ubiquitin ligase targets C/EBPα for ubiquitin mediated proteasome degradation and thereby negatively modulates its functions. Wild type E6AP promotes ubiquitin dependent proteasome degradation of C/EBPα, while catalytically inactive E6AP-C843A rather stabilizes it. Further, these two proteins physically associate both in non-myeloid (over expressed HEK293T) and myeloid cells. We show that E6AP mediated degradation of C/EBPα protein expression curtails its transactivation potential on its target genes. Noticeably, E6AP degrades both wild type p42C/EBPα and mutant isoform p30C/EBPα, this may explain perturbed p42C/EBPα/p30C/EBPα ratio often observed in AML. We show that over expression of catalytically inactive E6AP-C843A in C/EBPα inducible K562- p42C/EBPα-ER cells inhibits E2 induced C/EBPα degradation leading to enhanced granulocytic differentiation. This enhanced granulocytic differentiation upon E2 induced activation of C/EBPα in C/EBPα stably transfected cells (K562-C/EBPα-p42-ERcells) was further substantiated by siE6AP mediated knock down of E6AP in both K562-C/EBPα-p42-ER and 32dcl3 cells. Taken together, our data suggest that E6AP targeted C/EBPα protein degradation may provide a possible explanation for both loss of expression and/or functional inactivation of C/EBPα often experienced in myeloid leukemia