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

CCAAT/enhancer-binding protein alpha (C/EBP alpha) is an important transcription factor involved in granulocytic differentiation. Here, for the first time we demonstrate that E6-associated protein (E6AP), an E3 ubiquitin ligase targets C/EBP alpha for ubiquitin-mediated proteasome degradation and thereby negatively modulates its functions. Wild-type E6AP promotes ubiquitin dependent proteasome degradation of C/EBP alpha, while catalytically inactive E6-associated protein having cysteine replaced with alanine at amino-acid position 843 (E6AP-C843A) rather stabilizes it. Further, these two proteins physically associate both in non-myeloid (overexpressed human embryonic kidney epithelium) and myeloid cells. We show that E6AP-mediated degradation of C/EBP alpha protein expression curtails its transactivation potential on its target genes. Noticeably, E6AP degrades both wild-type 42 kDa CCAAT-enhancer-binding protein alpha (p42C/EBP alpha) and mutant isoform 30 kDa CCAAT-enhancer-binding protein alpha (p30C/EBP alpha), this may explain perturbed p42C/EBP alpha/p30C/EBP alpha ratio often observed in acute myeloid leukemia (AML). We show that overexpression of catalytically inactive E6AP-C843A in C/EBP alpha inducible K562- p42C/EBP alpha-estrogen receptor (ER) cells inhibits beta-estradiol (E2)-induced C/EBP alpha degradation leading to enhanced granulocytic differentiation. This enhanced granulocytic differentiation upon E2-induced activation of C/EBP alpha in C/EBP alpha stably transfected cells (beta-estradiol inducible K562 cells stably expressing p42C/EBP alpha-ER (K562-C/EBP alpha-p42-ER)) was further substantiated by siE6AP-mediated knockdown of E6AP in both K562-C/EBP alpha-p42-ER and 32dcl3 (32D clone 3, a cell line widely used model for in vitro study of hematopoietic cell proliferation, differentiation, and apoptosis) cells. Taken together, our data suggest that E6AP targeted C/EBP alpha protein degradation may provide a possible explanation for both loss of expression and/or functional inactivation of C/EBP alpha often experienced in myeloid leukemia. Cell Death and Disease (2013) 4, e590; doi:10.1038/cddis.2013.120; published online 18 April 2013