title: 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 E6-associated protein (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 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/EBPα protein expression curtails its transactivation potential on its target genes. Noticeably, E6AP degrades both wild-type 42 kDa CCAAT-enhancer-binding protein alpha (p42C/EBPα) and mutant isoform 30 kDa CCAAT-enhancer-binding protein alpha (p30C/EBPα), this may explain perturbed p42C/EBPα/p30C/EBPα ratio often observed in acute myeloid leukemia (AML). We show that overexpression of catalytically inactive E6AP-C843A in C/EBPα inducible K562- p42C/EBPα-estrogen receptor (ER) cells inhibits β-estradiol (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 (β-estradiol inducible K562 cells stably expressing p42C/EBPα-ER (K562-C/EBPα-p42-ER)) was further substantiated by siE6AP-mediated knockdown of E6AP in both K562-C/EBPα-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/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.
authors.affiliation: Drug Target Discovery and Development Division, CSIR-Central Drug Research Institute, Sector-10, Jankipuram Extension, Lucknow, UP 226021, India.
authors.forename: P
authors.initials: P
authors.lastname: Pal
authors.forename: S
authors.initials: S
authors.lastname: Lochab
authors.forename: J K
authors.initials: JK
authors.lastname: Kanaujiya
authors.forename: I
authors.initials: I
authors.lastname: Kapoor
authors.forename: S
authors.initials: S
authors.lastname: Sanyal
authors.forename: G
authors.initials: G
authors.lastname: Behre
authors.forename: A K
authors.initials: AK
authors.lastname: Trivedi
mesh_terms: D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D004958:Estradiol / Q000494:pharmacology; D006098:Granulocytes / Q000166:cytology* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020014:K562 Cells; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism / Q000473:pathology; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015533:Transcriptional Activation; D020298:U937 Cells; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D022763:CCAAT-Enhancer-Binding Protein-alpha; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D025801:Ubiquitin; D004958:Estradiol; C471639:UBE3A protein, human; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex