Proteomic discovery of MNT as a novel interacting partner of E3 ubiquitin ligase E6AP and a key mediator of myeloid differentiation

Abstract

// Isha Kapoor 1 , Jitendra Kanaujiya 1 , Yogesh Kumar 1 , Jagadeshwar Reddy Thota 3 , Madan L.B. Bhatt 2 , Naibedya Chattopadhyay 4 , Sabyasachi Sanyal 1 and Arun Kumar Trivedi 1 1 Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow, UP, India 2 Department of Radiotherapy, King George’s Medical University, Lucknow, UP, India 3 SAIF Division, CSIR-Central Drug Research Institute, Lucknow, UP, India 4 Division of Endocrinology and Center for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute (CSIR-CDRI), Lucknow, UP, India Correspondence to: Arun Kumar Trivedi, email: // Keywords : MNT, E6AP, ATRA, mass spectrometry, APL Received : May 10, 2015 Accepted : September 30, 2015 Published : October 25, 2015 Abstract Perturbed stability of regulatory proteins is a major cause of transformations leading to cancer, including several leukemia subtypes. Here, for the first time we demonstrate that E6-associated protein (E6AP), an E3 ubiquitin ligase negatively targets MAX binding protein MNT for ubiquitin-mediated proteasome degradation and impedes ATRA mediated myeloid cell differentiation. MNT is a member of the Myc/Max/Mad network of transcription factor that regulates cell proliferation, differentiation, cellular transformation and tumorigenesis. Wild-type E6AP promoted proteasome dependent degradation of MNT, while catalytically inactive E6AP having cysteine replaced with alanine at amino-acid 843 position (E6APC843A) rather stabilized it. Further, these proteins physically associated with each other both in non-myeloid (HEK293T) and myeloid cells. MNT overexpression induced G0-G1 growth arrest and promoted myeloid differentiation while its knockdown mitigated even ATRA induced differentiation suggesting MNT to be crucial for myeloid differentiation. We further showed that ATRA inhibited E6AP and stabilized MNT expression by protecting it from E6AP mediated ubiquitin-proteasome degradation. Notably, E6AP knockdown in HL60 cells restored MNT expression and promoted myeloid differentiation. Taken together, our data demonstrated that E6AP negatively regulates granulocytic differentiation by targeting MNT for degradation which is required for growth arrest and subsequent myeloid differentiation by various differentiation inducing agents.