BTK blocks the inhibitory effects of MDM2 on p53 activity

// Miran Rada 1 , Mohammad Althubiti 1, 2 , Akang E. Ekpenyong-Akiba 1 , Koon-Guan Lee 3 , Kong Peng Lam 3 , Olga Fedorova 4 , Nickolai A. Barlev 4 and Salvador Macip 1 1 Department of Molecular and Cell Biology, Mechanisms of Cancer and Aging Laboratory, University of Leicester, Leicester, UK 2 Department of Biochemistry, Faculty of Medicine, Umm Al-Qura University, Mecca, Saudi Arabia 3 Bioprocessing Technology Institute, A*STAR, Singapore 4 Institute of Cytology, RAS, Saint-Petersburg, Russia Correspondence to: Nickolai A. Barlev, email: nick.a.barlev@gmail.com Salvador Macip, email: sm460@leicester.ac.uk Keywords: BTK; p53; MDM2; phosphorylation; ubiquitination Received: August 25, 2017      Accepted: October 30, 2017      Published: November 20, 2017 ABSTRACT p53 is a tumour suppressor that is activated in response to various types of stress. It is regulated by a complex pattern of over 50 different post-translational modifications, including ubiquitination by the E3 ligase MDM2, which leads to its proteasomal degradation. We have previously reported that expression of Bruton’s Tyrosine Kinase (BTK) induces phosphorylation of p53 at the N-terminus, including Serine 15, and increases its protein levels and activity. The mechanisms involved in this process are not completely understood. Here, we show that BTK also increases MDM2 and is necessary for MDM2 upregulation after DNA damage, consistent with what we have shown for other p53 target genes. Moreover, we found that BTK binds to MDM2 on its PH domain and induces its phosphorylation. This suggested a negative regulation of MDM2 functions by BTK, supported by the fact BTK expression rescued the inhibitory effects of MDM2 on p53 transcriptional activity. Indeed, we observed that BTK mediated the loss of the ubiquitination activity of MDM2, a process that was dependent on the phosphorylation functions of BTK. Our data together shows that the kinase activity of BTK plays an important role in disrupting the MDM2-p53 negative feedback loop by acting at different levels, including binding to and inactivation of MDM2. This study provides a potential mechanism to explain how BTK modulates p53 functions.