DNA damage-induced ubiquitylation of proteasome controls its proteolytic activity
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Tatiana N. Moiseeva1,2,3, Andrew Bottrill4, Gerry Melino2,5 and Nickolai A. Barlev1,2,4
1 Institute of Cytology, Russian Academy of Sciences, St-Petersburg, Russia
2 Molecular Pharmacology Laboratory, Techological University, St-Petersburg, Russia
3 Department of Radiation Oncology, University of Pittsburgh School of Medicine, Hillman Cancer Center, Pittsburgh, PA
4 Department of Biochemistry, University of Leicester, Lancaster Road, Leicester, UK
5 MRC Toxicology Unit, Leicester University, Lancaster Road, Leicester, UK
Nickolai A. Barlev, email:
Keywords: proteasome, DNA damage, proteolytic activities, doxorubicin, ubiquitylation.
Received: May 29, 2013 Accepted: June 27, 2013 Published: June 29, 2013
Stability of proteins is largely controlled by post-translational covalent modifications. Among those, ubiquitylation plays a central role as it marks the proteins for proteasome-dependent degradation. Proteolytic activities of proteasomes are critical for execution of various cellular processes, including DNA damage signaling and repair. However, very little is known about the regulation of proteasomal activity in cells during genotoxic stress. Here we investigated post-translational modifications of the 20S proteasomal subunits upon DNA damage induced by doxorubicin. Using mass-spectrometry, we found novel sites of phosphorylation and ubiquitylation in multiple proteasome subunits upon doxorubicin treatment. Ectopic co-expression of proteasome subunits and tagged ubiquitin confirmed the presence of ubiquitylated forms of PSMA5, PSMA1, PSMA3 and PSMB5 in cells. Moreover, we demonstrated that ubiquitylation in vitro inhibited chymotrypsin-like and caspase-like activities of proteasomes. In vivo, doxorubicin increased the activity of proteasomes, paralleling with attenuation of the overall level of proteasome ubiquitylation. Collectively, our results suggest a novel mechanism whereby the proteolytic activities of proteasomes are dynamically regulated by ubiquitylation upon DNA damage.
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