title: DNA damage-induced ubiquitylation of proteasome controls its proteolytic activity.
abstract: 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.
authors.affiliation: Institute of Cytology, Russian Academy of Sciences, St-Petersburg, Russia.
authors.forename: Tatiana N
authors.initials: TN
authors.lastname: Moiseeva
authors.forename: Andrew
authors.initials: A
authors.lastname: Bottrill
authors.forename: Gerry
authors.initials: G
authors.lastname: Melino
authors.forename: Nickolai A
authors.initials: NA
authors.lastname: Barlev
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D045744:Cell Line, Tumor; D004249:DNA Damage; D004317:Doxorubicin / Q000494:pharmacology; D006801:Humans; D020014:K562 Cells; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000235:genetics* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D054875:Ubiquitination
chemical_list: D000903:Antibiotics, Antineoplastic; D004317:Doxorubicin; D046988:Proteasome Endopeptidase Complex
references.citation: J Biol Chem. 1985 Oct 5;260(22):12015-21
references.pmid: 2995356
references.citation: J Neurochem. 2007 Jun;101(6):1483-90
references.pmid: 17286585
references.citation: Science. 2007 May 25;316(5828):1160-6
references.pmid: 17525332
references.citation: Mol Cell. 2004 Dec 22;16(6):1027-34
references.pmid: 15610744
references.citation: Front Biosci. 2008 May 01;13:7184-92
references.pmid: 18508726
references.citation: Biochem Biophys Res Commun. 2011 Dec 16;416(3-4):258-65
references.pmid: 22079093
references.citation: Oncogene. 2013 Sep 26;32(39):4721-6
references.pmid: 23085759
references.citation: Nucleic Acids Res. 2004 Jan 1;32(Database issue):D115-9
references.pmid: 14681372
references.citation: Nat Biotechnol. 2005 Jan;23(1):94-101
references.pmid: 15592455
references.citation: Proc Jpn Acad Ser B Phys Biol Sci. 2009;85(1):12-36
references.pmid: 19145068
references.citation: Cell Cycle. 2010 Feb 15;9(4):840-9
references.pmid: 20139718
references.citation: J Biol Chem. 2000 Feb 18;275(7):4635-9
references.pmid: 10671491
references.citation: Arch Biochem Biophys. 2004 Jan 1;421(1):67-76
references.pmid: 14678786
references.citation: J Neurosci. 2003 Dec 17;23(37):11653-61
references.pmid: 14684867
references.citation: Am J Physiol Heart Circ Physiol. 2008 Dec;295(6):H2541-50
references.pmid: 18978187
references.citation: Cell Cycle. 2004 Apr;3(4):460-1
references.pmid: 14976429
references.citation: Science. 2006 Aug 25;313(5790):1122-6
references.pmid: 16931761
references.citation: Cell Div. 2012 Dec 26;7(1):26
references.pmid: 23268747
references.citation: Nat Rev Mol Cell Biol. 2001 Mar;2(3):195-201
references.pmid: 11265249
references.citation: Mol Cell. 2008 Aug 8;31(3):438-48
references.pmid: 18691976
references.citation: BMC Biochem. 2004 Dec 17;5(1):18
references.pmid: 15603592
references.citation: J Biol Chem. 2010 Apr 30;285(18):13580-8
references.pmid: 20189990
references.citation: Biochem J. 2004 Feb 15;378(Pt 1):177-84
references.pmid: 14583091
references.citation: Electrophoresis. 1999 Dec;20(18):3551-67
references.pmid: 10612281
references.citation: J Biol Chem. 2001 Aug 10;276(32):30057-63
references.pmid: 11375979
references.citation: Mol Cell Biol. 2000 Dec;20(23):8643-54
references.pmid: 11073966
references.citation: Int J Oncol. 2002 Nov;21(5):1081-5
references.pmid: 12370758
references.citation: Analyst. 2008 Sep;133(9):1128-38
references.pmid: 18709185
references.citation: FEBS Lett. 2007 Mar 6;581(5):885-90
references.pmid: 17289037
references.citation: Drugs. 2012 Oct 22;72(15):2023-32
references.pmid: 22994535
references.citation: Tsitologiia. 2010;52(12):1012-5
references.pmid: 21427980
references.citation: Science. 2009 Aug 14;325(5942):834-40
references.pmid: 19608861
references.citation: Nature. 2005 Oct 27;437(7063):1381-5
references.pmid: 16251969
references.citation: Mol Cell. 2011 Oct 21;44(2):325-40
references.pmid: 21906983
references.citation: Amino Acids. 2011 Jul;41(2):351-61
references.pmid: 20364280
references.citation: Mol Cell Proteomics. 2002 Aug;1(8):567-78
references.pmid: 12376572
references.citation: Mol Biol Cell. 2008 Mar;19(3):1072-82
references.pmid: 18162577
references.citation: Arch Biochem Biophys. 2003 Jul 1;415(1):1-5
references.pmid: 12801506
references.citation: Mol Cell Biol. 2011 Dec;31(24):4951-63
references.pmid: 21986495
references.citation: Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12130-5
references.pmid: 15302935
references.citation: Biochemistry. 2007 Mar 20;46(11):3553-65
references.pmid: 17323924
references.citation: Science. 1984 Oct 26;226(4673):466-8
references.pmid: 6093249
references.citation: Eur J Biochem. 2001 May;268(10):2773-8
references.pmid: 11358491
references.citation: Nat Biotechnol. 2006 Oct;24(10):1285-92
references.pmid: 16964243
references.citation: J Med Chem. 2012 Dec 13;55(23):10317-27
references.pmid: 22978849
references.citation: Cell Cycle. 2006 Dec;5(23):2778-86
references.pmid: 17172851
references.citation: Gene. 1999 Nov 1;239(2):301-8
references.pmid: 10548731
references.citation: Mol Cell. 2010 Feb 12;37(3):396-407
references.pmid: 20159558
references.citation: Biochem J. 1997 Aug 15;326 ( Pt 1):93-8
references.pmid: 9337855
references.citation: Int Rev Cell Mol Biol. 2008;267:59-124
references.pmid: 18544497
references.citation: J Clin Oncol. 2007 Sep 1;25(25):3892-901
references.pmid: 17679727
references.citation: Cell Cycle. 2012 Oct 1;11(19):3638-48
references.pmid: 22935697
references.citation: Arch Biochem Biophys. 2003 Jan 15;409(2):341-8
references.pmid: 12504901
references.citation: Cell Death Differ. 2010 Jan;17(1):1-3
references.pmid: 20010850
references.citation: Nat Rev Cancer. 2004 May;4(5):349-60
references.pmid: 15122206
references.citation: J Biol Chem. 2007 Jan 12;282(2):1152-60
references.pmid: 17105721