E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue.
Here, we show that E2-EPF ubiquitin carrier protein (UCP) elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL) and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196). A UCP mutant in which Cys118 was changed to alanine (UCPC118A) did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation.
Gene Therapy Research Unit, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
Jung Hwa
JH
Lim
Gene Therapy Research Unit, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.|University of Science and Technology, Daejeon, Republic of Korea.
Hee Won
HW
Shin
University of Science and Technology, Daejeon, Republic of Korea.|Genome Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
Kyung-Sook
KS
Chung
University of Science and Technology, Daejeon, Republic of Korea.|Genome Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
Nam-Soon
NS
Kim
Gene Therapy Research Unit, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
Ju Hee
JH
Kim
Gene Therapy Research Unit, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
Hong-Ryul
HR
Jung
Gene Therapy Research Unit, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
Dong-Soo
DS
Im
Gene Therapy Research Unit, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.|University of Science and Technology, Daejeon, Republic of Korea.
Cho-Rok
CR
Jung
Nat Med. 2006 Jul;12 (7):809-16
16819549
Mol Cell. 2003 Jan;11(1):249-59
12535537
Science. 2001 Apr 20;292(5516):468-72
11292861
J Biol Chem. 2010 Mar 19;285(12 ):8595-604
20061386
Prog Clin Biol Res. 1985;180:215
2994086
Biochemistry. 1997 Aug 26;36(34):10526-37
9265633
Proc Natl Acad Sci U S A. 2010 Jan 26;107(4):1355-60
20080579
Proc Natl Acad Sci U S A. 2009 Oct 27;106(43):18213-8
19822757
EMBO J. 1992 Aug;11(8):3091-8
1639076
Cell. 2014 May 8;157(4):910-21
24813613
Science. 2001 Apr 20;292(5516):464-8
11292862
Mol Cell Biol. 2003 Aug;23 (15):5388-400
12861024
J Biol Chem. 1996 Jan 5;271(1):376-9
8550590
Mol Interv. 2007 Jun;7(3):157-67
17609522
FASEB J. 2010 Apr;24(4):981-93
19940261
J Biol Chem. 1992 Aug 5;267(22):15829-35
1379239
BMC Cancer. 2015 Oct 26;15:800
26503325
Nat Cell Biol. 2009 Nov;11(11):1363-9
19820702
Cell. 2011 Mar 4;144(5):769-81
21376237
Mol Cell. 2010 Aug 13;39(3):477-84
20655260
Proc Natl Acad Sci U S A. 2010 Feb 16;107(7):2848-53
20133640
Proc Natl Acad Sci U S A. 1999 Oct 26;96(22):12436-41
10535940
Annu Rev Biochem. 2001;70:503-33
11395416
Nat Rev Mol Cell Biol. 2006 Sep;7(9):644-56
16896351
FEBS Lett. 1999 Jul 23;455(3):311-4
10437795
J Biol Chem. 1996 Feb 2;271(5):2817-22
8576260
J Biol Chem. 1993 Jan 15;268(2):955-60
8419375
Annu Rev Biochem. 2009;78:399-434
19489725
J Biol Chem. 1996 Feb 2;271(5):2823-31
8576261