Targeted silencing of Jab1/Csn5 in human cells downregulates SCF activity through reduction of F-box protein levels
Tóm tắt
SCF ubiquitin ligases target numerous proteins for ubiquitin dependent proteolysis, including p27 and cyclin E. SCF and other cullin-RING ligases (CRLs) are regulated by the ubiquitin-like protein Nedd8 that covalently modifies the cullin subunit. The removal of Nedd8 is catalyzed by the Jab1/MPN domain metalloenzyme (JAMM) motif within the Csn5 subunit of the Cop9 Signalosome. Here, we conditionally knock down Csn5 expression in HEK293 human cells using a doxycycline-inducible shRNA system. Cullin levels were not altered in CSN-deficient human cells, but the levels of multiple F-box proteins were decreased. Molecular analysis indicates that this decrease was due to increased Cul1- and proteasome-dependent turnover. Diminished F-box levels resulted in reduced SCF activity, as evidenced by accumulation of two substrates of the F-box protein Fbw7, cyclin E and c-myc, in Csn5-depleted cells. We propose that deneddylation of Cul1 is required to sustain optimal activity of SCF ubiquitin ligases by repressing 'autoubiquitination' of F-box proteins within SCF complexes, thereby rescuing them from premature degradation.
Tài liệu tham khảo
Pickart CM: Mechanisms underlying ubiquitination. Annu Rev Biochem. 2001, 70: 503-533. 10.1146/annurev.biochem.70.1.503.
Petroski MD, Deshaies RJ: Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol. 2005, 6 (1): 9-20. 10.1038/nrm1547.
Lyapina S, Cope G, Shevchenko A, Serino G, Tsuge T, Zhou C, Wolf DA, Wei N, Deshaies RJ: Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome. Science. 2001, 292 (5520): 1382-1385. 10.1126/science.1059780.
Groisman R, Polanowska J, Kuraoka I, Sawada J, Saijo M, Drapkin R, Kisselev AF, Tanaka K, Nakatani Y: The ubiquitin ligase activity in the DDB2 and CSA complexes is differentially regulated by the COP9 signalosome in response to DNA damage. Cell. 2003, 113 (3): 357-367. 10.1016/S0092-8674(03)00316-7.
Yang X, Menon S, Lykke-Andersen K, Tsuge T, Di X, Wang X, Rodriguez-Suarez RJ, Zhang H, Wei N: The COP9 signalosome inhibits p27(kip1) degradation and impedes G1-S phase progression via deneddylation of SCF Cul1. Curr Biol. 2002, 12 (8): 667-672. 10.1016/S0960-9822(02)00791-1.
Zhou C, Wee S, Rhee E, Naumann M, Dubiel W, Wolf DA: Fission Yeast COP9/Signalosome Suppresses Cullin Activity through Recruitment of the Deubiquitylating Enzyme Ubp12p. Mol Cell. 2003, 11 (4): 927-938. 10.1016/S1097-2765(03)00136-9.
Schwechheimer C, Serino G, Callis J, Crosby W, Lyapina S, Deshaies R, Gray W, Estelle M, Deng X-W: Interactions of the COP9 Signalosome with the E3 Ubiquitin Ligase SCFTIR1 in Mediating Auxin Response. Science. 2001
Podust VN, Brownell JE, Gladysheva TB, Luo RS, Wang C, Coggins MB, Pierce JW, Lightcap ES, Chau V: A Nedd8 conjugation pathway is essential for proteolytic targeting of p27Kip1 by ubiquitination. Proc Natl Acad Sci U S A. 2000, 97 (9): 4579-4584. 10.1073/pnas.090465597.
Wu K, Chen A, Pan ZQ: Conjugation of nedd8 to CUL1 enhances the ability of the ROC1-CUL1 complex to promote ubiquitin polymerization. J Biol Chem. 2000, 275 (41): 32317-32324. 10.1074/jbc.M004847200.
Kawakami T, Chiba T, Suzuki T, Iwai K, Yamanaka K, Minato N, Suzuki H, Shimbara N, Hidaka Y, Osaka F, et al.: NEDD8 recruits E2-ubiquitin to SCF E3 ligase. Embo J. 2001, 20 (15): 4003-4012. 10.1093/emboj/20.15.4003.
Liu J, Furukawa M, Matsumoto T, Xiong Y: NEDD8 modification of CUL1 dissociates p120(CAND1), an inhibitor of CUL1-SKP1 binding and SCF ligases. Mol Cell. 2002, 10 (6): 1511-1518. 10.1016/S1097-2765(02)00783-9.
Zheng J, Yang X, Harrell JM, Ryzhikov S, Shim EH, Lykke-Andersen K, Wei N, Sun H, Kobayashi R, Zhang H: CAND1 binds to unneddylated CUL1 and regulates the formation of SCF ubiquitin E3 ligase complex. Mol Cell. 2002, 10 (6): 1519-1526. 10.1016/S1097-2765(02)00784-0.
Wee S, Geyer RK, Toda T, Wolf DA: CSN facilitates Cullin-RING ubiquitin ligase function by counteracting autocatalytic adapter instability. Nat Cell Biol. 2005, 7 (4): 387-391. 10.1038/ncb1241.
Deng XW, Dubiel W, Wei N, Hofmann K, Mundt K, Colicelli J, Kato J, Naumann M, Segal D, Seeger M, et al.: Unified nomenclature for the COP9 signalosome and its subunits: an essential regulator of development. Trends Genet. 2000, 16 (5): 202-203. 10.1016/S0168-9525(00)01982-X.
Cope GA, Deshaies RJ: COP9 signalosome: a multifunctional regulator of SCF and other cullin-based ubiquitin ligases. Cell. 2003, 114 (6): 663-671. 10.1016/S0092-8674(03)00722-0.
Doronkin S, Djagaeva I, Beckendorf SK: CSN5/Jab1 mutations affect axis formation in the Drosophila oocyte by activating a meiotic checkpoint. Development. 2002, 129 (21): 5053-5064.
Oron E, Mannervik M, Rencus S, Harari-Steinberg O, Neuman-Silberberg S, Segal D, Chamovitz DA: COP9 signalosome subunits 4 and 5 regulate multiple pleiotropic pathways in Drosophila melanogaster. Development. 2002, 129 (19): 4399-4409.
Suh GS, Poeck B, Chouard T, Oron E, Segal D, Chamovitz DA, Zipursky SL: Drosophila JAB1/CSN5 acts in photoreceptor cells to induce glial cells. Neuron. 2002, 33 (1): 35-46. 10.1016/S0896-6273(01)00576-1.
Pintard L, Kurz T, Glaser S, Willis JH, Peter M, Bowerman B: Neddylation and Deneddylation of CUL-3 Is Required to Target MEI-1/Katanin for Degradation at the Meiosis-to-Mitosis Transition in C. elegans. Curr Biol. 2003, 13 (11): 911-921. 10.1016/S0960-9822(03)00336-1.
Cope GA, Suh GS, Aravind L, Schwarz SE, Zipursky SL, Koonin EV, Deshaies RJ: Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science. 2002, 298 (5593): 608-611. 10.1126/science.1075901.
Pintard L, Willis JH, Willems A, Johnson JL, Srayko M, Kurz T, Glaser S, Mains PE, Tyers M, Bowerman B, et al.: The BTB protein MEL-26 is a substrate-specific adaptor of the CUL-3 ubiquitin-ligase. Nature. 2003, 425 (6955): 311-316. 10.1038/nature01959.
Doronkin S, Djagaeva I, Beckendorf SK: The COP9 Signalosome Promotes Degradation of Cyclin E during Early Drosophila Oogenesis. Dev Cell. 2003, 4 (5): 699-710. 10.1016/S1534-5807(03)00121-7.
Wang X, Feng S, Nakayama N, Crosby WL, Irish V, Deng XW, Wei N: The COP9 Signalosome Interacts with SCF(UFO) and Participates in Arabidopsis Flower Development. Plant Cell. 2003, 15 (5): 1071-1082. 10.1105/tpc.009936.
Feng S, Ma L, Wang X, Xie D, Dinesh-Kumar SP, Wei N, Deng XW: The COP9 signalosome interacts physically with SCF COI1 and modulates jasmonate responses. Plant Cell. 2003, 15 (5): 1083-1094. 10.1105/tpc.010207.
Liu C, Powell KA, Mundt K, Wu L, Carr AM, Caspari T: Cop9/signalosome subunits and Pcu4 regulate ribonucleotide reductase by both checkpoint-dependent and -independent mechanisms. Genes Dev. 2003, 17 (9): 1130-1140. 10.1101/gad.1090803.
van de Wetering M, Oving I, Muncan V, Pon Fong MT, Brantjes H, van Leenen D, Holstege FC, Brummelkamp TR, Agami R, Clevers H: Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep. 2003, 4 (6): 609-615. 10.1038/sj.embor.embor865.
Wu K, Fuchs SY, Chen A, Tan P, Gomez C, Ronai Z, Pan ZQ: The SCF(HOS/beta-TRCP)-ROC1 E3 ubiquitin ligase utilizes two distinct domains within CUL1 for substrate targeting and ubiquitin ligation. Mol Cell Biol. 2000, 20 (4): 1382-1393. 10.1128/MCB.20.4.1382-1393.2000.
Spruck CH, Won KA, Reed SI: Deregulated cyclin E induces chromosome instability. Nature. 1999, 401 (6750): 297-300. 10.1038/45836.
Davis M, Hatzubai A, Andersen JS, Ben-Shushan E, Fisher GZ, Yaron A, Bauskin A, Mercurio F, Mann M, Ben-Neriah Y: Pseudosubstrate regulation of the SCF(beta-TrCP) ubiquitin ligase by hnRNP-U. Genes Dev. 2002, 16 (4): 439-451. 10.1101/gad.218702.
Rajagopalan H, Jallepalli PV, Rago C, Velculescu VE, Kinzler KW, Vogelstein B, Lengauer C: Inactivation of hCDC4 can cause chromosomal instability. Nature. 2004, 428 (6978): 77-81. 10.1038/nature02313.
He Q, Cheng P, Liu Y: The COP9 signalosome regulates the Neurospora circadian clock by controlling the stability of the SCFFWD-1 complex. Genes Dev. 2005, 19 (13): 1518-1531. 10.1101/gad.1322205.
Wu JT, Lin HC, Hu YC, Chien CT: Neddylation and deneddylation regulate Cul1 and Cul3 protein accumulation. Nat Cell Biol. 2005, 7 (10): 1014-1020. 10.1038/ncb1301.
Dohmann EM, Kuhnle C, Schwechheimer C: Loss of the CONSTITUTIVE PHOTOMORPHOGENIC9 Signalosome Subunit 5 Is Sufficient to Cause the cop/det/fus Mutant Phenotype in Arabidopsis. Plant Cell. 2005, 17 (7): 1967-1978. 10.1105/tpc.105.032870.
Lyapina SA, Correll CC, Kipreos ET, Deshaies RJ: Human CUL1 forms an evolutionarily conserved ubiquitin ligase complex (SCF) with SKP1 and an F-box protein. Proc Natl Acad Sci U S A. 1998, 95 (13): 7451-7456. 10.1073/pnas.95.13.7451.