CHARACTERIZATION OF THE FUNCTION AND REGULATION OF CULLIN RING E3 UBIQUITIN LIGASES CHOO YIN YIN B.Sc. (Honors), University of Malaya A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY !NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously.” _______________________________ CHOO YIN YIN 10th August 2013 ! ii! Acknowledgements First and foremost, I would like to deeply thank my supervisor Professor Thilo Hagen for his guidance and assistance through my Ph.D studies. Thilo provided an environment for me as a graduate student that exceeded all I could wish for. Without his help and encouragement, I definitely could not overcome so many obstacles in the projects. While my path towards graduation was not as smooth and straight as I would have preferred, I could not have navigated the bumps, curves, and changes in direction without your wealth of knowledge and seemingly unending support. His attitude and discipline will encourage me to continue the research work in the future. I am sincerely grateful to Thilo for giving me the opportunity to learn extensively in his lab and for providing me with everything I needed to help me strive to become a better scientist. Many thanks to my Thesis Advisory Committee members, Dr. Takao Inoue and Dr. Deng Lih Wen for their support, encouragement, and insight over the years. I would like to extend my sincere gratitude to Dr. Chew Eng Hui, who provided me help in my first few months in the lab when I first joined the Thilo’s lab. I would also like to thank members of the Thilo’s lab, past and present—Dr Boh Boon Kim, Christine Hu Zhi Wen, Chua Yee Liu, Daphne Wong Pei Wen, Wanpen Ponyeam, Tan Chia Yee, Hong Shin Yee, Ng Mei Ying, Natalie Weili Ng, Lucia Cordero Espinoza, Tan Li En, Regina Wong, Irena Tham, Natasha Vinanica, Chua Yee Shin, Jessica Leck, Jessica Lou, Jolane Eng, Tiffany Chai, Gan Fei Fei and Michelle Koh. They ! iii! provided an environment that always gave me tremendous insight into my research, helped me in the experiments and shared me with their experience. I am grateful for having the opportunity to work with so many exceptional colleagues. Finally, I would especially like to thank my family for their endless support, patience and numerous sacrifices all the time. Last but not the least, I would like to thank National University of Singapore for providing me chances of studying in Singapore. ! iv! Table of Contents i. Acknowledgement iii ii. Table of Contents v iii. Abstract x iv. List of Figures xiii v. xvii List of Abbreviations vi. List of Publications xix 1.0 Introduction 1.1 The Ubiquitin proteasome system 1.2 Ubiquitination 1.3 Proteasome 1.4 E3 Ubiquitin Ligases 1.5 Cullin RING E3 Ubiquitin Ligases !1.6 Structural characteristic of CRLs 1.7 Functions of CRLs 12 14-21 1.7.1 CRL1 1.7.2 CRL2 and CRL5 1.7.3 CRL3 1.7.4 CRL4 1.7.5 CRL7 ! 1.8 Ubiquitin-like protein Nedd8 22 1.9 COP9 Signalosome 24 1.10 CAND1 28 1.11 The Nedd8 Activating Enzyme (NAE1) inhibitor, MLN4924 30 2.0 Aims of The Study 34 3.0 Materials and Methods 35 v! 4.0 Chapter One: Characterization of the role of the COP9 signalosome in regulating cullin E3 ubiquitin ligase activity Introduction 44 Results 4.1.1 Overexpression of dominant negative form of Ubc12 (dnUbc12) decreases Cul1 neddylation. 49 4.1.2 Overexpression of dominant negative Ubc12 (dnUbc12) abolishes Cul5 neddylation. 50 4.1.3 Effect of dnUbc12 induction on cellular Nedd8 protein concentrations. 51 4.1.4 In vivo role of CSN. 53 Hypothesis 1: CSN promotes CRL activity by mediating cycle of neddylation and deneddylation. 4.1.4.1 Deneddylation rate of Cul1 and Cul2 in HEK293 cells. 54 4.1.4.2 Cul1 deneddylation is constitutive and not dependent on and coupled to substrate ubiquitination. 55 4.1.4.3 CAND1 siRNA does affect the Cul1 deneddylation rate. 59 4.1.4.4 Overexpression of Cdc34 does affect the Cul1 deneddylation rate 60 Hypothesis 2: CSN-mediated cullin deneddylation facilitates substrate- receptor exchange. ! 4.1.4.5 Role of CSN in promoting the exchange of the Cullin3 substrate receptor SPOP. 61 4.1.4.6 Role of CAND1 in promoting the exchange of the Cullin3 substrate receptor SPOP. 63 vi! Hypothesis 3: CSN prevents CAND1-mediated CRL disassembly. 4.1.4.7 CSN does not function to prevent binding of CAND1 to Cul1. 4.1.5 Cullin neddylation promotes CSN binding to cullin proteins in vivo. 4.1.6 65 66 Induction of Cullin deneddylation causes CSN dissociation from the CRL complex. 67 4.1.7 CSN5 preferentially binds to neddylated Cul1. 68 4.1.8 Cul2 and Cul3 C-terminal deletion mutants with constitutively active conformation show increased CSN binding in the absence of neddylation. 69 Preferential binding of CSN to active CRLs is not a consequence of increased amounts of bound polyubiquitinated substrates. 70 4.1.9 Discussion 72 5.0 Chapter Two: Mechanism of Cullin3 E3 Ubiquitination Ligase Dimerization ! Introduction 77 Results 5.1.1 Cul3 mutants that are unable to bind to the BTB substrate receptor protein exhibit markedly reduced Cul3-Cul3 association. 82 5.1.2 Cul3 mutants exhibit reduced binding to Keap1 in HEK293T cells. 83 5.1.3 Cul3-Cul3 binding is independent of the WH-B domain. 84 5.1.4 Cul2(NT)-Cul3(CT)-V5 is protected from the proteasome dependent degradation. 85 5.1.5 Cul3 N-terminus is necessary for Cul3-Cul3 binding. 87 vii! 5.1.6 Mutation of the neddylation site in Cul3 does not affect Cul3 dimerization. 88 5.1.7 Cullin neddylation is not involved in Cul3-Cul3 binding 89 5.1.8 Cul3-Cul3 binding is independent of cullin neddylation in vivo. 90 5.1.9 Two Cul3 proteins are involved in assembly of a CRL3-SPOP complex in vivo. 91 5.2.0 5.2.1 Two Cul3 proteins are involved in assembly of a CRL3-Keap1 complex in vivo. 93 Estimation of the proportion of Cul3 that exists in multimeric Cul3/Rbx1-BTB protein complexes in vivo. 94 Discussion 97 6.0 Chapter Three: Identification and characterization of SCFF-box protein substrates Introduction 102 Results ! 6.1.1 Flag-GKAP1 accumulates upon MLN4924 treatment 106 6.1.2 Flag-GKAP1 accumulates upon Cycloheximide treatment 107 6.1.3 Flag-GKAP1 does not bind to β-TrCP 108 6.2.1 Flag-ACBD5 accumulates upon MLN4924 treatment 110 6.2.2 Flag-ACBD5 is insensitive to MLN4924 111 6.3.1 Tulp1-Flag accumulates slightly upon MLN4924 treatment 112 6.3.2 Tulp1-Flag does not depend on the DSGXX(X)S recognition motif to bind to β-TrCP 113 viii! ! 6.4.1 SLBP accumulates upon MLN4924 treatment 115 6.4.2 SLBP accumulates upon Cyclin F siRNA knockdown treatment 115 6.4.3 Cyclin F does not regulate SLBP abundance in a cell cycle dependent manner 117 6.4.4 SLBP-V5 does not bind to HA-Cyclin F or Cul1 118 6.4.5 siRNA mediated knockdown of 41 different F-box does not lead to SLBP accumulation 120 6.4.6 Transfected dominant negative form of Cul1 (dnCul1-V5) increases SLBP protein expression slightly 121 6.4.7 V5-SLBP does not bind to endogenous Cul2, Cul3, Cul4, Cul5 or Cul7. 124 6.4.8 V5-SLBP does not bind to Cdh1. 124 6.4.9 Cycle Inhibiting Factor causes SLBP accumulation. 125 Discussion 127 7.0 General Conclusion 131 8.0 References 134 ix! Abstract Cullin RING ubiquitin ligases (CRLs) constitute the largest family of cellular ubiquitin ligases with diverse cellular functions. CRLs comprise of seven homologous cullin-based complexes. The cullin proteins serve as scaffolds for the assembly of the RING protein and substrate receptor subunits. CRLs are activated via the conjugation with the ubiquitin-like protein Nedd8 onto the cullin scaffold protein. Cullin neddylation leads to a conformational change in the cullin C-terminus/Rbx1 structure that is essential for facilitating the ubiquitin transfer onto the substrate. However, cullin neddylation is not permanent. It is reversed via the COP9 Signalosome (CSN). Although CSN-mediated cullin deneddylation inhibits CRL activity in vitro, it is important for CRL function in vivo. It has been suggested that cycles of neddylation and deneddylation are essential to regulate CRL activity in vivo. However, the mechanism through which CSN regulates CRL activity in vivo remains incompletely understood. In this study, we used a mammalian cellular system to study the mechanisms through which CRL activity is regulated by CSN and Nedd8 in vivo. We confirmed that the Nedd8 modification of cullin proteins is highly dynamic. We showed that CSN-mediated cullin deneddylation is not directly coupled to substrate polyubiquitination. We found that the CSN complex binds preferentially to the active form of CRLs that is in the neddylation-induced conformation. We propose that the binding of CSN to active CRLs may be important to recruit CSN-associated proteins that are essential to regulate CRL activity. CSN would subsequently mediate cullin deneddylation to promote its own ! x! Cope, G. A., Suh, G. S., Aravind, L., Schwarz, S. E., Zipursky, S. L., Koonin, E. V. and Deshaies, R. J. (2002). Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science 298, 608–611. Cope, G. A. and Deshaies, R. J. (2003). COP9 signalosome: a multifunctional regulator of SCF and other cullin-based ubiquitin ligases. Cell 114, 663–671. Correale, S., Pirone, L., Di Marcotullio, L., De Smaele, E., Greco, A., Mazzà, D., Moretti, M., Alterio, V., Vitagliano, L., Di Gaetano, S., Gulino, A. and Pedone, E. M. (2011). Molecular organization of the cullin E3 ligase adaptor KCTD11. Biochimie. 93, 715-724. Chuang, H. W., Zhang, W., Gray, W. M. (2004). Arabidopsis ETA2, an apparent ortholog of the human cullin-interacting protein CAND1, is required for auxin responses mediated by the SCF(TIR1) ubiquitin ligase. Plant Cell 16, 1883–1897. Cullinan, S. B., Gordan, J. D., Jin, J., Harper, J. W., and Diehl, J. A. (2004). The Keap1- BTB protein is an adaptor that bridges Nrf2 to a Cul3-based E3 ligase: oxidative stress sensing by a Cul3-Keap1 ligase. Mol Cell Biol. 24, 8477-8486. Dai, C. L., Shi, J., Chen, Y., Iqbal, K., Liu, F. and Gong, C.X. (2013) Inhibition of protein synthesis alters protein degradation through activation of protein kinase B (AKT). J Biol. Chem. In-press. Dealy, M. J., Nguyen, K. V., Lo, J., Gstaiger, M., Krek, W., Elson, D., Arbeit, J., Kipreos, E. T., Johnson, R. S. (1999). Loss of Cul1 results in early embryonic lethality and dysregulation of cyclin E. Nat. Genet. 23, 245-248. D'Angiolella, V., Esencay, M. and Pagano, M. (2012). A cyclin without cyclin-dependent kinases: cyclin F controls genome stability through ubiquitin-mediated proteolysis. Trends Cell Biol. 23, 135-140. Deng, H., Liang, H. and Jankovic, J. (2013) F-box only protein gene in parkinsonian-pyramidal disease. JAMA Neurol. 70, 20-24. Deng, L., Wang, C., Spencer, E., Yang, L., Braun, A., You, J., Slaughter, C., Pickart, C. and Chen, Z. J. (2000). Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351-361. ! 136! Dohmann, E. M., Levesque, M. P., De Veylder, L., Reichardt, I., Jürgens, G., Schmid, M. and Schwechheimer, C. (2008). The Arabidopsis COP9 signalosome is essential for G2 phase progression and genomic stability. Development 135, 2013–2022. Duan, S., Skaar, J. R., Kuchay, S., Toschi, A., Kanarek, N., Ben-Neriah, Y. and Pagano, M. (2011). mTOR Generates an Auto-Amplification Loop by Triggering the bTrCP- and CK1a-Dependent Degradation of DEPTOR. Mol. Cell 44, 317–324. Duda, D. M., Borg, L. A., Scott, D. C., Hunt, H. W., Hammel, M. and Schulman, B. A. (2008). Structural insights into NEDD8 activation of cullinRING ligases:conformational control of conjugation. Cell 134, 995–1006. Emberley, E. D., Mosadeghi, R. and Deshaies, R. J. (2012) Deconjugation of Nedd8 from Cul1 is directly regulated by Skp1-F-box and substrate, and the COP9 signalosome inhibits deneddylated SCF by a noncatalytic mechanism. J. Biol. Chem. 287, 29679-29689. Enchev, R. I., Scott, D. C., da Fonseca, P. C., Schreiber, A., Monda, J. K., Schulman, B. A., Peter, M. and Morris, E.P. (2012). Structural basis for a reciprocal regulation between SCF and CSN. Cell Rep. 2, 616-627. Feng, H., Zhong, W., Punkosdy, G., Gu, S., Zhou, L., Seabolt, E. K. and Kipreos, E. T. (1999). CUL-2 is required for the G1-to-S-phase transition and mitotic chromosome condensation in Caenorhabditis elegans. Nat. Cell Biol. 1, 486-492. Feng, S., Shen, Y., Sullivan, J. A., Rubio, V., Xiong, Y., Sun, T-P. and Deng, X. W. (2004). Arabidopsis CAND1, an unmodified CUL1-interacting protein, is involved in multiple developmental pathways controlled by ubiquitin/proteasome-mediated protein Degradation. Plant Cell 16, 1870– 1882. Freemont, P. S. (1993). The RING finger. A novel protein sequence motif related to the zinc finger. Ann N Y Acad Sci 684, 174-92. Fuchs, S.Y., Chen, A., Xiong, Y., Pan, Z. Q. and Ronai, Z. (1999). HOS, a human homolog of Slimb, forms an SCF complex with Skp1 and Cullin1 and targets the phosphorylation-dependent degradation of IkappaB and betacatenin. Oncogene 18, 2039-2046. Furukawa, M, He, Y. J., Borchers, C., and Xiong, Y. (2003). Targeting of protein ubiquitination. by BTB–Cullin 3–Roc1 ubiquitin ligases. Nat Cell Biol. 5, 1001–1007. ! 137! Gan-Erdene, T., Nagamalleswari, K., Yin, L., Wu, K., Pan, Z. Q. and Wilkinson, K. D. (2003). Identification and characterization of DEN1, a deneddylase of the ULP family. J. Biol. Chem. 278, 28892-28900. Gao, D., Inuzuka, H., Tan, M-K. M., Fukushima, H., Locasale, J. W., Liu, P., Wan, L., Zhai, B., Chin, Y. R., Shaik, S., Lyssiotis, C. A., Gygi, S. P., Toker, A., Cantley, L. C., Asara, J. M., Harper, J. W. and Wei, W. (2011). mTOR Drives Its Own Activation via SCFbTrCP-Dependent Degradation of the mTOR Inhibitor DEPTOR. Mol. Cell 44, 290–303. Glickman, M. H. and Ciechanover, A. (2002). The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol. Rev. 82, 373-428. Goldenberg, S. J., Cascio, T. C., Shumway, S. D., Garbutt, K. C., Liu, J., Xiong, Y. and Zheng, N. (2004). Structure of the Cand1-Cul1-Roc1 Complex Reveals Regulatory Mechanisms for the Assembly of the Multisubunit Cullin-Dependent Ubiquitin Ligases. Cell 119, 517–528. Goldstein, G., Scheid, M., Hammerling, U., Schlesinger, D. H., Niall, H. D. and Boyse E. A. (1975). Isolation of a polypeptide that has lymphocytedifferentiating properties and is probably represented universally in living cells. Proc. Natl. Acad. Sci. U S A. 72, 11-15. Gong, L. and Yeh, E. T. (1999). Identification of the activating and conjugating enzymes of the NEDD8 conjugation pathway. J. Biol. Chem. 274, 12036–12042. Guardavaccaro, D. and Pagano, M. (2004). Oncogenic aberrations of cullindependent ubiquitin ligases. Oncogene, 23, 2037-2049. Guardavaccaro, D. and Pagano, M. (2006). Stabilizers and destabilizers controlling cell cycle oscillators. Mol. Cell 22, 1-4. Haglund, K., Di Fiore, P. P., and Dikic, I. (2003). Distinct monoubiquitin signals in receptor endocytosis. Trends Biochem. Sci. 28, 598–603. Hao, B., Oehlmann, S., Sowa, M. E., Harper, J. W. and Pavletich, N.P. (2007) Structure of a Fbw7-Skp1-cyclin E complex: multisitephosphorylated substrate recognition by SCF ubiquitin ligases. 26, 131-143. Hayes, J. D. and McMahon, M. (2009). NRF2 and KEAP1 mutations: permanent activation of an adaptive response in cancer. Trends Biochem. Sci. 34, 176-188. ! 138! Hershko, A. and Ciechanover, A. (1998). The ubiquitin system. Annu. Rev. Biochem. 67, 425–479. Hetfeld, B. K., Helfrich, A., Kapelari, B., Scheel, H., Hofmann, K., Guterman, A., Glickman, M., Schade, R., Kloetzel, P. M. and Dubiel, W. (2005). The zinc finger of the CSN-associated deubiquitinating enzyme USP15 is essential to rescue the E3 ligase Rbx1. Curr. Biol. 15, 1217–1221. Hicke, L. (1999). Gettin' down with ubiquitin: turning off cell-surface receptors, transporters and channels. Trends Cell Biol, 1999. 9,107-112. Hicke, L., and Dunn, R. (2003). Regulation of membrane protein transport by ubiquitin and ubiquin-binding proteins. Annu. Rev. Cell Dev. Biol. 19, 141–172. Higa, L. A., Yang, X., Zheng, J., Banks, D., Wu, M., Ghosh, P., Sun, H. and Zhang, H. (2006) Involvement of CUL4 ubiquitin E3 ligases in regulating CDK inhibitors Dacapo/p27Kip1 and cyclin E degradation. Cell Cycle 5, 7177. Hori, T., F. Osaka, T. Chiba, C. Miyamoto, K. Okabayashi, N. Shimbara, S. Kato, and K. Tanaka. (1999). Covalent modification of all members of human cullin family proteins by NEDD8. Oncogene 18, 6829-34. Hsu, J. M., Lee, Y. C., Yu, C. T., Huang, C. Y. (2004) Fbx7 functions in the SCF complex regulating Cdk1-cyclin B-phosphorylated hepatoma upregulated protein (HURP) proteolysis by a proline-rich region. J Biol Chem. 279, 32592-32602. Hu, J., McCall, C. M., Ohta, T. and Xiong, Y. (2004). Targeted ubiquitination of CDT1 by the DDB1-CUL4A-ROC1 ligase in response to DNA damage. Nat Cell Biol. 6, 1003-1009. Huang, D. T., Ayrault, O., Hunt, H. W., Taherbhoy, A. M., Duda, D. M., Scott, D. C., Borg, L. A., Neale, G., Murray, P. J., Roussel, M. F. and Schulman, B. A. (2009). E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification. Mol. Cell 33, 483-495. Huber, C., Dias-Santagata, D., Glaser, A., O’Sullivan, J., Brauner, R., Wu, K., Xu, X., Pearce, K., Wang, R., Uzielli, M. L., Dagoneau, N., Chemaitilly, W., Superti-Furga, A., Dos Santos, H., Mégarbané, A., Morin, G., GillessenKaesbach, G., Hennekam, R., Van der Burgt, I., Black, G. C., Clayton, P. E., Read, A., Le Merrer, M., Scambler, P. J., Munnich, A., Pan, Z. Q., Winter, R. and Cormier-Daire, V. (2005). Identification of mutations in CUL7 in 3! 139! M syndrome. Nat. Genet. 37, 1119-1124. Huber, C., Delezoide, A. L., Guimiot, F., Baumann, C., Malan, V., Le Merrer, M., Da Silva, D. B., Bonneau, D., Chatelain, P., Chu, C., Clark, R., Cox, H., Edery, P., Edouard, T., Fano, V., Gibson, K., Gillessen-Kaesbach, G., Giovannucci-Uzielli, M. L., Graul-Neumann, L. M., van Hagen, J. M., van Hest, L., Horovitz, D., Melki, J., Partsch, C. J., Plauchu, H., Rajab, A., Rossi, M., Sillence, D., Steichen-Gersdorf, E. and Stewart, H., et al. (2009). A large-scale mutation search reveals genetic heterogeneity in 3M syndrome. Eur. J. Hum. Genet. 17, 395-400. Huibregtse, J. M., Scheffner, M. Beaudenon, S. and Howley, P. M. (1995). A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc. Natl. Acad. Sci. USA 92, 2563-2567. Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J. M., Lane, W. S. and Kaelin, W. G. Jr. (2001). HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 292, 464- 468. Jaakkola, P., Mole, D. R., Tian, Y. M., Wilson, M. I., Gielbert, J., Gaskell, S. J., Kriegsheim, Av., Hebestreit, H. F., Mukherji, M., Schofield, C. J., Maxwell, P. H., Pugh, C. W. and Ratcliffe, P. J. (2001). Targeting of HIFalpha to the von Hippel- Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292, 468-472. Jia, L., Li, H. and Sun, Y. (2011) Induction of p21-dependent senescence by an NAE inhibitor, MLN4924, as a mechanism of growth suppression. Neoplasia. 13, 561-569. Jin, J., Cardozo, T., Lovering, R. C., Elledge, S. J., Pagano, M. and Harper, J. W. (2004). Systematic analysis and nomenclature of mammalian F-box proteins. Genes & Dev. 18, 2573–2580. Jin, L., Williamson, A., Banerjee, S., Philipp, I. and Rape, M. (2008). Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex. Cell 133, 653-665. Johnson J. L., Lu C., Raharjo, E., McNally, K., McNally, F. J. and Mains, P. E. (2009). Levels of the ubiquitin ligase substrate adaptor MEL-26 are inversely correlated with MEI-1/katanin microtubule-severing activity during both meiosis and mitosis. Dev. Biol. 330, 349–357. Jubelin, G., Taieb, F., Duda, D. M., Hsu, Y., Samba-Louaka, A., Nobe, R., Penary, M., Watrin, C., Nougayrède, J. P., Schulman, B. A., Stebbins, C. E. ! 140! and Oswald, E. (2010). Pathogenic bacteria target NEDD8-conjugated cullins to hijack host-cell signaling pathways. PLoS Pathog. 6:e1001128. Kawakami, T., Chiba, T., Suzuki, T., Iwai, K., Yamanaka, K., Minato, N., Suzuki, H., Shimbara, N., Hidaka, Y., Osaka, F., Omata, M. and Tanaka, K. (2001). NEDD8 recruits E2-ubiquitin to SCF E3 ligase. EMBO J. 20, 4003– 4012. Kigoshi, Y., Tsuruta, F., and Chiba, T. (2011). Ubiquitin ligase activity of Cul3-KLHL7 protein is attenuated by autosomal dominant retinitis pigmentosa causative mutation. J Biol Chem. 286, 33613-33621. Kipreos, E. T., Lander, L. E., Wing, J. P., He, W. W. and Hedgecock, E. M. (1996). cul-1 is required for cell cycle exit in C. elegans and identifies a novel gene family. Cell 85, 829-839. Kohler, A., Cascio, P., Leggett, D. S., Woo, K. M., Goldberg, A. L. and Finley, D. (2001). The axial channel of the proteasome core particle is gated by the Rpt2 ATPase and controls both substrate entry and product release. Mol. Cell 7, 1143-1152. Kumar, S., Tomooka, Y. and Noda, M. (1992). Identification of a set of genes with developmentally down-regulated expression in the mouse brain. Biochem. Biophys. Res. Commun. 185, 1155-1161. Kurz, T., Pintard, L., Willis, J. H., Hamill, D. R., Gönczy, P., Peter, M. and Bowerman, B. (2002). Cytoskeletal regulation by the Nedd8 ubiquitin-like protein modification pathway. Science 295, 1294–1298 Kwon, J. E., La, M., Oh, K. H., Oh, Y. M., Kim, G. R., Seol, J. H., Baek, S. H., Chiba, T., Tanaka, K., Bang, O. S., Joe, C. O. and Chung, C. H. (2006). BTB domaincontaining speckle-type POZ protein (SPOP) serves as an adaptor of Daxx for ubiquitination by Cul3-based ubiquitin ligase. J. Biol. Chem. 281, 12664-12672. Levine, A. J. and Orena, M. (2009). The first 30 years of p53: growing ever more complex. Nat. Rev. Cancer. 9, 749-758. Lin, J. J., Milhollen, M. A., Smith, P. G., Narayanan, U. and Dutta, A. (2010) NEDD8-targeting drug MLN4924 elicits DNA rereplication by stabilizing Cdt1 in S phase, triggering checkpoint activation, apoptosis, and senescence in cancer cells. Cancer Res. 70, 10310-10320. Liu, J., Furukawa, M., Matsumoto, T. and Xiong Y. (2002). NEDD8 Modification of CUL1 Dissociates p120CAND1, an Inhibitor of CUL1! 141! SKP1 Binding and SCF Ligases. Mol. Cell 10, 1511–1518. Liu, J., Ghanim, M., Xue, L., Brown, C. D., Iossifov, I., Angeletti, C., Hua, S., Nègre, N., Ludwig, M., Stricker, T., Al-Ahmadie, H. A., Tretiakova, M., Camp, R. L., Perera- Alberto, M., Rimm, D. L., Xu, T., Rzhetsky, A., and White, K. P. (2009). Analysis of Drosophila segmentation network identifies a JNK pathway factor overexpressed in kidney cancer. Science. 323, 12181222. Liu, J., Vasudevan, S. and Kipreos, E. T. (2004). CUL-2 and ZYG-11 promote meiotic anaphase II and the proper placement of the anteriorposterior axis in C. elegans. Development 131, 3513-3525. Lo, S. C., Li, X., Henzl, M. T., Beamer, L. J., and Hannink, M. (2006). Structure of the Keap1:Nrf2 interface provides mechanistic insight into Nrf2 signaling. EMBO J. 25, 3605-3617. Lo, S. C. and Hannink, M. (2006). CAND1-mediated substrate adaptor recycling is required for efficient repression of Nrf2 by Keap1. Mol. Cell. Biol. 26, 1235–1244. Lonergan, K. M., Iliopoulos, O., Ohh, M., Kamura, T., Conaway, R. C., Conaway, J. W. and Kaelin, W. G. Jr. (1998). Regulation of hypoxiainducible mRNAs by the von Hippel-Lindau tumor suppressor protein requires binding to complexes containing elongins B/C and Cul2. Mol Cell Biol, 8, 732-41. Lyapina, S., Cope, G., Shevchenko, A., Serino, G., Tsuge, T., Zhou, C., Wolf, D. A., Wei, N., Shevchenko, A. and Deshaies, R. J. (2001). Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome. Science, 292, 1382–1385. Markovina, S., Callander, N. S., O'Connor, S. L., Kim, J., Werndli, J. E., Raschko, M., Leith, C. P., Kahl, B. S., Kim, K. and Miyamoto, S. (2008). Bortezomib-resistant nuclear factor-kappaB activity in multiple myeloma cells. Mol. Cancer Res. 6, 1356-1364. Märten A., Zeiss, N., Serba, S., Mehrle, S., von Lilienfeld-Toal, M. and Schmidt, J. (2008). Bortezomib is ineffective in an orthotopic mouse model of pancreatic adenocarcinoma. Mol. Cancer Ther. 7, 3624-31. Mathias, N., Johnson, S. L., Winey, M., Adams, A. E., Goetsch, L., Pringle, J. R., Byers, B. and Goebl, M. G. (1996). Cdc53p acts in concert with Cdc4p and Cdc34p to control the G1-to-S-phase transition and identifies a conserved family of proteins. Mol. Cell Biol. 16, 6634-6643. ! 142! Maytal-Kivity, V., Reis, N., Hofmann, K. and Glickman, M. H. (2002). MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and prokaryotes, is critical for Rpn11 function.BMC Biochem. 20:28. McKibbin, M., Ali, M., Mohamed, M. D., Booth, A. P., Bishop, F., Pal, B., Springell, K., Raashid, Y., Jafri, H. and Inglehearn, C.F. (2010). Genotypephenotype correlation for leber congenital amaurosis in Northern Pakistan. Arch. Ophthalmol. 128, 107-113. Min, K. W., Kwon, M. J., Park, H. S., Park, Y., Yoon, S. K. and Yoon, J. B. (2005). CAND1 enhances deneddylation of CUL1 by COP9 signalosome. Biochem. Biophys. Res. Commun. 334, 867–874. Miyauchi, Y., Kato, M., Tokunaga, F. and Iwai, K. (2008). The COP9/signalosome increases the efficiency of von Hippel-Lindau protein ubiquitin ligase- mediated hypoxia-inducible factor- ubiquitination. J. Biol. Chem. 283, 16622–16631. Moshe, Y., Bar-On, O., Ganoth, D. and Hershko, A. (2011). Regulation of the action of early mitotic inhibitor on the anaphase-promoting complex/cyclosome by cyclin-dependent kinases. J Biol Chem. 286, 1664716657. Nag, A., Bondar, T., Shiv, S. and Raychaudhuri, P. (2001). The xeroderma pigmentosum group E gene product DDB2 is a specific target of cullin 4A in mammalian cells. Mol Cell Biol. 21, 6738-6747. Nickell, S., Beck, F., Scheres, S.H., Korinek, A., Fo ̈rster, F., Lasker, K., Mihalache, O., Sun, N., Nagy, I., Sali, A., et al. (2009). Insights into the molecular architecture of the 26S proteasome. Proc. Natl. Acad. Sci. USA 106, 11943–11947. Nishitani, H., Sugimoto, N., Roukos, V., Nakanishi, Y., Saijo, M., Obuse, C., Tsurimoto, T., Nakayama, K. I., Nakayama, K., Fujita, M., Lygerou, Z. and Nishimoto, T. (2006). Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis. EMBO J. 25, 1126-1136. Orlicky, S., Tang, X., Neduva, V., Elowe, N., Brown, E. D., Sicheri, F., Tyers, M. (2010). An allosteric inhibitor of substrate recognition by the SCG(Cdc4) ubiquitin ligase. Nat Biotechnol. 28, 733-737. Orsborn, A. M., Li, W., McEwen, T. J., Mizuno, T., Kuzmin, E., Matsumoto, K. and Bennett, K. L. (2007). GLH-1, the C. elegans P granule protein, is ! 143! controlled by the JNK KGB-1 and by the COP9 subunit CSN-5. Development 134, 3383–3392. Petroski, M. D. and Deshaies, R. J. (2005). Function and regulation of cullinRING ubiquitin ligases. Nat Rev Mol Cell Biol 6, 19–20. Pickart, C. M. (2001). Mechanisms underlying ubiquitination. Annu. Rev. Biochem., 70, 503- 33. Pickart, C. M. and Fushman, D. (2004). Polyubiquitin chains: polymeric protein signals. Curr. Opin. Chem. Biol. 8, 610-616. Review. Pierce, N. W., Lee, J. E., Liu, X., Sweredoski, M. J., Graham, R. L., Larimore, E. A., Rome, M., Zheng, N., Clurman, B. E., Hess, S., Shan, S.O. and Deshaies, R. J. (2013). Cand1 promotes assembly of new SCF complexes through dynamic exchange of F box proteins. Cell 153, 206-215. Pintard, L., Kurz, T., Glaser, S., Willis, J. H., Peter, M., and Bowerman, B. (2003). Neddylation and deneddylation of CUL-3 is required to target MEI1/Katanin for degradation at the meiosis-to-mitosis transition in C. elegans. Curr. Biol. 13, 911-921. Pippucci, T., Savoia A., Perrotta, S., Pujol-Moix, N., Noris, P., Castegnaro, G., Pecci, A., Gnan, C., Punzo, F., Marconi, C., Gherardi, S., Loffredo, G., De Rocco, D., Scianguetta, S., Barozzi, S., Magini, P., Bozzi, V., Dezzani, L., Di Stazio, M., Ferraro, M., Perini, G., Seri, M. and Balduini, C. L. (2011) Mutations in the 5' UTR of ANKRD26, the ankirin repeat domain 26 gene, cause an autosomal-dominant form of inherited thrombocytopenia, THC2. Am. J Hum. Genet. 88, 115-120. Richardson, P. G., Mitsiades, C., Ghobrial, I. and Anderson, K. (2006). Beyond single-agent bortezomib: combination regimens in relapsed multiple myeloma. Curr. Opin. Oncol. 18, 598-608. Review. Richardson, P. G., Schlossman, R., Hideshima, T. and Anderson, K.C. (2005). New treatments for multiple myeloma. Oncology (Williston Park) 19, 1781-1792. Saha, A. and Deshaies, R. J. (2008). Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation. Mol. Cell 32, 21–31. Sarikas, A., Hartmann, T. and Pan, Z-Q. (2011). The cullin protein family. Genome Biol. 12, 220. Sarikas, A., Xu, X., Field, L. J. and Pan, Z, Q. (2008). The cullin7 E3 ! 144! ubiquitin ligase: a novel player in growth control. Cell Cycle 7, 3154-3161. Scheel, H. and Hofmann, K. (2005). Prediction of a common structural scaffold for proteasome lid, COP9-signalosome and eIF3 complexes. BMC Bioinformatics 24:71. Scheffner, M., U. Nuber, and J.M. Huibregtse. (1995). Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature 373, 813. Schmidt, M. W., McQuary, P. R., Wee, S., Hofmann, K. and Wolf, D. A. (2009). F-box-di- rected CRL complex assembly and regulation by the CSN and CAND1. Mol. Cell 35, 586–597. Schulman, B. A., Carrano, A. C., Jeffrey, P. D., Bowen, Z., Kinnucan, E. R., Finnin, M. S., Elledge, S. J., Harper, J. W., Pagano, M., Pavletich, N. P. (2000). Insights into SCF ubiquitin ligases from the structure of the Skp1Skp2 complex. Nature 408, 381- 386. Schulman, B. A. and Harper, J. W. (2009). Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. Nature Rev. Mol. Cell Biol. 10, 319–331. Schwartz, A.L. and Ciechanover, A. (1999). The ubiquitin-proteasome pathway and pathogenesis of human diseases. Annu. Rev. Med. 50, 57–74. Schwechheimer, C. and Deng, X. W. (2001). COP9 signalosome revisited: a novel mediator of protein degradation. Trends Cell Biol 11, 420-6. Shanker, A., Brooks, A. D., Tristan, C. A., Wine, J. W., Elliott, P. J., Yagita, H., Takeda, K., Smyth, M. J., Murphy, W. J. and Sayers, T. J. (2008). Treating metastatic solid tumors with bortezomib and a tumor necrosis factor-related apoptosis-inducing ligand receptor agonist antibody. J. Natl. Cancer Inst. 100, 649-662. Siergiejuk, E., Scott, D. C., Schulman, B. A., Hofmann, K., Kurz, T. and Peter, M. (2009). Cullin neddylation and substrate-adaptors counteract SCF inhibition by the CAND1-like protein Lag2 in Saccharomyces cerevisiae. EMBO J. 28, 3845-3856. Singer, J. D., Gurian-West, M., Clurman, B. and Roberts, J. M. (1999). Cullin-3 targets cyclin E for ubiquitination and controls S phase in mammalian cells. Genes Dev. 13, 2375–2387. Small, E., Eggler, A., and Mesecar, A. D. (2010). Development of an ! 145! efficient E. coli expression and purification system for a catalytically active, human Cullin3-RINGBox1 protein complex and elucidation of its quaternary structure with Keap1. Biochem Biophys Res Commun. 400, 471-475. Soucy, T. A., Smith, P. G., Milhollen, M. A., Berger, A. J., Gavin, J. M., Adhikari, S., Brownell, J. E., Burke, K. E., Cardin, D. P., Critchley, S., Cullis, C. A., Doucette, A., Garnsey, J. J., Gaulin, J. L., Gershman, R. E., Lublinsky, A. R., McDonald, A., Mizutani, H., Narayanan, U., Olhava, E. J., Peluso, S., Rezaei, M., Sintchak, M. D., Talreja, T., Thomas, M. P., Traore, T., Vyskocil, S., Weatherhead, G. S., Yu, J., Zhang, J., Dick, L. R., Claiborne, C. F., Rolfe, M., Bolen, J. B. & Langston, S. P. (2009). An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 458, 732–737. Soucy, T. A., Dick, L. R., Smith, P.G., Milhollen, M. A. and Brownell, J. E. (2010). The NEDD8 Conjugation Pathway and Its Relevance in Cancer Biology and Therapy. Genes Cancer. 1, 708-716. Spence, J., Sadis, S., Haas, A. L. and Finley, D. (1995). A ubiquitin mutant with specific defects in DNA repair and multiubiquitination. Mol Cell Biol 15, 1265-1273. Strous, G. J. and Govers, R. (1999). The ubiquitin-proteasome system and endocytosis. J. Cell Sci. 112, 1417-1423. Stogios, P. J., Downs, G. S., Jauhal, J. J., Nandra, S. K., and Privé, G. G. (2005) Sequence and structural analysis of BTB domain proteins. Genome Biol. 10, R82. Epub. Sullivan, K. D., Mullen, T. E., Marzluff, W. F. and Wagner, E. J. (2009). Knockdown of SLBP results in nuclear retention of histone mRNA. RNA 15, 459-472. Swords, R. T., Kelly, K. R., Smith, P. G., Garnsey, J. J., Mahalingam, D., Medina, E., Oberheu, K., Padmanabhan, S., O'Dwyer, M., Nawrocki, S. T., Giles, F. J. and Carew, J.S. (2010). Inhibition of NEDD8-activating enzyme: a novel approach for the treatment of acute myeloid leukemia. Blood 115, 3796-3800. Takagi, Y., Pause, A., Conaway, R. C. and Conaway, J. W. (1997) Identification of elongin C sequences required for interaction with the von Hippel-Lindau tumor suppressor protein. J. Biol. Chem., 272, 27444-27449. ! Tang, X., Orlicky, S., Lin, Z., Willems, A., Neculai, D., Ceccarelli, D., Mercurio, F., Shilton, B. H., Sicheri, F., and Tyers, M. (2007). Suprafacial 146! orientation of the SCFCdc4 dimer accommodates multiple geometries for substrate ubiquitination. Cell. 129, 1165- 1176. Tan, C. Y. and Hagen, T. (2013). Destabilization of CDC6 upon DNA damage is dependent on neddylation but independent of Cullin E3 ligases. Int. J Biochem. Cell Biol. 45, 1489-1498. Tsvetkov, L. M., Yeh, K. H., Lee, S. J., Sun, H. and Zhang, H. (1999). p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27. Curr. Biol. 9, 661–664. Vaziri, C., Saxena, S., Jeon, Y., Lee, C., Murata, K., Machida, Y., Wagle, N., Hwang, D. S. and Dutta A. (2003). A p53-dependent checkpoint pathway prevents rereplication. Mol. Cell. 11, 997-1008. Vodermaier, H. C. (2004) APC/C and SCF: controlling each other and the cell cycle. Curr. Biol. 14, 787-796. Wada, H., Yeh, E. T. and Kamitani, T. (2000). A dominant-negative UBC12 mutant sequesters NEDD8 and inhibits NEDD8 conjugation in vivo. J Biol Chem 275, 17008–17015. Wagner, E. J., Berkow, A. and Marzluff, W. F. (2005). Expression of an RNAi-resistant SLBP restores proper S-phase progression. Biochem Soc Trans 33, 471-473. Wang, Y., Penfold, S., Tang, X., Hattori, N., Riley, P., Harper, J. W., Cross, J. C. and Tyers, M. (1999). Deletion of the Cul1 gene in mice causes arrest in early embryogenesis and accumulation of cyclin E. Curr Biol. 9, 1191-1194. Wei, D., Li, H., Yu, J., Sebolt, J. T., Zhao, L., Lawrence, T. S., Smith, P. G., Morgan, M. A. and Sun, Y. (2012). Radiosensitization of human pancreatic cancer cells by MLN4924, an investigational NEDD8-activating enzyme inhibitor. Cancer Res. 72, 282-293. Wei, N. and Deng, X. W. (1992). COP9: a new genetic locus involved in light-regulated development and gene expression in Arabidopsis. Plant Cell 4, 1507–1518. Wei, N. and Deng, X. W. (2003) The COP9 signalosome. Annu Rev Cell Dev Biol. 19, 261-286. Wei, N., Serino, G. and Deng, X. W. (2008). The COP9 signalosome: more than a protease. Trends Biochem. Sci. 33, 592–600. ! 147! Whitfield, M. L., Zheng, L., Baldwin, A., Ohta, T., Hurt, M. M. and Marzluff, W. F. (2000). Stem-loop binding protein, the protein that binds the 3’ end of histone mRNA, is cell cycle regulated by both translational and posttranslational mechanisms. Mol Cell Biol 20, 4188-4198. Wilkinson, K. D., Urban, M. K. and Haas, A. L. (1980) Ubiquitin is the ATP-dependent proteolysis factor I of rabbit reticulocytes. J. Biol. Chem. 255,7529-7532. Willems A. R., Schwab, M. and Tyers, M. (2004). A hitchhiker's guide to the cullin ubiquitin ligases: SCF and its kin. Biochim Biophys Acta. 1695, 133170. Wimuttisuk, W., and Singer, J. D. (2007) The Cullin3 ubiquitin ligase functions as a Nedd8-bound heterodimer. Mol. Biol. Cell. 18, 899-909. Winston, J. T., Strack, P., Beer-Romero, P., Chu, C. Y., Elledge, S. J. and Harper, J. W. (1999). The SCFbeta-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IkappaBalpha and beta-catenin and stimulates IkappaB alpha ubiquitination in vitro. Genes Dev. 13, 270-283. Wu, S., Zhu, W., Nhan, T., Toth, J. I., Petroski, M. D. and Wolf, D. A. (2013) CAND1 controls in vivo dynamics of the cullin 1-RING ubiquitin ligase repertoire. Nat. Commun. 4:1642 Xirodimas, D. P., Sundqvist, A., Nakamura, A., Shen, L., Botting, C. and Hay, R.T. (2008). Ribosomal proteins are targets for the NEDD8 pathway. EMBO Rep. 9, 208-286. Yamoah, K., Oashi, T., Sarikas, A., Gazdoiu, S., Osman, R. and Pan, Z. Q. (2008). Autoinhibitory regulation of SCF-mediated ubiquitination by human cullin 1's C- terminal tail. Proc. Natl. Acad. Sci. USA 105, 12230–12235. Yang, D. T., Young, K. H., Kahl, B. S., Markovina, S. and Miyamoto, S. (2008). Prevalence of bortezomib-resistant constitutive NF-kappaB activity in mantle cell lymphoma. Mol Cancer. 19:40. Yang, D., Tan, M., Wang, G. and Sun, Y. (2012). The p21-dependent radiosensitization of human breast cancer cells by MLN4924, an investigational inhibitor of NEDD8 activating enzyme. PLoS One 7:e34079. Yaron, A., Gonen, H., Alkalay, I., Hatzubai, A., Jung, S., Beyth, S., Mercurio, F., Manning, A. M., Ciechanover, A. and Ben-Neriah, Y. (1997). ! 148! Inhibition of NF-kappa-B cellular function via specific targeting of the Ikappa-B-ubiquitin ligase. EMBO J. 16, 6486- 94. Ye, X., Nalepa, G., Welcker, M., Kessler, B. M., Spooner, E., Qin, J., Elledge, S. J., Clurman, B. E. and Harper, J. W. (2004) Recognition of phosphodegron motifs in human cyclin E by the SCF(Fbw7) ubiquitin ligase. 279, 5110-5119. Ye, Y. and Rape, M. (2009). Building ubiquitin chains: E2 enzymes at work. Nat. Rev. Mol. Cell Biol. 10, 755-764. Yen, H. C. and Elledge, S. J. (2008). Identification of SCF ubiquitin ligase substrates by global protein stability profiling. Science 322, 923-929. Yogosawa, S., Makino, Y, Yoshida, T., Kishimoto, T., Muramatsu, M. and Tamura, T. (1996). Molecular cloning of a novel 120-kDa TBP-interacting protein. Biochem. Biophys. Res. Commun. 229, 612-617. Yoshida, A., Yoneda-Kato, N., Panattoni, M., Pardi, R. and Kato, J. (2010). YCSN5/Jab1 controls multiple events in the mammalian cell cycle. FEBS Lett. 584, 4545–4552. Yu, H., King, R. W., Peters, J. M. and Kirschner, M.W. (1996). Identification of a novel ubiquitin-conjugating enzyme involved in mitotic cyclin degradation. Curr. Biol. 6, 455-466. Yu, X., Yu, Y., Liu, B., Luo, K. Kong, W. Mao, P. and Yu, X. F. (2003). Induction of APOBEC3G ubiquitination and degradation by an HIV-1 VifCul5-SCF complex. Science 302, 1056-1060. Yuasa, K., Omori, K. and Yanaka, N. (2000). Binding and phosphorylation of a novel male germ cell-specific cGMP-dependent protein kinaseanchoring protein by cGMP-dependent protein kinase Ialpha. J. Biol. Chem. 275, 4897-2905. Zemla, A., Thomas, Y., Kedziora, S., Knebel, A., Wood, N. T., Rabut, G. and Kurz, T. (2013). CSN- and CAND1-dependent remodelling of the budding yeast SCF complex. Nat. Commun. 4:1641. Zhao, Y., Xiong, X. and Sun, Y. (2011). DEPTOR, an mTOR Inhibitor, Is a Physiological Substrate of SCFβ-TrCP E3 Ubiquitin Ligase and Regulates Survival and Autophagy. Mol. Cell 44, 304–316. Zhang, D. D., Lo, S. C., Cross, J. V., Templeton, D. J., and Hannink, M. (2004). Keap1 is a redox-regulated substrate adaptor protein for a Cul3! 149! dependent ubiquitin ligase complex. Mol Cell Biol. 24, 10941-10953. Zhang, H., Gao, Z. Q., Wang, W. J., Liu, G. F., Shtykova, E. V., Xu, J. H., Li, L. F., Su, X. D. and Dong, Y. H. (2012) The crystal structure of the MPN domain from the COP9 signalosome subunit CSN6. FEBS Lett. 586, 11471153. Zhang, Q., Shi, Q., Chen, Y., Yue, T., Li, S., Wang, B. and Jiang, J. (2009). Multiple Ser/Thr-rich degrons mediate the degradation of Ci/Gli by the Cul3HIB/SPOP E3 ubiquitin ligase. Proc Natl Acad Sci U S A. 106, 2119121196. Zheng, J., Yang, X., Harrell, J. M., Ryzhikov, S., Shim, E-H., LykkeAndersen, K., Wei, N., Sun, H., Kobayashi, R. and Zhang, H. (2002). CAND1 Binds to Unneddylated CUL1 and Regulates the Formation of SCF Ubiquitin E3 Ligase Complex. Mol. Cell 10, 1519–1526. Zhong, W., Feng, H., Santiago, F. E. and Kipreos, E. T. (2003). CUL-4 ubiquitin ligase maintains genome stability by restraining DNA-replication licensing. Nature 423, 885-889. Zhou, C., Wee, S., Rhee, E., Naumann, M., Dubiel, W. and Wolf, D. A. (2003). Fission yeast COP9/signalosome suppresses cullin activity through recruitment of the deubiquitylating enzyme Ubp12p. Mol. Cell 11, 927-938. Zhu, X. F., Liu, Z. C., Xie, B. F., Feng, G. K., Zeng, Y. X. (2003). Ceramide induces cell cycle arrest and upregulates p27kip in nasopharyngeal carcinoma cells. Cancer Lett. 193, 149-154. Zhuang, M., Calabrese, M. F., Liu, J., Waddell, M. B., Nourse, A., Hammel, M., Miller, D. J., Walden, H., Duda, D. M., Seyedin, S. N., Hoggard, T., Harper, J. W., White, K. P. and Schulman, B. A. (2009). Structures of SPOPsubstrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases. Mol Cell 36, 39-50. Zipper, L. M. and Mulcahy, R. T. (2002). The Keap1 BTB/POZ dimerization function is required to sequester Nrf2 in cytoplasm. J. Biol. Chem. 277, 36544–36552. Zollman, S., Godt, D., Privé, G. G., Couderc, J. L. and Laski, F.A. (1994). The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc. Natl. Acad. Sci. U S A 91, 1071710721. ! 150! ! ! 151! [...]... domain or they serve as a scaffold protein that recruits specific substrate proteins There are numerous types of RINGcontaining E3 ubiquitin ligases One family of RING- containing E3 ligases that is well characterized is the Cullin RING E3 ubiquitin ligases 1.5 Cullin RING E3 Ubiquitin Ligases Cullins form an evolutionarily conserved gene family They were first discovered as mediators of ubiquitin dependent... degradation The E3 ubiquitin ligases recruit the ubiquitin- charged E2 enzyme through conserved HECT (Homologous to E6-AP Carboxy Terminus) or RING (Really Interesting New Gene) domains and mediate the formation of a polyubiquitin chain on the substrate The HECT ubiquitin ligase domain was originally found in the course of characterizing the mechanism of the p53 substrate ubiquitination by the E6-AP ubiquitin. .. manner The activated ubiquitin is then transferred to one of the several E2 conjugating enzymes and also forms a thioester bond between the E2 active-site cysteine and the activated ubiquitin Subsequently, with the collaboration of an E3 ligase, the activated ubiquitin is then transferred from the ubiquitin- charged E2 enzyme onto lysines of the substrate protein As a result, an isopeptide bond between the. .. glycine of ubiquitin and the terminal amino group of the target lysine is formed Reiteration of this catalytic cycle assembles a polyubiquitin chain where additional ubiquitin polypeptides are conjugated to any of the seven lysines residues of the ubiquitin molecule, thus leading to the formation of high molecular weight chains of ubiquitin attached to the target protein Figure 1.1 Ubiquitination Ubiquitin. .. levels of Mdm2 are known to inhibit the cell cycle arrest function of p53 (Reviewed in Levine and Oren, 2009) Although a wealth of knowledge has been built on the correlation between the regulation of the UPS and the development of certain diseases, the pathways leading to UPS malfunction in many of these pathological disorders are still unknown Therefore, it is important to provide more insight into the. .. affect the Cul1 deneddylation rate 59 4.11 CAND1 siRNA does not affect the Cul1 deneddylation rate 60 4.12 Overexpression of Cdc34 does not affect the Cul1 deneddylation rate 60 4.13 Role of CSN in promoting the exchange of the Cullin3 substrate receptor SPOP 63 4.14 Role of CAND1 in promoting the exchange of the Cullin3 substrate receptor SPOP 64 4.15 CSN does not function to prevent binding of CAND1... excellent tool for the functional characterization of cullin E3 ubiquitin ligases 1.2 Ubiquitination The UPS can be divided into two distinct phases: ubiquitin conjugation (ubiquitination) and proteasomal degradation Protein ubiquitination is a post-translational modification, which results in the conjugation of the 76 amino acid protein ubiquitin onto a target protein in a catalytic cascade of three enzymes... therapeutic agent that targets the E3 ubiquitin ligase specifically is the potent and selective inhibitor of the Nedd8 activating E1 enzyme (NAE), MLN4924 (Millennium Pharmaceutical) This drug inhibits the degradation of cullin E3 ubiquitin ligases specifically by preventing the cullin neddylation MLN4924 has been shown to inhibit the growth of numerous tumors, such as lung, breast and pancreas cancer as well... et al., 1980) The three enzymes that are involved in the ubiquitination process are ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin ! 3! ligating enzyme (E3) (Hershko and Ciechanover, 1998) In the first step, the E1 enzyme adenylates the Ubiquitin C–terminus and forms a thioester linkage between a C-terminal glycine of ubiquitin and a cysteine residue on the E1 catalytic... functions such as regulation of the cell cycle, signal transduction, transcription and development CRLs are ! 8! composed of several subunits, which contain one of the cullin homologs, the RING finger containing protein, Roc/Rbx1, the cullin homolog-specific adaptor and substrate recognition subunits The cullin homologs serve as scaffold proteins that bind to Roc/Rbx1 via their C-terminus whereas the . CHARACTERIZATION OF THE FUNCTION AND REGULATION OF CULLIN RING E3 UBIQUITIN LIGASES CHOO YIN YIN B.Sc. (Honors), University of Malaya A THESIS SUBMITTED. Proteasome 6 1.4 E3 Ubiquitin Ligases 7 1.5 Cullin RING E3 Ubiquitin Ligases 8 !1.6 Structural characteristic of CRLs 12 1.7 Functions of CRLs 14-21 1.7.1 CRL1 1.7.2 CRL2 and CRL5 1.7.3. Cullin RING ubiquitin ligases (CRLs) constitute the largest family of cellular ubiquitin ligases with diverse cellular functions. CRLs comprise of seven homologous cullin- based complexes. The