Characterization of the function and regulation of cullin ring e3 ubiquitin ligases

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. 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[...]... 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