Post translational regulation of the human TRIP br1 cell cycle protein

POST-TRANSLATIONAL REGULATION OF THE HUMAN TRIP-Br1 CELL CYCLE PROTEIN CHRISTOPHER YANG MAOLIN (B.Sc (Hons), University of Edinburgh, UK) NATIONAL UNIVERSITY OF SINGAPORE 2007 Christopher YANG Maolin Ph.D Thesis FOM, NUS 1999-2007 POST-TRANSLATIONAL REGULATION OF THE HUMAN TRIP-Br1 CELL CYCLE PROTEIN CHRISTOPHER YANG MAOLIN (B.Sc (Hons) Biological Sciences (Molecular Biology), University of Edinburgh, UK) A THESIS SUBMITTED FOR THE DEGREE OF PHILOSOPHICAL DOCTOR (PhD) DEPARTMENT OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2007 Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 ACKNOWLEDGEMENTS "Success is a journey, not a destination." - Ben Sweetland This journey of scientific and self-discovery has been a long ride During this rough journey, many life-turning events took place and I have learnt so much This thesis closes a very important chapter of my life, one that is unforgettable This thesis is dedicated in memory of my late mother, Susan, who provided me all the means to pursue my passion for science despite all odds since I was in school Her sacrifices of love, prudence, humility and helpfulness lie deep within the depths of my heart Alas, it is my only regret that she is not able to see the end of this life chapter I am indebted to the merciful Lord for His gentle guidance and keeping my spirit filled in times of greatest difficulties My family and parent-in-laws had been instrumental in support and encouragement over my postgraduate years My lovely wife, Cassandra, had been quietly encouraging and supporting my pursuit of science in every way possible, whilst coping with her own career and our family, often in my absence due to work She had been there before I started, and now, by the end of my journey she had gracefully brought children into our lives, my lovely princesses, Charlotte and Chloe, and their little brother, Caeden These children give me a refreshingly new perspective in life and a never-ending motivation to look forward for a better tomorrow I am also blessed with amazing parent-in-laws, aged 70 and 66 years, who take absolutely wonderful and loving care of my little angels Without them, I would not be able to immerse myself in science the way I i Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 I would also like to thank my supervisor and mentor, Dr Stephen Hsu, for creating the opportunities, allowing freedom in my training and development, and seeing to my postgraduate completion despite the many difficulties that were present My partners-in-science, Jit Kong, Shahidah, Chien Tei, Khe Guan, Sharon and Chui Sun, all deserved special mention for being part of the lab family It has been my greatest pleasure to work with you all over the years and for the friendship that remains Prof Bay Boon Huat, Vice-Dean Faculty of Medicine, deserves special mention and thanks for his understanding patience, advice and facilitation of the administrative hurdles in the final stages of the project To the many more relatives, family and close friends who are in my heart that I failed to acknowledge, your company, encouragement and friendship had indeed played a significant part in the development of who I am today I am so blessed to have people like you in my life “Many people will walk in and out of your life, But only true friends will leave footprints in your heart.” - Eleanor Roosevelt Thank you all for being a part of my life journey and God bless! Chris Yang @ MadScientist 2007 ii Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 TABLE OF CONTENTS Title i Acknowledgements ii Table of Contents iii List of Figures & Tables vii List of Abbreviations x Presentations & Publications arising from PhD Thesis xii Abstract Introduction The TRIP-Br (Transcriptional Regulator Interacting with the PHD-Bromodomain) family of regulatory proteins 1.1 Historical perspective 1.2 Structural features of the TRIP-Br proteins 1.3 The functional properties of the TRIP-Br proteins 11 1.3.1 Co-regulation of the E2F-1/DP-1 transcriptional activity 11 1.3.2 TRIP-Br proteins possess potent acidic transactivation domains 11 1.3.3 The unique ability to interact with PHD zinc finger- and/or bromodomain and its functional significance 12 1.3.4 The TRIP-Br proteins : a novel class of cell cycle regulators 13 1.3.5 Functional relationships between the TRIP-Br proteins and the E2F family of transcription factors 1.3.6 14 Cell cycle regulated expression of human TRIP-Br1 20 iii Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 1.3.7 Human TRIP-Br1, a CDK4-interacting regulatory protein 20 1.3.8 The model of TRIP-Br protein function in cell cycle regulation 21 Protein post-translational regulation and modification 2.1 Common regulatory mechanisms of protein activity 23 2.2 Protein sub-cellular localization 24 2.2.1 Nuclear import and export 24 2.2.2 Proteolysis 24 2.3 Post-translational modifications (PTM) 25 2.3.1 Phosphorylation 25 2.3.2 Acetylation 30 2.3.3 Ubiquitination and sumoylation 34 Materials and Methods Materials 3.1 39 3.2 Plasmid DNA and cDNA clones 39 3.3 Cell lines Biochemical reagents 40 Methods 4.1 Transformation and maintenance of plasmid DNA clones 40 4.2 Mini- and maxi-scale preparation of plasmid DNA from bacteria 41 4.3 Quantitation and purity assessment of DNA or RNA 41 4.4 Screening of transformants for positive clones 42 4.5 Agarose gel electrophoresis of DNA products 42 iv Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 4.6 Synthesis of proteins by in vitro translation 42 4.7 Preparation of proteins from tissue culture 43 4.8 Analysis of proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 43 4.9 Western blot for protein detection 43 4.10 Confocal microscopy 44 4.11 DNA Transfection and Sequential Dual Luciferase Assay 45 4.12 Semi-quantitative RT-PCR 46 4.13 Protein degradation analysis 48 4.14 Immunoprecipitation assay (IP) 48 Results TRIP-Br1 interacts with DP1 and other proteins in vivo 5.1 TRIP-Br1 interacts with DP1 in the E2F1/DP1 transcriptional complex in vivo 5.2 The DP1-binding region of TRIP-Br1 54 5.3 The TRIP-Br1 binding region of DP1 60 5.4 Other TRIP-Br1 binding partners - the p53 oncoprotein 61 5.5 50 Other TRIP-Br1 binding partners - the E6 oncoprotein 63 TRIP-Br1 is regulated post-translationally and degraded via the 26S proteosomal pathway 6.1 TRIP-Br1 is regulated by degradation, and is affected by DP1 overexpression 6.2 69 TRIP-Br1 is degraded via the 26S proteosomal pathway 76 v Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 6.3 77 6.4 TRIP-Br1 is regulated post-translationally by p53 Putative post-translational modification of TRIP-Br1 82 TRIP-Br1 is a nuclear protein and co-localizes DP1 via its interaction 7.1 TRIP-Br1 is a nuclear protein that aids in the co-localization of DP1 83 TRIP-Br1 is a transcriptional co-regulator of the E2F1/DP1 transcription complex 8.1 TRIP-Br1 and mutant co-activation of the E2F1/DP1 transcriptional regulator complex 8.2 89 A single residue conservative mutation is able to increase human TRIP-Br1 transcriptional activation in conjunction with E2F1/DP1 heterodimeric complex 8.3 90 The E2F1/DP1/TRIP-Br1 transcriptional complex if further coactivated by HPV E6 oncoprotein 94 Discussion 96 Bibliography & References 122 vi Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 LIST OF FIGURES AND TABLES FIGURE Schematic diagram of the structure of the C4HC3 Plant Homeodomain (PHD) Zinc finger (Source: Capili, 2001) [3] .4 FIGURE Ribbon 3D structure representation of the bromodomain ZABC alpha helices (Source: Marmorstein, 2001) [4] FIGURE Putative domain structure of human TRIPBr1 AND TRIP-Br2 FIGURE PEST analysis of human TRIP-Br1 primary sequence [10, 11] FIGURE Sequence alignment of human TRIP-Br1 domains with respective domains in other similar proteins using ClustalW program Adapted from [17] 10 FIGURE Domain organization of members of the E2F family [35] 16 FIGURE The model of the TRIP-Br Protein Functions 22 FIGURE Phosphorylation sites of human p53 [56, 63] 28 FIGURE Schematic diagram showing the functional domains and phosphorylation events impinging on MDM2 [58] 29 FIGURE 10 Acetylated Lysine 33 FIGURE 11 Structure comparison of Ubiquitin and human SUMO-1 [82] 36 FIGURE 12 Signalling functions of SUMO [80] 37 FIGURE 13 The SUMO conjugation pathway [80] 38 FIGURE 14 DP1 and E2F1 co-immunoprecipitate with hTRIP-Br-HA pulldown using an anti-HA antibody 52 FIGURE 15 DP1 and E2F1 co-immunoprecipitate with hTRIP-Br-HA pulldown using an anti-HA antibody (MG132) 53 FIGURE 16 Schematic drawing of hTRIP-Br1 domain structure and truncation mutants generated 56 FIGURE 17 hTRIP-Br1-HA ∆12-73 and ∆12-121 mutants cannot coimmunoprecipitate DP1 57 vii Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 FIGURE 18 hTRIP-Br1-HA ∆12-50 and ∆12-90 mutants cannot coimmunoprecipitate DP-1 58 FIGURE 19 hTRIP-Br1-HA and its mutants cannot co-immunoprecipitate DP1 ∆205-277 59 FIGURE 20 E2F1, DP1, hTRIP-Br1-HA and p53 are co-immunoprecipitation with hTRIP-Br1-HA in a quarternary complex with MG132 treatment 64 FIGURE 21 Co-expression of p53 affects the expressed levels of hTRIP-Br1-HA 65 FIGURE 22 Co-expression of p53 affects the interaction of hTRIP-Br1-HA with DP1 (in the presence of MG132) 66 FIGURE 23 p53 does not interact directly with hTRIP-Br1-HA in vitro 67 FIGURE 24 p53 co-immunoprecipitation with hTRIP-Br1-HA is observed with MG132 treatment 68 FIGURE 25 hTRIP-Br1-HA protein stability is increased by DP-1 overexpression 72 FIGURE 26 hTRIP-Br1 protein stability is influenced by DP-1 and/or E2F-1 overexpression 73 FIGURE 27 hTRIP-Br1 protein stability is specifically affected by DP1 and E2F1 overexpression in a dose-dependent manner 74 FIGURE 28 hTRIP-Br1 deletion mutants exhibit differences in protein stability when co-overexpressed with DP1 75 FIGURE 29 hTRIP-Br1 is degraded by the 26S proteosomal pathway 79 FIGURE 30 hTRIP-Br1-HA degradation is inhibited by the 26S proteosome inhibitors MG132 and Lactacystin .80 FIGURE 31 Co-overexpression of p53 is associated with more rapid hTRIP-Br1HA degradation 81 FIGURE 32 hTRIP-Br1 proteins are nuclear proteins that aid in DP-1 colocalization into the nucleus 85 FIGURE 33 hTRIP-Br1-HA C-terminal truncation mutant proteins retain the ability to localize in the nucleus 86 FIGURE 34 Intracellular localization of the hTRIP-Br1-HA N-terminal truncation mutant proteins 88 viii Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 degradation (possibly through steric hindrance of post-translational modifications to mark hTRIP-Br1 for degradation) The same direct interaction allows hTRIP-Br1 to translocate DP1 into the nucleus Within the nucleus, the hTRIP-Br1-DP1 complex can then recruit available E2F1 and other co-regulators to form a larger multi-protein transcription complex that regulates E2F-reesponsive genes This hypothesis is analogous to the reported finding that the E2F1/DP1 complex is differentially regulated by ARF (p14ARF) [36] Ectopically expressed T7-DP1 was also shown to be cytoplasmic, while and co-expression of E2F1 led to the colocalization of T7-DP1 into the nucleus Furthermore, co-transfection with ARF resulted in T7-DP1 nucleolar localization and subsequent degradation ARF was also shown to regulate E2F1 in a similar manner as DP1 However, when E2F1 and DP1 were co-expressed, ARF was unable to co-localize either into the nucleolus and/or effect protein degradation It appears that ARF regulates both E2F1 and DP1 protein levels to ensure that there is a physiological balance between these two proteins Taken together, an integrated model for the regulation of the E2F1, DP1 and hTRIPBr1 transcriptional regulator proteins can be proposed In this model (Figure 41), DP1 is cytoplasmic and can be translocated into the nucleus by its interactions with either E2F1 and/or hTRIP-Br1, both of which have the independent ability to localize into the nucleus Within the nucleus, free E2F1 is co-localized into the nucleolus and rapidly degraded through its interaction with ARF If DP1 is available, there would be a preferential formation of the E2F1/DP1 heterodimer complex, and consequent activation of transcription given appropriate 114 Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 conditions Free hTRIP-Br1 within the nucleus is degraded rapidly, through the 26S proteosomal pathway It is also possible that hTRIP-Br1 can interact with the preformed E2F1/DP1 complexes and the ternary E2F1/DP1/hTRIP-Br1 complex confers transcriptional activation through the previously proposed “integrator model” [6, 94] However, DP1 may also be able to translocate into the nucleus by its interaction with hTRIP-Br1 In the event of hTRIP-Br1/DP1 complex formation and translocation into the nucleus, hTRIP-Br1 is stabilized and accumulates until E2F1 (or possibly ARF) is present Furthermore, when the E2F1/DP1/hTRIP-Br1 ternary complex is activated and performs its transcriptional activities, the complex is then rapidly degraded in a feedback loop-control mechanism This self-attenuation mechanism is in place to regulate the activity of the transcriptional complexes activity, and may involve ARF-associated proteolytic degradation Table summarizes the properties of hTRIP-Br1-HA that were characterized in this thesis From the data, it is evident that the C-terminal half of hTRIP-Br1-HA may play a minor role in protein regulation The main function appears to correlate with the putative domain structure, whereby the C-terminal half contains the transactivation domain and primarily regulates the transcriptional co-regulation properties of hTRIP-Br1-HA, as shown by the transcriptional assays Notably, the Nterminal half of hTRIP-Br1-HA was demonstrated to contain putative domains involved in protein-protein interaction and the regulation of protein function through mechanisms such as direct effects on protein activity, stabilization and localization The protein-protein interaction with DP1 was narrowed to a region spanning the heptad repeat of hTRIP-Br1-HA (also similar to the SERTA domain), as 115 Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 demonstrated by the DP1 interaction with the N-terminal deletion mutants This interaction is envisaged to aid in protein stabilization since the loss of protein-protein interaction between hTRIP-Br1-HA and DP1 resulted in protein instability except for the hTRIP-Br1-HA ∆12-73 mutant, which was proposed to lack a degradation domain within residues 40-73 The localization of hTRIP-Br1-HA and DP1 was also demonstrated to be interdependent on each binding partner Protein-protein interaction, as determined by co-immunoprecipitation assays, was a requirement for T7-DP1 nuclear co-localization when co-overexpressed with hTRIP-Br1-HA (Table 3, ∆12-50 to ∆12-121 mutants) On the other hand, protein activity, which was assayed by transcriptional co-activation together with E2F1/DP1, demonstrated that the C-terminal half of hTRIP-Br1-HA was essential Furthermore, deletion of the putative ‘degradation domain’ in ∆12-73 (and to a lesser extent in ∆12-50 and ∆1290) augmented hTRIP-Br1-HA transcriptional co-activation activity (Figure 35) The observations and data support a model in which hTRIP-Br1-HA is posttranslationally regulated This regulation is fundamentally influenced by the ability of hTRIP-Br1-HA to interact with DP1 through protein-protein mediated by the proposed interacting domains within each binding partner, namely the SERTA/heptad repeat domain in hTRIP-Br1-HA and the DCB1 domain in DP1 The binding between DP1 and hTRIP-Br1-HA were observed to have at least two effects Firstly, DP1 binding to hTRIP-Br1-HA allowed the latter to be stabilized within the cells This stabilization effect imposed on hTRIP-Br1-HA by DP1 interaction when cooverexpressed was demonstrated to be specific Notably, hTRIP-Br1-HA stabilization (by DP1 co-overexpression) was concomitant with the presence of a 116 Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 slightly higher molecular weight band of hTRIP-Br1-HA The increase in molecular weight suggests that there may have been a post-translational modification (PTM) where there was an addition of chemical groups to the protein Since this band was also observed with MG132 treatment, it is likely that the PTM was ubiquitin, although it was not demonstrated in these experiments Preliminary experiments aimed at identifying post-translational modifications on hTRIP-Br1-HA using an array of antibodies specific for phospho-tyrosine and phospho-threonine, as well as antibodies that recognize N-acetyl, SUMO and ubiquitin groups, failed to identify the nature of the putative post-translational modification(s) of hTRIP-Br1-HA There are several limitations to this study Firstly, the experiments were done without the availability of an antibody to endogenous hTRIP-Br1 Therefore, a HA tagged version of hTRIP-Br1 was constructed for investigational purposes Detection of hTRIP-Br1 was based on the presence of the HA tag, which may conceivably have given rise to artifact by altering the properties of endogenous hTRIP-Br1 This phenomenon, though possible, is presumed to be remote To further validate this presumption and confirm the observations presented earlier, similar experiments may be performed with a monoclonal antibody specific to hTRIP-Br1, which was only recently generated in our laboratory group Ours is the only group to date that has successfully produced a mouse monoclonal antibody against hTRIP-Br1 after multiple failed attempts owing to the high degree of homology between TRIP-Br1 of murine and human origin All other groups have similarly published data based on the overexpression of tagged TRIP-Br proteins 117 Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 Secondly, although deletion mutants in hTRIP-Br1 and DP1 have been isolated that failed to interact, further investigation may be carried out to comprehensively define the minimal binding region and, with single nucleotide mutations, determine critical residues involved in the interaction between hTRIP-Br1 and DP1 Furthermore, the hTRIP-Br1 interaction domain on DP1 was identified based on a combination of deduction based on a careful review of published data to date and our own empirical observations Validation of the proposal that the DCB1 domain of DP1 is the hTRIPBr1 binding domain may be confirmed experimentally by co-immunoprecipitation studies using an isolated HA- or FLAG-tagged DCB1 domain Next, E2F1 and DP1 function as heterodimers, and since hTRIP-Br1 was observed to have functional activities with DP1 almost exclusively, it leaves the question of the influence of E2F1 interaction with DP1 on the DP1-hTRIP-Br1 interaction and consequent activities Clearly, E2F1 has exhibited significant effects in the turnover of hTRIP-Br1-HA (Figures 27 and 28) Delineating the effects of E2F1 would allow greater insights into the role and relationship between hTRIP-Br1 and DP1 Perhaps this can be achieved through the use of a DP1 mutant that is able to bind with hTRIPBr1 but is unable to bind E2F1 Additionally, p53 was found to interact with TRIP-Br1 in the in vivo coimmunoprecipitation studies However, direct binding was not observed with the IVT pull-down assay It was previously reported that there was no interaction between hTRIP-Br1 and p53 Hence, the relationship between p53 and hTRIP-Br1 is currently inconclusive and warrants further investigations 118 Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 Lastly, it was noted that hTRIP-Br1-HA exhibited a protein band of higher molecular weight when co-overexpressed with DP1 Attempts to identify this band with respect to potential PTMs using antibodies specific for phospho-tyrosine, phospho-threonine, N-acetyl, SUMO and Ubiquitin post-translational modification groups failed to identify the nature of the putative post-translational modification(s) of hTRIP-Br1HA Direct labeling with exogenous tagged-ubiquitin and ubiquitin labeling assays could be used as a better test for ubiquitination of hTRIP-Br1 in future experiments Other alternative approaches would be to use mass spectrometry to identify any potential PTMs, followed by additional experiments (e.g site-directed mutagenesis to block specific PTMs) to provide confirmatory functional data 119 Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 FIGURE 41 Proposed model for post-translational regulation of hTRIP-Br1 by DP1 integrated with proposed models of E2F1 and DP1 regulation by ARF Legend: hTB1: hTRIP-Br1, ARF: p14ARF(alternate reading frame) Cytoplasmic DP1 can bind to E2F1 (a) or hTRIP-Br1 (b) When bound to E2F1, the E2F1/DP1 heterodimeric complex is formed and DP1 is translocated into the nucleus by its association with E2F1 (c) The E2F1/DP1 complex can then bind DNA and/or hTRIP-Br1, to perform transcriptional activities as an activated transcriptional complex (g) The activated complex self-attenuates after transcriptional activation and is broken down by the proteosomal pathway (h) hTRIP-Br1 by itself has the intrinsic ability to translocate into the nucleus (d), and when it is in free form, it is rapidly broken down, presumably by the proteosomal pathway When DP1 is abundant, it binds hTRIP-Br1 (b) which then translocates DP1 into the nucleus (i) and the hTRIP-Br1/DP1 complex is stabilized due to DP1 binding This complex is able to bind E2F1 (j) and form the active ternary E2F1/DP1/hTRIP-Br1 complex E2F1 (and DP1) by itself would be bound by ARF and then co-localized into the nucleolus where the protein is degraded rapidly This is a form of regulation to control and check the activities of E2F1 120 Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 TABLE Summary of the properties hTRIP-Br1-HA proteins (wild-type and mutants) DP1 interaction hTRIP-Br1 WT hTRIP-Br1 1-222 hTRIP-Br1 1-186 hTRIP-Br1 1-121 hTRIP-Br1 ∆12-121 hTRIP-Br1 ∆12-90 hTRIP-Br1 ∆12-73 hTRIP-Br1 ∆12-50 hTRIP-Br1 ∆12-40 * ** NA Transcriptional DP1augmentation induced stabilization of E2F1/DP1 Nuclear localization of hTRIP-Br1-HA Nuclear co-localization of (T7-)DP1 + + + +++ +++ + + - +++ NA + + +++ NA + + +++ NA - - + + - - NA ++ + - - +++ * +++ + +/- +/- NA + + + + - ** - ++ + ∆12-73 mutant was stabilized independently and as a result of the deletion of residues 12-73 ∆12-40 mutant was highly unstable and was undetectable after 10 minutes with cycloheximide treatment Data currently unavailable 121 Christopher YANG Maolin Ph.D Thesis Yong Loo Lin School of Medicine, NUS 1999-2007 BIBLIOGRAPHY & REFERENCES 10 11 12 13 14 Aasland, R., Gibson, T J., and Stewart, A F., The PHD finger: implications for chromatin-mediated transcriptional regulation Trends Biochem Sci, 1995 20: p 56-59 Koken, M H M., Saib, A., and de The, H., A C4HC3 zinc finger motif C R Acad Sci III, 1995 318: p 733-739 Capili, A D., Schultz, D C., Rauscher, F J III, and Borden, K L B., Solution structure of the PHD domain from the KAP-1 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TRIP- Br1 (mTRIP -Br1) orthologue is 86% identical to hTRIP -Br1 in amino acid sequence The other TRIP- Br family member, the human TRIP- Br2 (hTRIP-Br2)... binding region of DP1 60 5.4 Other TRIP- Br1 binding partners - the p53 oncoprotein 61 5.5 50 Other TRIP- Br1 binding partners - the E6 oncoprotein 63 TRIP- Br1 is regulated post- translationally... structure of human TRIPBr1 AND TRIP- Br2 The extent of homology between the THDs of hTRIP -Br1 and hTRIP-Br2 proteins are shown as percentages The PHD-Bromodomain interacting region was identified at the