F-Box Proteins, Cell Cycle and Cancer

F-box proteins, cell cycle and cancer

Jihoon Kim

ISBN: 978-94-6203-741-0 Printing: CPI – Koninklijke Wöhrmann, Zutphen Copyright © 2014 by J. Kim. All rights reserved. No parts of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means, without prior permission of the author.

F-box proteins, cell cycle and cancer

F-box eiwitten, de celcyclus en kanker (met een samenvatting in het Nederlands)

Proefschrift

ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. G.J. van der Zwaan, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op dinsdag 13 januari 2015 des middags te 12.45 uur

door

Jihoon Kim geboren op 17 juni 1981 te Suwon, zuid-Korea Promotor:

Prof. dr. E. P. J. G Cuppen

Copromotor:

Dr. D. Guardavaccaro

Contents

Chapter 1 Introduction 9

Chapter 2 USP17- and SCF TrCP- 27 Regulated Degradationβ of DEC1 Controls the DNA Damage Response

Chapter 3 The SCF-mediated 49 Degradation of TFAP4 Regulates Progression Through the S and G2 Phases of the Cell Cycle

Chapter 4 SCFFBXW4 Targets DEC2 for 61 Proteasome-dependent Degradation.

Chapter 5 Other F-box Proteins and 73 Cancer

Chapter 6 Discussion 101

addendum Nederlandse samenvatting 107 Acknowledgements Curriculum vitae publication list

Chapter 1

Introduction

The ubiquitin-proteasome system (UPS) is the major pathway for regulated degradation of intracellular proteins (1, 2). It is well established that protein degradation by the UPS controls a broad array of crucial cellular processes. Not surprisingly, failure or dysfunction of the UPS is implicated in the pathogenesis of many human diseases, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, immune system related diseases, and cancer (3-5). Here I 1 describe the latest insights into the ubiquitin-proteasome system, with a special emphasis on SCF ubiquitin ligases, molecular machines that control cell cycle progression and whose aberrant regulation contributes to oncogenesis.

The UPS: ubiquitylation of the substrate protein

Degradation of intracellular proteins by the UPS involves two distinct steps. Proteins are first marked with ubiquitin and then degraded by the proteasome (6). Ubiquitin is an evolutionarily conserved protein composed of 76 amino acids. The amino acids sequence of ubiquitin is almost

Figure 1. The ubiquitin- proteasome system (UPS). Precursor forms of ubiquitin (Ub) are maturated by deubiquitylating enzymes (DUBs). Ubiquitin is then activated by an E1 ubiquitin- activating enzyme via the generation of a high-energy thioester bond and concomitant ATP hydrolysis. Ubiquitin is then transferred from the E1 to an E2 ubiquitin-conjugating enzyme. As a last step, ubiquitin is transferred onto a substrate lysine residue. HECT E3s form an intermediate with ubiquitin through a thioester bond. RING E3s instead interact with the E2 and help the transfer of ubiquitin onto the substrate. Polyubiquitylated substrates are then recognized by the 26S proteasome and degraded in short peptides.

Chapter 1

identical from yeast to human indicating that the structure of ubiquitin is an important factor for its evolutionarily conserved function. Conjugation of ubiquitin onto a substrate is catalyzed by three types of enzymes: E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases (7-9). Ubiquitin is first activated by the E1 ubiquitin-activating 1 enzyme in an ATP-dependent manner. The cysteine residue of the active site of the E1 enzyme forms a high-energy thioester bond with the C-terminus of ubiquitin. Ubiquitin is then transferred to the E2 enzyme. Finally, the E3 ubiquitin ligase helps the transfer of ubiquitin from the E2 to the -amino group of a lysine residue of the substrate forming an isopeptide bond. Ubiquitin conjugation can occur not only on a lysine residue of the substrate but also on ubiquitin moieties that are already attached to the substrate thus generating polyubiquitin chains (6). Ubiquitin contains seven lysine residues (K6, K11, K27, K29, K33, K48 and K63) (10) (Fig. 2a). It is known that the outcome of ubiquitylation depends on which specific lysine residue of ubiquitin is used. Ubiquitin conjugation can occur in many ways, i.e. monoubiquitylation, multimonoubiquitylation, polyubiquitylation with homogenous ubiquitin chains, mixed ubiquitin chains, branched ubiquitin chains and unanchored ubiquitin chains (Fig. 2b-g). Monoubiquitylation is mostly used in signal transduction (11-13). Multimonoubiquitylation mainly regulates endocytosis. For instance, endocytosis of epidermal growth factor and platelet-derived growth factor receptors are driven by multimonoubiquitylation (14). Polyubiquitylation via K48-linked chains is the main signal for proteasomal degradation (15-17). The K11-linked chain also leads to degradation. Indeed, it has been recently shown that during mitosis the APC/C ubiquitin ligase and its E2 UBCH10 promote K11-linked chain polyubiquitylation of their mitotic substrates which are then degraded by the proteasome (18, 19). In

Figure 2. (A) Crystal structure of ubiquitin. Seven lysine residues are shown. (B-G) Different types of ubiquitin modifications have been reported: monoubiquitylation (B), multimonoubiquitylation (C), homogenous polyubiquitin chains (D), mixed ubiquitin chains (E), branched ubiquitin chains (F), and unanchored ubiquitin chains (G).

Introduction contrast, the K63-linked chain has mainly non-proteolytic functions. By affecting protein-protein interaction and subcellular localization, K63-linked polyubiquitylation regulates signal transduction (6). Ubiquitylation is a reversible process. Specific enzymes, known as deubiquitylases (DUBs), counteract ubiquitin conjugation by removing ubiquitin and polyubiquitin chains from substrate proteins (20). To date, more than 100 distinct DUBs have 1 been identified in mammalian cells. DUBs are subdivided into five families based on their catalytic domain architecture: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), ovarian tumor proteases (OTUs), Machado- Joseph disease proteases (MJDs) and JAB1/MPN/MOV34 proteases (JAMMs). These five families can be further classified into two subcategories based on their mechanism of action. UCHs, USPs, OTUs and MJDs are cysteine proteases in which the cysteine residue in the catalytic site catalyzes the nucleophilic attack on the carbonyl group of the scissile peptide or isopeptide bond. On the other hand, JAMMs are zinc metalloproteases. JAMMs use two zinc ions, one of which activates water molecules. Polarized water molecules then attacks the isopeptide bond. DUBs are essential not only for substrate deubiquitylation but also for the maturation of poly-ubiquitin precursors which produces ubiquitin (20).

The UPS: proteasomal degradation of the substrate protein

Once the substrate protein is polyubiquitylated, it is recognized and degraded by the 26S proteasome. The 26S proteasome is a 2.5 MDa multimeric protein complex composed of two distinct parts: the core particle (CP), also known as 20S proteasome, and one or two regulatory particles (RP), referred as 19S proteasome. The actual proteolytic activity is located in the CP whereas the RP acts as gatekeeper. The CP is a 700 kDa cylinder-shaped multisubunit complex containing seven subunits () and sevensubunits () (21-24). These subunits are arranged in four seven-membered rings with a  configuration which form a hollow and narrow chamber that blocks unregulated access of substrates. The -rings control the entry into the central channel and are usually in a closed conformation in the free core particle (24) and can be gated by the RP or activators (25-27). Proteolytic activity is located at three types of -subunits (1, 2 and 5) inside the -rings so that there are 6 catalytic sites in total (28) (Fig.3). These catalytic sites have low preference toward specific amino acid sequences thus making possible to cleave any sequence. The 900 kDa RP, which is required to open the -rings, is subdivided into two subcomplexes, the base and the lid (29). The base contains six ATPase subunits (regulatory particle triple A protein 1 (RPT1) – 6) and four non-ATPase subunits (regulatory particle non-ATPase 1 (RPN1), RPN2, RPN10 and RPN13). These six members of ATPase subunits are located nearby the CP’s -ring and are responsible for the ATP hydrolysis which induces substrate unfolding and brings the unfolded substrate into the CP’s central channel. The non-ATPase subunits RPN10 and RPN13 recognize the polyubiquitin chain of the substrate (30). The lid 11

Chapter 1

is composed of RPN11, RPN8 and seven scaffold proteins (RPN3, RPN5, RPN6, RPN7, RPN9, RPN12 and RPN15). The RPN11 has the deubiquitylating activity that is responsible for cleaving off ubiquitin chains from the substrate (31). Because the CP central channel is too narrow for folded substrates, substrates must have 1 an unstructured region to initiate proteolysis (32).

Figure 3. Left: schematic representation of the structure of the proteasome. Side (middle) and top (right) view of the crystal structure of the yeast proteasome. The 1, 2 and 5 subunits, which have enzymatic activity, are shown in yellow, green and limon.

E3 ubiquitin ligases

The human genome encodes for two E1 ubiquitin-activating enzymes (UBE1 and UBE1L2) (33, 34), around 40 E2 ubiquitin-conjugating enzymes (35) and more than 600 E3 ubiquitin ligases (36). This huge number of E3s is needed because these enzymes provide substrate specificity to the UPS by selectively interacting with substrate proteins. Two classes of E3s have been described, Homologous of E6-AP Carboxy Terminus (HECT) E3s and Really Interesting New Gene (RING) E3s. HECT E3 ubiquitin ligases receive ubiquitin from the E2-Ub complex forming HECT E3-Ub intermediates and then transfer ubiquitin to the substrate (37, 38). RING E3 ubiquitin ligases instead transfer ubiquitin from the E2 to the substrate without forming an E3-Ub intermediate (9). RING E3 ubiquitin ligases are the most abundant E3s in mammals (39, 40). They contain a zinc finger-type protein-protein interaction domain, known as a RING finger domain, which consists of 40 to 60 amino acids. RINGs use two zinc ions to form a cross-brace structure with consensus sequence Cys-X2-Cys-X9-39-Cys-X1-3- His-X2-3-Cys-X2-Cys-X4-48-Cys-X2-Cys (where X can be any amino acids; histidines and cysteines can be exchanged or substituted) (41). The cross-brace pattern

Introduction

Figure 4. Schematic structure of multisubunit RING E3 ubiquitin ligase complexes. Cullin-based E3 ubiquitin ligases are composed of four subunits: a RING protein (blue), a cullin scaffold (green), an adaptor protein (pale blue), and a substrate-binding subunit (red). In CRL3 a single polypeptide functions both as adaptor and substrate-binding subunit. In APC/C, the APC2 subunit contains a cullin-homology domain and APC11 contains a RING finger domain which mediates the E2 binding. provide s the proper platform to interact with the E2 ubiquitin-conjugating enzyme. RING E3 ubiquitin ligases can be further subdivided into three subclasses based on their molecular architecture (42): single-subunit RING E3s (e.g. SIAH and CBLL1), dimeric RING E3s (e.g. BRCA1-BARD1, MDM2-MDMX) and multisubunit RING E3s (e.g. cullin-RING ligases (CRLs), APC/C, FANC complex). Single- subunit RING E3s are composed of a single polypeptide which interacts with both the E2 enzyme and the substrate. Dimeric RING E3s can form homodimer or heterodimer. It is not fully understood why some RING E3s are active only when 13

Chapter 1

they form a dimer. The RING domain and the surrounding region form a surface for dimerization enhancing the E3 activity. It has been proposed that RING dimerization enhances the local concentration of the E2 ubiquitin-conjugating enzyme promoting catalysis. The largest family of multisubunit E3 ubiquitin ligases is the cullin-RING (CRL) 1 family (Fig. 4). CRLs are composed of a cullin subunit, an adapter protein, a RING protein and a substrate-binding factor (1). Seven distinct cullin proteins, namely CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5 and CUL7, function as rigid scaffolds. Distinct adaptor-substrate binding protein pairs have been described for each cullin: SKP1-F-box protein for CUL1 (CRL1 or SCF) and CUL7 (CRL7), Elongin B/C- SOCS box protein for CUL2 (CRL2) and CUL5 (CRL5), DDB1-DCAF protein for CUL4A and CUL4B (CRL4). In CRL3, a single polypeptide, the BTB protein, acts both as an adaptor and a substrate-binding protein. Two RING proteins, RBX1 (also known as ROC1 and RNF75) and RBX2 (also referred as ROC2 and RNF7), interact with both the cullin and the E2 enzyme. The Anaphase Promoting Complex/Cyclosome (APC/C), a key regulator of mitotic progression, is a multisubunit RING E3 ligase composed of 13 different subunits (APC1, APC2, APC3, APC4, APC5, APC6, APC7, APC8, APC10, APC11, APC13, APC16 and CDC26) and one co-activator (CDH20 or CDH1) (43-45). APC11 has a RING finger domain that mediates the association with the E2. APC2 instead is a cullin-like protein that serves as scaffold. Depending on which co-activator subunit is bound to the core complex (CDH1 or CDC20), APC/C targets different substrates (43, 44).

The SCF complex and the F-box protein family

The SCF complex is the best-characterized cullin-RING ubiquitin ligase. It is composed of SKP1 (S-phase kinase-associated protein 1), CUL1 (Cullin-1), RBX1 and one of many F-box proteins (FBPs) (Fig. 4A). Many functional and structural details have been uncovered for the SCF complex. In this complex, similarly to other CRLs, the cullin subunit CUL1 functions as a molecular scaffold that binds at the N-terminus the adaptor subunit SKP1 and at the C-terminus the RING-finger protein RBX1. SKP1, in turn, binds to one of many FBPs, the substrate-binding subunit of the SCF. FBPs are composed of an F-box domain, in most cases located close to the N-terminus, and different protein-protein interaction domains that mediate substrate binding. The F-box is a motif of about 40 amino acids that mediates the interaction with SKP1. The term F-box comes from the fact that it was first identified in cyclin F. Sixty-nine F-box proteins have been identified in humans. They are classified into three subfamilies, FBXWs, FBXLs and FBXOs, based on the composition of their protein-protein interaction domains (46, 47) (Fig. 5). FBXWs contain WD40 repeats, FBXLs contain leucine-rich repeats (LRRs) and FBXOs contain various (other) domains.

Introduction

Figure 5. Domain architecture of human F-box proteins (FBPs). FBPs are grouped into three classes based on protein-protein interaction domains that mediate substrate binding. FBXWs contain WD40 repeats, FBXLs contain leucine-rich repeats (LRRs) and FBXOs contain various (other) domains. Three F-box proteins, namely TrCP, SKP2, and FBXW7, have been extensively studied. TrCP1 (also called as FBXW1) and its paralog TrCP2 (also known as FBXW11) consist of an F-box domain, seven WD40 domains which form a beta-propeller structure and an -helical linker connecting the two (17). The crystal structure of SCFTrCP suggests that TrCP increases the local concentration of substrate lysine residues close to E2, contributing to lysine selectivity. TrCP binds to a motif within the substrate, known as phosphodegron with the consensus DSGXX(X)S (where X can be any amino acid). One of these serine residues can be substituted by glutamic acid or aspartic acid. Phosphorylation of the two serine residues is required for the interaction between TrCP and its substrate (46). Although early studies demonstrated that TrCP regulates Wnt signaling by targeting -catenin for degradation (48-50) and the NF-κB pathway by inducing the degradation of IκBα (51-55), later studies have implicated TrCP in the regulation of cell cycle progression. Indeed, it has been found by different groups that TrCP controls the destruction of key cell cycle regulators such as EMI1, Claspin, CDC25A, 15

Chapter 1

and WEE1 (47, 56). SKP2 (also known as FBXL1) has also a major role in cell cycle progression. The protein levels of SKP2 increase at the G1-S transition reaching maximal levels in S-G2 (57, 58). It is well established that SKP2 induces the degradation of the CDK inhibitor p27, which has to be destroyed at the G1-S transition to trigger CDK 1 activation (59). p27 is first phosphorylated on Thr187 by cyclin E/CDK2 complex, allowing p27 binding to SKP2. For the interaction between SKP2 and p27, the accessory protein CKS1 is required. CKS1 interacts with SKP2 and enhances its affinity for phosphorylated p27. The whole complex leads to p27 ubiquitylation and its consequent degradation by the proteasome (60, 61). A number of other studies have demonstrated that SKP2 targets for degradation many other regulators of the cell cycle, such as p21, ORC1, CDT1, p57, and p130 (47, 62-64). FBXW7 targets a number of positive regulators of cell cycle progression such as cyclin E, c-MYC, c-JUN and NOTCH (65-67). Similarly to TrCP and SKP2, FBXW7 interacts with its substrates following phosphorylation of specific serine and threonine residues (68). The best-characterized substrate of FBXW7 is cyclin E, a critical regulator of the G1-S transition. Phosphorylation of cyclin E on Thr380 and Ser384 is required for its binding to FBXW7. It has been reported that CDK2- mediated phosphorylation of cyclin E on Ser384 promotes its phosphorylation on Thr380 by GSK3. (69, 70). Another substrate of FBXW7 is c-MYC, a well-known oncoprotein that is involved in cell cycle regulation. Also in this case, the binding of c-MYC to FBXW7 requires phosphorylation on two residues, namely Thr58 and Ser62 (71, 72). Regulation of SCF ubiquitin ligases. Since the SCF complex is a critical regulator of numerous cellular processes, it is not surprising that its activity must be tightly controlled. Several mechanisms have been reported. Modification of the CUL1 C- terminus tail with the small ubiquitin-like protein Nedd8, known as neddylation, enhances the activity of the SCF by reducing the gap between the E2 and the substrate (73). Nedd8 is conjugated onto CUL1 by a series of enzymatic reactions catalyzed by specific E1, E2 and E3 enzymes, similarly to what occurs for ubiquitin conjugation (74-76). Deconjugating of Nedd8 is driven by a large octameric complex called COP9 signalosome (CSN), which inhibits the activity of the SCF (77). The activity of the SCF is also controlled by a large polypeptide known as CAND1 (78). It has been proposed that CAND1 binds to CUL1 preventing the association of SKP1 with CUL1. The CAND1-CUL1 interaction can be inhibited by CUL1 neddylation (79). Post-translational modification of the SCF target represents another key mechanism that controls the activity of the SCF. As already mentioned above, it is well established that phosphorylation of specific residue(s) of the substrate is required for its binding to certain F-box proteins, e.g. TrCP, SKP2 and FBXW7 (68, 80, 81) (82). Moreover, glycosylation of the substrate protein is in some cases required for its binding to the F-box protein. This has been reported for substrates of FBXO2 and FBXO6 (83, 84).

Introduction

Scope of this thesis

E3 ubiquitin ligases, the enzymes that confer specificity to proteolysis by directly binding substrate proteins, are encoded by over 650 human genes, exceeding the number of genes encoding for protein kinases. Accordingly, E3 ubiquitin ligases have been linked to the control of virtually all cellular processes. In spite of their 1 fundamental importance, our knowledge of the biological function and mechanism of action for most E3 ubiquitin ligases is still poor. Increasing our knowledge of the biology of ubiquitin ligases is fundamental because experimental and clinical data have shown that their abnormal functions is involved in the pathogenesis of many human cancers. The main objective of this thesis is the identification of novel substrates of SCF ubiquitin ligases and the elucidation of the molecular mechanisms by which deregulated functions of SCF ubiquitin ligases contribute to the unchecked proliferation typical of cancer cells. In chapters 2 and 3, I show that TrCP targets for degradation two bHLH transcription factors, namely DEC1 (chapter 2) and TFAP4 (chapter 3), and demonstrate that their degradation is required for mitotic entry during recovery from the DNA damage checkpoint (DEC1) and during unperturbed cell cycles (TFAP4). In chapter 4, I describe the identification of DEC2 as a putative substrate of the SCFFBXW4 ubiquitin ligase. In chapter 5, I analyzed the expression of all F-box proteins in human primary tumors and found that six FBPs are overexpressed in human cancers. Finally for two of these F-box proteins, I performed a proteomic screen aimed at the identification of their specific substrates.

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Chapter 1

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Introduction

55. Yaron A, Gonen H, Alkalay I, Hatzubai A, Jung S, Beyth S, et al. Inhibition of NF-kappa-B cellular function via specific targeting of the I-kappa-B-ubiquitin ligase. The EMBO journal. 1997;16(21):6486-94. 56. Margottin-Goguet F, Hsu JY, Loktev A, Hsieh HM, Reimann JD, Jackson PK. Prophase destruction of Emi1 by the SCF(betaTrCP/Slimb) ubiquitin ligase activates the anaphase promoting complex to allow progression beyond 1 prometaphase. Developmental cell. 2003;4(6):813-26. 57. Bashir T, Dorrello NV, Amador V, Guardavaccaro D, Pagano M. Control of the SCF(Skp2-Cks1) ubiquitin ligase by the APC/C(Cdh1) ubiquitin ligase. Nature. 2004;428(6979):190-3. 58. Wei W, Ayad NG, Wan Y, Zhang GJ, Kirschner MW, Kaelin WG, Jr. Degradation of the SCF component Skp2 in cell-cycle phase G1 by the anaphase- promoting complex. Nature. 2004;428(6979):194-8. 59. Bassermann F, Eichner R, Pagano M. The ubiquitin proteasome system - implications for cell cycle control and the targeted treatment of cancer. Biochimica et biophysica acta. 2014;1843(1):150-62. 60. Spruck C, Strohmaier H, Watson M, Smith AP, Ryan A, Krek TW, et al. A CDK-independent function of mammalian Cks1: targeting of SCF(Skp2) to the CDK inhibitor p27Kip1. Molecular cell. 2001;7(3):639-50. 61. Ganoth D, Bornstein G, Ko TK, Larsen B, Tyers M, Pagano M, et al. The cell-cycle regulatory protein Cks1 is required for SCF(Skp2)-mediated ubiquitinylation of p27. Nature cell biology. 2001;3(3):321-4. 62. Liu H, Cheng EH, Hsieh JJ. Bimodal degradation of MLL by SCFSkp2 and APCCdc20 assures cell cycle execution: a critical regulatory circuit lost in leukemogenic MLL fusions. Genes & development. 2007;21(19):2385-98. 63. Mendez J, Zou-Yang XH, Kim SY, Hidaka M, Tansey WP, Stillman B. Human origin recognition complex large subunit is degraded by ubiquitin-mediated proteolysis after initiation of DNA replication. Molecular cell. 2002;9(3):481-91. 64. Li X, Zhao Q, Liao R, Sun P, Wu X. The SCF(Skp2) ubiquitin ligase complex interacts with the human replication licensing factor Cdt1 and regulates Cdt1 degradation. The Journal of biological chemistry. 2003;278(33):30854-8. 65. Nakayama KI, Nakayama K. Ubiquitin ligases: cell-cycle control and cancer. Nature reviews Cancer. 2006;6(5):369-81. 66. Welcker M, Clurman BE. FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation. Nature reviews Cancer. 2008;8(2):83-93. 67. Crusio KM, King B, Reavie LB, Aifantis I. The ubiquitous nature of cancer: the role of the SCF(Fbw7) complex in development and transformation. Oncogene. 2010;29(35):4865-73. 68. Hao B, Oehlmann S, Sowa ME, Harper JW, Pavletich NP. Structure of a Fbw7-Skp1-cyclin E complex: multisite-phosphorylated substrate recognition by SCF ubiquitin ligases. Molecular cell. 2007;26(1):131-43. 69. Koepp DM, Schaefer LK, Ye X, Keyomarsi K, Chu C, Harper JW, et al. Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase. Science. 2001;294(5540):173-7. 70. Welcker M, Singer J, Loeb KR, Grim J, Bloecher A, Gurien-West M, et al. Multisite phosphorylation by Cdk2 and GSK3 controls cyclin E degradation.

Chapter 1

Molecular cell. 2003;12(2):381-92. 71. Yada M, Hatakeyama S, Kamura T, Nishiyama M, Tsunematsu R, Imaki H, et al. Phosphorylation-dependent degradation of c-Myc is mediated by the F- box protein Fbw7. The EMBO journal. 2004;23(10):2116-25. 72. Welcker M, Orian A, Jin J, Grim JE, Harper JW, Eisenman RN, et al. The 1 Fbw7 tumor suppressor regulates glycogen synthase kinase 3 phosphorylation- dependent c-Myc protein degradation. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(24):9085-90. 73. Saha A, Deshaies RJ. Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation. Molecular cell. 2008;32(1):21-31. 74. Tanaka T, Nakatani T, Kamitani T. Inhibition of NEDD8-conjugation pathway by novel molecules: potential approaches to anticancer therapy. Molecular oncology. 2012;6(3):267-75. 75. Kurz T, Ozlu N, Rudolf F, O'Rourke SM, Luke B, Hofmann K, et al. The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae. Nature. 2005;435(7046):1257-61. 76. Huang G, Kaufman AJ, Ramanathan Y, Singh B. SCCRO (DCUN1D1) promotes nuclear translocation and assembly of the neddylation E3 complex. The Journal of biological chemistry. 2011;286(12):10297-304. 77. Lyapina S, Cope G, Shevchenko A, Serino G, Tsuge T, Zhou C, et al. Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome. Science. 2001;292(5520):1382-5. 78. Zheng J, Yang X, Harrell JM, Ryzhikov S, Shim EH, Lykke-Andersen K, et al. CAND1 binds to unneddylated CUL1 and regulates the formation of SCF ubiquitin E3 ligase complex. Molecular cell. 2002;10(6):1519-26. 79. Liu J, Furukawa M, Matsumoto T, Xiong Y. NEDD8 modification of CUL1 dissociates p120(CAND1), an inhibitor of CUL1-SKP1 binding and SCF ligases. Molecular cell. 2002;10(6):1511-8. 80. Lin HK, Wang G, Chen Z, Teruya-Feldstein J, Liu Y, Chan CH, et al. Phosphorylation-dependent regulation of cytosolic localization and oncogenic function of Skp2 by Akt/PKB. Nature cell biology. 2009;11(4):420-32. 81. Hunter T. The age of crosstalk: phosphorylation, ubiquitination, and beyond. Molecular cell. 2007;28(5):730-8. 82. Kuchay S, Duan S, Schenkein E, Peschiaroli A, Saraf A, Florens L, et al. FBXL2- and PTPL1-mediated degradation of p110-free p85beta regulatory subunit controls the PI(3)K signalling cascade. Nature cell biology. 2013;15(5):472-80. 83. Nelson RF, Glenn KA, Zhang Y, Wen H, Knutson T, Gouvion CM, et al. Selective cochlear degeneration in mice lacking the F-box protein, Fbx2, a glycoprotein-specific ubiquitin ligase subunit. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2007;27(19):5163-71. 84. Yoshida Y, Tokunaga F, Chiba T, Iwai K, Tanaka K, Tai T. Fbs2 is a new member of the E3 ubiquitin ligase family that recognizes sugar chains. The Journal of biological chemistry. 2003;278(44):43877-84.

Introduction

Chapter 2

USP17- and SCFβTrCP-Regulated Degradation of DEC1 Controls the DNA Damage Response

M G2

Jihoon Kim, Sara D’Annibale, Roberto Magliozzi, Teck Yew Low, Petra Jansen, Indra A. Shaltiel, Shabaz Mohammed, Albert J. R. Heck, Rene H. Medema, and Daniele Guardavaccaro

USP17- and SCFβTrCP-Regulated Degradation of DEC1 Controls the DNA Damage Response

ABSTRACT In response to genotoxic stress, DNA-damage checkpoints maintain the integrity of the genome by delaying cell cycle progression to allow for DNA repair. Here we show that the degradation of the bHLH transcription factor DEC1, a critical regulator of cell fate and circadian rhythms, controls the DNA damage response. During unperturbed cell cycles, DEC1 is a highly unstable protein that is targeted for proteasome-dependent degradation by the SCFβTrCP ubiquitin ligase in cooperation with CK1. Upon DNA damage, DEC1 is rapidly induced in an ATM/ATR-dependent manner. DEC1 induction results from protein stabilization via a mechanism that requires the USP17 ubiquitin protease. USP17 binds and deubiquitylates DEC1 markedly extending its half-life. Subsequently, during checkpoint recovery, DEC1 2 proteolysis is reestablished through βTrCP-dependent ubiquitylation. Expression of a degradation-resistant DEC1 mutant prevents checkpoint recovery by inhibiting the downregulation of p53. These results indicate that the regulated degradation of DEC1 is a key factor controlling the DNA damage response.

INTRODUCTION

Cells respond to genotoxic stress by activating DNA damage checkpoints, molecular networks that monitor the integrity of the genome before cells commit to either duplicate their DNA in S phase or separate their chromosomes in mitosis. Once DNA damage is sensed, cells temporally stop cycling facilitating DNA repair. If the degree of the DNA lesions exceeds the capacity of repair processes, cells die by apoptosis or exit irreversibly the cell division cycle and undergo senescence. The molecular mechanisms controlling the DNA damage response are of considerable interest not only because unrepaired DNA damage underlies the development of cancer and checkpoints represent critical barriers to tumor formation, but also because DNA damage is employed therapeutically to kill cancer cells. Many studies have shown that upon DNA damage, two major molecular cascades activated by the sensory ATM/ATR/DNA-PK kinases are responsible for the arrest in the G2 phase of the cell cycle (1-4). They both converge to control the activity of the cyclin B/CDK1 complex, the main regulator of the G2/M transition. The first cascade, which rapidly prevents mitotic entry, involves the activation of the CHK kinases, which, in turn, phosphorylate and inactivate (or target for proteasome- dependent degradation) CDC25 phosphatases, leading to the inhibition of CDK1. The second slower cascade involves the phosphorylation of p53, which impairs its interaction to the MDM2 ubiquitin ligase, promoting both the accumulation and activation of p53. Once induced, p53 target genes, such as p21, 14-3-3, and GADD45, contribute to block the activity of cyclin B/CDK1 through multiple mechanisms. Basic helix-loop-helix (bHLH) transcription factors are key regulators of cell fate specification, apoptosis, cell proliferation and metabolism (5-7). DEC1 (differentiated embryo-chondrocyte expressed gene 1), also known as BHLHE40 (basic helix-loop-helix family, member e40), SHARP2 (enhancer of split and hairy

27 Chapter 2

related protein 2), and STRA13 (stimulated with retinoic acid 13), binds to E-boxes and functions as a transcriptional repressor through histone deacetylase- dependent and -independent mechanisms (8, 9). It was originally identified as a retinoic acid-inducible gene that inhibits mesodermal and promotes neuronal differentiation (10). Subsequently, DEC1 was shown to have an important role in the regulation of mammalian circadian rhythms by repressing CLOCK/BMAL- dependent transactivation of gene expression (11-13). Interestingly, DEC1 expression is induced by a variety of clock resetting stimuli such as light (in the suprachiasmatic nucleus), feeding (in the liver), serum shock, forskolin, TGFβ and 2

28 USP17- and SCFβTrCP-Regulated Degradation of DEC1 Controls the DNA Damage Response

PMA (in cultured cells), suggesting that DEC1 plays a key role in how the circadian clock senses the environment (13). Besides confirming that DEC1 controls the circadian clock in mammals (12), in vivo studies have demonstrated that DEC1 is essential for T cell activation-induced cell death (AICD). Indeed, DEC1 deficiency in mice results in defective clearance of activated T and B cells, which accumulate progressively, causing lymphoid organ hyperplasia and systemic autoimmune disease (14). Depending on the cellular context and the specific stimuli, DEC1 was also shown to mediate cell cycle arrest, senescence and apoptosis via p53-dependent and independent mechanisms (8, 14-16). 2 In this study, we show that DEC1 degradation plays a critical role in the DNA damage response. Genotoxic stress induces DEC1 stabilization via the USP17 ubiquitin protease. During recovery from the DNA damage checkpoint, DEC1 is targeted for proteasomal degradation by the SCFβTrCP ubiquitin ligase in cooperation with CK1. Importantly, inhibition of DEC1 degradation slows down recovery from the G2 DNA damage checkpoint by preventing p53 downregulation.

Figure 1. DEC1 protein levels are upregulated in response to genotoxic stress. (A, B) U2OS cells were treated with doxorubin (A) or etoposide (B) for the indicated times. Cells were collected and lysed. Whole cell extracts were analyzed by immunoblotting with antibodies for the indicated proteins. Actin is shown as loading control. (C) U2OS cells were treated with etoposide in the presence or absence of KU55933 (ATM inhibitor) or ATR-45 (ATR inhibitor) for the indicated times, then collected, lysed and analyzed by immunoblotting. (D) HCT116 cells were treated with etoposide in the presence or absence of caffeine for the indicated times, then collected, lysed and analyzed by immunoblotting. (E) HCT116 p53+/+ and HCT116 p53-/- cells were treated with etoposide and collected at the indicated times. Cells were lysed and analyzed by immunoblotting (l.e., long exposure; s.e., short exposure). (F) U2OS cells were treated with the indicated compounds, alone or in combination, and collected at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. CTR: DMSO-treated control. (G) U2OS cells were pulse-treated with etoposide. After the pulse, the inhibitor of protein synthesis cycloheximide (CHX) was added and cells were collected at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. DEC1 protein levels are quantified in the graphs below. (H) U2OS cells expressing HA-tagged DEC1 and MYC-tagged ubiquitin were treated with etoposide. Whole cell extracts were immunoprecipitated in denaturing conditions with an anti-HA resin. DEC1 immunoprecipitates were immunoblotted with an anti-MYC antibody to detect ubiquitylated DEC1. The bracket indicates a ladder of bands corresponding to poly-ubiquitylated DEC1. Phospho-Chk2 (Thr68) and p53 are shown as markers of checkpoint activation.

29 Chapter 2

RESULTS

DEC1 is induced by DNA damage in an ATM/ATR-dependent manner.

It has been shown that genotoxic stress triggers the induction of DEC1 (15-17). We first confirmed these results by showing that DEC1 is rapidly induced by doxorubicin and etoposide, two topoisomerase inhibitors that cause DNA double- 2 strand breaks (Fig. 1A and B). To extend our observations, we examined the effect of pharmacological inhibition of ATM and ATR, main effector kinases of the DNA damage response, on the induction of DEC1 in response to genotoxic stress. Inhibition of either ATM or ATR prevented the increase of DEC1 protein levels upon etoposide treatment (Fig. 1C). Similarly, treatment of cells with caffeine, which blocks the activation of the ATM and ATR kinases, inhibited the induction of DEC1 upon genotoxic stress (Fig. 1D). To test whether the increase in DEC1 following a DNA damage insult depends on p53, we compared the levels of DEC1 in etoposide-treated HCT116 p53+/+ with the ones in HCT116 p53-/- cells (Fig. 1E). Although the levels of DEC1 are overall lower in the absence of p53, DEC1 was induced with similar kinetics in HCT116 p53-/- and HCT116 p53+/+ cells (compare long and short exposure times of DEC1 immunoblotting). A number of factors suggests that the induction of DEC1 in response to genotoxic stress is due to regulation of its turnover. First, proteasome inhibition and DNA damage in U2OS cells cause a similar increase in DEC1 levels (Fig. 1F). Second, MG132 treatment of damaged cells does not cause a further increase of DEC1 levels (Fig. 1F). Third, and more importantly, the half-life of DEC1 is extended by etoposide treatment (Fig. 1G). DEC1 stabilization in response to genotoxic stress might be due to regulation of its ubiquitylation. To test this possibility, we analyzed the ubiquitylation of DEC1 in U2OS cells treated with etoposide. As shown in Fig. 1H, the activation of the DNA damage response by etoposide treatment [monitored by the induction of p53 and phospho-CHK2 (Thr68)] was associated with a decrease in DEC1 ubiquitylation.

USP17 is required for the stabilization of DEC1 in response to genotoxic stress.

To identify DEC1 interacting proteins that may play a role in the regulation of DEC1 ubiquitylation and degradation, FLAG-HA-tagged DEC1 was expressed in HEK293T cells, immunopurified, and analyzed by mass spectrometry. We recovered peptides corresponding to the USP17 deubiquitylating enzyme as well as the subunits of the SCFβTrCP ubiquitin ligase, i.e., SKP1, CUL1, RBX1, βTrCP1, and βTrCP2 (Table S1). We first confirmed the binding between USP17 and DEC1 (Fig. 2A). Next, to test whether USP17 controls the stability of DEC1, we expressed DEC1 along with USP17 in U2OS cells and blocked protein synthesis by cycloheximide treatment.

30 USP17- and SCFβTrCP-Regulated Degradation of DEC1 Controls the DNA Damage Response

31 Chapter 2

As shown in Fig. 2B, expression of USP17, but not of a USP17 mutant in which the catalytic cysteine has been replaced by serine (C89S), markedly stabilized DEC1. Expression of other DUBs such as USP8 or USP36 had no effect on DEC1 turnover (Fig. 2C). Moreover, wild type USP17, but not the catalytically inactive USP17(C89S) mutant, reduced the amount of ubiquitylated DEC1 in cultured cells (Fig. 2D). These results suggest that USP17 might be responsible for the stabilization of DEC1 observed in response to genotoxic stress. To test this hypothesis, we depleted USP17 using RNAi and examined the levels of DEC1 following DNA damage. The knockdown of USP17 in etoposide-treated cells inhibited the 2 induction of DEC1 (Fig. 2E), which was rescued by proteasome inhibition (Fig. 2F). Next, we tested whether the physical interaction between DEC1 and USP17 is regulated by genotoxic stress. Fig. 2G shows that treatment of U2OS cells with etoposide stimulates the association of USP17 with DEC1.

DEC1 is targeted for proteasome-dependent degradation by SCFβTrCP.

The identification of the SCFβTrCP subunits in the immunopurification of DEC1 (Table S1) prompted us to assess the involvement of SCFβTrCP in the regulation of DEC1 proteolysis. To confirm the interaction of DEC1 with βTrCP, we immunoprecipitated a panel of F-box proteins and tested their binding to

Figure 2. USP17 mediates the stabilization of DEC1 in response to DNA damage. (A) HEK293T cells were transfected with the indicated cDNAs. Cells were lysed and whole cell extracts were subjected to immunoprecipitation using anti-HA resin before immunoblotting with antibodies for the indicated proteins. (B, C) Cells were transfected with the indicated constructs. After 48 hours, cells were treated with cycloheximide (CHX) to block protein synthesis. Cells were collected at the indicated times and lysed. Whole cell extracts were subjected to immunoblotting with antibodies specific for the indicated proteins. DEC1 abundance is quantified in the graphs below. EV: empty vector. (D) HEK293T cells were transfected with the indicated constructs and culture in the presence of MG132 for 3 hours. Cells were collected and lysed. Whole cell extracts were prepared in denaturing conditions and subjected to immunoprecipitation with anti- HA resin. Immunocomplexes were the analyzed by immunoblotting with an anti-MYC antibody. The bracket indicates a ladder of bands corresponding to poly-ubiquitylated DEC1. (E, F) U2OS cells were transfected with constructs expressing either USP17 or control shRNAs. Cells were then treated with etoposide for the indicated times in the absence (E) or presence (F) of the proteasome inhibitor MG132 (where indicated). Cells were collected at the indicated times and lysed. Whole cell extracts were analyzed by immunoblotting with antibodies specific for the indicated proteins. (G) U2OS cells expressing MYC-tagged USP17 and HA-tagged DEC1 were treated with etoposide for the indicated times. Cells were lysed and whole cell extracts were immunoprecipitated with anti-HA resin. Immunoprecipitates were then analyzed by immunoblotting with antibodies specific for the indicated proteins.

32 USP17- and SCFβTrCP-Regulated Degradation of DEC1 Controls the DNA Damage Response

Figure 3. DEC1 degradation is controlled by the SCFβTrCP ubiquitin ligase. (A) βTrCP1 and βTrCP2 interact with DEC1. The indicated FLAG-tagged F-box proteins (FBPs) or an empty vector (EV) were expressed in HEK293T cells. Forty-eight hours after transfection, cells were treated for 5 hours with the proteasome inhibitor MG132, then harvested and lysed. Whole cell extracts were immunoprecipitated (IP) with anti- FLAG resin and immunoblotted with the indicated antibodies. (B) Arg447 in the WD40 repeat of βTrCP2 is required for βTrCP2 binding to DEC1. HEK293T cells were transfected as indicated. After 48 hours, cells were treated with MG132 for 5 hours, then harvested and lysed. Whole cell extracts (WCE) were subjected to immunoprecipitation with anti-FLAG resin, followed by immunoblotting with antibodies specific for the indicated proteins (WT: wild type). (C) DEC1 is ubiquitylated by SCFβTrCP in vitro. DEC1, Skp1, Cul1, and Rbx1 were expressed in HEK293T cells in the absence or presence of either FLAG-tagged βTrCP1 or a FLAG-tagged βTrCP1(∆F-box) mutant. After immunopurification with anti-FLAG resin, in vitro ubiquitylation of DEC1 was performed. Samples were analyzed by immunoblotting with an antibody anti-DEC1. The bracket indicates a ladder of bands corresponding to poly-ubiquitylated DEC1. (D) Cells were transfected with the indicated siRNA oligonucleotides and treated with cycloheximide. Cells were then collected at the indicated times and lysed. Whole cell extracts were subjected to immunoblotting with antibodies specific for the indicated proteins. Actin is shown as a loading control. DEC1 expression is quantified in the graph below.

33 Chapter 2

34 USP17- and SCFβTrCP-Regulated Degradation of DEC1 Controls the DNA Damage Response endogenous DEC1. As shown in Fig. 3A, only βTrCP1 and βTrCP2 were able to co-immunoprecipitate endogenous DEC1. Furthermore, mutation of Arg447, a residue within the WD40 repeats of βTrCP2 required for the interactions with its substrates (18, 19), prevented the binding of DEC1 to βTrCP (Fig. 3B). To test whether DEC1 is a substrate of SCFβTrCP, we reconstituted the ubiquitylation of DEC1 in vitro. βTrCP1, but not an inactive βTrCP1(∆F-box) mutant, was able to mediate DEC1 ubiquitylation in vitro (Fig. 3C). To examine whether DEC1 stability is regulated by βTrCP, we reduced the expression of both βTrCP1 and βTrCP2

Figure 4. CK1α-mediated phosphorylation of DEC1 on a conserved degron is required for DEC1 degradation. 2 (A) DEC1 contains three potential βTrCP-binding domains. The amino acid residues that were mutated to alanine are shown in bold. (B) HEK293T cells were transfected with an empty vector (EV), HA-tagged wild type DEC1, or the indicated HA-tagged DEC1 mutants. Forty-eight hours after transfection, cells were harvested and lysed. Whole cell extracts were subjected to immunoprecipitation (IP) with anti-HA resin, followed by immunoblotting with antibodies specific for the indicated proteins. (C) HEK293T cells were transfected with the indicated constructs. After 24 hours, cells were treated with the inhibitor of protein synthesis cycloheximide (CHX) and collected at the indicated times. Whole cell extracts were then immunoblotted with the indicated antibodies. DEC1 expression is quantified in the graph below. (D) Top: alignment of the amino acid regions corresponding to the βTrCP-binding motif (highlighted in black) in previously reported βTrCP substrates and DEC1 orthologs. The amino acidic sequence of the DEC1 double mutant is shown. Bottom: sequence of the phosphopeptide (top) and the corresponding unphosphorylated peptide (bottom) used to generate the anti-DEC1 phosphospecific antibody (Ser243). (E) U2OS cells were treated with the indicated kinase inhibitors. Cells were collected at the indicated times and lysed. Whole cell extracts were then immunoblotted with antibodies specific for the indicated proteins. Actin is shown as a loading control. DEC1 expression is quantified in the graph below. (F) U2OS cells were transduced with the indicated lentiviral shRNA vectors. Cells were then treated with cycloheximide (CHX) for the indicated times. Cells were collected and lysed. Whole cell extracts were analyzed by immunoblotting. DEC1 expression is quantified in the graph below. (G) Immunoprecipitated wild type DEC1 or the DEC1(S243A/E248A) mutant were analyzed by immunoblotting with antibodies specific for the indicated proteins. When indicated, immunocomplexes were treated with lambda phosphatase (λPP) for 30 minutes. (H) Immunoprecipitated wild type DEC1 or the DEC1(S243A/E248A) mutant were dephosphorylated by treatment with lambda phosphatase and then incubated with the indicated purified kinases in the presence of ATP. Reactions were stopped by adding Laemmli buffer and analyzed by immunoblotting with antibodies specific for the indicated proteins. (I) U2OS cells were transfected with HA-tagged wild type DEC1. Forty-eight hours after transfection, cells were treated with either D4476 or IC261 (CK1 pharmacological inhibitors). Cells were then harvested and lysed. Whole cell extracts were immunoprecipitated (IP) with anti-HA resin and analyzed by immunoblotting. The asterisk indicates a nonspecific band.

35 Chapter 2

using a previously validated siRNA (18). As shown in Fig. 3D, the knockdown of βTrCP caused DEC1 stabilization in HEK293T cells. Altogether these results indicate that DEC1 degradation is controlled by the SCFβTrCP ubiquitin ligase.

CK1α-mediated phosphorylation of DEC1 is required for its destruction.

The binding of βTrCP to its substrates requires the phosphorylation of serine residues within a degron sequence. Some substrates of βTrCP have one or both 2 serine residues replaced by either aspartic or glutamic acid (20, 21). DEC1 contains three putative βTrCP binding domains (Fig. 4A). To assess which of these three domains mediates the binding of DEC1 to βTrCP, we generated a number of serine/aspartic acid/glutamic acid to alanine DEC1 double mutants [all HA-tagged], and examined their ability to interact with endogenous βTrCP. As shown in Fig. 4B, mutation to alanine of Ser243 and Glu248 prevented the binding of DEC1 to βTrCP, whereas mutation of the other two motifs had no effect on the DEC1-βTrCP interaction. Accordingly, the DEC1(S243A/E248A) mutant is stable in cultured HEK293T cells (Fig. 4C). The βTrCP-binding domain in DEC1 is conserved in different species (Fig. 4D). DEC1 Ser243 is part of a conserved phosphorylation consensus site for casein kinase 1 (CK1). To test whether CK1 is involved in the degradation of DEC1, we treated cells with CK1 pharmacological inhibitors and analyzed the half-life of DEC1. Treatment of U2OS cells with D4476 (a CK1 inhibitor), but not TBB (a CK2 inhibitor) caused DEC1 stabilization (Fig. 4E). Furthermore, knockdown of CK1α by RNAi prevented DEC1 degradation in U2OS cells (Fig. 4F). Next, we generated a phospho-specific antibody that recognizes DEC1 only when it is phosphorylated on Ser243 (Fig. 4D and 4G). We employed this antibody to test whether CK1α phosphorylates DEC1 on Ser243. DEC1 was immunopurified from HEK293T cells, dephosphorylated, and then subjected to phosphorylation in vitro in the presence of different purified kinases. As a control, we used the DEC1(S243A/E248A) mutant. DEC1 phosphorylation on Ser243 was then assessed by immunoblotting using the phosphospecific antibody that recognizes DEC1 only when it is phosphorylated on Ser243. Figure 4H shows that DEC1 Ser243 is specifically phosphorylated by CK1α. Accordingly, pharmacological inhibition of CK1 blocked Ser243 phosphorylation in cultured cells (Fig. 4I).

SCFβTrCP and CK1α trigger the degradation of DEC1 during checkpoint recovery. Our data demonstrate that SCFβTrCP and CK1α control the degradation of DEC1 and suggest that they may be involved not only in the constitutive turnover of DEC1, but also in its destruction during checkpoint recovery to counteract the USP17- mediated and DNA damage-induced stabilization of DEC1. To test this possibility, we expressed physiological levels of wild type DEC1 and the DEC1(S243A/E248A) mutant unable to bind βTrCP in U2OS cells (Fig. 5A). Cells were then synchronized in G2 and pulsed with doxorubicin to activate the G2 DNA damage checkpoint. To

36 USP17- and SCFβTrCP-Regulated Degradation of DEC1 Controls the DNA Damage Response induce checkpoint recovery, cells were then treated with caffeine, which inhibits the checkpoint kinases ATM and ATR, turning off the checkpoint. As shown in Fig. 5B, whereas wild type DEC1 was rapidly degraded during caffeine-induced checkpoint recovery, the DEC1(S243A/E248A) mutant was stable. Similar results were obtained when cells were allowed to recover spontaneously from the G2 DNA damage checkpoint that was activated by a lower dose of doxorubicin (Fig. 5C). In contrast, the abundance of wild type DEC1 did not change when cells were released from the G1/S border without DNA damage (Fig. 5D). In addition, no significant changes in the levels of endogenous DEC1 were observed in cells synchronized in G0 by serum deprivation and released in serum-containing medium to allow synchronized progression through G1, S, G2 and mitosis (Fig. 5E). 2

Regulated degradation of DEC1 controls the G2 DNA damage checkpoint.

To study the biological effect of CK1α- and βTrCP-mediated degradation of DEC1 on checkpoint recovery, we analyzed the mitotic entry of hTERT-immortalized retinal pigment epithelial (RPE1) cells expressing either wild type DEC1 or the degradation-resistant DEC1(S243A/E248A) mutant after a pulse of doxorubicin. As shown in Fig. 5F, hTERT-RPE1 cells expressing DEC1(S243A/E248A) displayed defective checkpoint recovery when compared with control cells. Indeed, sixty hours after the doxorubicin pulse, approx. 50% of control cells have entered mitosis,

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whereas only 20% of cells expressing the degradation-resistant DEC1 mutant have reached mitosis at that time. Failure to degrade DEC1 during unperturbed cell cycles had a minimal effect on mitotic entry (Fig. 5G). It has been shown that DEC1 regulates the expression of p53 by directly interacting with p53, blocking its MDM2-mediated degradation (16). To test whether the defective recovery of cells expressing the non-degradable DEC1 mutant was associated with sustained levels of p53, we assessed the levels of p53 in cells expressing the degradation-resistant DEC1 mutant during checkpoint recovery. As shown in Fig. 5H, 24 hours after the DNA damage pulse, cells expressing DEC1(S243A/E248A) displayed increased levels of p53 when compared with cells 2 expressing wild type DEC1. To demonstrate that the defective checkpoint recovery in cells expressing the degradation-resistant DEC1 mutant was indeed caused by the inability of these Figure 5. DEC1 degradation is required for checkpoint recovery. (A) U2OS cells were transduced with retroviruses expressing HA-tagged wild type DEC1, HA-tagged DEC1(S243A/E248A) or with an empty virus (EV). Cells were then collected and analyzed by immunoblotting with an anti-DEC1 antibody. Staining of the PVDF membrane with Ponceau S shows equal loading. (B) U2OS cells, transduced with retroviruses expressing HA-tagged wild type DEC1 or HA-tagged DEC1(S243A/E248A), were treated according to the scheme below. Cells were then collected and analyzed by immunoblotting with antibodies for the indicated proteins. Actin is shown as a loading control. (C) U2OS cells were treated as in A, except that doxorubin was used at a lower dose (0.125 μM) and cells were left recovering spontaneously (without adding caffeine). Cells were then collected and analyzed by immunoblotting with antibodies for the indicated proteins. Actin is shown as a loading control. (D) U2OS cells expressing HA-tagged wild type DEC1 were first synchronized at the G1/S transition by thymidine block and then washed extensively and incubated in fresh medium (indicated as time 0) to allow progression through S, G2 and mitosis. (E) T98G cells (revertants from T98 glioblastoma cells that acquired the property to accumulate in G0 /G1 in low serum) were synchronized in G0 by serum deprivation (0) and released from the arrest by the addition of serum. AS: asynchronously growing cells. Cells were collected at the indicated time points and lysed. Whole cell extracts were analyzed by immunoblotting with antibodies specific for the indicated proteins. (F) hTERT-RPE1 cells expressing HA-tagged wild type DEC1, HA-tagged DEC1(S243A/E248A) or an empty vector were pulse-treated (1 hour) with 0.5 μM doxorubicin. Cells were followed by time-lapse microscopy and scored for mitotic entry. (G) Asynchronously growing hTERT-RPE1 cells expressing wild type DEC1, DEC1(S243A/E248A) or an empty vector were followed by time-lapse microscopy and scored for mitotic entry. (H) As in (F). Twenty-four hours after the pulse, cells were collected and analyzed by immunoblotting with antibodies specific for the indicated proteins. The expression of p53 is quantified in the graph below. (I, J) HCT116 p53+/+ (I) and HCT116 p53-/- (J) cells, expressing HA-tagged wild type DEC1, HA-tagged DEC1(S243A/E248A) or an empty vector, were pulse-treated (1 hour) with 0.5 μM doxorubicin. Cells were then followed by time-lapse microscopy and scored for mitotic entry.

38 USP17- and SCFβTrCP-Regulated Degradation of DEC1 Controls the DNA Damage Response cells to downregulate p53, we analyzed the effect of defective DEC1 degradation on checkpoint recovery of p53-deficient HCT116 cells. Figures 5I and 5J show that the recovery from the G2 DNA damage checkpoint is impaired by the expression of DEC1(S243A/E248A) in HCT116 p53+/+ cells, but not in HCT116 p53-/- cells. Finally, we examined the role of the induction of DEC1 upon DNA damage. To this aim, we silenced the expression of DEC1 by RNAi in hTERT-RPE1-FUCCI cells, which were then treated with a pulse of doxorubicin. These cells, which express both a red (RFP) and a green (GFP) fluorescent protein fused to the cell cycle regulators CDT1 and Geminin, respectively, allowed us to monitor the progression through cell cycle phases in response to genotoxic stress (22). Figures 6A-C indicate that the knockdown of DEC1 caused a defective G2 DNA damage 2 checkpoint as doxorubicin-treated cells in which DEC1 was silenced have a shorter

Figure 6. Role of the USP17-mediated induction of DEC1 in the G2 DNA damage checkpoint. (A) hTERT-RPE1-FUCCI cells were transfected with the indicated siRNA. Whole cell extracts were analyzed by immunoblotting with antibodies specific for the indicated proteins. (B, C) Cells as in (A) were pulsed-treated (1 hour) with doxorubicin and imaged by time- lapse microscopy. The duration of S/G2 (GFP-positive cells) was measured. The graph indicates the quantification of the S/G2 duration. Averages values (+/- S.D.) are indicated. (D) hTERT-RPE1-FUCCI cells were transfected with HA-tagged USP17 or an empty vector. Whole cell extracts were analyzed by immunoblotting with antibodies specific for the indicated proteins. (E) Cells as in (D) were pulsed-treated (1 hour) with doxorubicin and imaged by time- lapse microscopy. The duration of S/G2 (GFP-positive cells) was measured. The graph indicates the quantification of the S/G2 duration. Averages values (+/- S.D.) are indicated.

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G2 phase when compared to doxorubicin-treated control cells. In contrast, ectopic expression of USP17, which mediates the stabilization of DEC1 in response to DNA damage, leads to a prolonged G2 checkpoint following treatment with doxorubicin (Figures 6D-E).

DISCUSSION

In the present work, we show that the rapid induction of DEC1 in response to 2 genotoxic stress is dependent on the USP17 deubiquitylating enzyme and that the proteasomal degradation of DEC1, mediated by SCFβTrCP and CK1α, is required for efficient recovery from the G2 DNA damage checkpoint. In line with our findings, a recently performed large-scale comparative phosphoproteomic screen, aimed at analyzing changes in protein phosphorylation during checkpoint recovery and identifying factors required for this process, identified CK1α as a potential regulator of checkpoint recovery (23). Indeed, CK1α was found differentially phosphorylated during recovery from the G2 checkpoint caused by DNA damage and, importantly, cells in which CK1α was depleted by siRNA were unable to recover from the DNA damage-induced G2 arrest (23). A number of studies have uncovered a key function of SCFβTrCP in driving cells into mitosis during recovery from DNA replication and the DNA damage checkpoints. Indeed, during checkpoint recovery, SCFβTrCP, in cooperation with Polo-like kinase- 1, contributes to turn CDK1 activity on by targeting both Claspin and WEE1 for proteasomal degradation. Claspin is an adaptor protein that in response to DNA replication and DNA damage checkpoints promotes ATR-mediated phosphorylation and activation of CHK1. Claspin degradation triggered by βTrCP and Polo like kinase-1 leads to CHK1 inactivation and subsequent accumulation of CDC25A and activation of CDC25B and CDC25C. WEE1 is a direct inhibitor of CDK1 and its destruction by βTrCP and Polo like kinase-1 removes the break on CDK1 activity. These degradation events, despite being crucial, are insufficient to account for the whole checkpoint recovery process. In fact, inactivation of p53, a second important brake responsible for a sustained cell cycle arrest upon genotoxic stress, is essential for checkpoint recovery. Our study indicates that, by targeting DEC1 for degradation during recovery from the G2 checkpoint, SCFβTrCP is also responsible for the termination of the inhibitory action of p53 on G2 progression and mitotic entry. Interestingly, a genome-wide characterization of DEC1-regulated genes in T- lymphocytes has been recently reported (24). Gene ontology analysis revealed that cell cycle genes, and in particular genes that control the G2/M transition, were highly enriched (third highest category) among the DEC1-regulated genes, implying that, in addition to its effect on p53 expression, DEC1 regulated stability in response to DNA damage might modulate the expression of a multitude of genes controlling cell cycle progression. Finally, as DEC1 is a crucial regulator of circadian rhythms, our findings suggest that acute induction of DEC1 in response to genotoxic stress, carried out by the

40 USP17- and SCFβTrCP-Regulated Degradation of DEC1 Controls the DNA Damage Response coordinated actions of USP17 and CK1α/SCFβTrCP, might enable a rapid resetting of the circadian clock following DNA damage.

MATERIALS AND METHODS

Cell culture and drug treatment. U2OS, HEK293T, HEK293-GP2, HCT116, HCT116 p53 -/-, T98G, hTERT-RPE1, and hTERT-RPE1-FUCCI cells were maintained in Dulbecco’s modified Eagle’s 2 medium (Invitrogen) containing 10% fetal calf serum, 100 U/ml penicillin and streptomycin. The following drugs were used: etoposide (Sigma-Aldrich, 20 µg/ml), doxorubicin (Sigma-Aldrich; 0.125 μM for spontaneous recovery and 1 mM for caffeine-induced recovery), MG132 (Peptide Institute; 10 µM), TBB (EMD Millipore, 75 μM), D4476 (Sigma-Aldrich, 50 μM), IC261 (Sigma-Aldrich, 50 μM), cycloheximide (Sigma-Aldrich, 100 μg/ml), caffeine (Sigma-Aldrich, 5 µM), thymidine (Sigma-Aldrich, 2.5 µM), nocodazole (Sigma-Aldrich, 0.1 µg/ml), KU55933 (ATM inhibitor, EMD Millipore, 10 μM), ATR-45 (ATR inhibitor, Ohio State University, 2 μM).

Biochemical methods. Extract preparation, immunoprecipitation, and immunoblotting were previously described (20, 25). Mouse monoclonal antibodies were from Cell Signaling [phospho-p53(Ser15)], Invitrogen (CUL1), Sigma-Aldrich (FLAG), BD Transduction Laboratories (p27), Santa Cruz Biotechnology (Actin), and Covance (HA). Rabbit polyclonal antibodies were from Cell Signaling [βTrCP1, CK1α, p53, MYC, phospho-CHK2(Thr68)], Sigma-Aldrich (FLAG, USP8), Novus Biologicals (DEC1, USP17), Santa Cruz Biotechnology (cyclin A) and EMD Millipore [phospho-Histone H3 (Ser10)]. To generate the anti-DEC1 phosphospecific antibody (Ser243), rabbits were immunized with the SDTDTDpSGYGG phosphopeptide (pS = phospho-Ser243). The rabbit polyclonal antiserum was purified through a two-step purification process. First, the antiserum was passed through a column containing the unphosphorylated peptide (SDTDTDSGYGG) to remove antibodies that recognize the unphosphorylated peptide. The flow-through was then purified against the phosphopeptide to isolate antibodies that recognize DEC1 phosphorylated on Ser243. The final product was dialyzed with 1XPBS, concentrated and tested for phosphopeptide and phosphoprotein specificity.

Purification of DEC1 interactors. HEK293T cells were transfected with pcDNA3-FLAG-HA-DEC1 and treated with 10 μM MG132 for 5 hours. Cells were harvested and subsequently lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP40, plus protease and phosphatase inhibitors). DEC1 was immunopurified with anti-FLAG

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agarose resin (Sigma-Aldrich). After washing, proteins were eluted by competition with FLAG peptide (Sigma-Aldrich). The eluate was then subject to a second immunopurification with anti-HA resin (12CA5 monoclonal antibody crosslinked to protein G Sepharose; Invitrogen) prior to elution in Laemmli sample buffer. The final eluate was separated by SDS-PAGE, and proteins were visualized by Coomassie colloidal blue. Bands were sliced out from the gels and subjected to in-gel digestion. Gel pieces were then reduced, alkylated and digested according to a published protocol (26). For mass spectrometric analysis, peptides recovered from in-gel digestion were separated with a C18 column and introduced by nano-electrospray into the LTQ Orbitrap XL (Thermo Fisher) with a configuration as described (27). 2 Peak lists were generated from the MS/MS spectra using MaxQuant build 1.0.13.13 (28), and then searched against the IPI Human database (version 3.37, 69164 entries) using Mascot search engine (Matrix Science). Carbaminomethylation (+57 Da) was set as fixed modification and protein N-terminal acetylation and methionine oxidation as variable modifications. To filter out false positives, all MS/MS spectra were searched against a forward and a reverse (decoy) protein database. All peptide-spectrum matches (PSMs) containing both forward and decoy hits were then trained and validated with an algorithm named Percolator (29) which uses semi-supervised machine learning to discriminate between the correct and decoy matches. Finally, we only accepted PSM based on a high stringency filter of q = 0.01 or 1% false discovery rate (FDR). This 1% FDR filter is currently the standard for the proteomics community.

Plasmids and small hairpin RNAs. Mammalian expression plasmids for CK1α, His-USP17, MYC-USP17, USP8 and HA-USP36 were provided by H. Clevers, J. Johnston, K. Baek, M. Donzelli and M. Komada, respectively. DEC1 cDNAs (wild type and mutants) were cloned in pcDNA3.1. For retrovirus production, HA-tagged DEC1 and MYC-tagged USP17 cDNAs (wild type and mutants) were subcloned into pBABE-PURO. DEC1 mutants were generated using the QuickChange Site-directed Mutagenesis kit (Stratagene). shRNAs targeting human CK1α were provided by W. Wei (30). The shRNA for USP17, CCAAGACGTTAACTTTACA (construct 1) and GCAGGAAGATGCCCATGAA (construct 2) were in cloned in pSUPER. All constructs were sequenced.

Transient transfections, retrovirus- and lentivirus-mediated transfer. HEK293T and U2OS cells were transfected using the polyethylenimine (PEI) method. For retrovirus production, HEK293-GP2 cells were co-transfected with pBABE-puro-HA DEC1 and packaging vectors, and the virus-containing medium was collected after 48 hours and supplemented with 8 μg/ml polybrene. Lentivirus produced in HEK293T cells transfected with pLKO-CK1 and packaging vectors. Virus containing medium was collected 48 hours after transfection and served with 8 g/ml polybrene. Target cells were incubated with the virus for 6 hours.

Gene silencing by small interfering RNA.

42 USP17- and SCFβTrCP-Regulated Degradation of DEC1 Controls the DNA Damage Response

The sequence and validation of the oligonucleotides corresponding to βTrCP1 and βTrCP2 were previously published (20, 31). Cells were transfected with the oligonuclotides twice (24 and 48 hours after plating) using Oligofectamine (Invitrogen) according to manufacturer’s recommendations. Forty-eight hours after the last transfection, lysates were prepared and analyzed by SDS-PAGE and immunoblotting.

In vitro ubiquitylation assay. DEC1 ubiquitylation was performed in a volume of 10 µl containing SCFβTrCP- DEC1 immunocomplexes, 50 mM Tris pH 7.6, 5 mM MgCl2, 0.6 mM DTT, 2 mM ATP, 1.5 2 ng/µl E1 (Boston Biochem), 10 ng/µl UBC3, 2.5 µg/µl ubiquitin (Sigma-Aldrich), 1 µM ubiquitin aldehyde. The reactions were incubated at 30°C for 60 minutes and analyzed by immunoblotting.

Live cell microscopy. Time-lapse microscopy to analyze mitotic entry was performed as described (32, 33).

Statistical analysis. All data shown are from one representative experiment of at least three performed. Statistical analysis was performed using Student’s t-test. Results with p < 0.005 were considered to be statistically significant.

REFERENCES

1. Medema RH, Macurek L. Checkpoint control and cancer. Oncogene. 2012;31(21):2601-13. 2. Jackson SP, Bartek J. The DNA-damage response in human biology and disease. Nature. 2009;461(7267):1071-8. 3. Kastan MB, Bartek J. Cell-cycle checkpoints and cancer. Nature. 2004;432(7015):316-23. 4. Harper JW, Elledge SJ. The DNA damage response: ten years after. Molecular cell. 2007;28(5):739-45. 5. Murre C, McCaw PS, Vaessin H, Caudy M, Jan LY, Jan YN, et al. Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell. 1989;58(3):537-44. 6. Atchley WR, Fitch WM. A natural classification of the basic helix-loop- helix class of transcription factors. Proc Natl Acad Sci U S A. 1997;94(10):5172-6. 7. Ledent V, Paquet O, Vervoort M. Phylogenetic analysis of the human basic helix-loop-helix proteins. Genome Biol. 2002;3(6):RESEARCH0030. 8. Sun H, Taneja R. Stra13 expression is associated with growth arrest and represses transcription through histone deacetylase (HDAC)-dependent and HDAC-independent mechanisms. Proc Natl Acad Sci U S A. 2000;97(8):4058-63.

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9. St-Pierre B, Flock G, Zacksenhaus E, Egan SE. Stra13 homodimers repress transcription through class B E-box elements. J Biol Chem. 2002;277(48):46544-51. 10. Boudjelal M, Taneja R, Matsubara S, Bouillet P, Dolle P, Chambon P. Overexpression of Stra13, a novel retinoic acid-inducible gene of the basic helix- loop-helix family, inhibits mesodermal and promotes neuronal differentiation of P19 cells. Genes Dev. 1997;11(16):2052-65. 11. Honma S, Kawamoto T, Takagi Y, Fujimoto K, Sato F, Noshiro M, et al. Dec1 and Dec2 are regulators of the mammalian molecular clock. Nature. 2002;419(6909):841-4. 2 12. Nakashima A, Kawamoto T, Honda KK, Ueshima T, Noshiro M, Iwata T, et al. DEC1 modulates the circadian phase of clock gene expression. Mol Cell Biol. 2008;28(12):4080-92. 13. Kon N, Hirota T, Kawamoto T, Kato Y, Tsubota T, Fukada Y. Activation of TGF-beta/activin signalling resets the circadian clock through rapid induction of Dec1 transcripts. Nat Cell Biol. 2008;10(12):1463-9. 14. Sun H, Lu B, Li RQ, Flavell RA, Taneja R. Defective T cell activation and autoimmune disorder in Stra13-deficient mice. Nat Immunol. 2001;2(11):1040-7. 15. Qian Y, Zhang J, Yan B, Chen X. DEC1, a basic helix-loop-helix transcription factor and a novel target gene of the p53 family, mediates p53- dependent premature senescence. J Biol Chem. 2008;283(5):2896-905. 16. Thin TH, Li L, Chung TK, Sun H, Taneja R. Stra13 is induced by genotoxic stress and regulates ionizing-radiation-induced apoptosis. EMBO Rep. 2007;8(4):401-7. 17. Qian Y, Jung YS, Chen X. Differentiated embryo-chondrocyte expressed gene 1 regulates p53-dependent cell survival versus cell death through macrophage inhibitory cytokine-1. Proc Natl Acad Sci U S A. 2012;109(28):11300-5. 18. Kruiswijk F, Yuniati L, Magliozzi R, Low TY, Lim R, Bolder R, et al. Coupled Activation and Degradation of eEF2K Regulates Protein Synthesis in Response to Genotoxic Stress. Science Signaling. 2012;5(227):ra40. 19. Magliozzi R, Low TY, Weijts BG, Cheng T, Spanjaard E, Mohammed S, et al. Control of Epithelial Cell Migration and Invasion by the IKKbeta- and CK1alpha-Mediated Degradation of RAPGEF2. Dev Cell. 2013;27(5):574-85. 20. Guardavaccaro D, Frescas D, Dorrello NV, Peschiaroli A, Multani AS, Cardozo T, et al. Control of chromosome stability by the beta-TrCP-REST-Mad2 axis. Nature. 2008;452(7185):365-9. 21. Watanabe N, Arai H, Nishihara Y, Taniguchi M, Hunter T, Osada H. M- phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFbeta-TrCP. Proc Natl Acad Sci U S A. 2004;101(13):4419-24. 22. Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell. 2008;132(3):487-98. 23. Halim VA, Alvarez-Fernandez M, Xu YJ, Aprelia M, van den Toorn HW, Heck AJ, et al. Comparative phosphoproteomic analysis of checkpoint recovery identifies new regulators of the DNA damage response. Sci Signal. 2013;6(272):rs9.

44 USP17- and SCFβTrCP-Regulated Degradation of DEC1 Controls the DNA Damage Response

24. Martinez-Llordella M, Esensten JH, Bailey-Bucktrout SL, Lipsky RH, Marini A, Chen J, et al. CD28-inducible transcription factor DEC1 is required for efficient autoreactive CD4+ T cell response. J Exp Med. 2013;210(8):1603-19. 25. Ping Z, Lim R, Bashir T, Pagano M, Guardavaccaro D. APC/C (Cdh1) controls the proteasome-mediated degradation of E2F3 during cell cycle exit. Cell Cycle. 2012;11(10):1999-2005. 26. Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 1996;68(5):850-8. 27. Raijmakers R, Berkers CR, de Jong A, Ovaa H, Heck AJ, Mohammed S. Automated online sequential isotope labeling for protein quantitation applied to 2 proteasome tissue-specific diversity. Mol Cell Proteomics. 2008;7(9):1755-62. 28. Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008;26(12):1367-72. 29. Kall L, Canterbury JD, Weston J, Noble WS, MacCoss MJ. Semi- supervised learning for peptide identification from shotgun proteomics datasets. Nat Methods. 2007;4(11):923-5. 30. Gao D, Inuzuka H, Tan MK, Fukushima H, Locasale JW, Liu P, et al. mTOR drives its own activation via SCF(betaTrCP)-dependent degradation of the mTOR inhibitor DEPTOR. Molecular cell. 2011;44(2):290-303. 31. D'Annibale S, Kim J, Magliozzi R, Low TY, Mohammed S, Heck AJ, et al. Proteasome-dependent Degradation of Transcription Factor AP4 (TFAP4) Controls Mitotic Division. J Biol Chem. 2014. 32. Lindqvist A, de Bruijn M, Macurek L, Bras A, Mensinga A, Bruinsma W, et al. Wip1 confers G2 checkpoint recovery competence by counteracting p53- dependent transcriptional repression. EMBO J. 2009;28(20):3196-206. 33. Alvarez-Fernandez M, Halim VA, Krenning L, Aprelia M, Mohammed S, Heck AJ, et al. Recovery from a DNA-damage-induced G2 arrest requires Cdk- dependent activation of FoxM1. EMBO Rep. 2010;11(6):452-8.

Chapter 3

The SCF-mediated Degradation of TFAP4 Regulates Progression Through the S and G2 Phases of the Cell Cycle

AP4

Sara D’Annibale, Jihoon Kim, Roberto Magliozzi, Teck Yew Low, Shabaz Mohammed, Albert J. R. Heck, and Daniele Guardavaccaro.

The SCF-mediated Degradation of TFAP4 Regulates Progression Through the S and G2 Phases of the Cell Cycle ABSTRACT

The transcription factor TFAP4 (Transcription factor activating enhancer-binding protein 4) is a basic helix-loop-helix leucine-zipper transcription factor. It has been reported that TFAP4 is a c-MYC target gene that can either activate or suppress gene expression. TFAP4 is implicated in many crucial cellular processes such as regulation of stemness, epithelial-mesenchymal transition, neuronal differentiation, cell proliferation. Importantly, TFAP4 is up-regulated in human colorectal cancer and other cancer types. We have recently demonstrated that the abundance of TFAP4 in the cell is controlled by proteasomal degradation and is mediated by the SCFTrCP ubiquitin ligase. Here, I have studied the biological function of TFAP4 degradation by expressing a degradation-resistant TFAP4 mutant in cells and characterizing the effect on cell cycle progression. I have found that failure to degrade TFAP4 leads to accumulation of cells in S and G2. Moreover, by using the fluorescence-based cell cycle tracking system FUCCI, I was able to show that 3 inhibition of TFAP4 degradation leads to a delayed progression through S/G2.

INTRODUCTION

TFAP4, a ubiquitously expressed member of the family of basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factors, binds to the symmetric E-box element 5’-CAGCTG-3' (1). Through its multiple protein-protein interaction domains, i.e. bHLH, LZ1 and LZ2, TFAP4 specifically forms homodimers (2). TFAP4 was originally identified as a cellular factor that activates viral genes from the simian virus 40 (SV40) enhancer (3). Thereafter, several reports have described additional roles of TFAP4. The Hermeking lab has demonstrated that TFAP4 regulates cell proliferation by suppressing the expression of the cyclin-dependent kinase inhibitors p16 and p21 (4). Indeed, it has been found that the direct interaction of TFAP4 with the conserved E-box elements present in the promoters of p16 and p21 results in inhibition of cellular senescence. TFAP4 was also shown to control epithelial-mesenchymal transition (EMT) by regulating the expression of a large number of genes implicated in EMT, such as SNAIL, E-cadherin, OCLN, VIM, FN1, CLDN1, CLDN4, CLDN7, miR-15a and miR- 16-1 (5, 6). Moreover, it has been reported that the p53-induced microRNAs miR- 15a and miR16-1 together with TFAP4 form a double negative feedback loop to regulate EMT in colon cancer (6). Others have reported that TFAP4 regulates apoptosis by binding the promoter of caspase-9 (7), however, TFAP4 overexpression and knock down do not dramatically affect caspase-9 expression indicating that TFAP4 might have a role in maintaining the basal expression of caspase-9. Moreover, TFAP4 cooperates with other factors, namely, geminin, SMRT and HDAC3, in repressing the neuron-specific genes such as PAHX-AP1 in non-neuronal cells thus contributing to the developmental expression of neuronal genes in the brain (8). Remarkably, TFAP4 expression is up-regulated in colorectal cancer, hepatocellular carcinoma, gastric cancer and pancreatic cancer (5, 9-12).

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In spite of the reported key roles of TFAP4 in the control of crucial cellular processes linked to cancer development, very little is known about the regulation of TFAP4 protein levels.

RESULTS

TFAP4 is degraded by SCFTrCP in the G2 phase of the cell division cycle.

In Guardavaccaro lab, TFAP4 was recently found in a mass spectrometry-based screen aimed at the identification of new substrates of SCFTrCP (13). We confirmed 3 that TFAP4 specifically binds to TrCP1 and its paralog TrCP2 and not to other F- box proteins (FBXW2, FBXW4, FBXW5, FBXW7 and FBXW8) or the APC/C activators CDH1 and CDC20 (13). Moreover, mutation of arginine 474, a residue within the WD40 repeats of TrCP crucial for substrate binding (14), prevents TFAP4 binding to TrCP (13). Furthermore, we demonstrated that TrCP, but not a TrCP-ΔF mutant that lacks the F-box domain, mediates the ubiquitylation of TFAP4 in vitro (13). We also identified, downstream of the TFAP4 bHLH domain, a conserved phosphodegron that is required for the interaction of TFAP4 with TrCP. Indeed, replacing glutamic acid 135 and serine 139 with alanine abolishes the binding of TFAP4 with TrCP in co-immunoprecipitation assays and leads to TFAP4 stabilization in half-life experiments (13). Altogether these results demonstrate that TFAP4 is a substrate of SCFTrCP. Next, we determined the biological condition under which TFAP4 is targeted for proteasomal degradation. We found that TFAP4 protein levels decrease during the S and G2 phases of the cell cycle and that this degradation depends on the proteasome and TrCP because it is abolished by the proteasome inhibitor MG132 or by TrCP RNAi (13). Importantly, the TFAP4(E135A/S139A) mutant unable to bind TrCP is not degraded in S/G2 (13). Taken together, these data indicate that TFAP4 is targeted for proteasomal degradation by SCFTrCP during the S and G2 phases of the cell cycle.

Inhibition of TFAP4 degradation leads to accumulation of S/G2 cells.

To study the effect of the TrCP-mediated degradation of TFAP4 on cell cycle progression, we expressed wild type TFAP4 and the non-degradable TFAP4(E135A/S139A) mutant in cells. To this aim, we employed a doxycycline inducible system in HCT116 colorectal cancer cells (Fig. 1A). This system allowed us to culture the cells without the effect of inhibiting TFAP4 degradation until the beginning of the experiment. When asynchronously growing cells were treated with

50 The SCF-mediated Degradation of TFAP4 Regulates Progression Through the S and G2 Phases of the Cell Cycle

Figure 1. Effect of inhibiting the degradation of TFAP4 on the cell cycle. (A) HCT116 cells were pulse-treated with doxycycline. At the indicated times after the pulse, cells were then collected and lysed. Whole cell lysate were analyzed by immunoblotting with antibodies specific for the indicated proteins. (B) HCT116 cells as in (A) were fixed, stained with propidium iodide and analyzed by flow cytometry. (C) Hela (C) and HCT116 p53-/- cells (D) expressing with wild type TFAP4, TFAP4(E135A/S139A) or an empty vector were treated as in (B). doxycycline, the expression of wild type TFAP4 and the non-degradable TFAP4(E135A/S139A) mutant was induced (Fig. 1A). We then analyzed the cell cycle profile by propodium iodide (PI) staining followed by flow cytometry analysis. Cells expressing TFAP4-wild type, the TFAP4(E135A/S139A) mutant or an empty vector were fixed with methanol and washed. Cells were then stained with PI, treated with RNAse and analyzed with flow cytometry. Figure 1B shows that cells expressing the degradation-resistant TFAP4 mutant show a larger population of S and G2 cells when compared to cells expressing wild type TFAP4. Interestingly, the accumulation of S/G2 cells caused by the expression of the non-degradable TFAP4 mutant was not observed in p53-deficient HCT116 cells (Fig. 1C) or in HeLa in which p53 is inactive (Fig. 1D). These data indicate that the TrCP-mediated degradation of TFAP4 is required for normal cell cycle progression and that the accumulation of S/G2 cells upon inhibition of TFAP4 degradation depends on the intact function of p53.

Generation of cells expressing FUCCI and the non-degradable TFAP4 mutant.

51 Chapter 3

To characterize the phenotype caused by the inhibition of TFAP4 degradation, we established cell lines in which the inducible expression of wild type TFAP4 or the degradation-resistant mutant was coupled to the expression of fluorescent ubiquitination-based cell cycle indicator (FUCCI), a fluorescent, two-color sensor that allows to monitor cell cycle progression and division of living cells in real time (15). In this system, red fluorescent protein (RFP) and green fluorescent protein (GFP) are fused to human CDT1 and Geminin, respectively. CDT1 is present only in G1 because is targeted for degradation by the SCFSKP2 ubiquitin ligase specifically in S/G2/M, whereas Geminin is present only in S/G2 because is targeted for degradation by APC/CCDH1 specifically in late M and G1. As a result, these two chimeric proteins, RFP-CDT1 and GFP-Geminin, accumulate reciprocally in the nuclei of transfected cells during the cell cycle, labeling red the

Figure 2. Generation of cells expressing FUCCI and 3 the non-degradable TFAP4 mutant. (A) Schematic presentation of the FUCCI system. See text for details. (B) HCT116 cells in which the expression of TFAP4 (wild type or E135A/S139A) can be induce by doxycycline treatment were transduced by FUCCI lentiviruses (see text for detail) and sorted for high expression of GFP and RFP double positive. (C) Time course experiment of FUCCI system established HCT116 inducible cell line. Cells expressing color cell cycle dependent manner. The single asterisk marks a green cell dividing into two daughter cells 8 hours after filming. The double asterisk marks a cell turning color from red to green 8 hours after filming.

nuclei of G1 cells and green those of S/G2 cells green (Fig. 2A). We first delivered these constructs by lentiviral transduction into cells that can express wild type TFAP4 or the non-degradable mutant in an inducible manner (upon doxycyclin treatment) and then sorted the cells that expressed high levels of GFP and RFP by flow cytometry (Fig. 2B and 2C).

52 The SCF-mediated Degradation of TFAP4 Regulates Progression Through the S and G2 Phases of the Cell Cycle Failure to degrade TFAP4 delays progression through the S and G2 phases.

Once established the FUCCI-HCT116 cells in which the expression of the non- degradable TFAP4 mutant can be induced by treatment with doxycycline, we analyzed the effect of inhibiting the degradation of TFAP4. We pulse-induced the expression of wild type TFAP4 and TFAP4(E135A/S139A) and monitored cell cycle progression by time-lapse microscopy. Figure 3 shows that the duration of the CDT1-positive G1 phase of HCT116 cells expressing wild type TFAP4 was comparable with the one of cells expressing the TFAP4(E135A/S139A) mutant.

Figure 3. Effect of inhibiting TFAP4 degradation on cell cycle progression. (A) FUCCI-HCT116 cells were treated with doxycycline to induce the expression of wilt type TFAP4 (top) or the non-degradable mutant (bottom). Cells were then filmed for 14 hours and introduced time-lapse microscopy analysis. Fluorescent images using GFP and RFP filters as well as phase contrast images were taken every ten minutes. Sixty-minute interval time points are shown. (C) Quantification of the duration of each cell phase shown in (B). Average values (± SD) are indicated. However, cells expressing TFAP4(E135A/S139A) exhibited a prolonged geminin- positive S/G2 (9.9 hours) when compared with cells expressing wild type TFAP4 (6.8 hours). Altogether, our data indicate that failure to degrade TFAP4 leads to accumulation of cells in S/G2 and that this is due to a delayed S/G2 progression.

DISCUSSION

53 Chapter 3

In this study we have shown that the βTrCP-dependent degradation of TFAP4 is required for the progression through the S and G2 phases of the cell cycle. Indeed, expression of a TFAP4 mutant that is resistant to degradation leads to accumulation of S/G2 cells. We have also demonstrated that this accumulation is caused by a delayed progression through S and G2. Additional studies are required to identify the TFAP4 target genes whose misregulation is responsible for the defective progression through the S and G2 cell cycle phases. Interestingly, it has been recently demonstrated that TFAP4 represses the expression of the CDK inhibitor p21 (10). In this regard, several labs have shown that during unperturbed cell cycles, p21 has two peaks of expression, the first in G1 and the second in G2, needed to establish the two cell cycle pauses (16-18). According to these studies, the βTrCP-mediated degradation of TFAP4 in S/G2 would allow the derepression of p21 expression in G2. Although we have not confirmed this hypothesis, the repression of p21 caused by the expression of the 3 degradation-resistant TFAP4 mutant is expected to result in increased CDK activity and faster (rather than slower) G2 progression compared to cells expressing wild type TFAP4. However, it is worth mentioning that hyperphosphorylated p21 in G2 was shown to play a positive role (rather than negative) in promoting the G2/M transition (19). According to this study, the interaction between p21 and cyclin B1- CDK1 appears to promote (instead of inhibiting) the activity of cyclin B1-CDK1 at G2/M. Indeed, p21-deficient cells were shown to have a delayed activation of cyclin B1-CDK1 kinase activity and a delayed G2 progression compared to cells expressing wild type p21. An alternative model is based on the fact that the G2 expression of p21 is required to promote a transient pause that contributes to the integration of the G2 cell cycle checkpoints. According to this scenario, the βTrCP-dependent degradation of TFAP4 in G2, which remains low in the following mitosis and G1, would be required to keep the cells competent for the G2 as well as the next G1 DNA damage checkpoint. Indeed, in case of genotoxic stress, TFAP4 degradation would remove a break for p21 induction to enable temporary or permanent (senescence) cell cycle arrest. Further experiments are needed to test these hypotheses.

MATERIALS AND METHODS

Cell culture, synchronization and drug treatment. HEK293T, HeLa, HCT116, HCT116p53-/- and HCT116-FUCCI cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) containing 10% fetal calf serum. Doxycycline (1 g/ml) was added to induce the expression of TFAP4.

Plasmids The TFAP4 non-degradable mutant was generated using the QuickChange Site- directed Mutagenesis kit (Stratagene). For retrovirus production, both wild type

54 The SCF-mediated Degradation of TFAP4 Regulates Progression Through the S and G2 Phases of the Cell Cycle

TFAP4 and the non-degradable TFAP4 mutant were subcloned into the retroviral vector LZRSpBMN-GFP (pallino). For lentivirus production, both wild type TFAP4 and the TFAP4 mutant were subcloned into the lentiviral vector pHAGE2-EF1α. To generate the FUCCI cells, the constructs pCSII-EF-mKO2-hCdt1 (30/120) and pCSII-EF-mAG-hGEM (1/110) were used. For stable transfection, wild type TFAP4 and TFAP4(E135A/S139A) were subcloned into pcDNA 4/TO inducible vector. All cDNAs were sequenced.

Transient transfections, retrovirus- and lentivirus-mediated gene transfer HEK293T cells were transfected using the calcium phosphate method as described (20). Retrovirus-mediated gene transfer was previously described (20, 21). For lentivirus production, HEK293T cells were co-transfected with pHAGE2-EF1α and packaging vectors by using PEI (Polyethylenimine). Virus-containing medium was collected 48 hours after transfection and supplemented with 8 μg/ml polybrene. Cells were incubated with virus-containing medium for 6 hours for 2 consecutive 3 days.

Antibodies Mouse monoclonal antibodies were from Santa Cruz Biotechnology (Actin) and Covance (HA).

Live cell imaging Cells were plated and cultured in six-well plates in complete medium. Microscope was equipped with an incubating chamber at 37°C and a CO2 supply. Images were taken at 10 minutes intervals for 3 days.

REFERENCES

1. Jung P, Hermeking H. The c-MYC-AP4-p21 cascade. Cell cycle. 2009;8(7):982-9. 2. Hu YF, Luscher B, Admon A, Mermod N, Tjian R. Transcription factor AP- 4 contains multiple dimerization domains that regulate dimer specificity. Genes & development. 1990;4(10):1741-52. 3. Mermod N, Williams TJ, Tjian R. Enhancer binding factors AP-4 and AP- 1 act in concert to activate SV40 late transcription in vitro. Nature. 1988;332(6164):557-61. 4. Jackstadt R, Jung P, Hermeking H. AP4 directly downregulates p16 and p21 to suppress senescence and mediate transformation. Cell death & disease. 2013;4:e775. 5. Jackstadt R, Roh S, Neumann J, Jung P, Hoffmann R, Horst D, et al. AP4 is a mediator of epithelial-mesenchymal transition and metastasis in colorectal cancer. The Journal of experimental medicine. 2013;210(7):1331-50. 6. Shi L, Jackstadt R, Siemens H, Li H, Kirchner T, Hermeking H. p53-

55 Chapter 3

induced miR-15a/16-1 and AP4 form a double-negative feedback loop to regulate epithelial-mesenchymal transition and metastasis in colorectal cancer. Cancer research. 2014;74(2):532-42. 7. Tsujimoto K, Ono T, Sato M, Nishida T, Oguma T, Tadakuma T. Regulation of the expression of caspase-9 by the transcription factor activator protein-4 in glucocorticoid-induced apoptosis. The Journal of biological chemistry. 2005;280(30):27638-44. 8. Kim MY, Jeong BC, Lee JH, Kee HJ, Kook H, Kim NS, et al. A repressor complex, AP4 transcription factor and geminin, negatively regulates expression of target genes in nonneuronal cells. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(35):13074-9. 9. Friess H, Ding J, Kleeff J, Fenkell L, Rosinski JA, Guweidhi A, et al. Microarray-based identification of differentially expressed growth- and metastasis- associated genes in pancreatic cancer. Cellular and molecular life sciences : CMLS. 2003;60(6):1180-99. 3 10. Jung P, Menssen A, Mayr D, Hermeking H. AP4 encodes a c-MYC- inducible repressor of p21. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(39):15046-51. 11. Hu BS, Zhao G, Yu HF, Chen K, Dong JH, Tan JW. High expression of AP- 4 predicts poor prognosis for hepatocellular carcinoma after curative hepatectomy. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 2013;34(1):271-6. 12. Xinghua L, Bo Z, Yan G, Lei W, Changyao W, Qi L, et al. The overexpression of AP-4 as a prognostic indicator for gastric carcinoma. Medical oncology. 2012;29(2):871-7. 13. D'Annibale S, Kim J, Magliozzi R, Low TY, Mohammed S, Heck AJ, et al. Proteasome-dependent degradation of transcription factor activating enhancer- binding protein 4 (TFAP4) controls mitotic division. The Journal of biological chemistry. 2014;289(11):7730-7. 14. Wu G, Xu G, Schulman BA, Jeffrey PD, Harper JW, Pavletich NP. Structure of a beta-TrCP1-Skp1-beta-catenin complex: destruction motif binding and lysine specificity of the SCF(beta-TrCP1) ubiquitin ligase. Molecular cell. 2003;11(6):1445-56. 15. Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell. 2008;132(3):487-98. 16. Dulic V, Stein GH, Far DF, Reed SI. Nuclear accumulation of p21Cip1 at the onset of mitosis: a role at the G2/M-phase transition. Molecular and cellular biology. 1998;18(1):546-57. 17. Amador V, Ge S, Santamaria PG, Guardavaccaro D, Pagano M. APC/C(Cdc20) controls the ubiquitin-mediated degradation of p21 in prometaphase. Molecular cell. 2007;27(3):462-73. 18. Medema RH, Klompmaker R, Smits VA, Rijksen G. p21waf1 can block cells at two points in the cell cycle, but does not interfere with processive DNA- replication or stress-activated kinases. Oncogene. 1998;16(4):431-41. 19. Dash BC, El-Deiry WS. Phosphorylation of p21 in G2/M promotes cyclin B-Cdc2 kinase activity. Molecular and cellular biology. 2005;25(8):3364-87.

56 The SCF-mediated Degradation of TFAP4 Regulates Progression Through the S and G2 Phases of the Cell Cycle

20. Guardavaccaro D, Frescas D, Dorrello NV, Peschiaroli A, Multani AS, Cardozo T, et al. Control of chromosome stability by the beta-TrCP-REST-Mad2 axis. Nature. 2008;452(7185):365-9. 21. Dorrello NV, Peschiaroli A, Guardavaccaro D, Colburn NH, Sherman NE, Pagano M. S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth. Science. 2006;314(5798):467-71.

FBXW4 DEC2

SCFFBXW4 Targets DEC2 for Proteasome-dependent Degradation

Abstract

The DEC1 paralog DEC2 is a basic helix-loop-helix (bHLH) transcription factor that binds to E-box elements. It is known that DEC2 regulates cell cycle, response to hypoxia, apoptosis, circadian rhythm, and differentiation. Moreover, the expression of DEC2 is altered in several cancer types, such as ovarian, lung, and pancreatic cancer. Although DEC2 is involved in a number of crucial cellular processes, nothing is known about its posttranslational regulation. Here, we show that FBXW4, the substrate-binding component of the SCFFBXW4 ubiquitin ligase, regulates DEC2 protein levels in a proteasome-dependent manner.

Introduction

The basic helix-loop-helix transcription factor DEC2 (also known as Bhlhe41 and Sharp1) is a paralog of DEC1 (1, 2). Like other bHLH proteins, DEC2 binds to the E-box element CAXXTG and regulates the expression of genes involved in several important biological processes (3). The best-characterized function of DEC2 is its regulation of the mammalian 4 circadian rhythm (4). It has been reported that DEC2 and its paralog DEC1 repress the transactivation of gene expression mediated by CLOCK/BMAL, a heterodimeric transcription factor complex, which represents the core molecular complex of the circadian rhythm molecular network (5, 6). Two possible mechanisms of regulation of CLOCK/BMAL-dependent transactivation of gene expression by DEC2 have been proposed. DEC2 might inhibit CLOCK/BMAL by directly binding to the complex or by competing for the same E-box element that is bound by CLOCK/BMAL (4). Genetic studies in mice unveiled that both single and double DEC1/DEC2 knock- out animals have defective (i) period length, (ii) CLOCK/BMAL target gene expression and (iii) re-setting of the circadian rhythm (7). Unlike mice deficient for Cryptochrome (CRY), an additional protein involved in the circadian clock molecular network, DEC1 and DEC2 knock-out mice still have rhythmicity, suggesting that DEC1 and DEC2 have a role in fine-tuning the circadian rhythms (7, 8). Remarkably, DEC2 is implicated in the regulation of sleep length in human. Indeed, it has been recently reported that a mutation in DEC2 (DEC2-P385R) is associated with a human short sleep phenotype (9). Shortened sleep length and more vigilance time were also confirmed in mice carrying the equivalent hDEC2-P385R mutation. The authors suggested that this mutation has a dominant negative function in mammals. Another well-studied function of DEC2 is its regulation of apoptosis. Whereas a number of studies have shown that DEC1 has a pro-apoptotic function, DEC2 seems to be anti-apoptotic (10-12). Biochemical studies have demonstrated that DEC2 suppresses the expression of Bim, a negative regulator of the pro-survival protein Bcl-2, thus leading to increased cell viability.

61 Chapter 4

A number of studies have implicated DEC2 in human cancer. Montagner and colleagues have recently found that DEC2 suppresses metastasis of triple negative breast cancer by triggering the proteasome-dependent degradation of the hypoxia- inducible factors, HIF-1 and HIF-2(13). Moreover, It has been reported that ectopic expression of DEC2 inhibits migration and invasion of pancreatic cancer cells. In these cells, DEC2 regulates the expression of Slug, a key transcription factor involved in epithelial-mesenchymal transition (EMT), by binding the E-box element present in its promoter region. F-box/WD repeat-containing protein 4 (FBXW4) is member of the family of F-box proteins, substrate-recognizing subunits of SCF ubiquitin ligases (14). As all other F-box proteins, FBXW4 contains an F-box motif that interacts with the adaptor protein SKP1 and five WD40 repeats, which provide the substrate-interacting interface. It has been recently shown that FBXW4 interacts with the other SCF components SKP1, CUL1, RBX1 and mediates protein ubiquitylation. Moreover, FBXW4 can be co-immunoprecipitated with the components of the COP9 signalosome (15). The function of FBXW4 has been studied in zebrafish and mice (16-18). The Hopkins lab has shown that mutation of hagoromo (the zebrafish ortholog of FBXW4) leads to defective stripe patterning in fish (16). In this study, the authors 4 employed insertional mutagenesis and found that the defective stripe patterning results from mutation in the hag (hagoromo) gene. Defects in the formation of iridophores was found to be the primary cause of the observed phenotype. Furthermore, disruption of Dactylin, the FBXW4 ortholog in the mouse, results in the absence of central digits, underdeveloped or absent metacarpal/metatarsal bones and syndactyly (18). This phenotype is remarkably similar to the human split hand-split foot malformation (SHFM), also known as ectrodactyly, a congenital limb malformation, characterized by a deep median cleft of the hand and/or foot (19). A significant decrease of FBXW4/Dactylin transcript in several individuals affected by SHFM has been reported (20). However, it is worth to mention that there is much debate in the field as to whether or not loss of FBXW4 is actually the causal lesion leading to SHFM in humans or mice. In fact some in the field believe that it is the alteration of the adjacent Fgf8 gene that causes the SHFM malformations. It has been reported that FBXW4 has tumor suppressive functions. FBXW4 is often mutated, lost or deleted and expressed at low levels in many human cancer cell lines. Approximately 25% of human cancer cell lines show lost/deletion of the FBXW4 locus with more than 40% in certain types of cancer cell lines (skin, breast, central nervous system and lung). Moreover, patients who have low levels of FBXW4 show less survival rate compared to patients with higher FBXW4 expression (15). Despite the described functions of FBXW4 in development and oncogenesis, its specific substrates as well as its function in the regulation of downstream signaling pathways are currently unknown.

Results

62 SCFFBXW4 Targets DEC2 for Proteasome-dependent Degradation

Figure 1. DEC2 binds to FBXW4. FLAG epitope-tagged F-box proteins and the APC/C co-activator CDC20 were expressed in HEK293T cells. Forty- eight hours after transfection, cells were treated with the proteasome inhibitor MG132 for 5 hours. Cells were harvested and lysed. Whole cell extracts were subjected to immunoprecipitation (IP) with a FLAG antibody and immunoblotted with antibodies for the indicated proteins. In the previous study (Chapter 2), I found that DEC1, a paralog of DEC2, is targeted 4 by SCFTrCP for proteasome-dependent degradation (21). While I was investigating the functional role of the TrCP-mediated degradation of DEC1, I wondered how the abundance of DEC2 is regulated. The sequence homology between the two paralog proteins suggested that also DEC2, similarly to DEC1, may be targeted by SCFTrCP for proteasome-dependent degradation. To test this hypothesis, HEK293T cells were transfected with a number of FLAG epitope-tagged F-box proteins and CDC20, the substrate-binding protein of the APC/C ubiquitin ligase. Cells were then treated with the proteasome inhibitor MG132 before lysis. Whole cell extracts were then immunoprecipitated with a FLAG antibody. Immunoprecipitated complexes were separated by SDS-PAGE and immunoblotted with antibodies specific for DEC1 or DEC2. Figure 1 shows that whereas DEC1 is specifically pulled down by TrCP1 and TrCP2, DEC2 co-immunoprecipitates with FBXW4 but not with TrCP1 or TrCP2. These results indicate that FBXW4 interacts specifically with DEC2 in HEK293T cells and suggest that FBXW4 might target DEC2 for proteasomal degradation. Next, I employed a CUL1 deletion mutant (CUL1-N385) that acts in a dominant negative manner since it binds SKP1-F-box protein complexes but it is unable to bind RBX1 and the E2 ubiquitin-conjugating enzyme (22) (Fig. 2A). If DEC2 is targeted for proteasomal degradation by an SCF ubiquitin ligase, i.e. SCFFBXW4, DEC2 protein levels are expected to increase upon overexpression of CUL1-N385. Figure 2B shows that DEC2, as well as established SCF substrates such as WEE1 and catenin, accumulated upon overexpression of the CUL1-N385 mutant. To test whether DEC2 abundance is controlled by the proteasome, we analyzed the levels of endogenous DEC2 in cells treated with the proteasome inhibitor MG132. Endogenous DEC2 accumulated in response to MG132 treatment (Fig. 3) suggesting that the turnover of DEC2 is regulated by proteasome-dependent

63 Chapter 4

degradation.

Figure 2. DEC2 is an SCF substrate. (A) Schematic representation of the molecular mechanism underlying the dominant- negative function of the CUL1 deletion mutant (CUL1-N385). (B) HEK293T cells were transiently transfected with either an empty vector or CUL1-N385. Two days later, cells were harvested and lysed. Whole cell extracts were analyzed by immunoblotting with antibodies specific for the indicated proteins. 4

Discussion

We have demonstrated that the F-box protein FBXW4 interacts specifically with the bHLH transcription factor DEC2. We have also shown that overexpression of a CUL1 deletion mutant (CUL1-N385), which functions in a dominant negative fashion because it interacts with Skp1 and the F-box protein but it does not to bind to RBX1, leads to accumulation of DEC2. Finally we have found that proteasome inhibition causes accumulation of DEC2 in cells. Taken together these results suggest that DEC2 is targeted by SCFFBXW4 for proteasome-dependent degradation. Additional experiments are needed to confirm this model. First, it is necessary to verify whether DEC2 is ubiquitylated by SCFFBXW4 in vitro. It would be also important to test if FBXW4 controls the turnover of DEC2. To address this question, two different loss-of-function approaches could be used. Knock-down of FBXW4 or overexpression of FBXW4 mutant lacking the F-box motif are expected to cause DEC2 accumulation. Also, it would be key to identify the specific regions in DEC2 that are required for the binding to FBXW4 and DEC2 proteolysis. DEC2 mutants that are unable to bind FBXW4 and resistant to degradation would be precious tools to understand the biological function of DEC2 degradation. A possible scenario is that FBXW4 may mediate the periodic ubiquitylation and degradation of DEC2 during its circadian oscillation. In this regard, it has been shown that a number of F-box proteins are responsible for the oscillation of core molecules of the circadian clock molecular network. For instance, FBXL3 and FBXL21 have been shown to target CRY1 and CRY2 for proteasome-dependent degradation in a circadian manner. In addition, TrCP induces the ubiquitylation

64 SCFFBXW4 Targets DEC2 for Proteasome-dependent Degradation

Figure 3. Proteasome- dependent degradation of DEC2. U2OS cells were treated with the proteasome inhibitor MG132 for indicated times. Cells were harvested and lysed. 2 Whole cell extracts 1.8 were analyzed by 1.6 immunoblotting with antibodies for the Fold 1.4 indicated proteins. The 1.2 graph illustrates the 1 quantification of DEC2 0 0.5 1 2 4 abundance relative to the amount at time 0. Time (hr) and degradation of PER1 and PER2 regulating their circadian periodicity. If FBXW4 is responsible for the periodic ubiquitylation and degradation of DEC2, the 4 abundance of DEC2 mutants that are unable to interact with FBXW4 are expected to be constant during circadian rhythms. Moreover, blocking this oscillation might have an effect on the circadian rhythms of these cells. FBXW4-dependent periodic ubiquitylation and degradation of DEC2 could be achieved in different ways, for instance by opposite periodic oscillation of FBXW4, or by periodic changes of those posttranslational modifications of DEC2 that are likely required (on the basis of the analogy with other FBXW substrates) for its binding to FBXW4 (e.g. phosphorylation). It would be interesting to test whether the FBXW4-dependent ubiquitylation and degradation of DEC2 is responsible for the decreased levels of DEC2 observed in metastatic TNBC (13). Do the decreased levels of DEC2 in metastatic TNBC result from increased FBXW4-mediated degradation during epithelial-mesenchymal transition of TNBC cells? Does expression of the DEC2 non-degradable mutant prevent epithelial-mesenchymal transition of TNBC cells? Interestingly, an increase in the incidence of breast cancer has been reported in women who work night shifts, and the incidence is higher among women who spend more hours per week and years working at night. These findings suggest that perturbed circadian rhythmicity is linked to cancer development. In conclusion, characterizing the molecular mechanisms by which FBXW4 targets DEC2 for proteasomal degradation and studying its biological significance will shed light on possible links between the circadian clock and cancer.

MATERIALS AND METHODS.

65 Chapter 4

Tissue culture and drug treatment. HEK293T and U2OS cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal calf serum, 100 U/ml penicillin and streptomycin. MG132 (Peptide Institute; 10 M) was used to inhibit the proteasome.

Biochemical methods. Whole cell extracts preparation, immunoprecipitation and immunoblotting were previously described (23, 24). Mouse monoclonal antibodies were purchased from Sigma-Aldrich (FLAG) and Santa Cruz (Actin). The polyclonal anti-DEC2 antibody was from Santa Cruz Biotechnology.

Plasmids and transient transfection. The cDNAs for FLAG-tagged F-box proteins and CDC20 were cloned in pcDNA3.1 and sequenced. The C-terminal truncated mutant of CUL1 (CUL1-N385) was subcloned into pcDNA3.1. Cells were transfected using the polyethylenimine (PEI) method. 4 References

1. Fujimoto K, Shen M, Noshiro M, Matsubara K, Shingu S, Honda K, et al. Molecular cloning and characterization of DEC2, a new member of basic helix-loop- helix proteins. Biochemical and biophysical research communications. 2001;280(1):164-71. 2. Rossner MJ, Dorr J, Gass P, Schwab MH, Nave KA. SHARPs: mammalian enhancer-of-split- and hairy-related proteins coupled to neuronal stimulation. Molecular and cellular neurosciences. 1997;10(3-4):460-75. 3. Massari ME, Murre C. Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms. Molecular and cellular biology. 2000;20(2):429-40. 4. Honma S, Kawamoto T, Takagi Y, Fujimoto K, Sato F, Noshiro M, et al. Dec1 and Dec2 are regulators of the mammalian molecular clock. Nature. 2002;419(6909):841-4. 5. Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, et al. Role of the CLOCK protein in the mammalian circadian mechanism. Science. 1998;280(5369):1564-9. 6. Darlington TK, Wager-Smith K, Ceriani MF, Staknis D, Gekakis N, Steeves TD, et al. Closing the circadian loop: CLOCK-induced transcription of its own inhibitors per and tim. Science. 1998;280(5369):1599-603. 7. Rossner MJ, Oster H, Wichert SP, Reinecke L, Wehr MC, Reinecke J, et al. Disturbed clockwork resetting in Sharp-1 and Sharp-2 single and double mutant mice. PloS one. 2008;3(7):e2762. 8. van der Horst GT, Muijtjens M, Kobayashi K, Takano R, Kanno S, Takao M, et al. Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature. 1999;398(6728):627-30.

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9. He Y, Jones CR, Fujiki N, Xu Y, Guo B, Holder JL, Jr., et al. The transcriptional repressor DEC2 regulates sleep length in mammals. Science. 2009;325(5942):866-70. 10. Wu Y, Sato F, Bhawal UK, Kawamoto T, Fujimoto K, Noshiro M, et al. BHLH transcription factor DEC2 regulates pro-apoptotic factor Bim in human oral cancer HSC-3 cells. Biomedical research. 2012;33(2):75-82. 11. Wu Y, Sato F, Bhawal UK, Kawamoto T, Fujimoto K, Noshiro M, et al. Basic helix-loop-helix transcription factors DEC1 and DEC2 regulate the paclitaxel- induced apoptotic pathway of MCF-7 human breast cancer cells. International journal of molecular medicine. 2011;27(4):491-5. 12. Liu Y, Sato F, Kawamoto T, Fujimoto K, Morohashi S, Akasaka H, et al. Anti-apoptotic effect of the basic helix-loop-helix (bHLH) transcription factor DEC2 in human breast cancer cells. Genes to cells : devoted to molecular & cellular mechanisms. 2010;15(4):315-25. 13. Montagner M, Enzo E, Forcato M, Zanconato F, Parenti A, Rampazzo E, et al. SHARP1 suppresses breast cancer metastasis by promoting degradation of hypoxia-inducible factors. Nature. 2012;487(7407):380-4. 14. Skaar JR, Pagan JK, Pagano M. Mechanisms and function of substrate recruitment by F-box proteins. Nature reviews Molecular cell biology. 2013;14(6):369-81. 4 15. Lockwood WW, Chandel SK, Stewart GL, Erdjument-Bromage H, Beverly LJ. The novel ubiquitin ligase complex, SCF(Fbxw4), interacts with the COP9 signalosome in an F-box dependent manner, is mutated, lost and under-expressed in human cancers. PloS one. 2013;8(5):e63610. 16. Kawakami K, Amsterdam A, Shimoda N, Becker T, Mugg J, Shima A, et al. Proviral insertions in the zebrafish hagoromo gene, encoding an F-box/WD40- repeat protein, cause stripe pattern anomalies. Current biology : CB. 2000;10(8):463-6. 17. Friedli M, Nikolaev S, Lyle R, Arcangeli M, Duboule D, Spitz F, et al. Characterization of mouse Dactylaplasia mutations: a model for human ectrodactyly SHFM3. Mammalian genome : official journal of the International Mammalian Genome Society. 2008;19(4):272-8. 18. Sidow A, Bulotsky MS, Kerrebrock AW, Birren BW, Altshuler D, Jaenisch R, et al. A novel member of the F-box/WD40 gene family, encoding dactylin, is disrupted in the mouse dactylaplasia mutant. Nature genetics. 1999;23(1):104-7. 19. Duijf PH, van Bokhoven H, Brunner HG. Pathogenesis of split-hand/split- foot malformation. Human molecular genetics. 2003;12 Spec No 1:R51-60. 20. Basel D, DePaepe A, Kilpatrick MW, Tsipouras P. Split hand foot malformation is associated with a reduced level of Dactylin gene expression. Clinical genetics. 2003;64(4):350-4. 21. Kim J, D'Annibale S, Magliozzi R, Low TY, Jansen P, Shaltiel IA, et al. USP17- and SCFbetaTrCP-Regulated Degradation of DEC1 Controls the DNA Damage Response. Molecular and cellular biology. 2014. 22. Piva R, Liu J, Chiarle R, Podda A, Pagano M, Inghirami G. In vivo interference with Skp1 function leads to genetic instability and neoplastic transformation. Molecular and cellular biology. 2002;22(23):8375-87. 23. Kruiswijk F, Yuniati L, Magliozzi R, Low TY, Lim R, Bolder R, et al. Coupled

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activation and degradation of eEF2K regulates protein synthesis in response to genotoxic stress. Science signaling. 2012;5(227):ra40. 24. Guardavaccaro D, Frescas D, Dorrello NV, Peschiaroli A, Multani AS, Cardozo T, et al. Control of chromosome stability by the beta-TrCP-REST-Mad2 axis. Nature. 2008;452(7185):365-9.

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Chapter 5

Other F-box Proteins and Cancer

Jihoon Kim and Daniele Guardavaccaro

Other F-box Proteins and Cancer

ABSTRACT

F-box proteins (FBPs), substrate-binding subunits of SCF ubiquitin ligases, are encoded by 69 different genes in humans but only for a few of them biological roles and substrates are known. It is well established that a handful of FBPs, which target for degradation cancer-related substrates, are either overexpressed or inactivated in many types of cancer. Here we performed a systematic analysis of the gene expression profile of most members of the FBP family in human primary tumors. According to our analysis, at least 6 additional FBPs are highly overexpressed in cancer. We further studied two of these overexpressed FBPs, namely FBXW9 and FBXO6, which were immunopurified and analyzed by mass spectrometry to identify their specific substrates and regulators.

INTRODUCTION

The SCF (SKP1-CUL1-F-box protein) complex is composed of four major subunits, SKP1, CUL1, RBX1 and one of many F-box proteins (FBPs). The human genome encodes for 69 distinct F-box proteins which selectively interact with their substrates (1, 2). F-box proteins are grouped into three classes based on the architecture of their protein-protein interaction domains which mediate substrate binding: FBXWs, FBXLs and FBXOs containing, respectively, WD40 domains, leucine-rich repeats (LRR) and various (other) domains. F-box proteins recognize short motifs within the substrate, which, in many cases, have to be post- 5 translationally modified (phosphorylation, glycosylation) to be able to interact with the F-box protein. It is well established that a number of F-box proteins, i.e. FBXW7, SKP2 and βTrCP, have oncogenic or tumor suppressive functions (1). FBXW7, for instance, is a renowned tumor suppressor mutated in approximately 6% of overall human cancers and in some cases (cholangiocarcinomas and T-cell acute lymphocytic leukemia) the mutation frequency reaches more than 30%. There are two known mutation hotspots (Arg465 and Arg479) on FBXW7 occurring approximately in 43% of overall mutations. Structural studies of FBXW7 in complex with its substrate cyclin E have revealed that Arg465 and Arg479, located in the third WD40 domain of FBXW7, form hydrogen bonds with phosphorylated Thr380 of cyclin E (3, 4). This finding explains why mutations of Arg465 and Arg479 lead to accumulation of FBXW7 oncogenic substrates, namely, MYC, Cyclin E, JUN, TRF1, NOTCH1, NOTCH4, and MCL1 (5, 6). In addition, genetic studies have revealed that FBXW7 deficiency in mice contributes to tumor initiation (7-10). Conditional knock-out of FBXW7 in murine bone marrow drives pancytopenia and abnormal proliferation of hematopoietic stem cells (HSCs). Moreover most of these mice develop leukaemia that has been attributed to accumulation of MYC (8). Many studies have demonstrated that SKP2, also known as FBXL1, is an oncoprotein. SKP2 overexpression was observed in various cancers such as

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74 Other F-box Proteins and Cancer lymphoma, prostate cancer, melanoma, nasopharyngeal carcinoma, gastric cancer, colon cancer, and breast cancer (11-18). The oncogenic functions of SKP2 are attributed to its role in the ubiquitylation of tumor suppressor substrates such as p27, p21, and FOXO1, whose degradation is accelerated in tumor cells overexpressing SKP2 (1). The tumor suppressor function of SKP2 is also indicated by the fact that its expression is reversely correlated with prognosis in breast cancer, gastric cancer and melanoma (17, 19, 20). Mouse genetic studies support SKP2 oncogenic functions. Constitutive expression of SKP2 in mouse prostate promotes marked overproliferation, which result in hyperplasia, dysplasia and low-grade carcinoma of the prostate gland, and, in agreement with its role in p27 proteolysis, leads to downregulation of p27. Analogously, SKP2 overexpression in the T- lymphoid lineage cooperates with N-ras in lymphomagenesis (21).

Due to the diversity of its targets, βTrCP is expected to have both oncogenic and tumour suppressor functions, however, accumulating data suggest that βTrCP displays mostly an oncogenic role. In fact, it has been shown that βTrCP is overexpressed in many human tumors. For instance, βTrCP1 mRNA and protein levels are increased in human colorectal cancer and that this increase is associated with reduced apoptosis and poor prognosis (22). Moreover, pancreatic carcinoma cells display increased expression of βTrCP1 and constitutive activation of NFκB (23). βTrCP1 levels are increased in human hepatoblastomas (24), whereas βTrCP2 overexpression is found in human breast cancers (25). Accordingly, targeted expression of βTrCP1 in murine mammary epithelia leads to increased proliferation, and formation of breast carcinomas in 38% of these mice (26). Notably, ectopic expression of βTrCP in lymphoid organs does not lead to any evident 5 phenotype, indicating that βTrCP-mediated oncogenesis is tissue specific (26). FBXO11 provides another example of FBP with tumor suppressive function. It has been recently found that FBXO11 triggers the proteasome-dependent degradation of BCL-6, an oncoprotein involved in the pathogenesis of human B-cell lymphomas. Moreover, it has been demonstrated that the gene encoding FBXO11 is deleted or mutated in aggressive diffuse large B-cell lymphoma cell lines and primary tumors and that these mutations lead to BCL6 stabilization (27-31). The aim of this study is to systematically analyze the expression of all F-box proteins in human primary tumors. We found that the expression of two FBPs, namely FBXW9 and FBXO6, is elevated in many cancer types. We then performed affinity purification followed by mass spectrometry analysis for both FBPs for an initial characterization of their interactomes. Figure 1. Gene expression analysis of F-box proteins. (A) Expression of FBPs (βTrCP2, SKP2 and EMI1) that are known to be up-regulated in cancer. Each red dot indicates an individual sample from a cancer patient. FBP expression is shown in different cancer types and in normal tissues. (B) FBP expression of six differentially expressed FBPs in cancer. The gene expression profile of all FBPs is shown in Fig. S1. (C) Schematic representation of the domain architecture of FBXO6 and FBXW9. RESULTS

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Expression profiling of FBPs in human primary tumors

Although many studies have implicated abnormal functions of a number of F-box proteins in oncogenesis, a large-scale analysis is still necessary. We decided to perform a systematic analysis of FBP gene expression from primary tumor samples. We used primary tumor microarray data sets (GEO microarray experiment ID: GSE2109), which contain the following cancer samples: 292 colon cancers, 125 breast cancers, 87 melanomas, 72 lung cancers, 70 cervical cancers, 38 gastric cancers, 276 ovarian cancers and 79 renal tumors. To test the reliability of this approach, we first analyzed the gene expression in numerous cancer types and in normal tissues of FBXO5, FBXL1/SKP2 and TrCP, which are known to be overexpressed in many human cancers. As shown in Fig 1A, the gene expression of FBXO5, FBXL1 and TrCP2 is highly increased in various cancer types when compared with the one in normal tissues. Next, we assessed the gene expression of additional 64 F-box proteins in the same primary tumor microarray data set (Fig. S1). We found that FBXW9, FBXW12, FBXL7, FBXL15, FBXO6 and FBXO22 are either highly overexpressed or underexpressed in some or most cancer types (Fig. 1B). For instance, the expression levels of FBXO6, FBXO22 and FBXL15 are increased (if compared with normal tissues) in most cancer types. Interestingly, FBXW9 was highly expressed in specific cancers, typically breast cancer, ovarian cancer and melanomas. On the other hands, FBXL7 and FBXW12 were underexpressed specifically in pancreatic cancer. 5 Identification of FBXW9 and FBXO6 interactors

As FBXW9 and FBXO6 are highly expressed in most of cancer types, we decided to focus on these two F-box proteins. From a structural point of view, in addition to the F-box domain, FBXW9 contains WD40 repeats while FBXO6 contains an FBA (F-box associated domain) domain (Fig. 1C). Since very little is known about the function of FBXW9 and FBXO6, we decided to immunopurify them from cells and perform mass spectrometry analysis for an initial characterization of their interactomes and possible substrate candidates. To this aim, we generated constructs (for expression in mammalian cells) of FBXW9 and FBXO6 bearing double FLAG and HA epitope tags. FBXW9 was stably expressed in MCF7 breast cancer cells and FBXO6 was transiently expressed in HEK293T cells. Five hours before harvesting, cells were treated with MG132 to block proteasome-dependent degradation. Cells were then lysed and two sequential immunoprecipitations (FLAG and HA) were performed. The final eluate was then separated and proteins were analyzed by mass spectrometry. We recovered hundreds of hits including components of the SCF complex, i.e. Cul1, SKP1 and the FBP baits FBXW9 and FBXO6. After removing common contaminants (proteins also found in the control sample (EV) where no bait was present), we overall identified 44 putative interactors for FBXW9 and 418 for FBXO6 (Table S1).

76 Other F-box Proteins and Cancer

Figure 2. Identification of FBXW9 and FBXO6 interactors. FBXW9 and FBXO6 were expressed in MCF7 or HEK293T cells. Five hours before collecting the cells, MG132 added to block the function of the proteasome. Cells were then lysed. Whole cell extracts were then subjected to FLAG and HA sequential immunoprecipitations. Immunocomplexes were then analyzed by mass spectrometry analysis. The top 10 proteins in our mass-spectrometry analysis list are presented based on the number of unique spectra. The complete lists of proteins used for categorization are available in Table S1. SCF components were highlighted in green. Functional categorization of biological processes of FBXW9 and FBXO6 interacting proteins are shown in the bar graph below the table.

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Next, we categorized these interacting proteins based on gene ontology terms. As shown in Fig. 2, the top listed biological processes are metabolic (GO: 0008152, cellular amino acid, lipid, protein metabolic process, glycolysis), cellular (GO: 0009987, cell communication, cell cycle, cytokinesis, cellular component movement and chromosome segregation), biological regulation (GO: 0065007, homeostatic process, regulation of biological process and regulation of molecular function), developmental (GO: 0032502, anatomical structure morphogenesis, cell differentiation, system development) and localization (GO: 0051179, RNA, protein localization and transport) or immune system process (GO: 0002376, B cell mediated immunity and response to interferon-gamma).

Validation of FBXW9 interactors

According to our mass spectrometry analysis data, Adenomatosis Polyposis Coli-2 (APC2, also known as APCL) is pulled down specifically with FBXW9. APC2 is a paralog of the Adenomatosis Polyposis Coli (APC) tumor suppressor which is frequently mutated in colon cancer and other cancer types (32-36). These APC mutations lead to stabilization of the - catenin oncoprotein resulting in uncontrolled cell proliferation. APC, 5 Fig 3. Validation of the APC2- together with axin/axin2, glycogen synthase FBXW9 interaction. FLAG epitope- kinase 3 beta (GSK3) and casein kinase 1 tagged F-box proteins, namely alpha (CK1), forms the -catenin FBXW6, FBXW9, FBXW11, FBXWO6 destruction complex (37, 38). Similarly to and FBXO21, were co-expressed with MYC-tagged APC2 in HEK293T APC, APC2 has a conserved domain cells. Cells were collected 48 hours architecture that makes it possible to after transfection. F-box proteins interact with -catenin and possibly with were immunoprecipitated (IP) with a axin/axin2 (39). Interestingly, unlike APC, mouse monoclonal anti-FLAG APC2 mutations have not been reported in antibody. Immunocomplexes were cancer. subjected to SDS-PAGE, transferred onto a PVDF membrane and blotted To confirm the interaction of FBXW9 with with an anti-MYC antibody to detect APC2, we performed a co- APC2 and an anti-FLAG to detect the immunoprecipitation assay followed by immunoprecipitated F-box proteins. immunoblotting. We transiently co- overexpressed MYC-tagged APC2 along

with FLAG-tagged FBXW6, FBXW9, FBXW11, FBXO6 or FBXO21 in HEK293T cells. Forty-eight hours later, cells were harvested and lysed. Whole cell extracts were immunopurified with a FLAG resin and APC2 was detected with a MYC antibody. As shown in Fig. 3, APC2 is specifically pulled down by FBXW9 but not by other FBPs indicating that APC2 selectively interacts with FBXW9. Additional experiments are needed to demonstrate that APC2 is a substrate of the SCFFBXW9 ubiquitin ligase. It is necessary to assess whether SCFFBXW9 is able to

78 Other F-box Proteins and Cancer ubiquitylate APC2 in vitro. It would be also crucial to test if APC2 is degraded in an ubiquitin- and proteasome-dependent manner and if its turnover is mediated by FBXW9. Loss-of-function and gain of function approaches could be used. Moreover, it is important to identify the specific regions in APC2 that mediate APC2 binding to FBXW9. APC2 mutants that are unable to bind FBXW9 and resistant to degradation could then be expressed in cells to study the biological function of APC2 degradation.

DISCUSSION

A growing body of evidences indicates that mutational inactivation or overexpression of FBPs as well as mutations of FBP substrates contribute to cancer development. In this study, we provide new insights on novel FBPs and their possible binding partners which might have functional roles in cancer development. We propose that a number of FBPs, namely FBXW9, FBXW12, FBXL7, FBXL15, FBXO6 and FBXO22, might be good candidates for more detailed studies as they are differentially expressed in either all or specific cancer types when compared to normal tissues. We performed additional studies on FBXW9 and FBXO6. By using affinity chromatography followed by mass spectrometry analysis, we identified many candidate interacting proteins of FBXW9 and FBXO6 and confirmed that one of them, APC2, is a specific binding partner of FBXW9. 5

MATERIALS AND METHODS

Tissue culture and drug treatment. Human embryonic kidney 293T cells and breast adenocarcinoma MCF7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) or DMEM:Nutrient Mixture-F12 (DMEM/F-12) containing 10% fetal calf serum, 100 U/ml penicillin and streptomycin. MG132 (Peptide institute; 10 M) was added for 5 hours to inhibit the proteasome.

Biochemical methods. Cells were lysed in triton lysis buffer (50 mM Tris-HCl at pH 7.4, 1 mM EDTA, 250 mM NaCl, 0.1% Triton Tx-100 50 mM NaF, 1 mM DTT and 0.1 mM Na3VO4) with additional protease and phosphatase inhibitors (Sigma-Aldrich) and incubated on ice for 30 min. Whole cell extracts (WCE) were then centrifuged for 20 min at 4oC. Protein concentration was measured with Bio-Rad Dc protein assay. For immunoprecipitation, WCE was pre-cleared with Protein G-Sepharose 4B (Invitrogen) for 1 hour at 4oC. Pre-cleared WCE were incubated with mouse monoclonal FLAG antibody for 2 hours at 4oC. Protein G-Sepharose 4B beads were

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added and incubated more 45 min at 4oC. Beads were washed 5 times with lysis buffer. All proteins were then eluted with Laemmli buffer. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (PVDF, Millipore) overnight at 4oC.

Antibodies. Mouse monoclonal antibodies were purchased from Sigma-Aldrich (FLAG). Rabbit monoclonal antibodies were from Cell Signaling (MYC).

Plasmids. Human FBXW9 (isoform 3) and FBXO6 cDNAs were purchased from imaGENE. FBXO6 cDNA was then subcloned into pcDNA3 with 2XFLAG and 2XHA epitope- tags at its N-terminus. FBXW9 cDNA was subcloned in LZRSpBMN-GFP (pallino) vector with 2XFLAG and 2XHA epitope-tags at its N-terminus. All constructs were sequenced. MYC-tagged human APC2 was a generous gift from Dr. Hidewaki Nakagawa.

Transient transfection and retroviral infection. The FBXO6 expression vector was transiently transfected in HEK293T cells by using the polyethylenimine (PEi) transfection method. The retroviral FBXW9 construct was transfected into HEK293-GP2 cells along with packaging vectors. Forty-eight hours after transfection, the virus containing medium was collected and 5 supplied to MCF7 cells with polybrene (8 g/ml).

Mass spectrometry analysis. FBXO6 was transiently expressed in HEK293T cells. MCF7 cells stably expressing FBXW9 were visualized with GFP to assess the efficiency of FBXW9 expression. To inhibit proteasome-dependent degradation, MG132 was added 6 hours before collecting the cells. Harvested cells were then lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP40, plus protease and phosphatase inhibitors). FBPs were immunopurified with anti-FLAG agarose resin (Sigma- Aldrich). After washing, proteins were eluted by competition with a FLAG peptide (Sigma-Aldrich). The eluate was then subjected to a second immunopurification with anti-HA resin (12CA5 monoclonal antibody cross-linked to protein G Sepharose; Invitrogen) prior to elution in Laemmli sample buffer. The final eluate was separated by SDS-PAGE. Proteins were visualized by Coomassie colloidal blue. Bands were sliced out from the gels and subjected to in-gel digestion. Gel pieces were then reduced, alkylated and digested according to a published protocol (40). For mass spectrometric analysis, peptides recovered from in-gel digestion were separated with a C18 column and introduced by nano-electrospray into the LTQ Orbitrap XL (Thermo Fisher) with a configuration as described (41). Peak lists were generated from the MS/MS spectra using MaxQuant build 1.0.13.13, and then searched against the IPI Human database (version 3.37, 69164 entries) using Mascot search engine (Matrix Science). Carbaminomethylation (+57 Da) was set

80 Other F-box Proteins and Cancer as fixed modification and protein N-terminal acetylation and methionine oxidation as variable modifications. To filter out false positives, all MS/MS spectra were searched against a forward and a reverse (decoy) protein database. All peptide- spectrum matches (PSMs) containing both forward and decoy hits were then trained and validated with an algorithm named Percolator which uses semi- supervised machine learning to discriminate between the correct and decoy matches. Finally, we only accepted PSM based on a high stringency filter of q = 0.01 or 1% false discovery rate (FDR). This 1% FDR filter is currently the standard for the proteomics community.

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gene expression in gastric carcinoma: modulation of malignant phenotype by Skp2 overexpression, possibly via p27 proteolysis. Cancer research. 2002;62(13):3819- 25. 12. Masuda TA, Inoue H, Nishida K, Sonoda H, Yoshikawa Y, Kakeji Y, et al. Cyclin-dependent kinase 1 gene expression is associated with poor prognosis in gastric carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research. 2003;9(15):5693-8. 13. Shapira M, Ben-Izhak O, Linn S, Futerman B, Minkov I, Hershko DD. The prognostic impact of the ubiquitin ligase subunits Skp2 and Cks1 in colorectal carcinoma. Cancer. 2005;103(7):1336-46. 14. Signoretti S, Di Marcotullio L, Richardson A, Ramaswamy S, Isaac B, Rue M, et al. Oncogenic role of the ubiquitin ligase subunit Skp2 in human breast cancer. The Journal of clinical investigation. 2002;110(5):633-41. 15. Seki R, Ohshima K, Fujisaki T, Uike N, Kawano F, Gondo H, et al. Prognostic significance of S-phase kinase-associated protein 2 and p27kip1 in patients with diffuse large B-cell lymphoma: effects of rituximab. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO. 2010;21(4):833-41. 16. Wang Z, Gao D, Fukushima H, Inuzuka H, Liu P, Wan L, et al. Skp2: a novel potential therapeutic target for prostate cancer. Biochimica et biophysica acta. 2012;1825(1):11-7. 17. Rose AE, Wang G, Hanniford D, Monni S, Tu T, Shapiro RL, et al. Clinical relevance of SKP2 alterations in metastatic melanoma. Pigment cell & melanoma research. 2011;24(1):197-206. 5 18. Fang FM, Chien CY, Li CF, Shiu WY, Chen CH, Huang HY. Effect of S- phase kinase-associated protein 2 expression on distant metastasis and survival in nasopharyngeal carcinoma patients. International journal of radiation oncology, biology, physics. 2009;73(1):202-7. 19. Chan CH, Li CF, Yang WL, Gao Y, Lee SW, Feng Z, et al. The Skp2-SCF E3 ligase regulates Akt ubiquitination, glycolysis, herceptin sensitivity, and tumorigenesis. Cell. 2012;149(5):1098-111. 20. Ma XM, Liu Y, Guo JW, Liu JH, Zuo LF. Relation of overexpression of S phase kinase-associated protein 2 with reduced expression of p27 and PTEN in human gastric carcinoma. World journal of gastroenterology : WJG. 2005;11(42):6716-21. 21. Latres E, Chiarle R, Schulman BA, Pavletich NP, Pellicer A, Inghirami G, et al. Role of the F-box protein Skp2 in lymphomagenesis. Proceedings of the National Academy of Sciences of the United States of America. 2001;98(5):2515- 20. 22. Ougolkov A, Zhang B, Yamashita K, Bilim V, Mai M, Fuchs SY, et al. Associations among beta-TrCP, an E3 ubiquitin ligase receptor, beta-catenin, and NF-kappaB in colorectal cancer. Journal of the National Cancer Institute. 2004;96(15):1161-70. 23. Muerkoster S, Arlt A, Sipos B, Witt M, Grossmann M, Kloppel G, et al. Increased expression of the E3-ubiquitin ligase receptor subunit betaTRCP1 relates to constitutive nuclear factor-kappaB activation and chemoresistance in pancreatic carcinoma cells. Cancer research. 2005;65(4):1316-24.

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24. Koch A, Waha A, Hartmann W, Hrychyk A, Schuller U, Waha A, et al. Elevated expression of Wnt antagonists is a common event in hepatoblastomas. Clinical cancer research : an official journal of the American Association for Cancer Research. 2005;11(12):4295-304. 25. Spiegelman VS, Tang W, Chan AM, Igarashi M, Aaronson SA, Sassoon DA, et al. Induction of homologue of Slimb ubiquitin ligase receptor by mitogen signaling. The Journal of biological chemistry. 2002;277(39):36624-30. 26. Kudo Y, Guardavaccaro D, Santamaria PG, Koyama-Nasu R, Latres E, Bronson R, et al. Role of F-box protein betaTrcp1 in mammary gland development and tumorigenesis. Molecular and cellular biology. 2004;24(18):8184-94. 27. Duan S, Cermak L, Pagan JK, Rossi M, Martinengo C, di Celle PF, et al. FBXO11 targets BCL6 for degradation and is inactivated in diffuse large B-cell lymphomas. Nature. 2012;481(7379):90-3. 28. Lohr JG, Stojanov P, Lawrence MS, Auclair D, Chapuy B, Sougnez C, et al. Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing. Proceedings of the National Academy of Sciences of the United States of America. 2012;109(10):3879-84. 29. Cancer Genome Atlas Research N. Integrated genomic analyses of ovarian carcinoma. Nature. 2011;474(7353):609-15. 30. Stransky N, Egloff AM, Tward AD, Kostic AD, Cibulskis K, Sivachenko A, et al. The mutational landscape of head and neck squamous cell carcinoma. Science. 2011;333(6046):1157-60. 31. Kan Z, Jaiswal BS, Stinson J, Janakiraman V, Bhatt D, Stern HM, et al. Diverse somatic mutation patterns and pathway alterations in human cancers. Nature. 2010;466(7308):869-73. 5 32. Fearon ER. Molecular genetics of colorectal cancer. Annual review of pathology. 2011;6:479-507. 33. Cancer Genome Atlas N. Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487(7407):330-7. 34. Kuraguchi M, Ohene-Baah NY, Sonkin D, Bronson RT, Kucherlapati R. Genetic mechanisms in Apc-mediated mammary tumorigenesis. PLoS genetics. 2009;5(2):e1000367. 35. Groves C, Lamlum H, Crabtree M, Williamson J, Taylor C, Bass S, et al. Mutation cluster region, association between germline and somatic mutations and genotype-phenotype correlation in upper gastrointestinal familial adenomatous polyposis. The American journal of pathology. 2002;160(6):2055-61. 36. Gaujoux S, Pinson S, Gimenez-Roqueplo AP, Amar L, Ragazzon B, Launay P, et al. Inactivation of the APC gene is constant in adrenocortical tumors from patients with familial adenomatous polyposis but not frequent in sporadic adrenocortical cancers. Clinical cancer research : an official journal of the American Association for Cancer Research. 2010;16(21):5133-41. 37. MacDonald BT, Tamai K, He X. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Developmental cell. 2009;17(1):9-26. 38. Clevers H, Nusse R. Wnt/beta-catenin signaling and disease. Cell. 2012;149(6):1192-205. 39. Schneikert J, Vijaya Chandra SH, Ruppert JG, Ray S, Wenzel EM, Behrens J. Functional comparison of human adenomatous polyposis coli (APC)

83 Chapter 5

and APC-like in targeting beta-catenin for degradation. PloS one. 2013;8(7):e68072. 40. Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Analytical chemistry. 1996;68(5):850-8. 41. Raijmakers R, Berkers CR, de Jong A, Ovaa H, Heck AJ, Mohammed S. Automated online sequential isotope labeling for protein quantitation applied to proteasome tissue-specific diversity. Molecular & cellular proteomics : MCP. 2008;7(9):1755-62.

84 Other F-box Proteins and Cancer

85 Chapter 5

86 Other F-box Proteins and Cancer

87 Chapter 5

88 Other F-box Proteins and Cancer

89 Chapter 5

90 Other F-box Proteins and Cancer

91 Chapter 5

92 Other F-box Proteins and Cancer

93 Chapter 5

94 Other F-box Proteins and Cancer

95 Chapter 5

96 Other F-box Proteins and Cancer

Table S1.

Chapter 6

Discussion

Intro

DEC1 AP4

DEC2 FBPs

Discussion

General discussion

In this thesis, I have studied SCF-mediated protein degradation by using biochemical and cell biological approaches as well as mass-spectrometry analysis. I have identified two previously unknown substrates of SCFTrCP, DEC1 and TFAP4, and uncovered the importance of their degradation in cell cycle progression. In addition, I started exploring how the SCF-mediated degradation of DEC2, a paralog of DEC1, is regulated. Finally, I have performed a large-scale analysis of the gene expression of most FBPs in human primary tumors and focused on two FBPs that I have found overexpressed in cancer. In chapter 2, I showed that SCFTrCP targets for proteasomal degradation the basic helix-loop-helix (bHLH) transcription factor DEC1. In particular, I have found that the regulated proteolysis of DEC1 controls the DNA damage checkpoint. I have demonstrated that in response to DNA damage, DEC1 is stabilized by the USP17 deubiquitylating enzyme and that during recovery from the G2 DNA damage checkpoint DEC1 is targeted for degradation by SCFTrCP in collaboration with CK1. In chapter 3, I have shown that the same SCF ubiquitin ligase, SCFTrCP, is responsible for the destruction of another bHLH transcription factor, namely TFAP4, and demonstrated that the SCFTrCP-mediated degradation of TFAP4 is required for efficient G2 progression and mitotic entry. In chapter 4, I have shown that DEC2, a paralog of DEC1, is targeted for degradation by SCFFBXW4 and that this degradation is mediated by the proteasome. Finally, in chapter 5, I have performed a systematic analysis of the gene expression profile of most members of the FBP family in human primary tumors. I have found that six FBPs are either overexpressed or underexpressed in some specific or most of cancer types. I have then focused on two FBPs, FBXW9 and FBXO6, which are highly overexpressed in most cancer types. To provide initial insights into the function of these two FBPs, I have immunopurified them from cells and performed mass spectrometry analysis. I believe that this study increases our understanding of the key roles of SCF 6 ubiquitin ligases in the cell division cycle and their links to malignancies. This knowledge is needed to explore the possibility of modulating the activity of specific SCF ubiquitin ligases as a way of developing novel mechanism-based, approaches to fight cancer. The clinical success of the proteasome inhibitor Bortezomib provided the proof-of-concept of targeting the ubiquitin-proteasome system as an anti-cancer therapeutic approach (1-3). Indeed, this drug has striking efficacy against multiple myeloma and is used for the treatment of mantle cell lymphoma. Moreover, an inhibitor of NAE-E1 (MLN4924), the enzyme needed for the activation of Cullin-RING ubiquitin ligases, has entered clinical trials for the treatment of multiple myeloma and non-Hodgkin’s lymphoma (4). I expect that inhibition of the activity of individual ubiquitin ligases will cause fewer side effects and have a better therapeutic ratio than general inhibition of the proteasome or a whole class of ubiquitin ligases. A small molecule capable of inhibiting the ubiquitin ligase specific for a negative regulator of cell proliferation is expected to cause an increase in the cellular levels of such a negative regulator leading to a block in cellular proliferation, and consequently in cancer progression (5, 6). This is especially true in those tumors

101 Chapter 6

that have low levels of negative regulators as a result of a "hyperactivated" ubiquitylation pathway. The example of the Nutlins, small-molecule inhibitors that target protein-protein interaction between the tumor suppressor p53 and its ubiquitin ligase MDM2, serves as proof-of-principles of selectively targeting ubiquitin ligases (7-9). Depending on the molecular characteristics of the different tumors, one could envisage targeting specific ubiquitin ligases in different cancers as a valid approach in clinical oncology. Importantly, βTrCP and other F-box proteins promote proteasome-dependent degradation in cooperation with protein kinases for which targeted drugs have been already approved for clinical use (10, 11). Elucidating the phosphorylation events and identifying the kinases involved in the degradation of a particular substrate can lead to a rational therapeutic strategy in times relatively shorter than the ones needed to develop a novel small molecule inhibitor of an E3 ligase.

References

1. Nowis D, Maczewski M, Mackiewicz U, Kujawa M, Ratajska A, Wieckowski MR, et al. Cardiotoxicity of the anticancer therapeutic agent bortezomib. The American journal of pathology. 2010;176(6):2658-68. 2. Orlowski RZ, Stinchcombe TE, Mitchell BS, Shea TC, Baldwin AS, Stahl S, et al. Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2002;20(22):4420-7. 3. Molineaux SM. Molecular pathways: targeting proteasomal protein degradation in cancer. Clinical cancer research : an official journal of the American Association for Cancer Research. 2012;18(1):15-20. 4. Soucy TA, Smith PG, Milhollen MA, Berger AJ, Gavin JM, Adhikari S, et 6 al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature. 2009;458(7239):732-6. 5. Wang Z, Liu P, Inuzuka H, Wei W. Roles of F-box proteins in cancer. Nature reviews Cancer. 2014;14(4):233-47. 6. Ciechanover A. Intracellular protein degradation: from a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting. Biochimica et biophysica acta. 2012;1824(1):3-13. 7. Vassilev LT. MDM2 inhibitors for cancer therapy. Trends in molecular medicine. 2007;13(1):23-31. 8. Yuan Y, Liao YM, Hsueh CT, Mirshahidi HR. Novel targeted therapeutics: inhibitors of MDM2, ALK and PARP. Journal of hematology & oncology. 2011;4:16. 9. Tovar C, Rosinski J, Filipovic Z, Higgins B, Kolinsky K, Hilton H, et al. Small-molecule MDM2 antagonists reveal aberrant p53 signaling in cancer: implications for therapy. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(6):1888-93. 10. Lill JR, Wertz IE. Toward understanding ubiquitin-modifying enzymes: from pharmacological targeting to proteomics. Trends in pharmacological sciences. 2014;35(4):187-207.

102 Discussion

11. Mattern MR, Wu J, Nicholson B. Ubiquitin-based anticancer therapy: carpet bombing with proteasome inhibitors vs surgical strikes with E1, E2, E3, or DUB inhibitors. Biochimica et biophysica acta. 2012;1823(11):2014-21. 1. Nowis D, Maczewski M, Mackiewicz U, Kujawa M, Ratajska A, Wieckowski MR, et al. Cardiotoxicity of the anticancer therapeutic agent bortezomib. The American journal of pathology. 2010;176(6):2658-68. 2. Orlowski RZ, Stinchcombe TE, Mitchell BS, Shea TC, Baldwin AS, Stahl S, et al. Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2002;20(22):4420-7. 3. Molineaux SM. Molecular pathways: targeting proteasomal protein degradation in cancer. Clinical cancer research : an official journal of the American Association for Cancer Research. 2012;18(1):15-20. 4. Soucy TA, Smith PG, Milhollen MA, Berger AJ, Gavin JM, Adhikari S, et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature. 2009;458(7239):732-6. 5. Wang Z, Liu P, Inuzuka H, Wei W. Roles of F-box proteins in cancer. Nature reviews Cancer. 2014;14(4):233-47. 6. Ciechanover A. Intracellular protein degradation: from a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting. Biochimica et biophysica acta. 2012;1824(1):3-13. 7. Vassilev LT. MDM2 inhibitors for cancer therapy. Trends in molecular medicine. 2007;13(1):23-31. 8. Yuan Y, Liao YM, Hsueh CT, Mirshahidi HR. Novel targeted therapeutics: inhibitors of MDM2, ALK and PARP. Journal of hematology & oncology. 2011;4:16. 9. Tovar C, Rosinski J, Filipovic Z, Higgins B, Kolinsky K, Hilton H, et al. Small-molecule MDM2 antagonists reveal aberrant p53 signaling in cancer: implications for therapy. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(6):1888-93. 10. Lill JR, Wertz IE. Toward understanding ubiquitin-modifying enzymes: 6 from pharmacological targeting to proteomics. Trends in pharmacological sciences. 2014;35(4):187-207. 11. Mattern MR, Wu J, Nicholson B. Ubiquitin-based anticancer therapy: carpet bombing with proteasome inhibitors vs surgical strikes with E1, E2, E3, or DUB inhibitors. Biochimica et biophysica acta. 2012;1823(11):2014-21.

103

Addendum

Nederlandse samenvatting Acknowledgements List of publications Curriculum vitae

Nederlandse Samenvatting

Nederlandse samenvatting

In deze thesis heb ik de degradatie van eiwitten door SCF ubiquitine ligases bestudeerd met behulp van biochemische en celbiologische methodes en massaspectrometrie. Daarnaast heb ik de gen expressie van zevenenzestig F-Box eiwitten in primaire tumoren geanalyseerd en verdiepend onderzoek gedaan naar twee F-box eiwitten, FBXW9 en FBXO6, beide zeer overexpressief in de meeste kankersoorten.

In Hoofdstuk 1 vat ik de laatste inzichten samen in de belangrijkste moleculaire spelers van het ubiquitine-proteasoom systeem met een speciale nadruk op SCF ubiquitine-ligases. Dit zijn moleculaire machines die celcyclus progressie controleren en waarvan deregulatie bijdraagt aan oncogenese.

In Hoofdstuk 2 beschrijf ik de SCF-gemedieerde degradatie van de bHLH trancriptie factor DEC1, een kritische regulator van cel determinatie en circadiaanse ritmes. Tijdens onverstoorde cel cycli is DEC1 een zeer instabiel eiwit wat het doel is van proteasoom-afhankelijke degradatie door de SCFβTrCP ubiquitine ligase in samenwerking met CK1. DNA schade resulteert in de een ATM/ATR afhankelijke inductie van DEC1. DEC1 inductie resulteert in eiwit stabilisatie via een mechanisme dat via het USP17 ubiquitine protease gaat. Vervolgens wordt tijdens checkpoint recovery DEC1 proteolyse hersteld via βTrCP-afhankelijke ubiquitilatie. Belangrijk is dat de expressie van een niet-degradeerbare DEC1 mutant checkpoint recovery voorkomt door de negatieve regulatie van p53 te inhiberen

In Hoofdstuk 3 beschrijf ik het onderzoek naar de biologische functie van de SCF- afhankelijke degradatie van TFAP4, een transcriptie factor die in vele cruciale cellulaire processen geïmpliceerd wordt zoals regulatie van stemness, epitheliale- mesenchymale transitie, neurale differentiatie en cel proliferatie. Uit mijn onderzoek blijkt dat het niet degraderen van TFAP4 tot de accumulatie leidt van cellen in de S en G2 fases van de celdelingscyclus. Door het gebruiken van het FUCCI systeem & heb ik ook kunnen aantonen dat inhibitie van TFAP4 degradatie tot een vertraagde progressie door S/G2 leidt.

In Hoofdstuk 4 schets ik de degradatie van DEC2, een paraloog van DEC1, die de cel cyclus, de respons op hypoxie, apoptose, circadiaans ritme en differentiatie reguleert en wiens expressie veranderd is in verschillende kankersoorten als eierstok-, long-, en alvleesklierkanker. Mijn onderzoek wijst uit dat de stabiliteit van DEC2 gecontroleerd wordt door FBXW4, de substraat bindende component van de SCFFBXW4 ubiquitine ligase.

In Hoofdstuk 5 beschrijf ik de systematische analyse van het gen expressie profiel

107 Addendum

van bijna alle humane F-box eiwitten in primaire tumoren. Ik heb ontdekt dat tenminste zes F-box eiwitten zeer tot overexpressie komen in kanker. Ik heb FBXW9 en FBXO6 verder bestudeerd door deze te zuiveren en vervolgens te analyseren met massaspectrometrie om hun specifieke substraten en regulatoren te identificeren.

In Hoofdstuk 6 vat ik mijn onderzoeken samen en bediscussieer ik de belangrijkste vindingen. Daarnaast beschrijf ik de therapeutische implicaties van dit werk door verschillende manieren te schetsen waarop het ubiquitine-proteasoom systeem kan dienen als doel voor anti-kanker therapie.

108 Acknowledgements

Acknowledgements

So far, you guys read boring numbers, bands, figures and graphs. Now I’m 100 % sure this part is the easiest part of my thesis.

It is really amazing that I already finished my PhD and writing my thesis. It was really lucky that I contacted to Daniele and started my PhD here in Netherlands. I cannot forget the day when I received an e-mail introducing research of DG lab. That was the topic I wanted to study but it was big challenge for me to start because I’ve never lived outside of my country. I worried my social and academic life in Netherlands so I have to say thank you for all people who helped me a lot and spent great time with me. It is obvious that I could not survive without great help from those people.

First, I should start with Daniele. It was great pleasure to work with you. Without your unlimited support, I could not make anything for my PhD. Do you remember the legendary day when you found out small pH change can stimulate endogenous DEC1 promoter? Your inspiration was so incredible and I thought you might have some metaphysical power like a force of Jedi from the movie STARWARS. I will never forget things you taught me and thank you for giving me an opportunity to study in DG lab. Grazie infinite.

Dear my great friends in DG lab...

Flore, the tallest and the only Dutch person in DG lab. I could get used to Netherlands much easier because of your help. You taught me Netherlands is not just a country with crazy weather. Dank u wel!

Roberto, my great Italian teacher. You are really awesome. Five Italian bad word combination will be always in my mind forever. Although I cannot use it in this thesis. You know we always be in the lab even during the weekend. It would be really & boring and cannot be tolerable if you were not there. La ringrazio tanto!!

Sara, we started our PhD course at the same time. So you were my great friend, colleague and competitor. I wish your future life with Diego and baby will be fantastic. I migliori auguri!!!

Ratna, thank you for your lab help and invitations for BBQ. It was amazing time. You make my social and lab life so happy. Terima kasih!!!!

Former DG group members Tina, Ragna, Yuni, Koen, Sharif, Trang and Petra.

109 Addendum

We didn’t spend several years but I think we shared pretty good time while you guys were in the lab. Thank you for making my lab life so funny. Ragna, thanks for your translating skill and kindness. I will never forget your cookies and cakes. It was better than commercial one. Yes... I’m pretty sure. Petra, you were the only student who I supervised during my PhD. I know I was pretty harsh while I was teaching you. Sorry for that and I wish you remember it as a good memory.

Teck, I could do lots of purifications with your help. Thanks a lot. Stefan, it is pretty clear that I bothered you quite much. Without your help, I could not make such a nice FACS data so easily. Wytse, from Medema’s group. Lots of thanks for your help for establishing FUCCI system.

Hubrecht people. John, Monica, Miriam, Jeroen, Alex and Suma. A big thank to you guys for being a good neighbor. Giuliano and Fabrizio, thank you for making my lab life so funny. You guys are my second Italian teacher. The legendary word that you taught me... Porco… you know the next haha... Salvo, it was happy to know you and I hope your plan after PhD will be great. Sylvia, my great Catalonian friend. With your amazing suggestions, I could remember Barcelona as a great city. Turan, Yuva and Onur. Thank you my nice Turkish friends. Roel, good luck to your project and PhD.

It is pretty sure I might missed some people. Sorry for that and thanks again for all people who shared great time with me.

Hereafter, for Korean people.

Bon-Kyoung. 형, 덕분에 이렇게 외국에서 오래 살면서 공부할 수 있는 좋은 기회도 얻고 연구소 생활이 더 재미있을 수 있었어요. 실험하는데도 도움 많 이 얻고 생활하는데도 진짜 큰 도움이 되었어요. 형, 형수님 그리고 아이들과 같이 다닌 여행들도 진짜 잊지 못할거에요. 형네 식구들 덕분에 네덜란드 생 활이 덜 외로울 수 있었어요. 진짜 감사드립니다. & Minchul. 민철이형! 우리 그래도 꽤 재미있는 네덜란드 생활을 같이 즐긴거 같죠? 형과 본경이형 저 셋이서 참~~ 많은 일들이 있었어요.. 그렇죠? 자세한 이야기는 쓰지 않을랍니다~ 진짜 덕분에 여러가지 도움도 많이 받고 즐거운 네덜란드 생활이 될 수 있었어요. 감사했습니다~

Bon-Kyoung’s wife and Mincul’s wife. 원래는 그냥 형들쓰는데 같이 쓰려 했 는데 생각해보니 두분 형수님께는 이렇게 따로 공간을 마련해놔야 할 것 같더 군요. 형수님들께 얻어먹은 것들과 도움받은게 진짜 엄청나게 많으니까요. 두 분 형수님 덕분에 한국 음식도 자주 얻어 먹을 수 있었고 외로울때마다 불러 서 같이 놀아 주시고.. 진짜 감사했습니다~ 물론~!! 맨날 여자도 소개 안해주

110 Acknowledgements

시면서 결혼은 언제하냐고 물어보시는 것만 빼면요~~ 농담인거 아시죠? 진짜 진짜 형들보다도 더(?) 감사했습니다.

Inha, 함께 있었던 시간은 그리 길지 않았지만 그래도 나의 네덜란드 생활의 마지막을 같이 보내줘서 고마웠어~ 니가 안왔으면 진짜 연구소에 한국인은 나 밖에 안남아서 더 외로웠을텐데… 같이 차도 마시고 밥도 먹어주고.. 고마웠다 친구! 주철민 박사님도 참 고마웠다고 전해주길~

My family. 마지막으로 나의 가족들에게. 친구들은 다들 취직하고 결혼하고 하 는데 이때까지 공부한다고 남들 다하는 것들을 하나도 못하고 지금에 이르렀 네요. 그저 지금껏 믿어주시고 밀어주신 것에 대해 감사할 따름입니다. 할머니, 할아버지. 손주며느리 음식도 가르치셔야하고 증손자까지 돌봐 주셔야죠? 하 실 일이 아직 많~이 있습니다. 오래오래 사세요~ 부모님, 대학까지 공부시켜주 시고 서포트 해주신 것 감사합니다. 이 나이까지 철없이 저혼자 좋다고 공부 만 하고 있네요. 아마도.. 앞으로도 이럴꺼 같아 죄송하네요.. 동생아 너라도 일찍 장가가고 돈 많이 벌어라~ 형은 그런 것과는 좀 거리가 있는듯하다~ 삼 촌들, 외숙모들, 이모들과 이모부, 드디어 제가 박사가 되긴 되나봅니다. 이런 걸 쓰고 있다니요.. 어렷을때부터 관심가져 주시고 감사합니다~. 사촌동생들아!! 말좀 잘 듣고 행복해라~.

111 Addendum

Curriculum vitae

Jihoon Kim was born on 17th of June 1981 in Suwon, South Korea. In 2001, he started bachelor’s degree in Biology at Konkuk University in Seoul, South Korea. He obtained this degree in 2007 and continued to study Structural Biology at Pohang University of Science and Technology in Pohang, South Korea. In January of 2010, he started his PhD at Hubrecht Institute under supervision of dr. Daniele Guardavaccaro. All the results obtained during PhD are described in this thesis.

List of Publications

Kim J, D'Annibale S, Magliozzi R, Low TY, Jansen P, Shaltiel IA, et al. USP17- and SCFbetaTrCP-Regulated Degradation of DEC1 Controls the DNA Damage Response. Molecular and cellular biology. 2014.

D'Annibale S, Kim J, Magliozzi R, Low TY, Mohammed S, Heck AJ, et al. Proteasome-dependent degradation of transcription factor activating enhancer- binding protein 4 (TFAP4) controls mitotic division. The Journal of biological chemistry. 2014;289(11):7730-7.

Magliozzi R, Kim J, Low TY, Heck AJ, Guardavaccaro D. Degradation of Tiam1 by Casein Kinase 1 and the SCFbetaTrCP Ubiquitin Ligase Controls the Duration of mTOR-S6K Signaling. The Journal of biological chemistry. 2014;289(40):27400-9.

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