The roles of ubiquitin modifying enzymes in neoplastic disease

BBA - Reviews on Cancer 1868 (2017) 456–483

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BBA - Reviews on Cancer journal homepage: www.elsevier.com/locate/bbacan

Review

The roles of ubiquitin modifying enzymes in neoplastic disease a,b

a

a,b,c

MARK a

Nishi Kumari , Patrick William Jaynes , Azad Saei , Prasanna Vasudevan Iyengar , John Lalith Charles Richarda, Pieter Johan Adam Eichhorna,b,⁎ a b c

Cancer Science Institute of Singapore, National University of Singapore, 117599, Singapore Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, 117597, Singapore Genome Institute of Singapore, A*STAR, Singapore

A R T I C L E I N F O

A B S T R A C T

Keywords: Ubiquitin E3 ligases Deubiquitinating enzymes Cancer Dub inhibitors

The initial experiments performed by Rose, Hershko, and Ciechanover describing the identification of a specific degradation signal in short-lived proteins paved the way to the discovery of the ubiquitin mediated regulation of numerous physiological functions required for cellular homeostasis. Since their discovery of ubiquitin and ubiquitin function over 30 years ago it has become wholly apparent that ubiquitin and their respective ubiquitin modifying enzymes are key players in tumorigenesis. The human genome encodes approximately 600 putative E3 ligases and 80 deubiquitinating enzymes and in the majority of cases these enzymes exhibit specificity in sustaining either pro-tumorigenic or tumour repressive responses. In this review, we highlight the known oncogenic and tumour suppressive effects of ubiquitin modifying enzymes in cancer relevant pathways with specific focus on PI3K, MAPK, TGFβ, WNT, and YAP pathways. Moreover, we discuss the capacity of targeting DUBs as a novel anticancer therapeutic strategy.

1. Introduction The initial concept of protein turnover was viewed as a minor component in cellular homeostasis for it was thought that proteins in general were intrinsically stable and were only subjected to degradation when proteins were damage by prolonged “wear and tear”. It is of course now recognized that ubiquitination mediated proteosomal degradation of proteins is far more wide-spread and dynamically regulated then was originally imagined. Furthermore, developments over the last two decades has led to a better understanding of the versatile functions of ubiquitination. We now understand that ubiquitin is able to form eight structurally and functionally distinct polymers which can affect not only protein stability but also affect protein activity and cellular localization. Accumulating evidence indicates that perturbations affecting the activity of ubiquitin modifying enzymes are important in tumour cell growth and survival and therefore these enzymes have been suggested to be potential targets for pharmacological intervention. Clinical validation of this was initially observed with the approval of the proteasome inhibitor Bortezomib in multiple myeloma with patients exhibiting significant responses to the treatment [1]. The involvement of E3 ligases in crucial signalling pathways implicated in tumour progression is well documented and a number of excellent reviews have been written on the role of E3 ligases in cancer which we invite the reviewer to read [2–5]. However, the role of deubiquitinating ⁎

enzymes (DUBs) as tumour suppressors or oncogenes has also been an area of intense research. In addition to E3 ligase function here we provide a portrait of DUB mutational profiles and their respective substrate specificity in contribution to tumorigenesis. The ubiquitin moiety is a highly conserved 76 amino acid polypeptide and ubiquitination of target proteins by ubiquitin and ubiquitin like molecules serves as mechanism to regulate a myriad of protein functions including protein stability, subcellular localisation, and activity. The ubiquitination cascade comprises of coordinated action of a E1 activating enzyme, a E2 ubiquitin-conjugating enzyme, and the E3 ubiquitin protein ligase. E1 ligases act as ubiquitin sponges recruiting free ubiquitin inside the cell and subsequently transferring ubiquitin to the E2 ligase. The major component of the ubiquitin cascade is the E3 ligases of which the human genome encodes over 600 allowing a high degree of substrate specificity. Three main families of E3 ligases exist: homologues to E6-associated protein carboxy terminus (HECT) family, really interesting new gene (RING) family, which are the most abundant in the genome, and RING-Between-RING (RBR) family. All three families link E2s with substrates however, ubiquitin transfer is inherently unique between the E3s. In the case of HECT domain ligases ubiquitin transfer involves the formation of a thioester intermediate formed between ubiquitin and the catalytic cysteine of the HECT E3 prior to transfer of ubiquitin to its recruited substrate [2]. Unlike the HECT domain ligases, RING ligases appear to act as scaffolds permitting

Corresponding author at: Cancer Science Institute of Singapore, Centre for Translational Medicine, 14 Medical Drive 12-01, 117599, Singapore. E-mail address: [email protected] (P.J.A. Eichhorn).

http://dx.doi.org/10.1016/j.bbcan.2017.09.002 Received 4 August 2017; Received in revised form 11 September 2017; Accepted 12 September 2017 Available online 18 September 2017 0304-419X/ © 2017 Elsevier B.V. All rights reserved.

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Fig. 1. Copy number variation of deubiquitinating enzymes in cancer. Individual DUB expression in cancer subtypes extrapolated from cBioPortal (www.cBioPortal.org) derived from the following datasets, [304–317], Adrenocortical Carcinoma (TCGA), Colorectal Adenocarcinoma (TCGA), Cervical Squamous Cell Carcinoma (TCGA), Uveal Melanoma (TCGA), Hepatocellular carcinoma (TCGA), B-cell Lymphoma (TCGA), Ovarian adenocarcinoma (TCGA), Pancreatic Adenocarcinoma (TCGA), Skin Cutaneous Melanoma (TCGA), Testicular Germ Cell Cancer (TCGA), Uterine Carcinosarcoma (TCGA). Expression was annotated in tumours if DUBs expression was shown to be altered in more than 5 individual cases.

ubiquitin chains. Although the mechanism of chain formation is unknown the majority of experimental evidence indicates that chains are built sequentially on the substrate (Reviewed in Ye et al. [6]). However, a second mechanism of chain formation has been explored whereby ubiquitin chains are sewn together on the E2's and E3's and transferred as a single block to its cognate substrate. Polyubiquitin chain topology is determined by the varying ubiquitin–ubiquitin linkages which may occur. Ubiquitin chains can occur through specific K6, K11, K27, K29, K33, K48, or K63 linkages. Unlike HECT domain ligases where the transfer of ubiquitin to the substrate occurs directly from the HECTs own catalytic cysteine, the E2 ligase is accountable for the transfer of ubiquitin to the substrate when RING ligases are involved. Therefore, it stands to reason that the type of ubiquitination that occurs is determined largely by the characteristics of the E2. Many RING ligases can bind a number of E2s producing several types of ubiquitination products. This may explain the recent

direct transfer of ubiquitin from the E2 to the substrate. However, it has been proposed that RING ligases may also function by luring ubiquitin away from the E2 through allosteric mechanisms independent of its catalytic site [3,4]. The RBR family of ligases share many of the biochemical features present in RING and HECT function including recruiting ubiquitin conjugated E2 to RING1 domains. The bound E2 then transfers the ubiquitin to the second C-terminal RING domain whereby the ubiquitin bound at the intrinsic catalytic cysteine is transferred to the substrate. The transfer of ubiquitin to the substrate primarily occurs through the formation of a isopeptide bond between the carboxylic acid group of glycine 76 (Gly76) of ubiquitin to the epsilon amino group in the target lysine. Substrates may be modified at a single lysine residues (monoubiquitnation) or at multiple lysine residues simultaneously (multimonoubiquitination). Similarly, Gly76 of ubiquitin may attach to lysine residues on other ubiquitin molecules permitting the formation of poly-

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Therefore, a better understanding of the dynamic interactions of DUBs and the pathways they regulate is essential. This review focuses on the tumour suppressor and oncogenic specific functions of E3 ligases and DUBS in the major pathways involved in cancer progression including TGFβ, YAP, MAPK, PI3K, and WNT pathways. In reference to the role of ubiquitin modifying enzymes in the NFKB pathway we refer to the excellent review by Harhaj et al. [17].

observation that chain formation may either be homotypic (the links in the chain all occur at a single lysine residue, for example K48) or heterotypic (the links in the chain may switch for example from K11 to K27). Furthermore, some ubiquitin chains have been described to contain SUMO moieties while others have exhibited a branched formation. All of this indicates that chain topology is eventually likely to be dependent on the three-dimensional interface between ligase and substrate. The function of these distorted structures remains to be determined but their discovery opens up a new window into the basic biology of ubiquitin function. Lastly, linear ubiquitin chains, also known as M1 linked chains, can be generated through the formation of a covalent bond between the carboxy terminal glycine of ubiquitin and the amino terminal methionine of the substrate bound ubiquitin [7]. It is well described that monoubiquitination and polyubiquitination resulting from the various assortment of linkages described above convey distinct structural and functional information. For the most part polyubiquitination is still epitomized by two ubiquitin chain topologies, K48 and K63. K48-linked chains serve to act as the prototypical degradation signal targeting the protein for proteasome mediated degradation while K63-linked chains perform a number of non-proteolytic functions including cellular signalling, DNA damage repair, intracellular trafficking and ribosomal biogenesis. Due to the relative infrequency of the other chain topologies they have been termed atypical linkages with their cellular functions remaining largely elusive [8]. Similar to the way that protein phosphatases oppose the action of protein kinases, ubiquitination is a process that is also reversible, having a family of enzymes that counteract the role of the ligase. Ubiquitin moieties can be removed from polypeptides by DUBs. The human genome encodes approximately 80 DUBs which are grouped into six distinct families: ubiquitin-specific protease (USP, 57 members), ubiquitin C-terminal hydrolase (UCH, 4 members), otubain protease, (OTU, 14 members), motif interacting with Ub-containing novel DUB family (MINDY, potential 4 members, only 1 is discuss in this review), and machado-joseph disease protease (MJD, 4 members). USPs make up by far the largest family of these thiol proteases. The sixth DUB family, is distinct from the other 5 DUB families in that they possess a JAMM (JAB1/MPN/Mov34 metalloenzyme, 11 members) domain to carry out their catalytic function [9,10]. The deubiquitination ability and substrate specificity of a particular DUB is determined by chain topology, sub cellular localization and protein complex formation. It is feasible that any disequilibrium in the ubiquitination-deubiquitination balance can act as a major contributing factor to tumour formation. To further understand the potential role of DUBS in tumourgenesis we performed expression analysis of all the known DUBs across 20 tissue specific datasets using cBioPortal (Fig. 1). Our analysis reveals that across many tumour types, there appears to be a general trend towards DUB amplification. Given that gene copy number alterations are a common phenomenon in cancer our observations could merely be a result of this. It is, however, conceivable that the overexpression of K48 specific DUBs, by virtue of stabilizing oncogenes, could potentially be critical determinants in tumorigenesis. Undoubtedly, a much more indepth analysis will be required before this can be claimed with any certainty. The exception to this is observed in brain and prostate cancer where deletion of a number of DUBs facilitate tumorigenesis suggesting that in these cancer types these DUBs operate as potential tumour suppressors. Nevertheless, in the majority of cases DUB expression and function (tumour suppressor or oncogene) appears to be tissue and context specific. For example, USP9X has been shown to stabilize the oncogenes β-catenin, MCL-1, estrogen receptor, Ets-1, among others, promoting cancer progression [11–14]. In contrast, USP9X expression is lost in renal cell carcinoma and pancreatic ductal adenocarcinoma with downregulation of USP9X corresponding with increased YAP and RAS signalling, respectively [15,16]. This apparent contradiction in function has restricted the development of novel inhibitors targeting DUBS and limited the progression of DUB inhibitors through clinical development.

2. The role of ubiquitin modifying enzymes in the TGFβ pathway The Transforming Growth Factor β (TGFβ) pathway is crucial for regulating embryonic development as well as maintaining tissue homeostasis in adult tissues [18–20]. The majority of human cell types are responsive to TGFβ, through which it regulates cell proliferation, differentiation, migration, adhesion, apoptosis and various aspects of the microenvironment [18–20]. TGFβ is a member of a large family of cytokines divided into two distinct branches: TGFβ, activin, lefty, nodal and myostatin form one arm, whereas the other comprises of bone morphogenetic proteins (BMPs), anti-muellerian hormone (AMH) and other growth and differentiation factors (GDFs) [19]. These classifications are simply based on sequence homology and the distinct pathways that each activates [21]. Activation of the TGFβ pathway requires the ligand induced formation of a tetrameric complex comprised of a pair of TGFβ receptor I (TβRI) subunits as well as a pair of TGFβ receptor II (TβRII) subunits [22]. TβRII is a constitutively active serine/threonine kinase and upon ligand binding it is able to phosphorylate TβRI at a distinct location preceding the kinase domain called the GS domain [23]. This results in the activation of the TβRI serine/threonine kinase allowing for effector molecules, Receptor–SMADs (R-SMADs), to be phosphorylated [18–20]. The R-SMADs belonging to the TGFβ arm of the pathway include SMAD2 and SMAD3, whereas SMAD1, SMAD5 and SMAD8 are attributed to the BMP pathway [18,19]. SMAD proteins are made up of two globular Mad-homology (MH) domains, MH1 and MH2, separated by a highly regulated linker region [24]. The MH1 domain contains the conserved DNA binding motif capable of recognizing the 51-AGAC-31 DNA sequence, more commonly known as the SMAD-Binding-Element (SBE). MH2 is highly conserved and mediates complex binding with other SMADs or SMAD nuclear complexes. The variable linker region is enriched in proline residues and contains multiple phosphorylation residues which can be phosphorylated by various kinases in response to stimuli such as MAPK, or CDK8/9 [25]. R-SMADs are phosphorylated at a precise-SSXS c-terminal motif creating an interaction interface permitting the association with the coSMAD, SMAD4. Upon entry to the nucleus the R-SMAD/co-SMAD complex binds to the SMAD binding element (SBE) sequence on DNA permitting appropriate transcriptional responses [18,20,21].The RSMAD/co-SMAD complex has only a weak affinity for the SBE response elements, thus full transcriptional activation requires the association of additional transcription factors [18–21]. This deceptive weakness in this family of transcription factors appears to be overcome by the overall flexible nature of the linker region whereby it is thought that the distinct phosphorylation patterns in the R-SMAD linker region are likely to result in varying conformational differences which will allow the various permutations of available transcription factors and the subsequent recruitment of co-repressors or co-activators to dictate whether a gene will be repressed or activated, respectively, by TGFβ stimulation [18–20]. Therefore, it is the versatility of the R-SMAD/co-SMAD complex in binding a variety of other transcription factors which accounts for the pleiotropy response to TGFβ. Given the fundamental cellular processes that TGFβ regulates one can speculate that should this signalling pathway be deregulated, aberrations in cellular function would therefore occur promoting tumorigenesis. To investigate the validity of this, one must first understand how TGFβ signalling is regulated. As this review will reveal, the 458

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Fig. 2. Schematic overview of regulation of TGFβ pathway by Ubiquitin ligases and deubiquitinating enzymes. (A) Factors that turn off the TGFβ pathway: At the receptor level AIP4/ Itch, in a non-catalytic manner, brings SMAD7 and TGFβRI into close proximity preventing the association of SMAD2 with the latter and thus prevents signalling. SMAD7 itself brings HECT E3 ligases SMURF1, SMURF2, Tuil1/WWP1 and NEDD4-2 to the receptor complex resulting in complex ubiquitination and degradation. The DUB, USP26, facilitates this event by increasing the stability of SMAD7 by removing degradative ubiquitin chains. TGFβ pathway signal mediators are also regulated by degradative ubiquitin: SMURF2 targets SMAD2 for degradation whereas SMAD3 is targeted by U-Box E3 ubiquitin ligase, CHIP; HECT E3 ligase, NEDD4L; and SCF E3 ligase ROC1-SCFFbw1a. Co-SMAD, SMAD4, is regulated by a number of HECT E3 ligases, namely: SMURF1, SMURF2, Tuil1/WWP1, and NEDD4-2. These ligases associate with and ubiquitinate SMAD4 indirectly by forming a complex with SMAD7, SMAD6 or activated SMAD2, resulting in the degradation of the former. In many cancers SMAD4 harbors point mutations. In this context these protein variants are targeted for degradation by SCF E3 ligases, SCFTrCP1β and SCFSkp2. In addition to promoting degradation, ubiquitination is also able to disrupt protein complexes: SMURF2 multi-monoubiquitinates SMAD3 in either the cytoplasm or the nucleus resulting in the dissociation of the R-SMAD/SMAD4 complex and hence disruption of TGFβ signalling. A similar reaction occurs in the nucleus as E3 ligase Ectodermin/Tif1γ monoubiquitinates SMAD4 also resulting in the dissociation of the R-SMAD/SMAD4 complex. Once the R-SMAD/SMAD4 complex enters the nucleus to initiate transcription, here SMAD2 is targeted for degradation as repressive DNA binding proteins SnoN and TGIF direct NEDD4-2 and Tiul/WWP1, respectively, to this R-SMAD, resulting in the termination of signalling. It has also been shown that the action of an undetermined ligase is able to mono-ubiquitinate the R-SMADs in the R-SMAD/SMAD4 complex thus precluding complex binding to the DNA. (B) Factors the turn the on the TGFβ pathway: At the receptor level, USP4 is able to interact directly with TGFβRI resulting in its relative persistence at the plasma membrane level therefore promoting TGFβ signalling. DUBs USP11, USP15 and UCH37 all augment TGFβ signalling by associating with SMAD7, which effects a plasma membrane localization and therefore stabilizes the receptor complex by way of degradative-ubiquitin removal. SMAD7, itself an antagonist to TGFβ signalling, is degraded by ubiquitination by three difference ligases: Arkadia, AIP4/Itch and RNF12. Further downstream, AIP4/Itch has also been observed to ubiquitinate SMAD2, resulting in an augmentation of TGFβ signalling. The mechanism for this is unclear but this ubiquitination ostensibly promotes SMAD2 and TGFβRI proximity as an AIP4/Itch, SMAD2 and TGFβRI trimeric complex have been observed in TGFβ stimulated cells (not depicted). Stability of SMAD2/3 is enhanced by the action of OTUB1, which prevents E2 ligase transfer of ubiquitin to the R-SMADs. USP9X promotes the formation of the R-SMAD/SMAD4 complex by removing the ubiquitin moiety that is attached to SMAD4 by Ectodermin/Tif1γ, therefore relieving steric inhibition. Once in the nucleus the R-SMAD/SMAD4 complex facilitates the transcription of TGFβ target genes by inducing the destruction of transcriptional repressor SnoN: Arkadia, SMURF2 and CDH1APC bind to SMAD2/3 in the nucleus bringing the ligases into close proximity to SnoN allowing for ubiquitin mediated degradation. Finally, as mono-ubiquitination of R-SMADs precludes the R-SMAD/SMAD4 complex from associating with the DNA, USP15 therefore promotes TGFβ dependent transcription by removing this ubiquitin moiety.

found to be a ubiquitin ligase for SMAD1 resulting in the degradation of SMAD1 and loss of BMP signalling [27]. Subsequent analysis found, however, that it did not bind to SMAD2, indicating that another E3 ligase was responsible for the negative feedback mechanism originally identified by Lo and Massague [28]. The SMURF1 homologue, SMURF2 was subsequently shown to bind SMAD2 in a TGFβ dependent manner, targeting SMAD2 for proteasomal degradation [29,30]. However, both Bonni et al. and Zhang et al. contest the notion that SMURF2 can degrade SMAD2 with Zhang et al. conceding that SMURF2 can degrade SMAD2 but only when ectopic expression of SMURF2 is high [31,32]. It is important to note that it remains inconclusive if endogenous SMAD2 can be degraded by SMURF2 [32,34]. Both SMURF1 and SMURF2 are C2-WW-HECT-domain ligases capable of binding SMADs through an intermolecular interaction between the SMURFs WW domain and the PPXY sequence (PY) motif in SMADs [35]. SMURF1 has also been

dynamic ubiquitination/deubiquitinating process plays a crucial role in regulating overall TGFβ signalling at all nodes of the pathway (Fig. 2). It will also provide pertinent examples of how aberrations in this process can lead to cancer formation. 2.1. E3 ligases in the TGFβ pathway The earliest observation that TGFβ signalling is regulated by ubiquitination was in 1999 by Lo and Massague where they observed that prolonged TGFβ exposure reduced levels of total SMAD2 in a proteasome dependent manner. Their discovery has led to almost 20 years of research in ubiquitin dependent regulation of the TGFβ pathway [26]. With regard to ubiquitin mediated regulation the TGFβ superfamily as a whole, the first major discovery was the identification of the HECT E3 ligase SMAD-specific E3 ubiquitin-protein ligase 1 (SMURF1). This was 459

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of the TβR complex hence removing the tumour suppressor effect of TGFβ signalling [44]. Yet another HECT E3 ligase, NEDD4-2 (neural precursor cell expressed developmentally down-regulated 4-2), has been found to ubiquitinate and degrade SMAD2 [46]. This ubiquitination and degradation occurs in the presence of activated TGFβ signalling and since NEDD4-2 can bind nuclear TGFβ repressor SnoN (see later regarding SnoN) via its association with SMAD2 this suggests that this process again takes place in the nucleus [46]. NEDD4-2 therefore may contribute along with Tiul1 to the negative feedback instigated due to prolonged TGFβ exposure. It has been observed that SMAD3 is also regulated by ubiquitination. This ubiquitination is mediated by the RING E3 ligase ROC1-SCFFbw1a where this complex binds the MH2 domain of SMAD3 [47]. SCF complexes are comprised of three proteins, namely, SKP1, Cullins and F-box proteins, where F-box proteins are important for substrate specificity [48]. In the aforementioned complex, a RING finger protein ROC1 associates with the SCF and is crucial for the ubiquitination function of the entire complex [47]. The degradation of SMAD3 by ROC1-SCF-Fbw1a is TGFβ dependent. Interestingly, SMAD3 is exported out of the nucleus by ROC1-SCF-Fbw1a where it is presumed that in the cytoplasm degradation occurs [47]. This is in contrast to what is observed with SMAD2, as mentioned earlier. Of note, it has been subsequently found that U box E3 ligase, CHIP, is able to target SMAD3 irrespective of phosphorylation status [49]. In addition to targeting the R-SMADs, ubiquitin based regulatory mechanisms also exist for co-SMAD, SMAD4. Here again we see that a negative feedback mechanisms are built in to the regulation of this protein [50]. When TGFβ ligand is added, the F-box protein β-TrCP1 putatively recognizes a phosphorylated “DLSGLTLQS” motif on SMAD4 resulting in the degradation of the latter [50]. Post translational modification of this motif remains unproven but it has been postulated that Jun activation domain binding protein 1 (JAB1) may promote phosphorylation of SMAD4 at this motif [50]. Importantly, JAB1 has been observed to interact with and induce degradation of SMAD4 [51]. SMAD4 is prominently inactivated at the genomic level in pancreatic cancer and is considered a key driver for tumorigenesis in this context [52,53]. Furthermore, SMAD4 point mutations isolated from pancreatic tumours appear to be correlated with errant ubiquitination patterns, and overall increased protein turnover [54]. This is most likely due to misfolding of the mutant protein and subsequent ‘quality control’ ubiquitination [54]. Furthermore F-box protein SKP2 which has been demonstrated to preferentially bind and degrade cancer derived SMAD4 mutants [54]. It interesting to note here that unlike SMAD4, SMAD2 and SMAD3 are rarely mutated in cancer suggesting that this type of ubiquitin mediated degradation of SMAD2/3 is unlikely to operate in cancer [19]. Degradative poly-ubiquitination of SMAD4 can also be mediated by various HECT E3 ligases, including SMURF1/2, WWP1 and NEDD4-2 [54]. It has been proposed that these ligases do not directly interact with SMAD4 but rather regulate SMAD4 physiological levels through complex formation with either inhibitory SMADs (I-SMADs) or SMAD2 [54]. As this interaction appears to occur in the cytoplasm this may suggest a mechanism of quashing latent SMAD4 which can no longer be recycled following re-entry into the cytoplasm from the nucleus, thus maintaining tolerable SMAD4 levels quantities for R-SMAD/Co- SMAD binding. Conversely, Ectodermin/Tif1γ (ECTO) was identified to be a mono-ubiquitin ligase for SMAD4 specifically ubiquitinating at the K519 site thus preventing the formation of SMAD4/R-SMAD4 heterotrimer complex and inhibiting chromatin binding [57]. With ubiquitination occurring in the nucleus the monoubiquitinated form of SMAD4 is exported to the cytoplasm. Interestingly, it has been hypothesized that the acetylation of histones in close proximity to the chromatin bound SMAD complexes increases the affinity for ECTO thus expelling the SMAD complex from the promoter regions on the DNA resulting in the loss of transcription of downstream TGF-β target genes.

shown to ubiquitinate and degrade SMAD5, while SMURF2 also ubiquitinates SMAD1 under steady-state conditions [32,36]. NEDD4-like or NEDD4-L is another well-studied E3 ligase, which belongs to the same E3 ubiquitin ligase family as the SMURF proteins. NEDD4-L specifically recognizes a threonine phosphorylation site directly prior to the PY motif in the linker region of R-SMADs allowing protein-protein recognition and marking SMAD2/3 for proteasomal degradation. Ostensibly, therefore certain phosphorylation residues in the linker region enhance SMAD transcriptional action before being marked for degradation by NEDD4L. Controversially, recent results by Tang et al. clearly indicate that SMURF2 does not regulate protein stability of these aforementioned SMAD proteins. Intriguingly, they demonstrate that in engineered Smurf2 (−/−) mouse embryonic fibroblasts turnover rates of SMAD2/ 3 were equivalent compared to wild type fibroblasts. However, loss of SMURF2 did appear to alter monoubiquitination at multiple lysine residues in the MH2 domain of SMAD3. Monoubiquitination of the K333, K378, and K409 in the MH2 domain of SMAD3 blocks formation of both homotrimeric SMAD3 and heterotrimeric SMAD3-SMAD4 complexes invariably limiting these complexes in binding to SMAD motifs on the DNA [37]. The interesting finding that SMURF2 targets SMAD3 for monoubiquitination is in line with recent results that show that NEDD4 family members (of which SMURF1 and SMURF2 are included) predominantly assemble K63 linkages [38]. The fact that SMURF proteins may only form K63 chains and not K48 chains as was once believed leads to a host of questions regarding the role of SMURF2 targeting proteins for degradation. It may be that SMURF proteins may only prime their targets for entry into early endosomes whereby other ligases targeted these proteins for degradation. On the other hand, SMURF proteins might function in a dual role: priming targets through monoubiquitination and later catalysing polyubiquitinated chains depending on other regulatory proteins bound in the complexes. Tiul1/WWP1 is another member of the HECT E3 ligase family and has also been found to promote degradation of SMAD2 [39]. Upon stimulation with TGFβ, Tiul1/WWP1 interacts with Transforming growth factor beta-inducing factor 1 (TGIF) and facilitates the formation of a repressor complex eventually leading to the ubiquitination and degradation of SMAD2 [40]. Lo and Massague have previously observed that SMAD2 is degraded specifically in the nucleus. This observation coupled with the fact that Tiul1/WWP1 mediated degradation of SMAD2 is induced by prolonged TGFβ ligand (16 h) suggests that this ligase may be responsible for the TGFβ induced negative feedback loop first observed by Lo and Massague [28]. Importantly, in breast cancer the chromosomal band 8q21 is often amplified [41]. Tiul1/WWP1 gene is present within this band and it was found that copy number gain was present in approximately 51% of breast cancer cell lines and 41% of primary breast tumours [41]. As already described Tiul1/WWP1 is a negative regulator of TGFβ signalling and further biochemical analysis confirmed that siRNA targeting of Tiul1/WWP1 in breast cancer lines enhanced TGFβ mediated responses such as cytostasis and apoptosis [41]. This implies that elevated Tiul1/WWP1 provides an early proliferative advantage in breast cancer and in particular, in estrogen receptor positive breast cancers [41,42]. A similar phenomenon is observed in prostate cancer with 44% of prostate cancer xenografts and cell lines and 31% of prostate clinical samples possessing a gain in Tiul1/WWP1 copy number [43]. Similar to breast cancer, siRNA targeting of Tiul1/WWP1 in a prostate cancer cell line enhanced TGFβ induced cytostasis, again implying that overexpression of Tiul1/WWP1 has oncogenic capacity [43]. In addition, hyperactivity of Tiul1/WWP1 has also been noted in prostate cancer [44].To limit the intrinsic autocatalytic activity inherent to HECT E3 ligases intramolecular interactions between the C2 and HECT domains maintain the ligase in closed inactive confirmation [41,44,45] Interestingly, prostate cancer derived point mutant WWP1- E798V was found to be hyperactive due to the disruption of the aforementioned auto-inhibition mechanism. This disruption lead to hyper-degradation 460

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negative regulators for proteasomal degradation including, SMAD7, cSki, and SnoN [73,74]. As previously mentioned HECT E3 ligases bind to PY domain of SMAD7, in contrast, ARKADIA binds to the MH2 domain of SMAD7 and induces the polyubiquitination and degradation of SMAD7 leading to enhanced TGFβ signalling [74]. This uncovers perhaps an interesting insight to how whereabouts of binding to SMAD7 influences whether a particular ligase will degrade SMAD7 or simply uses it as an adaptor protein. C-Ski and SnoN are related transcriptional co-repressors which associate with the R-SMAD-SMAD4 complexes and recruit histone deacetylases (HDACs) to prevent transcription of TGFβ target genes [31]. SnoN levels are partially regulated through ligand induced phosphoSMAD2 whereby phospho-SMAD2 translocates to the nucleus forming a complex with ARKADIA targeting SMAD2 bound SnoN for ubiquitin mediated degradation. Interestingly, ARKADIA binds and ubiquitinates SnoN in the absence of TGFβ signalling [76]. Nevertheless, it is only upon TGFβ pathway activation that efficient ARKADIA induced SnoN degradation occurs due the formation of a phospho-SMAD2 -SnoNARKADIA complex [76]. This insinuates that phospho-SMAD2 is required for proteasome targeting [76]. Interestingly, under conditions where ARKADIA expression is repressed phospho-SMAD2/3 accumulates in the nucleus but appears to be non-functional. Furthermore, reconstitution of ARKADIA in null embryonic stem (ES) cells results in a restoration of SMAD2/3 transcriptional activity. However, counterintuitively, under these conditions phospho-SMADs gets degraded in the process [77]. This coupling of activation with degradation has been suggested to provide a mechanism to regulate appropriate transcriptional responses to ensure that active transcription is transient and that transcription factors do not perpetually remain on promoter sequences. Thus, once transcription has been initiated utilized SMAD complexes will be targeted for degradation or recycling, allowing new SMAD complexes to bind to the DNA. This termination signal is then one mechanism to ensure that the level of extracellular TGFβ ligand produces the desired physiological responses. Whether the accumulation of nuclear SMADs in ARKADIA null ES cells is the result of increased SnoN expression and the inhibition of binding of SMAD complexes to DNA remains inconclusive but is likely to be part of the process. Nagano et al. report that ARKADIA can bind and degrade SnoN, as well as c-Ski, a SnoN related repressor, independently of TGFβ signalling, contrasting previous observations [73,78]. Incidentally SMURF2 has also been demonstrated to target SnoN via SMAD2 [79]. SMAD2 is essential for this process as the utilization of SMAD2 deleted for its PY motif limited the ability of SMURF2 to bind SnoN [31]. Interestingly, SMAD3 also functions to degrade SnoN but in this scenario degradation occurs via the RING E3 ligase, Anaphase Promoting Complex (APC)-CDH1 complex. CDH1 acts as an adaptor for specific substrate recognition [80]. It is reported that this action is also TGFβ dependent as APC only interacts weakly with SnoN in the absence of ligand. This can be explained by the fact that destruction box motif (D- Box) within SnoN, which the CDH1 subunit recognizes, is not perfect thereby limiting CDH1-SnoN affinity [80]. SMAD3 is therefore required for efficient association of the APC complex with SnoN [80]. Recalling the observation made by Bonni et al. that phospho-SMAD2 is required for SMURF2 recruitment to degrade SnoN, employing a SMAD3 mutant incapable of SMURF2 binding similarly led to reduced degradation of SnoN [79,80]. This suggests that both SMURF2 and APC mediated degradation of SnoN operates concurrently for maximal effect [80]. The contextual importance of the TGFβ negative regulator SMURF2 repressing SnoN, another negative regulator remains to be determined. ARKADIA may also be involved in endocytosis of TβR as it has been shown to bind and ubiquitinate the μ2 subunit of AP-2, which is involved in the formation of clathrin coated pits [81]. ARKADIA's overall importance in terms of cancer remains inconclusive. Although the loss of ARKADIA is rare in human cancers a number of missense mutations have been localized in colorectal cancer patients correlating with SnoN

In addition to E3 ligases acting downstream in the TGF-β pathway, they also mediate TGF-β kinetics at the receptor level. As part of a transcriptionally regulated negative feedback loop BMP and TGFβ signalling induces the expression of the inhibitor adaptor SMADs (I-SMAD) SMAD6 and SMAD7, respectively. I-SMADs are distinct from the RSMADs in that they possess weak homology with respect to the SMAD1 MH1 domain [58–60]. The function of these SMADs is principally to alleviate TGFβ output [58–60]. SMAD6 has been shown to associate with type 1 receptors TβRI and BMP receptor IB (BMPRIB), mitigating the levels of p-SMAD2 and p-SMAD1, respectively [59]. It has also been reported that SMAD6 inhibits BMP signalling by competing with SMAD1 for physical association with SMAD4 [61]. SMAD7 has two distinct mechanisms by which it inhibits TGFβ signalling. Firstly, SMAD7 binds preferentially to activated TβRI hence competing with RSMADs and subsequently mitigating pathway activation [58]. However, a potentially more important role for SMAD7 appears that it is the key node for ubiquitin mediated regulation of the TGF-β pathway. Mechanistically, SMAD7 serves as scaffold to recruit SMURF2 and the E2 ligase UBCH7 to the TGF-β receptor complex to facilitate receptor polyubiquitination and complex degradation [62]. The interaction of SMAD7 to SMURF2 also has a secondary function. As previously mentioned, SMURF2 possesses autocatalytic activity when the protein is unfolded and therefore to maintain its stability and constrain unwanted activity towards its substrates the C2 and HECT domains remain in a tightly closed confirmation. The binding of SMAD7 to the HECT domain of SMURF2 abrogates these inhibitory intramolecular interactions between these domains facilitating SMURF2 ubiquitin ligase activity [63]. Besides acting as a scaffold for SMURF2 it performs a similar function for the E3 ligases NEDD4-2, SMURF1, and its homologue WW Domain Containing E3 Ubiquitin Protein Ligase 1 (WWP1) also known as Tiul1 (TGIF interacting ubiquitin ligase 1), which also target the type I TGF-β receptors for ubiquitin mediated degradation [39,64–66]. The existence of this apparent redundancy in function between the HECT E3 ligase family is unclear and may ultimately be context dependent. Furthermore, CD109 also binds to the SMURF2/SMAD7 complex and regulates receptor ubiquitination and degradation in a ligand dependent manner [67]. It has been observed in renal cell carcinoma that TGFβ signalling is depressed when compared with normal renal tissues, with a decrease in signalling being correlated with a post-translational decrease of TβRII protein levels [68]. In fact, in renal cell carcinoma tissues the levels of SMURF2 protein are inversely correlated with TGFβRII [68]. High levels of SMURF2 also correlate with poor prognosis in Esophageal Squamous Cell Carcinoma [69]. It was found that in this cancer, SMURF2 is overexpressed compared with normal tissues and this high expression was correlated with depth of invasion, and lymph node metastasis [69]. Unlike renal cell carcinoma, the main substrate of SMURF2 in this context appeared to be pSMAD2. Atrophin 1-interacting protein 4/ITCH(AIP4/Itch) subsequently to be referred to as ITCH is another HECT E3 ligase that acts to inhibit TGFβ signalling [70]. In contrast to other members of the HECT family, in this context, ITCH operates independently of its ligase activity [70]. ITCH appears to strengthen the association between SMAD7 and activated TGFβRI (without affecting turnover of TGFβRI) and in this way blocks R-SMAD access to the receptor kinase domain [70]. In a contrasting study, Bai et al. observed that ITCH enhances pSMAD2 levels by ubiquitination of SMAD2. They observe the formation of a trimeric complex between ITCH, SMAD2 and TGFβRI in stimulated cells and therefore postulate that this ubiquitination promotes SMAD2 proximity with TGFβRI thus facilitating overall signalling [71]. In addition, ITCH has also been shown to act as a positive regulator of the TGFβ pathway by ubiquitinating and inducing degradation of SMAD7 [72]. It is unclear why ITCH facilitates such opposing effects with respect to TGFβRI activity but it is likely to be context dependent. The ubiquitin-proteasome system can also positively regulate the TGFβ cascade. ARKADIA, a RING-finger containing E3, targets multiple 461

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of course leaves us with the question of what is the E3 ligase that monoubiquitinates the lysine residues within the MH1 domain. The fact that TGFβ enhances USP15 proteins levels but at the same time dissociates USP15 from the SMAD7/SMURF2 complex is in intriguing enigma. Recently, it has been demonstrated that USP15 binds to and stabilizes the transcription factor p53 [89]. Under physiological conditions p53 can form complex with SMAD2/3 to co-activate transcription of an assortment of genes involved in tumour suppression. It may therefore be that under certain conditions USP15 acts as a general regulator of the TGFβ tumour suppressor function by deubiquitinating both R-SMADs and its co-transcriptional regulator p53 enhancing TGFβ mediated transcription of genes. However, in scenarios where p53 is mutated or lost and the levels of USP15 are abnormally high, this may switch the binding of SMAD transcription factors from the promoters of tumour suppressor genes to those governing more pro- proliferative functions. This appears to be the case in certain cancers as USP15 gene was demonstrated to be amplified in glioblastoma, breast and ovarian cancers Using a number of independent samples sets, Eichhorn et al. was able to confirm that USP15 expression is highly correlated with TGFβRI and pSMAD2 in these cancers. Furthermore, glioblastoma patients with relatively high copy numbers of USP15 have a significantly poorer prognosis compared to those with lower copy numbers [91]. As previously mentioned a number of DUBs including USP4, 11, and 19 have been identified as potent regulators of the signalling cascade. All of these DUBs bound to the TβRI directly resulting in decreased levels of TβRI ubiquitination and stabilization. The existence of DUBs with apparent overlapping functions can only attest to the level of complexity required for internalization and compartmentalization of receptors at the cell surface. Interestingly, the fate of TGFβR is dependent upon a continuous interplay between ligases and DUBS to regulate ubiquitination and internalization. This is not only determined by transitory ubiquitination state of the receptor but of all components of the receptor complex including the ligases and DUBs themselves. Preliminary results by Ten Dijke and colleagues add weight to this theory whereby they identified that the ability of USP15 to deubiquitinate TβRI requires USP4, as USP15 was unable to perform this function in USP4 deficient cells [85]. Furthermore, they demonstrate that AKT phosphorylation of USP4 enhances the binding of USP4 to USP15 and that overexpression of USP15 increases USP4 stability. Under these conditions AKT hijacks USP4 to stabilize the TβRI and reinforces the pro-tumorigenic responses of the TGFβ pathway. Notably, USP4 is overexpressed in breast cancer and may function as an oncogene to maintain the TGFβ breast cancer progression [92]. Regarding the cross talk observed between enzymes regulating ubiquitination and internalization of receptors it would be of great surprise if AKT did not regulate other components of this complex. Not unlike USP15, two other DUBS USP11 and UCH37 are able to bind SMAD7 and target the TGFβ receptor for deubiquitination [62,93]. In the case of USP11, SMAD7 does not appear to act exclusively as a scaffold for as SMAD7 does not appear to mediate the interaction between USP11 and TβRI, despite the fact that SMAD7 and USP11 being able to form a complex. In addition, the interaction between USP11 and SMAD7does not appear to be greatly affected by TGFβ ligand. However, besides acting as TβRI deubiquitinases both USP15 and USP11 are able to regulate their counterpart the E3 ligase SMURF2 albeit through apparently divergent mechanisms [88]. USP15 appears to target key residues within the HECT domain of SMURF2 thereby limiting the catalytic activity of SMURF2 towards it's substrates, while USP11 appears to increase overall SMURF2 ubiquitination. The effect of USP11 in this context is as of yet undetermined but it may suggest that USP11 may indirectly regulate SMURF2 stability. All the DUBs mentioned thus far, bar USP4 and USP11, utilize SMAD7 as a scaffold protein to mediate their effects. Recently, we identified that as part of a negative feedback loop TGF-β not only enhances the expression of SMAD7 but also of USP26, whereby USP26 acts as a SMAD7 K48 ubiquitin chain specific DUB rescuing SMAD7

stabilization thus potentially confirming the role of ARKADIA as an tumour suppressor [82] [83]. Furthermore, mice heterozygous for ARKADIA possess tumours with enhanced nuclear SnoN and decreased TGFβ signalling leading to colorectal cancer development [82]. Finally, it has been found that in the esophageal cancer cell line (SEG1) which exhibits low levels of ARKADIA expression, degradation of SnoN is also subsequently impaired [76]. In addition to activation of R-SMADs through the canonical TGFβ signalling pathway, TGFβ also the recruits tumour-necrosis factor receptor (TNFR)-associated factor TRAF4 to the TGFβ receptor complex restraining the E3 ligase activity of SMURF2 towards the receptor complex thereby limiting ubiquitination and degradation and thus maintaining TGFβ activity. Furthermore, TRAF4 is also able to recruit the deubiquitinating enzyme USP15 to aid in this process (see below). RNF12 has also been identified as a ligase for SMAD7 degradation [84]. 2.2. DUBs in the TGFβ pathway Considering the importance of the TGFβ pathway it is not surprising that TGFβ signalling is heavily regulated by the ubiquitination process, which is involved in crucial negative feedback loops, quality control mechanisms and constitutive degradation to maintain homeostasis. Ubiquitination is a reversible process and this adds another level of regulation to the TGFβ pathway. This reversal process mediated by deubiquitinating enzymes (DUBs) provides further opportunities for aberrations to arise and facilitate cancer progression. TβR stability and turnover functions as a principle juncture for the downregulation of the pathway. Counteracting the ubiquitination and degradation of the TβR complex six DUBs (USP4, USP11, USP15, USP19, and UCH37) have been identified that directly affect TβR deubiquitination and stability [62,85–87]. Making use of genome wide DUB libraries three independent groups identified USP15 as a critical component of the TGF-β pathway. Interestingly, akin to many of the E3 ligases USP15 appears to target a number of different nodes in the TGFβ pathway. At the receptor level USP15 forms a complex with SMAD7 and SMURF2 with USP15 opposing the effects of the SMURF2 ligase on TβR stabilization [87,88]. In this context, the scaffold protein SMAD7 interacts with two enzymes harbouring contrasting activities resulting in a constant balancing act regulating TGF-β output. Recently, it has been shown that TGFβ upregulates the translation of USP15 suggesting a positive feedback loop, however, the extent of the TGFβ activity also regulates the access of USP15 to the SMAD7-SMURF2 complex [87,89]. As a result, when the TGFβ signal is low SMAD7 engages both SMURF2 and USP15 to maintain TβR stability thus retaining low levels of TGFβ output. However, when excessive levels of TGFβ is present USP15 is dissociated from the SMAD7-SMURF2 complex leading to enhanced ubiquitination of the TβR and degradation of the complex. This generates an elegant rheostat whereby TGFβ regulates its own activity preventing hyperactivation of the signal cascade. USP15 also appears to regulate other nodes within the canonical TGF-β pathway. As well as acting as a DUB for the TβR, USP15 has recently been identified to deubiquitinate monoubiquitinated and polyubiquitinated isoforms of R-SMADs [90]. Post translational modifications within the DNA binding domains of R-SMADs prevents promoter recognition and aberrant TGFβ signalling. As previously discussed, Tang et al. demonstrated that SMURF2 knockout MEFs, rather than displaying the expectant stabilization of known substrates merely exhibited decreased mono-ubiquitinated isoforms of SMAD3 [37]. These specifically occurred at K333, K378, and K409 within the MH2 domain. In contrast, USP15 deubiquitinates mono-ubiquitinated SMAD3 at K81 and to a lesser degree at K33 and K53 suggesting that USP15 deubiquitinates R-SMADs in the MH1 domain at sites independent of SMURF2 function [90]. Notably, all of these ubiquitination sites disrupt the R-SMAD/SMAD4 heterodimer from binding to the chromatin. USP15 reverses this modification permitting SMAD transcription factor binding and full TGF-β transcriptional responses. This 462

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Fig. 3. Schematic overview of regulation of Hippo pathway by Ubiquitin ligases and deubiquitinating enzymes. Activation of the Hippo pathway results through multiple extracellular mediums including cell-cell contact or mechanical tension leading to the downstream activation of two key core inhibitory kinase modules. The first of these comprises of MST1 and MST2 and the second module comprises of LATS1 and LATS2 (Drosophila Hippo and Warts, respectively). MST1/2 in combination with its binding partner Sav1 permits phosphorylation and kinase activation of LATS1/2 and the LATs co-factors MOB1A and MOB1B. Downstream transcription is mediated by YAP and its paralogue TAZ. These two transcription factors shuttle back and forth between the cytoplasm and the nucleus where they regulate transcription of a multitude of genes primarily through TEA domain family members (TEAD). LATS1/2 phosphorylation of the YAP/TAZ complex displaces YAP/TAZ to cytoplasm inhibiting YAP/TAZ mediated transcription. Phosphorylation of YAP/TAZ by LATS1/2 occurs in a unique phosphodegron site inducing the recruitment of the E3 ligase β-TrCP resulting in polyubiquitnation and degradation of YAP/TAZ. Similar to LATS1/2, Angiomotin (AMOT) regulates hippo pathway activation by directly binding to YAP/TAZ resulting in cytoplasmic retention of both proteins. Ubiquitination regulates numerous proteins in the hippo pathway. AMOT itself undergoes polyubiquitination and degradation by the E3 ligases NEDD4 and ITCH inhibiting AMOT regulation of YAP/TAZ. USP9X has been shown revert this effect. NEDD4, ITCH, its HECT family member WWP1, and SIAH2 also ubiquitinate and degrade LATS1/2, and NEDD4 has also been demonstrated to perform a similar function towards SAV1. The E3 ligase PRAJA2 ubiquitinates and inhibits the stability of MOB1. All of which result in downregulation of hippo pathway activation and enhanced YAP/TAZ transcription. The deubiquitinating enzyme DUB3 deubiquitinates and stabilizes multiple components of the hippo pathway including the negative regulator ITCH resulting in AMOT and LATS1/2 degradation. Remarkably, DUB3 can also deubiquitinate AMOT and LATS enhancing hippo activity.

By modulating the activity of co-SMAD, SMAD4, USP9X/FAM has also been found to modulate the TGFβ pathway [57]. To counteract the monoubiquitination of SMAD4 by Ectodermin at K519 USP9x/FAM acts to remove the ubiquitin moiety in the cytoplasm permitting SMAD4 to once again form a complex with phosphorylated forms of SMAD2. The juxtaposed activities of ECTO and USP9X function as a feedback loop to regulate overall SMAD transcriptional output by initially permitting SMAD2/SMAD4 complex formation and then by terminating transcriptional output. Recent results have demonstrated the importance of this as abrogation of ECTO expression impeded proper embryonic development [103]. Loss of SMAD4 is a common event in pancreatic cancer and colon cancer, and loss of USP9X has recently been shown to collaborate with KRAS to induce pancreatic cancer. Although the role of USP9X in this scenario may likely be due to the stabilization of YAP it cannot be discounted that inhibition of the TGFβ plays an integral role in this process.

from degradation. This then permits SMAD7 to remain in stable confirmation with SMURF2, permitting SMURF2 recruitment to the TGF-β receptor complex potentiating complex degradation [94]. Other ISMAD regulatory mechanisms involve two members of the deubiquitinating enzyme JAMM subfamily, associated molecule with the SH3 domain of STAM (AMSH) and its homologue AMSH-2 sequester SMAD6 and SMAD7, respectively, suppressing the inhibitory action of these ISMADs towards their targets although it has never been conclusively determined if their deubiquitinating enzyme activity was required for this function [95,96]. The exact targets of these DUBs are also unknown but it is thought that AMSH is required for the regulation of TβR turnover by the endosomal sorting complexes required for transport (ESCRT) formation [97]. R-SMADs are also regulated by DUBs. OTUB1 has been found to modulate TGFβ signalling by promoting the stabilization of only the phosphorylated form of SMAD2/3 [98]. Interestingly, it was found that the mechanism is non-canonical and non-catalytic: instead of OTUB1 directly deubiquitinating SMAD2/3 rather it interacts with the E2 ligases prevents the transfer of ubiquitin from the E2 ligase to the E3. This non-canonical functionality of OTUB1 has been observed prior on multiple occasions [99–102]. Importantly, OTUB1's role in modulating TGFβ output has been established as a knockdown resulted in an inhibition of TGFβ induced migration [98] This lends support of a potential role of OTUB1 in cancer, though this is yet to be verified.

3. The role of ubiquitin modifying enzymes in the hippo pathway The importance of the Hippo pathway was original recognized in Drosophila through the use of genetic mosaic screens whereby random loss-of-function mutations in components of this pathway gave rise to severe organ overgrowth. It was later elucidated that these observed effects were the result of increased cellular proliferation, decreased 463

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adaptor SAV1 concomitantly ubiquitinating and degrading both SAV1 and LATS. Interestingly, the upstream kinase MST1 negatively regulates the ability of NEDD4 to target SAV1 but in a disconnected fashion is unable to inhibit NEDD4 mediated degradation of LATS [110]. Furthermore, the authors indicate that the proficiency of MST1 to inhibit NEDD4 activity towards SAV1 is highly dependent on confluency of the cells. In such a scenario high cell density activates the Hippo pathway by increasing phosphorylated active MST1 releasing the negative inhibition of NEDD4 towards SAV1 and silencing YAP transcription. Incidentally, of the NEDD4 family members only NEDD4 is capable of binding SAV1 indicating that NEDD4, ITCH, and WWP1 interact with other adaptor proteins to regulate overall LATS1/2 levels. Similarly, in response to hypoxia the E3 ubiquitin ligase SIAH2 ubiquitinates LATS2, resulting in its destabilization [114]. Finally, the RING ligase PRAJA2 induces proteasome mediated degradation of MOB1 [115]. Mechanotransduction and the associated actin dynamics translate the cells kinetic physical signals with the required downstream chemical cues to direct both short term and long term signalling. How actin specifically plays a role in downstream YAP and TAZ signalling remains to be elucidated. Angiomotin (AMOT) is a filamentous actin binding protein that predominantly localizes to cellular junctions. AMOT and two AMOT-like proteins, AMOTL1 and AMOTL2 regulate Hippo pathway activation by directly binding YAP and TAZ resulting in cytoplasmic retention of both proteins [116]. Interestingly, the interaction between AMOT and YAP or TAZ occurs independent of YAP and TAZ phosphorylation by LATS even though LATS enhances complex formation between these proteins by directly phosphorylating AMOT itself [117]. This indicates that LATS inhibits the nuclear localisation of YAP and TAZ by two independent mechanisms; enhanced AMOT complex formation and targeting YAP and TAZ for proteasomal mediated degradation. AMOT proteins themselves have been shown to be ubiquitinated on multiple residues with opposing effects on protein function mediated by different ubiquitin topologies. Mono-ubiquitination of K347 and K408 is required for AMOTL2 to bind to the LATS1/2 complex and may act as a scaffold to permit LATS phosphorylation and inactivation of YAP and TAZ [118]. In contrast, polyubiquitination at K496 of AMOT targets AMOT for degradation resulting in YAP nuclear localization [15]. Curiously however, both of these studies identify USP9X as the deubiquitinating enzyme regulating both the monoubiquitination and polyubiquitination of AMOT. This contradictory result would suggest that deubiquitination of monoubiquitinated AMOTL2 by USP9X would inhibit the ability of LATS to phosphorylated YAP and TAZ, permitting YAP and TAZ to translocate to the nucleus and induce target gene expression. In contrast, however, Nguyen and colleagues demonstrate that USP9X deubiquitinates polyubiquitinated AMOT stabilizing AMOT and sequestering YAP and TAZ in the cytoplasm [15]. Further work will be needed to address the differences between these two studies and identify the function of USP9X in the YAP pathway. Recently, several groups have shown the importance of a functional YAP1 pathway in KRAS mediated oncogenesis [108,119–121]. In particular two independent studies revealed that loss of YAP1 expression prevented cancer progression in KRAS driven mouse models of pancreatic ductal adenocarcinoma and lung cancer [122,123]. Data by Nguyen et al. indicate that USP9X transcript levels are significantly lower in thyroid, prostate, liver, hepatocellular carcinoma and kidney renal clear cell carcinoma with low USP9X expression correlating with significantly worse disease free survival in these later patients [15]. Also, USP9X appears to be a major tumour suppressor gene in pancreatic ductal adenocarcinoma, as genetic abalation of USP9X co-operated with KRAS to accelerate pancreatic tumourigenesis [16]. Furthermore, USP9X has been identified to mutated or deleted in 4% of pancreatic adenocarcinomas. Ostensibly in the above pre-clinical contexts, loss of USP9X stabilizes YAP therefore indirectly enhancing RAS driven oncogenesis through YAP activation. As YAP inhibitors are currently being tested in early phase clinical trials the potential that USP9X may be a biomarker for response for YAP inhibition must be

apoptosis and enhanced progenitor stem cell renewal. The observation that this pathway is also conserved in mammals has resulted in a flurry of intensive research. The integrity of the Hippo pathway is absolutely essential in early development. In some adult organs however, Hippo activity is expendable for normal homeostasis but is indispensable for tissue repair and regeneration upon damage. Furthermore, experimental evidence has indicated a strong correlation between abnormal Hippo pathway activation and oncogenesis. The disparity between the requirement of Hippo signalling in cancer and its relative dispensability in adult human tissues has led to the Hippo pathway being considered as a very attractive therapeutic target in cancer. Several early phase clinical trials have been initiated and it will be interesting to see if any clinical responses will be observed in patients receiving these compounds. The canonical Hippo pathway consists of two central components, regulatory and transcriptional, which control overall Hippo activity. The core regulatory component is a kinase cassette comprised of the Mammalian sterile-20-like (MST1/2, orthologue of Drosophila Hippo) and the adaptor protein Salvador family WW domain-containing protein 1 (SAV1). Complex formation of MST1/2 and SAV1 permits phosphorylation and activation of large tumour suppressor 1(LATS1) and LATS2 and the LATS co-factor MOB kinase activator protein 1A (MOB1A) or MOB1B (reviewed in [104]). The transcriptional component of the pathway is composed of the yes-associated protein (YAP) and its paralogue, transcriptional co-activator with PDZ-binding motif (TAZ). These two transcription factors shuttle back and forth between the cytoplasm and the nucleus where they regulate transcription of a multitude of genes primarily through TEA domain family members (TEAD). When the Hippo pathway is activated the LATS/MOB complex directly phosphorylates and inhibits the transcriptional co-activators YAP and TAZ through 14–3-3 mediated cytoplasmic retention. The Hippo pathway is further regulated by numerous upstream molecules, which regulate Hippo signalling through a multitude of post-translational modifications (reviewed extensively in [105–107]). Additionally, the mechanism of action of ubiquitination and deubiquitination in the Hippo pathway is starting to fall into place (Fig. 3). The most documented of these is the priming of YAP and TAZ for proteasomal mediated degradation following phosphorylation by LATS. LATS phosphorylation of YAP and TAZ serves to retain YAP and TAZ in the cytoplasm while concomitantly priming them for degradation. The phosphorylation by LATS occurs in a unique phosphodegron site permitting recognition by the β-TrCP/SCF ubiquitin ligase complex. Recently, it has also been demonstrated that the RAS oncogene may serve to inhibit YAP protein turnover through two independent mechanisms. RAS has been shown to downregulate the ubiquitin ligase complex substrate recognition factors SOCS5/6 which serves to target YAP to the B/C-Cullin 5 ubiquitin ligase for proteasomal degradation. Furthermore, the RAS isoform, H-RAS, induces the formation of MST1/ MST2 heterodimers muffling the kinase activity of MST1 towards LATS resulting in downregulation of the pathway and YAP/TAZ nuclear localization [108]. While degradation of YAP and TAZ is critical to the overall tumour suppressor activity of the Hippo pathway, ubiquitin mediated regulation of pathway has been demonstrated to act at multiple nodes along its axis. A number of members of the Neural Precursor Cell Expressed, Developmentally Down-Regulated 4 (NEDD4) family of E3 ubiquitin ligases including the E3 ubiquitin-protein ligase Itchy homologue (ITCH), WW Domain Containing E3 Ubiquitin Protein Ligase 1(WWP1), and NEDD4 itself have been shown to regulate the stability and abundance of LATS1 kinase [109–112]. Although these homologues share a common functional architecture suggesting redundancy in their activity recent work has begun to identify the mechanisms that underlie their specificity [113]. As such their activity towards LATS may be likely attributed to particular extracellular signals leading to specific complex formation. An example of this has been shown to specifically occur with NEDD4. NEDD4 directly binds to the previously mentioned LATS 464

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in contrast, TGF-α reduces tyrosine phosphorylation [131]. This phosphorylation of USP8 at S680 permits complex formation with 14-3-3 proteins [131]. 14-3-3 proteins are highly important for regulation of various pathways in cells including cell cycle progression, DNA damage, apoptosis, transcriptional regulation of genes etc. It has been postulated that 14-3-3 binding to USP8 may result in: alterations in its enzymatic activity, changes in subcellular localisation or 14-3-3 may in fact mediate the binding of other proteins affecting USP8 activity in an undefined way [132]. However, some studies have shown the role of USP8 in deubiquitinating and stabilizing a RING finger containing E3 Ubiquitin ligase called Nrdp1 which is involved in degradation of ERBB3 and ERBB4 receptor tyrosine kinases [133]. These results are opposite of the results seen by Niendorf et al. [130] showing that deletion of USP8 leads to lower level of ERBB3. Further investigation is needed to completely characterize the role of USP8 in regulation of ERBB receptor tyrosine kinase family. Possibly, the functional network between USP8, Nrdp1 and their targets such as EGFR family receptors is tissue and context specific dependent. Similarly, it has been shown that ubiquitin specific protease 2A (USP2A) is localized in endosomes and prevents EGFR degradation [134]. Liu et al. showed that overexpressing wild type USP2A, but not mutant USP2A, led to stabilization of EGFR and enhanced downstream signalling. Furthermore, overexpressing USP2A in human bladder cells significantly extended the duration of response to Heparin binding EGF like growth factor (HB-EGF) [135]. USP2A overexpressed cells could maintain high level of p-ERK from 5 to 60 min after treatment with HBEGF while control cells showed only a transient increase in p-ERK level. This was probably due to the potential role of USP2A in deubiquitinating and stabilizing EGFR receptor. Insulin Growth Factor recptor-1 (IGF-1) has been shown as an important initiator of MAPK and PI3K pathway activation. Intriguingly, mutant K1003R IGF-1R, which is not able to bind to ATP, is markedly less ubiquitinated than wild type IGF-1R upon stimulation with IGF, suggesting that ubiquitination of IGF-1R is dependent on its autophosphorylation [136]. Also, C-terminal domain of IGF-1R is needed for ubiquitination and IGF induced ERK activation. C-terminal truncated IGF-1R (Δ1245) is not ubiquitinated after IGF treatment and does not induce MAPK pathway activation while no change was reported in PI3K pathway activation [136]. Moreover, treating 3T3-like fibroblasts with lysosome inhibitor (Chloroquine) completely rescued IGF-1R protein level from degradation while proteasome inhibitor (epoxomicine) was only able to partly rescue this receptor [136]. Although, degradation of truncated Δ1245 IGF-1R, which is not ubiquitinated, was completely rescued by lysosome inhibitor, further studies are needed to investigate whether ubiquitination of IGF-1R has any role in degradation of this receptor. Receptor Tyrosine Kinases (RTKs) such as EGFR and IGF-1R bind growth factor receptor bound protein-2 (GRB2) following ligand induced activation. GRB2 subsequently recruits the nucleotide exchange factor son of sevenless (SOS). The activity of downstream GTPase protein, RAS, depends on the ability of SOS to convert inactive RASGDP to the activate RAS-GTP. Activated RAS leads to phosphorylation and consequently sequential activation of the MAPKKK, MAPKK, and MAPKs. c-CBL is a RING finger E3 ligase involved in the regulation of RTKs and contains three different domains in its N-terminal region including 4 helix bundle domains (4H), EF hand like domain (EF) and SH2 like domain. Together these three domains form what is known as a Tyrosine Kinase Binding (TKB) domain. The TKB supports the binding of c-CBL to phosphotyrosine sites (Y1045 in EGFR) on receptor tyrosine kinases such as EGFR, ERBB2 and ERBB4 [137,138]. c-CBL is also recruited to EGFR through an adaptor protein, GRB2 which binds to phosphorylated tyrosine sites on EGFR (mainly Y1068 and Y1086) promoting ubiquitination and degradation of this receptor [137,138]. Upon complex formation with the RTK's c-CBL is itself phosphorylated at tyrosine Y371 allowing the previously guarded E2 binding domain of c-CBL to become exposed and thus permitting full activation of the E3

established. The stability of AMOT does not only appear to be regulated by USP9X, but recent data has also shown that the deubiquitinating enzyme DUB3 plays integral role in AMOT stability. However, like a number of DUBs in the TGFβ pathway DUB3 appears to act at several levels to regulate Hippo pathway activation. First, DUB3 deubiquitinates and stabilizes the E3 ligase ITCH. As, ITCH mediated ubiquitination has been shown to promote the turnover of LATS and AMOT proteins this suggests that DUB3 acts to enhance YAP activity. Interestingly DUB3 also interacts with and deubiquitinates AMOT, AMOTL1, and the kinases LATS1 and LATS2 to promote their stability and in so doing decrease YAP activity [124]. The authors hypothesis the primary function of DUB3 is to stabilize the scaffold protein AMOT permitting an unperturbed complex formation between AMOT and the LATS kinases to downregulate YAP. The purpose of the seemingly contradictory function of DUB3 with respect to ITCH in this setting remains elusive but it is feasible that DUB3 binding to the AMOT/LATS complex may be regulated by post translational modifications whereby in the event that loss of binding to AMOT occurs, DUB3 can then act as a potent activator of the pathway by stabilizing ITCH subsequently leading to degradation of AMOT and LATS. 4. The role of ubiquitin modifying enzymes in the MAPK pathway Mitogen-activated protein kinases (MAPKs) are protein Ser/Thr kinases that convert proliferative signals at the cell surface receptors into a wide range of cellular responses. The importance of this pathway lies in maintaining a sensor dependent dynamic communication between cells and their surrounding environment. Many studies have emphasized sustained proliferative signalling due to hyperactive MAPK pathway as one of the main hallmarks of neoplastic diseases [125]. In normal cells the classical MAPK cascade is sequentially relayed through RAS induced phosphorlyation of MAPKKKs A-RAF, B-RAF, and C-RAF (also known as RAF1) following to MAPKKs MEK1 and MEK2, and eventually MAPKs ERK1 and ERK2. The culmination of ERK signalling results in regulation of genes involved in activation of cell cycle, proliferation, survival, cell migration among others. In order to maintain a systemic balance and to ensure that extracellular signals elicit appropriate downstream responses a number of mechanisms have evolved to regulate ERK activation, including ubiquitination (Fig. 4). The fact that ubiquitination is reversible and that ubiquitinated proteins are known to interact with a plethora of ubiquitin interacting proteins involved in signalling highlights the influence of ubiquitination as not only a degradative signal. In no other context is this more evident than in the crucial process of endocytosis. Initial activation of most pathways is stimulated by ligand induced dimerization of cell surface receptors resulting in autophosphorylation of Tyr residues in the intercellular domain creating docking sites for protein adaptors. This autophosphorylation event is more often than not associated with concomitant ubiquitination of the receptors. The addition of the ubiquitin moiety is required for cargo internalization and acts as a pilot shuttling the internalized receptor towards active signalling nodes, recycling chutes, or a degradative fate. As a number of excellent reviews on the role of ubiquitination in endocytosis has been written we will focus this segment on the ubiquitination enzymes which effect canonical downstream MAPK signalling [126,127]. Briefly, however, it must be noted that regulation of internalization and endosomal sorting involves the concerted action of an assembly of ligases and DUBs including for example USP8, USP2A, USP9X, AMSH, and Cezanne [126,128]. USP8 has been reported as an important factor for rescuing transmembrane receptors such as EGFR, HGFR (c-MET), ERBB2 and ERBB3 receptors from endocytosis mediated lysosomal degradation thereby further mediating downstream MAPK activation [129,130]. Interestingly, overexpression of constitutively active mutant SRC (SRC Y527A) significantly increases USP8 tyrosine phosphorylation [131]. Moreover, EGF stimulation of EGFR induces USP8 tyrosine phosphorylation while, 465

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Fig. 4. Schematic overview of regulation of MAPK pathway by Ubiquitin ligases and deubiquitinating enzymes. (A) Activation of the pathway through binding of ligand binding to receptor tyrosine kinase leads to phosphorylation of tyrosine sites on intracellular components of the receptor creating docking sites for GRB2. Upon binding of GRB2 to the receptor, SOS converts inactive GDP-RAS to active GTP-RAS. Active RAS induces RAF activation which leads to phosphorylation and activation of downstream MEK and ERK. Phosphorylated ERK translocates to the nucleus and induces transcription. Sprouty can also bind to phosphorylated sites on receptor and competitively inhibits the binding of the negative regulator c-CBL and rescues the receptor from degradation. Both USP2A and USP8 can also independently deubiquitinate and stabilize the receptor. Activation of the pathway is maintained by through SMURF2 and USP15. SMURF2 ubiquitinates and degrades the RAS negative regulator β-TrCP while USP15 deubiquitinates and stabilizes RAF. (B) The MAPK pathway is negatively regulated through a number of different ubiquitin mediated mechanisms. Upon binding of ligand and receptor phosphorylation the E3 ligases c-CBL and Nrdp1 ubiquitinate the receptor targeting the receptor for degradation. SPROUTY blocks the interaction of SOS and RAS and SPROUTY's interaction to RAF-1 blocks its kinase activity. β-TrCP ubiquitinates and degrades RAS. Rabex-5 monoubiquitinates H-RAS and N-RAS and prevents their endosomal recycling into the membrane. IMP is a known ubiquitin ligase which targets the scaffold protein KSR leading to its degradation. IMP autoubiquitination is reversed by USP15 stabilizing the protein. Several ubiquitin ligases and chaperones are responsible for degradation of RAF including RNF149, HUWE1, XIAP and HSP90. Also, MEKK1 ligase is known to add ubiquitin chains to ERK and degrade this protein in proteasome dependent manner. The overall outcome of these events is downregulation of pathway and inhibition of ERK mediated transcription.

previously been described as having both overlapping and non-redundant functions. Under physiological conditions RAS proteins continuously cycle between an active GTP-bound state and inactive GDPbound form which allows RAS proteins to bind and activate its various downstream substrates. The majority of RAS mutations typically affect hotspots within codons 12 and 13 most notably G12 V for KRAS. On a molecular level these mutations impair GTP-GDP hydrolysis which in turn leads to permanent activation of RAS and persistent stimulation of downstream signalling pathways. The abundance and subcellular localisation of all isoforms of RAS are regulated by ubiquitination. In light of this, though attempts to effectively pharmacologically inhibit K-RAS have been unsuccessful, recent data suggests that targeted inhibition of ubiquitin modifying enzymes may lead to downregulation of K-RAS function. The F-box protein β-transducin repeat–containing protein (βTrCP) mediates polyubiquitination of all RAS isoforms, leading to proteasome-dependent degradation of RAS [148]. Interestingly, this process is partly mediated by Wnt/β-catenin signalling pathway whereby Axin and Adenomatous Polyposis Coli (APC) enhance the binding of the WD40 domain of β-TrCP with H-RAS leading to downregulation of the transforming capability of H-RAS. These results emphasize regulation of the RAS proteins by the WNT pathway. β-TrCP itself undergoes ubiquitin mediated degradation. The E3 ligase SMURF2 monoubiquitinates its cognate ubiquitin-conjugating enzyme (E2), UBCH5/UBE2D, leading to the active E3:E2 complex formation resulting in the polyubiquitination and degradation of βTrCP [149]. Thus SMURF2 activity leads to the stabilization of K-RAS. Conversely, loss of SMURF2 expression by genetic means stabilized βTrCP and degraded K-RAS. Interestingly, the authors also observed that K-RAS mediated ubiquitination occurred at a higher level in mutant

ligase activity of c-CBL towards its bound RTK leading to proteosomal mediated degradation of the RTKs [139]. The adaptor protein SPROUTY has been commonly regarded as a negative regulator of MAPK pathway though negative regulation appears contextual; dependant on the specific RTK activated ligand. Like c-CBL SPROUTY contains a TKB binding motif with an affinity for phosphorylated EGFR tyrosine Y55 and in the event of EGF stimulation SPROUTY is able to bind instead of c-CBL inhibiting c-CBL ubiquitination of EGFR [140]. Therefore, SPROUTY acts as a competitive inhibitor preventing c-CBL interaction with EGFR hence enhancing MAPK signalling pathway [141]. In contrast, SPROUTY tends to act as a negative regulator following exposure to fibroblast growth factor (FGF), vascular-endothelial growth factor (VEGF), platelet- derived growth factor (PDGF), hepatocyte growth factor (HGF), glial-derived growth factor (GDGF), or nerve growth factor [142]. It does so by acting as a decoy adaptor for GRB2 preventing activation of RAS by inhibiting interaction of GRB2 with RAS [143]. Secondly, SPROUTY can bind to the cysteine rich domain to C-RAF kinase and blocking its kinase activity [144,145]. Interestingly, SPROUTY expression is induced by MAPK pathway activation eluding to the existence of a negative feedback loop [144]. In about one third of all human cancers one of three isoforms of RAS (H-RAS, N-RAS and K-RAS) are mutated asserting the critical importance of these small GTPases in proliferation and cell survival [146]. The primary difference in three isoforms of RAS lies in their subcellular localization which is mediated by a 25 amino acid C-terminus hypervariable region (HVR) containing sites for farnesylation and palmitoyilation for H-RAS and N-RAS and only farnesylation site (in the case of K-RAS) [147]. H-, N- and K-RAS, are ubiquitously expressed and have 466

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inhibitor of apoptosis (ML-IAP) using siRNA leads to stabilization of CRAF and promotes migration [157,158]. Although, IAP family members contains a RING E3 Ubiquitin ligase domain, XIAP and c-IAP depletion stabilized C-RAF level (≈2fold) in a RING domain independent manner [157]. Moreover, the same study showed the role of chaperons and cochaperons (mainly CHIP) in ubiquitination and degradation of C-RAF. Carboxy terminal Hsc70 interacting protein, CHIP, is one of the important co-chaperons which conjugates E2 ubiquitin ligases to C-RAF. However, interaction of C-RAF and CHIP requires XIAP as an adaptor protein which explains the increased ubiquitination and degradation of C-RAF by overexpression of the mutant RING domain deleted XIAP [157]. Similar to C-RAF, A-RAF and mutated B-RAF V600E (but not wildtype B-RAF) are stabilized through HSP90 chaperone machinery [159]. HSP90 is required for C-RAF stability and membrane localization. Geldanamycin, a HSP90 binding antibiotic, disrupts complex formation of C-RAF and HSP90 and induces ubiquitin dependent degradation of CRAF [160]. C-RAF mediated downstream signalling was inhibited by Geldanamycine possibly through reduction of C-RAF level. However, the signalling and C-RAF level was maintained in presence of proteasome inhibitors, even in presence of Glendamycine [161]. Also, mutant B-RAF V600E, which is frequently mutated in different types of cancer including melanoma, colorectal and thyroid cancer, is a HSP90 client [159]. Although, HSP90-cdc37 complex was involved in degradation of different B-RAF mutants (V600E, V600D, G465V, G468A), there was no change in level of wild type B-RAF after 17-AGG (HSP90 inhibitor) treatment, consistently shown in different melanoma cell lines [159]. Interestingly, RNF149 was reported as an E3 ligase responsible for degradation of wild type B-RAF, but not mutant B-RAF [162]. siRNA mediated downregulation of RNF149 leads to stabilization of wild type B-RAF and simvastatin treatment which induces RNF149 expression leads to lower level of wildtype B-RAF, without altering mutant V600E B-RAF level [162]. Interestingly, some studies have investigated the role of RAF proteins and dysregulation of its HSP90 dependent degradation in the non-cancer context of metabolic diseases such as diabetes [163]. It has been shown that, methylglyoxal, a physiological metabolite of glucose which is accumulated in serum of diabetic patients, significantly reduces C-RAF level through ubiquitin dependent degradation [163]. Extracellular signal related kinases (ERKs) are a family of proteins which are responsible for transmitting the extracellular signal into nucleus through phosphorylation or regulation of various transcription factors including ETS, ELK, MNK, RSK etc. ERK family kinases are classified into two categories depending on scaffold dependence for activation and function. C-terminal domain containing MAPKs such as ERK3 and ERK7/8 can exist in individual subunits independent of scaffold binding. The second category includes the classical ERK family members ERK1 and ERK2. The signalling axis of this pathway requires scaffolding proteins for their proper activation. As an example KSR (Kinase suppressor of RAS) is needed as a scaffold protein to preassemble and engage the multiple components of the MAPK cascade for efficient ERK1/2 activation [153]. ERKs have been shown to be regulated by ubiquitination/deubiquitination system. Upstream of ERK, MEK kinase1 (MEKK1) is able to activate MEKs in both JNK and ERK1/2 pathways. PHD (plant homeodomain) domain of MEKK1 which is a Ring finger like domain shows E3 ubiquitin ligase activity and is responsible for ubiquitination and degradation of ERK1/2 [164]. Therefore, MEKK1 activates and downregulates ERK1/2 through its kinase and PHD domain, respectively [164]. The PHD domain in MEKK1 contains 7 cysteines and a histidine which resembles the structure of other known E3 ubiquitin ligases. It has also been reported that the PHD domain is found in many proteins which are involved in chromatin mediated transcriptional regulation [164]. ERK1/2 maintains homeostasis by inhibiting apoptosis. Treatment with sorbitol results in cellular hyperosmotic conditions. This increases the interaction of MEKK1 and ERK1/2 leading to

forms of the protein, probably owing to its persistent GTP activation. This data would suggest that effective targeting of SMURF2 may be a unique strategy to degrade mutant K-RAS in cancer cells. However, one must consider that SMURF2 acts as a negative regulator of TGF-β signalling and therefore targeting of SMURF2 may activate the pro-oncogenic effects of TGF-β while simultaneously deregulating K-RAS. The endosomally localized Rabex5 (also known as RabGEF1) functions partly as an E3 ubiquitin ligase and promotes mono- and di-ubiquitination of H-RAS and N-RAS, leading to the anchoring of RAS at the endosomes and reducing downstream signalling [150,151]. It has been shown that Rabex-5 contains an A20 like Zing finger ubiquitin ligase domain (ZnF) which mediates the interaction of Rabex-5 with H-RAS and N-RAS. Interestingly, mutant H-RAS which is not able to be ubiquitinated is significantly more effective than wild type H-RAS in its activation of MAPK pathway highlighting the role of non-degradative ubiquitination of RAS proteins in downregulation of MAPK pathway [147]. The overall fate of endosomal bound RAS remains undetermined, most likely is it either targeted for endosomal mediated recycling or shunted towards the proteasomal degradation pathway. Furthermore, reversal of this ubiquitination at this location by an endosomal localized deubiquitinating enzyme has not yet been described. Individual RAS family members can also undergo monoubiquitination at K117 and K147. It has been proposed that targeted ubiquitination of K-RAS specifically at K147 impairs regulator-mediated GTP hydrolysis leading to markedly increased activation of MAPK pathway. In contrast, monoubiquitination of H-RAS at K117 accelerates intrinsic nucleotide exchange promoting GTP loading [152]. Interestingly, these hyperactivated forms of RAS demonstrate enhanced interaction binding with downstream effectors and overall activation of the pathway through monoubiquitination at distinct sites eliciting distinct mechanisms of action. Though the identity of both the E3 ligase and the counteracting DUB that regulates this site remains elusive, it is tempting to speculate that a balance exists between E3 ligase activity and DUB activity to regulate overall RAS activity through stabilization and localization of the protein. Impedes mitogenic signal propagation (IMP) is a Ring finger E3 ligase which negatively regulates KSR and inhibits MAPK pathway activation through the prevention of RAF and MEK complex formation. Binding of extracellular ligands to RTKs and activation of RAS leads to activation of IMP E3 ligase activity and its autoubiquitination and subsequent degradation. Therefore, increased IMP autoubiquitination following RAS activation leads to KSR stabilization and higher level of phosphorylated ERK1/2 [153,154]. How activation of RAS causes increased E3 ligase activity of IMP remains an unanswered question which requires further investigation. Recently, some studies have reported the role of USP15 in deubiquitinating and rescuing IMP from degradation [155]. Although, USP15 and USP4 both are involved in deubiquitination of IMP, depletion of only USP15, not USP4, destabilizes IMP promoting its proteasomal dependent degradation [155]. Hayes et al. showed that the interaction of USP15 and IMP is mediated through DUSP-UBL domain of USP15 and coiled coil region of IMP. Moreover, the same study showed the role of USP15 in stabilizing CRAF [155]. Although, USP15 depletion reduced the level of C-RAF, it had no effect on the level of B-RAF and p-MEK in B-RAFV600E harbouring melanoma cells [155]. HUWE1 a HECT family E3 ligase has recently been demonstrated to regulate of C-RAF protein level, but not B-RAF and A-RAF levels [156]. HUWE1 binds indirectly to C-RAF through a scaffold protein called SHOC2 and ubiquitinates both SHOC2 and C-RAF proteins. As expected, depletion of SHOC2 abrogates HUWE1 mediated C-RAF ubiquitination and degradation [156]. Downregulation HUWE1 expression by shRNA stabilized C-RAF and SHOC2 and increased p-ERK levels in different cells lines, demonstrating HUWE1 as a negative regulator of MAPK signalling. Some studies have shown that silencing X linked Inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis (c-IAP) or melanoma 467

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highlights the complex role of ubiquitination towards its targeted substrates and may suggest a tissue specific regulatory function of p85 towards PI3K activation. Like p85, IRS-1 is itself a target of proteasomal mediated degradation. CUL7 E3 ligase is a component of the SCF complex including FBW8, SKP1, and the ROC RING finger protein. As part of negative feedback loop S6K activation downstream of mTOR phosphorylates IRS-1 at S307 stimulating the interaction of IRS-1 with the substrate recognition subunit FBW8 and recruiting the SCF complex consequently resulting in IRS-1 ubiquitination and degradation. Treatment with the mTOR inhibitor rapamycin repressed IRS-1 phosphorylation and inhibited the CUL7 E3 ligase mediated degradation of IRS-1 revealing the importance of mTOR/S6 activities in ubiquitin mediated upstream regulation of IRS-1 by the SCF complex [171]. Likewise, SOCS containing E3 ligases has been reported to block insulin signalling by inducing ubiquitin mediated degradation of IRS1 and IRS2 and have been implicated in the mechanism for inflammation-induced insulin resistance [172]. Several other ubiquitin ligases suppress IGF/insulin signalling by inducing ubiquitin mediated proteasomal degradation of IRS-1/2 [173,174] whereas mono-ubiquitination of IRS-2 at single or multiple sites by NEDD4 has been reported to enhance IGF signalling in a manner dependent on the ubiquitin binding protein Epsin1 [175]. Ubiquitination of IRS proteins has been shown to be reversed by deubiquitinating enzyme USP7. USP7 forms a complex with IRS1/2 deubiquitinating and stabilizing IRS1/2. Interestingly, complex formation between IRS and USP7 is modulated by PI3K signalling as the addition of insulin led to the dissociation between USP7 and IRS1/2 enhancing IRS1/2 degradation [176]. This dissociation was prevented by treatment with the pan PI3K inhibitor LY294002. This suggests that IRS1/2 mediated activation of the PI3K pathway following insulin exposure induces a dual feedback mechanism targeting IRS1/2 stability. Following activation of the PI3K pathway S6K phosphorylates IRS1/2 recruiting the SCF ligase complex targeting IRS1/2 for ubiquitination. Concomitantly, PI3K activation dissociates USP7 from the IRS1/2 complex ensuring a rapid downregulation of activated IRS1/2.

downregulation of ERK1/2 through proteasome mediated degradation mechanism allowing for apoptosis to occur [164]. This demonstrates the crucial nature of ubiquitination along the ERK1/2 axis in maintaining homeostasis at the organism level. 5. The role of ubiquitin modifying enzymes in the PI3K pathway P13K-AKT-mTOR (PAM) pathway is a critical signalling pathway involved in cellular growth and survival. Genetic aberrations that lead to constitutive activation of the pathway are frequently observed in human cancers. However, due its aberrant behavior and irregular crosstalk with other signalling pathways targeted therapy with single agent PI3K pathway inhibitors has generated disappointing results clinically. Renewed enthusiasm analyzing various combination therapies, including strategies targeting CDK4/6 with PI3K inhibitors has revived hope that effective downregulation of the pathway will lead to prolonged responses in patients. Nevertheless, a better understanding of the PI3K pathway is essential for this effort. PI3Ks as the major downstream effector of receptor tyrosine kinases (RTKs) and G protein coupled receptors (GPCRs), leads to the formation of a potent second messenger, PIP3 (Phosphatidylinositol (3,4,5)-triphosphate) which transduces the signals from various growth factors and cytokines and assists in AKT activation. The serine threonine kinase AKT once activated plays a vital role in the regulation of cell survival in a variety of human neoplastic diseases and polices a range of cellular processes, including cell survival, cell cycle progression, cytoskeletal organization, vesicle trafficking, glucose transport, and platelet function. Along with a number of activating mutations within components of the PAM pathway a number of inhibitory mutations have also been identified in the tumour suppressor phosphatase and tensin homologue deleted from chromosome 10 (PTEN). PTEN is a dual specific protein and lipid phosphatase whose primary function is to degrade the phosphoinositide products of PI3K hydrolyzing (3,4,5)-trisphosphate (PIP3) to phosphatidylinositol (4,5)-bisphosphate (PIP2) [165,166]. In terms of enzymatic activity of the various component of PI3K-AKT-mTOR pathway including AKT and PTEN the role of phosphorylation has been well established however, very little is known about the role ubiquitin modifying enzymes in this process (Fig. 5).

5.2. Ubiquitin mediated regulation of AKT Substrate ubiquitination and targeting of proteins to the proteasome is a dynamic process and can have a great impact on progression of oncogenesis. Recent studies suggest that ubiquitination is equally important as phosphorylation for activation of PAM pathway components and as such it is unsurprising that pharmaceutical inhibition of a number of ubiquitin modifying enzymes with respect to PI3K activation is being explored, including a number of enzymes targeting AKT. AKT is a member of the AGC family of kinases, the members of which possess a pleckstrin homology (PH) domain at its N-terminus. As indicated above PI3K phosphorylates the inositol ring of PI(4,5)P2 at the D-3 position to form PI(3,4,5)P3, which is required for the binding of the PH-domain of AKT to the plasma membrane. This permits AKT to be phosphorylated by phosphoinositol-dependent kinase 1 (PDK1) within its catalytic domain at T308. Following pathway activation AKT is subsequently phosphorylated at S473 by ribosome-associated RICTOR-mTOR complex (mTORC2). Phosphorylation of both T308 and S473 is required for maximum kinase activity of AKT. Similarly, AKT kinase activity can be both regulated by K63-linked ubiquitination and K48 linked ubiquitination. TRAF6, TRAF4, NEDD4, and SKP2 all regulate AKT signalling by promoting K63 linked ubiquitination. K63-linked ubiquitination has been revealed to be critical for activation of AKT by enabling its membrane recruitment. TRAF6 ubiquitinates AKT at two lysines (K8 and K14) within its PH domain promoting translocation of AKT to plasma membrane and enhancing AKT T308 phosphorylation and activation [177]. Curiously, K63 ubiquitination at these two residues is not required for PIP3 binding even though the K14 residue resides within the PIP3 binding pocket. It has therefore been speculated that ubiquitination may enhance a conformational change within the

5.1. Ubiquitin mediated regulation of PI3K and IRS Phosphatidylinositol 3-kinase (PI3K) is a conserved hetero-dimer intracellular lipid kinase consisting of an 85-kD regulatory subunit bound to a 110-kD catalytic subunit. The phosphoinositol-3-kinase family is grouped into three different classes: Class I, Class II and Class III. In response to various growth factors class IA PI3Ks get activated either through direct binding with the activated receptors or through adapter molecules such as phosphotyrosyl insulin receptor substrate (IRS). The p85 subunit of PI3K has been shown to be regulated by several E3 ligases including SCF-FBXL2, CHIP, and CBL. SCF-FBXL2 catalyses ubiquitination and hence degradation of monomeric p85 [167]. Curiously, FBXL2 mediated degradation of p85 appears to enhance PI3K activation rather the inhibit it. The authors rationalize that FBXL2 preferentially targets excess free monomeric p85 thereby limiting the ability of the free p85 to outcompete the p85-p110 heterodimer for binding to IRS1 [168]. Recently, it has been demonstrated that the ErbB3-binding protein 1 (EBP1) couples p85 to the HSP70/CHIP complex. The chaperone-dependent ubiquitin ligase (CHIP) drives p85 ubiquitination and ultimately proteasomal mediated degradation of p85 [169]. It has been also reported that CBL family ubiquitin ligases not only mediates polyubiquitination of p85 in T cells but under certain conditions can regulate p85 compartmentalization and protein-protein interactions at the cell membrane [170]. CBL-B recruits p85 to CD28 and T cell antigen receptor and negatively regulates p85 in a proteolysis-independent manner. The contrasting effects of ubiquitin mediated degradation and regulation of p85 on downstream PI3K activity in these three studies 468

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Fig. 5. Schematic overview of regulation of PI3K pathway by Ubiquitin ligases and deubiquitinating enzymes. (A). Ubiquitin mediated downregulation of the PI3K pathway. PI3Ks are downstream effectors of receptor tyrosine kinases (RTKs) and G protein coupled receptors (GPCRs). Phosphatidylinositol 3-kinase (PI3K) is a hetero-dimer conserved intracellular lipid kinase consisting of an 85-KD regulatory subunit bound to a 110-KD catalytic subunit. CHIP and CBL regulate stability and function of p85 respectively through ubiquitination. Like p85, the RTK interacting protein IRS undergoes ubiquitination mediated degradation through CUL7 and SOCS. The downstream serine/threonine kinase AKT has been shown to undergo K48 ubiquitinated by the ligases MULAN, TTC3, BRCA1 and CHIP. These ligases degrade AKT downregulating PI3K pathway activity. Likewise, the DUB CYLD prevents activation of AKT by counteracting K63 linked ubiquitination and inhibiting AKT recruitment to membrane. Furthermore, the phosphatase PHLPP1 functions in complex with FKBP51 to dephosphorylate and inhibit AKT. USP49 stabilizes FKBP1 further enhancing AKT pathway inhibition by promoting interaction of PHLPP1 to AKT. Cul4-DDB1-Fbw5 mediates TSC2 stability and inhibiting TSC1/TSC2 complex turnover resulting in constitutive downregulation of the mTOR activator, RHEB. mTOR is the key component of mTORC1 (mTOR, mLST8, RAPTOR) and mTORC2 (mTOR, mLST8, RICTOR) whereby mTORC1 enhances phosphorylation of downstream S6K1 and eIF4E while mTORC2 acts through a positive feedback loop to phosphorylate AKT at S473 leading to full activation of AKT. Cul1-Skp-Fbw7 and USP9X negatively regulate mTOR in ubiquitin dependent manner, however, the manner through which USP9X regulates mTOR stability is unknown. The mTORC2 component RICTOR has been shown to be ubiquitinated and degraded by SCF-FBXL7. Interestingly mROC1 and mTORC2 complex formation is regulated by a number of ubiquitin modifying enzymes. mLST8, another component of both mTORC complexes, undergoes K63 mediated ubiquitination by TRAF2 hindering mTORC2 formation while increasing mTORC1. A mechanism offset by the DUB OTUD7B. In a similar fashion CUL4-DDB1 ubiquitinates RAPTOR limiting mTORC1 formation but enhancing mTORC2 formation. A mechanism reversed in this case by UCH-L1. The tumour suppressor PTEN negatively regulates PI3K pathway by inhibiting formation of PIP3 but also plays an undefined role as a tumour suppressor in the nucleus. PTEN also undergo various post translational modification including ubiquitination. PTEN was shown to be monoubiquitinated by NEDD4-1 enhancing its nuclear translocation. Furthermore, two DUBs USP13 and OTUD3 deubiquitinate and stabilize PTEN thereby aiding in its tumour suppressor activities. (B) Ubiquitin mediated upregulation of the PI3K pathway. The E3 ligase FBXL2 mediates degradation of p85 but interestingly this degradation enhances PI3K activity by preferentially targeting excess free monomeric p85 thereby limiting the ability of free p85 to outcompete the p85-p1110 heterodimer for binding to IRS1. NEDD4 has been reported to enhance IGF signalling by monoubiquitinating IRS at single or multiple sites. Similarly, USP7 counteracts ubiquitination of and stabilizes IRS1/2. PDK1 which phosphorylates and activates AKT is monoubiquitinated and undergoes deubiquitinated by USP4. Although the exact function of this DUB with respect to PDK1 needs to be explored. Membrane recruitment of AKT is a critical step for its activation. Various ligases such as TRAF6, SKP2, NEDD4 and TRAF4 all mediate K63 linked ubiquitination of AKT recruiting AKT to the plasma membrane. The E3 ligase SCF-β-TrCP ubiquitinates and degrades the AKT negative regulator PHLPP1. Similarly, UCH-L1 which is a deubiquitinating enzyme suppress the level of PHLPP1 thus enhancing AKT pathway. Deptor, thought to be a negative regulator of mTORC activity undergoes ubiquitination and degradation by the E3 ligase β-TrCP. S6K1, which is downstream of mTORC1 also undergoes ubiquitin mediated modification by ROC1. ROC1 might be responsible for turnover of S6K protein. PTEN itself is also negatively regulate by a number of ligases including WWP1, XIAP, CHIP, NEDD4-1 targeting PTEN polyubiquitination and degrading PTEN. RFP also polyubiquitinates PTEN but rather then inducing degradation it functions by inhibiting its activity. USP7 as a deubiquitinating enzyme counteract monoubiquitination of PTEN and causes its nuclear exclusion.

degradation, the SCF complex (SKP2, CUL, FBOX), mediates K63 ubiquitination of AKT [180]. For the most part SCF complexes promote protein degradation through the transfer of K48 ubiquitin chains. Why in this case SKP2-SCF complex promotes a non-proteolytic K63 linked ubiquitination of AKT remains a bit of mystery and this issue, undoubtedly, will need to be probed into further. It is important to note that all three of these ligases have been reported to promote cancer progression. Activating mutations in AKT itself are rarely observed, with the exception of a single point mutation E17K within the PH domain [181]. Interestingly, this cancer associated mutant displays hyperubiquitination of AKT, increased PIP3 binding and increased

protein permitting complex formation with a ubiquitin binding factor resulting in the relocalisation of AKT from the cytosol to the plasma membrane. Interestingly, AKT mediated ubiquitination by TRAF6 is enhanced following growth factor stimuli. In fact, it has been noted that AKT K63 ubiquitination is mediated by a diverse set of E3 ligases depending on the stimulus, with both TRAF6 and the neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4) having been demonstrated to regulate AKT activity through K63 linked ubiquitination following IGF-1 signalling whereas SKP2 and TRAF4 function downstream of ErbB receptors [178,179]. Curiously, unlike IRS1/2 where the SCF complex targets IRS1/2 for proteosomal mediated

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component in two distinct signalling complexes mTORC1 and mTORC2. mTORC1 is comprised of mTOR, the regulatory associated protein of mTOR (RAPTOR), mammalian lethal with Sec13 protein 8 (mLST8), the proline rich AKT substrate 40 kDA (PRAS40), and DEP-domain-containing mTOR-interacting protein (DEPTOR). mTORC2 comprises of six different proteins including rapamycin-insensitive companion of mTOR (RICTOR), mammalian stress-activated protein kinase interacting protein (mSIN1), protein observed with RICTOR-1 (Protor-1), mLST8, and DEPTOR [194]. Due to the importance of mTORC1/2 in cell growth and metabolism it is unsurprising that deregulation of mTOR signalling is associated with various diseases some of which appear to be determined by aberrant ubiquitination of mTOR components. Even though both mTORC1 and mTORC2 contain a number of common components activation of mTORC1 and mTORC2 occurs through unique stimuli including growth factors or nutrients and PI3K signalling, respectively. Correspondingly, mTORC1 and mTORC2 appear to be regulated through both analogous and distinctive ubiquitination schemes. mTORC1 primarily functions downstream of AKT to regulate protein translation and cell growth by phosphorylating S6K1 and EIF4EBP1 Eukaryotic translation initiation factor 4E-binding protein 1 (EIF4E-BP1) [195]. It has been reported that DEPTOR is a naturally occurring inhibitor of both mTORC1 and mTORC2 [196]. DEPTOR is targeted for ubiquitin mediated degradation by the SCF (Skp1-Cullin-F box proteins)- βTrCP E3 ligase in response to serum stimulation, thereby promoting mTOR activation and cellular proliferation [197–199]. Similarly, RAPTOR and mLST8 interact with CUL4-DDB1, a core component of CUL4-DDB1-WDR ubiquitin E3 ligase complex, resulting in enhanced mTOR mediated signalling. Loss of CUL4B and DDB1 diminished mTORC1-dependent phosphorylation of S6K and 4EBP1 [200]. Interestingly, CUL4-DDB1 function does not appear to regulate RAPTOR or mLST8 stability directly but functions to enhance mTORC1 assembly. Counteracting this process ubiquitination of RAPTOR by CUL4-DDB1 is offset by the deubiquitinating enzyme UCHL1 [201]. In either setting a direct consequence of either CUL4B-DDB1 or UCH-L1 function is the reorganization and assembly of competing mTOR complexes. CUL4B-DDB1 supports mTORC1 complex formation while limiting mTORC2 while UCH-LI destabilizes mTORC1 enhancing mTORC2 assembly. Comparably, the E3 ligase TRAF2 has recently been shown to promote K63 mediated ubiquitination of mLST8 hindering complex formation with the mTORC2 specific component SIN1 thereby promoting mTORC1 formation, a process reversed by the deubiquitinating enzyme OTUD7B (also known as CEZANNE) [202]. As such it would be expected that both CUL4B-DDB1 and TRAF2 enhance mTORC1 dependent S6K and 4E-BP1 phosphorylation while UCH-L1 and OTUD7B enhance mTORC2 dependent AKT phosphorylation at S473. mTOR signalling can further be downregulated through a number of ubiquitin dependent mechanisms with mTOR itself being negatively regulated by both Cul1-Skp-Fbw7 E3 ligase and the DUB USP9X [203,204]. The targeting of the Cul1-Skp-Fbw7 complex to mTOR results in the latter's ubiquitination and degradation while the mechanism through which USP9X inhibits mTOR signalling remains ill defined. In contrast, Cul4-DDB1-Fbw5 E3 ligase regulates TSC1/TSC2 complex turnover increasing in the constitutive activation of the downstream mTOR activator RHEB. Downstream of mTOR ribosomal protein S6 kinase has been demonstrated to be ubiquitinated and targeted for degradation by the ligase ROC1 resulting in the inhibition of S6K activity [205]. In addition to the key ubiquitin modifying enzymes which regulate mTORC1 a number of specific mTORC2 ubiquitin mediated activators and repressors have been identified. RICTOR is distinctively ubiquitinated and degraded by SCF-FBXL7 highlighting one of the more interesting feedback loops regulating overall PI3K activity. As observed with a large proportion of ubiquitination reactions catalyzed by E3 ligases, ubiquitination is mediated by prior phosphorylation of the substrate. Likewise, phosphorylation of RICTOR is prerequisite for its interaction

membrane localization indicative of the importance of PH domain ubiquitination and AKT activation. Counteracting these ligases the deubiquitinating enzyme CYLD acts as a tumour suppressor and negative regulator of AKT activation by apparently directly deubiquitinating K63 linked ubiquitin chains within the PH domain, terminating AKT activation [182,183]. Similar results were observed in TRAF6 depleted cells. As it has previously been demonstrated that CYLD deubiquitinates TRAF6 these results suggests that CYLD deubiquitinates and suppresses the activity of both the E3 ligase and its kindred substrate [184]. An effect which has been frequently observed [88,185]. Interestingly, K14 but not K8 was the critical residue for CYLD mediated deubiquitination. Proteasomal mediated degradation of AKT is regulated by a plethora of ubiquitin ligases including tetratricopeptide repeat domain 3 (TTC3), breast cancer susceptibility gene 1 (BRCA1), chaperon-associated ubiquitin ligase (CHIP), and mitochondrial ubiquitin ligase activator of NFκB (MULAN) [186–189]. In the majority of these cases phosphorylation and activation of AKT is a prerequisite determinant for ligase binding, AKT K48 ubiquitination, and subsequent degradation. TTC3 has been shown to preferentially bind to phosphorylated forms of AKT. Interestingly, as part of a self-regulation mechanism, AKT phosphorylates TTC3 at S378, a site elucidated to be required for TTC3 activity as mutation of TTC3 inhibited TTC3 mediated ubiquitination and degradation of AKT. The tumour suppressor BRCA1 is frequently mutated in breast and ovarian cancers. BRCA1 activity regulates a number of signalling pathways involved in genome stability including DNA damage repair, cell cycle check point control, chromatin remodeling, transcriptional regulation, apoptosis, and ubiquitination. Like TTC3, BRCA1 is recruited to phosphorylated isoforms of AKT and targets active AKT inducing K48 ubiquitin chain formation and degradation of the later [186]. This work highlights the ubiquitin mediated tumour suppressive function of BRCA1 in tumorigenesis. Similarly, the mitochondrial associated ubiquitin ligase MULAN and CHIP target phosphorylated AKT. Interestingly, K48 ubiquitination of phosphorylated AKT can occur both in the nucleus (BRCA1, TTC3) and in the cytoplasm (MULAN, CHIP) eluding to the potential role of AKT substrates in negative feedback loops in the recruitment of these ligases in both the nucleus and cytoplasm to downregulate active AKT. It also suggests that to prevent nuclear exclusion of AKT through its degradation in the cytoplasm by ubiquitin ligases a small proportion of phosphorylated AKT may be specifically recruited to the nucleus to perform its nuclear functions. It will be interesting to determine if the hyperactivated form of AKT(E17K) functions primarily in the cytoplasm or the nucleus and ubiquitin mediated degradation of AKT (E17K) is altered at either of these locales. 5.3. Ubiquitin mediated regulation of PDK1 and mTOR AKT is phosphorylated at T308 by PDK1 and subsequently at S473 by mTORC2 resulting in full activation of the protein. PDK1 (3phospho-inositide-dependent kinase 1) is a serine/threonine kinase which phosphorylates and activates approximately twenty-three of the AGC family of protein kinases including the proto-oncogene AKT, Protein Kinase C (PKC) and the p70 S6 kinase (S6K) [190–192]. PDK1 plays critical role in cell proliferation and metabolism. Mono-ubiquitinated isoforms of PDK1 have been reported in a various human cell lines suggesting PDK1 ubiquitination is a common process. The deubiquitinating enzyme USP4 co-localizes with PDK1 at the plasmamembrane and has been shown to deubiquitinate monoubiquitinated PDK1 both in vitro and in vivo [193]. However, the role of PDK1 ubiquitination remains unclear. It is yet to be determined if ubiquitination is required for recruitment of PDK1 to the membrane or if PDK1 ubiquitination is required for AKT activation. The mammalian target of rapamycin (mTOR) protein is a serinethreonine kinase that belongs to the PI3K family. mTOR is the key 470

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expression and localization of PTEN in a Nedd4-1-deficient mouse model signifying that NEDD4-1 may not be a relevant E3 ligase for PTEN ubiquitination in the murine system [214]. Counteracting the effects of NEDD4-1 ubiquitin-specific protease Herpesvirus-associated ubiquitin-specific protease (HAUSP), also known as USP7, removes ubiquitin from PTEN in the nucleus resulting PTEN nuclear exclusion [215]. Critically, absence of nuclear PTEN has been linked with tumorigenesis [210]. In correlation with PTEN nuclear exclusion, USP7 may therefore be regarded as a potential onco-protein a point further strengthened as USP7 has been observed to be overexpressed in prostate cancer [215]. X-linked inhibitor of apoptosis protein (XIAP), a RING domain E3 ligase has been reported to regulate PTEN stability both in vitro and in vivo. XIAP has been shown to enhance PTEN turnover through polyubiquitination but similarly ectopic expression of XIAP did not induce mono-ubiquitination of PTEN [216]. In contrast, knockdown of XIAP using shRNA in 293T and MCF-7 cells reduced both mono, multi-mono and poly-ubiquitination forms of PTEN resulting in decreased PTEN expression in the nucleus followed by a corresponding accumulation of PTEN in cytoplasm suggesting involvement of another E3 ligase regulating PTEN mono-ubiquitination. On the other hand, both WWP2 (also known as AIP2), a member of NEDD4 family of E3 ligases and CHIP, the chaperone-associated E3 ligase, have been proposed to mediate PTEN degradation and demonstrated to be required for neoplastic formation [217,218]. Along with stability, the phosphatase activity of PTEN has also been demonstrated to be regulated by ubiquitination. Ret Finger Protein (RFP) or TRIM27, a member of the tripartite motif (TRIM) family, promotes poly-ubiquitination of PTEN but rather then altering PTEN stabilization or localization RFP significantly inhibits PTEN phosphatase activity leading to PI3K pathway hyperactivation [219]. Apart from ubiquitination, de-ubiquitination of PTEN has also been determined to be a critical factor in tumorigenesis. Utilising a genome wide library encompassing all the known deubiquitinating enzymes Zhang et al. performed a PTEN interacting DUB screen through which they identified USP8, USP10, USP13, and USP39 as novel interactors of PTEN [220]. They highlight USP13 as a deubiquitinating enzyme of PTEN whereby USP13 stabilizes PTEN by counteracting ubiquitinmediated degradation of PTEN. Furthermore, they demonstrate that USP13 expression is downregulated in breast cancer correlating with the expectant loss of PTEN expression. OTUD3, plays a similar role in regulation of PTEN, and tumour progression in breast cancer [221]. DUBs have has been also shown to control PTEN at transcription level. The DUB Ataxin-3, a Josephin family DUB has been reported to restrict PTEN transcription in lung cancer cells [222].

to SCF-FBXL7 and eventual degradation. Furthermore, the phosphorylation of RICTOR limits RICTOR binding to AKT. The phosphorylation of RICTOR is mediated by GSK3β which itself is inhibited by AKT generating a positive feedback loop when AKT is activated. Meanwhile, GSK3β can also phosphorylate the serine/threonine phosphatase PHLPP1 promoting the interaction of PHLPP1 with the SCF-βTrCP ligase complex targeting PHLPP1 for degradation. PHLPP1 dephosphorylates AKT limiting the activity of AKT. As such under certain conditions GSK3β can activate AKT by degrading its negative regulator PHLPP1. Similarly, the deubiquitinating enzyme UCHL1 has been shown to enhance AKT pathway activation by suppressing the levels of PHLPP1 an effect found to drive the development of lymphoma in vivo [206]. In contrast, another DUB, USP49, inhibits AKT pathway by promoting interaction of PHLPP1 to AKT, thus facilitating the dephosphorylation of AKT. USP49 does so by deubiquitinating and stabilizing the scaffold protein FKBP51, which enhances PHLPP1/AKT interaction. 5.4. Ubiquitin mediated regulation of PTEN The lipid phosphatase PTEN plays an indispensable role in controlling PI3K pathway activation by inhibiting formation of the upstream signalling molecule PIP3. PTEN has also recently been shown to negatively regulate EGFR activity by affecting stabilization of the EGFR complex with the ubiquitin ligase CBL [207]. The overall outcome of PTEN function in the cytoplasm is the inactivation of downstream oncogenic AKT mediated signalling. Unsurprisingly, post translational modifications including ubiquitination has been shown to play a huge role in regulation of PTEN stability and activity as ubiquitinated isoforms of PTEN have been reported in a plethora of cancers [208].As indicated PTEN is primarily located in the cytosol with only a small fraction of PTEN recruited to plasma membrane to convert PIP3 to PIP2. This recruitment of PTEN to the membrane is dependent upon on an open accessible motif on the surface of PTEN generating a membrane binding regulatory interface. A phosphorylated C-terminal region of PTEN forms intra molecular interactions with this interface blocking PTEN binding to lipid membrane. Upon dephosphorylation of C-terminal residues PTEN reverts to the open conformation permitting membrane binding and PTEN mediated dephosphorylation of PIP3 to PIP2. Importantly, the open conformation of PTEN also leads to nuclear translocation of the protein where PTEN exerts a tumour suppressive role in the regulation of DNA repair and genome stability. Mutations at K13 and K289 of PTEN, are frequently observed in Cowdens disease and glioblastoma and are regulatory ubiquitination sites for PTEN function [209,210]. Interestingly, these disease associated PTEN mutants display normal phosphatase activity. However, in both cases the nuclear translocation of the enzyme is perturbed [210]. The E3 ligase neural precursor cell expressed developmentally downregulated protein 4-1 (NEDD4-1) functions in a bi-modal manner to mediate both mono- and poly-ubiquitination of PTEN with K289 acting as a major site for NEDD4-1 activity. K289 is located within the C2 domain of PTEN and it has been suggested that mutations at these sites may disrupt the C2 domain from binding the plasma membrane thus maintaining high levels of PIP3 [211]. Following the catalyses of PIP3 to PIP2 PTEN is likely polyubiquitinated by NEDD4-1 at K289 to confinement of PTEN in cytoplasm and its subsequent degradation via the proteasome. In contrast, mono-ubiquitination of PTEN by NEDD4-1 at K13 and K289 leads to a regulatory modification promoting its nuclear translocation [212]. Thus, NEDD4-1 acts as both an oncogene and a tumour-supressor. NEDD4-1 is amplified in a number of cancers and expression of NEDD4-1 correlates with low PTEN levels in lung cancer [213]. In the majority of cases PTEN associated cancer mutations are present in the C2 domain, however, in some cases the C2 domain remains intact, the latter situation potentially acting as a predictive biomarker for cancers that may respond to NEDD4-1 inhibition. It must be noted that data from an independent group revealed no changes in the

6. The role of ubiquitin modifying enzymes in the WNT pathway The Wnt signalling pathway plays a key role during embryogenesis/ development. It has been known to be involved in cell proliferation, cell migration and cell fate determination. Unfortunately, aberrant activation of the Wnt pathway is frequently observed in tumorigenesis. The canonical Wnt signalling pathway undergoes activation following Wnt ligand binding to a seven-pass transmembrane Frizzled (Fz) family of receptors and its co-receptors LRP5/6 at the cell surface resulting in the recruitment of the adaptor protein Dishevelled to the receptor complex. LRP5/6 is subsequently phosphorylated by Casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3) resulting in a high affinity binding site for AXIN. This then thwarts AXIN complex mediated phosphorylation of β-catenin leading to an overall increase in the cytoplasmic levels of β-catenin and permitting β-catenin to accumulate in the nucleus where it activates T-cell factor (TCF) and Lymphoid enhancer factor (LEF) leading to the transcription of key target genes such as Cyclin D1 and c-Myc [223,224]. The ubiquitin-proteasome system has previously been described to tightly regulate key components of Wnt pathway (Fig. 6). Cytosolic β471

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Fig. 6. Schematic overview of regulation of Wnt pathway by Ubiquitin ligases and deubiquitinating enzymes. (A) Wnt off: In the absence of Wnt ligand, the Axin-GSK-APC-CK1 destruction complex targets β-catenin for phosphorylation, priming β-catenin for ubiquitination by β-TRCP and resulting in β-catenin degradation. GSK can also target the E3 ligase RNF220 inhibiting recruitment of USP7 and deubiquitination of β-catenin. USP15 can enhance stabilization of the destruction complex by deubiquitinating APC. The pro Wnt pathway activator Dishevelled is ubiquitinated by either the E3 ligases ITCH, NEDD4L or CUL-3 reducing its stability and thus limiting Dishevelled recruitment to LRP5/6 and Wnt activation. In some scenarios Dishevelled can act as a negative regulator of Wnt signalling by binding ZNRF3/RNF43. ZNRF3/RNF43 functions to downregulate Wnt signalling by limiting Frizzled recruitment to the membrane. ZNRF3/RNF43 also plays a role downstream of β-catenin by sequestering the transcription factor TCF4 to the nuclear membrane. The E3 ligases CBL and JADE-1 ubiquitinate and destabilise nuclear β-catenin. Taken together multiple ubiquitin modifying enzymes are involved in the downregulation of Wnt signalling. (B) Wnt on: Upon Wnt ligand binding to the Fzd receptor and the coreceptor Lpr5/6, Dishevelled is recruited to the receptor complex. LRP5/6 is subsequently phosphorylated by CK1 and GSK resulting in a high affinity binding site for Axin. The binding of Axin to the receptor complex results in the demolition of the destruction complex resulting β-catenin stability and translocation to the nucleus where it binds to the TCF/LEF family of transcription factors to induce transcription. A number of DUBs act to stabilize the receptor complex including USP8 which deubiquitinates Fzd and USP14 which deubiquitinates Dishevelled. In contrast, the E3 ligases RNF146 and SMURF2 both ubiquitinate and degrade Axin. Axin itself is also regulated by the SMURF2 homologue SMURF1 however, SMURF1 mediated ubiquitination of Axin results in enhanced K29 ubiquitination of the protein and downregulation of Axin activity. USP34 also deubiquitinates Axin leading to stabilization of Axin however, the authors also noted that downregulation of USP34 downregulated β-catenin induced transcription of Wnt target genes. Four DUBs bind and deubiquitinate β-catenin resulting in its stability: USP4, USP9X, USP35 AND USP47. A fifth DUB, USP7, performs a similar function but does so in complex with RNF220 to limit β-catenin ubiquitination. Also the E3 ligase RAD6B functions in the nucleus to stabilize β-catenin.

phosphorylated forms of β-catenin therefore regulating β-catenin stabilization in both the Wnt-off and Wnt-on phases [229]. β-catenin can also be regulated by the E3 ligase MULE with ectopic expression of MULE decreasing β-catenin stabilization. Interestingly, MULE appears to function through a Wnt ligand dependent negative feedback loop whereby MULE targets both β-catenin and its upstream regulator Dishevelled [230]. As such under low Wnt signalling activation of MULE is restricted however, under conditions of cellular hyperproliferation promoting constitutive Wnt signalling MULE expression limits β-catenin stability. In Apcmin mice exhibiting enhanced βcatenin expression MULE deficiency further accelerated adenoma development compared to Apcmin mutation alone [231]. Of note, other E3 ubiquitin ligases targeting Dishevelled have also been described. The Cullin-3 ubiquitin ligase with the Broad complex, tramtrack and Bric- a- Brac (BTB) protein Kelch-like 12 (KLHL12) was one of the first E3 ubiquitin ligases found to ubiquitinate and degrade Dishevelled thereby antagonizing Wnt pathway [232]. Furthermore, two of the HECT domain containing-NEDD4 family of E3 ubiquitin ligases have been shown to regulate levels of Dishevelled. ITCH ubiquitinates phosphorylated forms of Dishevelled, thereby promoting its proteasome mediated degradation and attenuating Wnt signalling

catenin and its regulation by Wnt is the essence of Wnt signalling and along with its original discovery it was noted that β-catenin levels were policed by various degradative mechanisms. The AXIN complex is made up of the scaffolding protein AXIN which contains dispersed binding sites to interact with the tumour suppressor adenomatous polyposis coli gene product (APC), casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3). GSK3 and CK1 coordinate sequential phosphorylation of β-catenin at S45 (CK1) and subsequently at T41, S37 and S33 (GSK3) [225]. The primary E3 ubiquitin ligase responsible in the regulation of β-catenin stability is β-TrCP. GSK3 phosphorylation of β-catenin at S33 and S37 generates a binding site for β-TrCP, leading to β-catenin ubiquitination and degradation [226]. GSK3 and CK1 can also phosphorylate AXIN and APC increasing the association of these components to β-catenin further enhancing β-catenin degradation. Another RING finger containing E3 ubiquitin ligase, c-CBL has been described to ubiquitinate and degrade β-catenin in the nucleus [227]. The authors demonstrate that wild-type c-CBL suppresses while a ligase deficient mutant of c-CBL enhances Wnt signalling. Although, the authors describe the UBA domain on c-CBL dimerizes with β-catenin, it was not until recently that phosphorylation was found to be involved [228]. In contrast, JADE-1 ubiquitylates both phosphorylated and non472

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ligase RNF146 directly interacts with poly(ADP-ribose) enhancing tankyrase dependent PARsylation of AXIN promoting its degradation [241]. AXIN is also regulated by the E3 ligase SMURF2 [242]. SMURF2 ubiquitinates K505 of AXIN leading to protein degradation. AXIN has a number of putative PPXY motifs however, it remains undetermined if the WW domains of SMURF2 are required for this interaction. Similarly, the SMURF2 homologue SMURF1 interaction with AXIN is independent of its WW domain. Rather the C2 domain of SMURF1 is required for K29 linked polyubiquitination of AXIN mediating AXIN SMURF1 localisation at the plasma membrane, an effect which appears to be cell cycle regulated [243]. Overall, these three E3 ligases RNF146, SMURF1, and SMURF2 regulate either AXIN stability or function depending on the distinct ubiquitin chain topology. Opposing the tankyrase-dependent ubiquitination and degradation of AXIN is the deubiquitinating enzyme USP34. USP34 was found to associate with AXIN containing protein complexes through liquid chromatography-tandem mass spectrometry. In line with previous reports depletion of USP34 led to the polyubiquitnation and degradation of AXIN. However, the authors also suggest that loss of USP34 inhibited β-catenin mediated transcription indicating that USP34 may function downstream of β-catenin to regulate nuclear accumulation of AXIN [244]. Although AXIN has been prescribed as a key component of the βcatenin destruction complex AXIN is also known to shuttle between both the cytoplasm and nucleus where nuclear AXIN expression has been demonstrated to be greatly enriched in diverse cancers. Nevertheless, the precise function of how nuclear AXIN enhances β-catenin transcription remains unknown. Reflecting on the overall function of the known ubiquitin modifying enzymes that regulate AXIN it becomes apparent that multiple post translational modifications and unique chain topologies are required to tightly regulate AXIN homeostasis in the cells. TRABID which belongs to the OTU domain containing family of DUBs has been described as a positive regulator of the Wnt signalling [245]. TRABID binds APC but rather then antagonizing the proteasomal turnover of APC TRABID inhibits APC activity through preferential deubiquitination of K63 linked ubiquitin chains. Furthermore, the effect of TRABID on APC appears to function downstream β-catenin stabilization with the authors postulating that APC may function to decrease the rate of TCF-β-catenin complex formation, a process seemingly dependent upon K63 ubiquitination of APC and one which is reverted by TRABID. Recently, Ubiquitin specific protease 4 (USP4) was identified as a DUB targeting β-catenin. Deubiquitination of β-catenin by USP4 reverses the ubiquitination mediated degradation of β-catenin, upregulating Wnt signalling. Unsurprisingly, a positive co-relation between levels of USP4 and β-catenin was observed in tissues from patients diagnosed with colorectal cancer [246]. In a separate study, USP4 was also identified in an RNAi screen performed in the colorectal cancer cell line SW480 using the Wnt-reporter TOPFlash reporter assay [247]. However, in contrast to the previous work RNAi depletion of USP4 caused an up-regulation of Wnt signalling. In this case, USP4 was observed to interact with Wnt regulators Nemo-like kinase (NLK) and TCF4. USP4 deubiquitinated a subpopulation of TCF4. However, it remains unclear if TCF4 ubiquitination is required for TCF4/LEF mediated transcription. Interestingly, NLK promoted nuclear accumulation USP4. As NLK positively regulates Wnt signalling it appears that either NLK regulates USP4 nuclear localization as part of negative feedback loop to downregulate TCF or that NLK enhances USP4 mediated stabilization of β-catenin further promoting TCF mediated transcription. Even though, both the studies were performed using colorectal cancer cell lines, caution must be taken to carefully understand the role of USP4 in both scenarios. USP15 and USP47 have also been identified as positive regulators of Wnt signalling. Both USP15 and USP47 form a complex with β-catenin leading to its deubiquitination and stabilization [248,249]. USP7, a closely related member to USP47 has been shown to function along

[233]. Expectedly, RNAi mediated knockdown of ITCH stabilized Dishevelled and upregulated Wnt signalling. This group also mapped two regions on Dishevelled, a prototypical PPXY motif and DEP domain through which it interacts with ITCH. Mutation within the PPXY motif at Y568F or deletion of the DEP domain led to reduced affinity for ITCH. NEDD4L, which also belongs to the NEDD4 family of HECT containing E3 ubiquitin ligases has been shown to target Dishevelled and promote its proteasome mediated degradation [234]. Interestingly, NEDD4L mediated degradation of Dishevelled involves atypical ubiquitin chain formation including K6, K27 and K29 but not the canonical K48 ubiquitin chain topology, usually associated with degradation. Dishevelled can also be regulated through K63 polyubiquitination a modification regulated by the deubiquitinating enzyme CYLD. CYLD directly deubiquitinates Dishevelled but the direct function of K63 ubiquitination or the ligase involved in the process remains unknown. Nevertheless, it remains clear that depletion of CYLD enhanced Wnt induced β-catenin accumulation in the nucleus and activation of β-catenin induced transcription [235]. Curiously, Dishevelled has also been demonstrated to be required for ZNRF3/RNF43 mediated ubiquitination and degradation of the FZD receptor. Depletion of either Dishevelled or ZNRF3/RNF43 enhanced cell surface levels of FZD and LRP5/6 indicating that Dishevelled acts as an intermediary for the recruitment of these E3 ligase to the receptor complex [236]. This also places Dishevelled at a crossroads for both the activation and inhibition of the pathway by acting as a scaffold protein required for both AXIN downregulation and FZD stabilization. Further experimentation will no doubt be required to tease out the organization of Dishevelled in these two phases of Wnt regulation. ZNRF3/RNF43 can also function downstream of β-catenin to inhibit Wnt pathway activation by physically interacting with the TCF4 transcription factor and tethering TCF4 to the nuclear membrane [237]. Importantly, oncogenic mutations within RNF43 associated with human gastrointestinal tumours disrupted this inhibitory mechanism resulting in transactivation of the Wnt pathway. Counteracting these processes, a number of ubiquitin modifying enzymes have been identified which activate Wnt signalling by regulating proteins along multiple nodes in the pathway. USP8/UBPY along with USP6 were identified as a deubiquitinating enzymes for the Frizzled receptor. Ubiquitination of receptors at the plasma membrane induces receptor endocytosis and localization of receptors into lysosomes. Within these lysosomes receptors can either be recycled back to cell membrane or targeted for degradation in a ubiquitin dependent manner. USP6 and USP8 were found to deubiquitinate FZD stimulating FZD recycling and increase FXD expression at the plasma membrane [238,239]. Another deubiquitinating enzyme that has been shown to positively regulate Wnt signalling is USP14. Interestingly, both USP14 and the aforementioned CYLD deubiquitinate K63 polyubiquitinated isoforms of Dishevelled. However, in contrast to CYLD, which acts as a negative regulator of the pathway, USP14 positively enhanced Dishevelled function as genetic and chemical suppression of USP14 impaired downstream Wnt signalling. This disparity maybe explained by the targeting of specific ubiquitination sites by both enzymes. CYLD deubiquitinates K46 and K50 within the DIX domain of Dishevelled which mediates the dynamic polymerization of the protein enhancing binding sites for Wnt signalling partners [240]. This suggests that K63 mediated ubiquitination at K46 and K50 is required for polymerization to occur for the binding to Wnt signalling partners, potentially AXIN, an effect downregulated by CYLD. In contrast, USP14 deubiquitinates K444 and K451 within the DEP domain of Dishevelled, a motif required membrane translocation for the protein. Thus, K63 ubiquitination at these residues in the DEP domain appears to have a negative regulatory role by inhibiting the recruitment of Dishevelled to the membrane. A number of components of the AXIN-APC-GSK-CK1 destruction complex are also regulated by ubiquitination. AXIN stability is primarily regulated by the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2, whereby both enzymes interact with a highly conserved domain of AXIN targeting AXIN for degradation. In this setting the E3 473

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with an E3 ubiquitin ligase RNF220 to stabilize β-catenin. Although RNF220 is an E3 ubiquitin ligase, knockdown of USP7 renders the former inactive. Knockdown of either RNF220 or USP7 impairs overall Wnt signalling in colon cancer cells [250]. Like the β-catenin oppressor MULE, RAD6B is a transcriptional target of β-catenin and functions through a feedback loop to regulate overall β-catenin levels. However, unlike MULE, RAD6B mediated polyubiquitnation of β-catenin renders β-catenin insensitive to proteasomal degradation. RAD6B is a 17 kDA ubiquitin conjugating enzyme which induces K63 linked β-catenin polyubiquitination. Depletion of RAD6B decreases β-catenin polyubiquitnation and stability limiting βcatenin transcription. Unsurprisingly, β-catenin and RAD6B expression is positively correlated in breast carcinoma with RAD6B levels significantly enhanced in breast cancer compared to normal controls. Taken together it becomes wholly apparent that a multitude of post translational modifications are required for effective regulation of Wnt signalling with manipulation of ubiquitin modifying enzymes offering an exciting opportunity for cancer therapy and regenerative medicine.

degradation of oxidized proteins and enhanced resistance towards oxidative stress [254]. UCH-L1 has been linked to Parkinson's disease and has been identified as a biomarker for aggressive multiple myeloma and enhances HIF-1 mediated metastasis [257,258]. LDN-57444, a molecule which belongs to a class of isatin O-acyl oximes was identified by a high throughput screen to find inhibitors against UCH-L1. Though the molecule can inhibit UCH-L1, it was found that proliferation was increased in H1299 lung tumour cell line though this effect was not exhibited a in lung tumour lines that did not express UCH-L1. RNAi based approaches also had similar proliferative effects and suggests that UCH-L1 enzymatic activity is antiproliferative and UCH-L1 expression may be a adaptive response to enhanced tumour growth [259]. In a second study, LDN-57444 treatment enhanced apoptosis by increasing the levels of highly ubiquitinated proteins activating unfolded protein response, a protective response that promotes apoptosis when cellular stress continues to persist [260]. Similarly, LDN91946, a moderately potent inhibitor of UCH-L1 was identified using an assay measuring the hydrolysis of the fluorogenic substrate Ub-AMC. Subsequent work analyzing structural activity relationship studies and kinetics revealed that this compound is a noncompetitive inhibitor of UCH-L1 but not other cysteine hydrolases tested including such as UCH-L3, USP5 and caspase 3 [261]. Again, downregulation of UCH-L1 by LDN91946 enhanced cell death indicating that under certain conditions downregulation of UCHL1 may be used to optimize patient responses [262]. USP7 is probably the most well studied of all the known deubiquitinating enzymes targeting a plethora of cancer relevant genes including both tumour suppressors (p53, PTEN) and oncogenes (MDM2, IRS1). Nevertheless, USP7 may be considered a promising therapeutic target in cancer. With the help of biochemical assays and activity based protein profiling small molecule antagonists with high specificity have been identified against USP7. Both P02207 and P5091 target USP7 with the added function that P5091 simultaneously targets USP47 directing the cell fate towards apoptosis. Interestingly P5091 exhibited this effect in multiple myeloma cells resistant to bortezomib suggesting that P5091 may be used in bortezomib resistant myeloma patients as preliminary results indicate that P5091 is well tolerated in animal models and inhibits tumour growth and prolongs survival [263,264]. Incidentally, blocking HDM2 and p21 abrogated the cytotoxicity induced by P5091. Further research has led to the development of a potent analogue of P5091 called cdp14, against both USP7 and USP47, which has enhanced potency, solubility and a strong metabolic reactivity profile [264]. Finally, HBX19818 has also been identified as a USP7 inhibitor. Interestingly, HBX19818 covalently modifies the active Cys233 residue of USP7 limiting overall activity of the enzyme [265]. Colon cancer cells treated with this molecule produced a G1 arrest mimicking results observed in cell lines following USP7 shRNA knockdown, showcasing its high anti tumorigenic potential [265]. The USP1/UAF complex is involved in the translesion synthesis through the deubiquitination of FANCD2 and FANCI [266]. Two important domains in UAF1 regulate FANC pathway activation. The SLD2 region of UAF1 binds FANCI and the WD40 domain of UAF1 binds and stimulates USP1. Disruption of either of these domains promotes FAND2/FANCI ubiquitination and enhanced DNA repair defects [267]. Utilising a high throughput screen, pimozide, an anti-psychotic drug and GW7647, a PPAR-α-agonist were shown to act as potent inhibitors of USP1/UAF activity [268]. These inhibitors have been shown to target the USP and WD40 repeat protein complex. Furthermore, pimozide and GW7647 act synergistically with cisplatin in inhibiting proliferation of non-small cell lung cancer cells resistant to cisplatin [268]. LS1 was also recently identified as a potent inhibitor of UCH-L3. Applying a FRET based approach the authors analyzed the synthesis of ubiquitin bioconjugates to substrates [269]. Ubiquitinated peptides were labelled with a pair of FRET labels and were used to screen a library comprising about 1000 compounds against UCH-L3.

7. Therapeutic intervention of dubs Following the approval of Bortezomib, a proteasome inhibitor, as an anti-cancer therapeutic for the treatment of multiple myeloma, inhibitors of the ubiquitin-proteasome system (UPS) has received much attention and focus as a potential and attractive drug target for cancer therapy. However, to date no inhibitors targeting the ubiquitin proteasome system other than those that inhibit proteasome function have been approved for clinical use. Deubiquitinating enzymes of course are an integral part of the UPS, functioning to remove ubiquitin moieties from polyubiquitin chains or target proteins. The intrinsic isopeptidase activity of DUBs results in the cleavage of the carboxyl terminus of ubiquitin destabilizing ubiquitin substrate binding. Taking into account that the majority of cancers demonstrate enhanced expression of DUBs which ostensibly act as oncogenes in a number of cancer relevant pathways, DUB inhibition may be a considered as a valid cancer therapeutic strategy. The majority of the DUBs are cysteine proteases (the exception being the JAMM family), wherein the catalytic cysteine residues sheltered in the active sites of DUBs are highly reactive towards electrophiles. Most ubiquitination reactions involve the formation of thioester bonds between the c-terminal tail of ubiquitin and its substrate whereby the nucleophile cysteine containing thiol side chains can then attack this reaction. Revolving around this property, most of the DUB inhibitors that contain α, β-unsaturated ketones have been demonstrated to possess DUB inhibitory activity by trapping these thiol side chains. Towards the development of such DUB inhibitors, a wide range of compounds ranging from synthetic small molecules to natural products with DUB inhibitory activity have been isolated. This section reviews effective strategies to target DUBs. We would also like the readers to refer to the comprehensive review by D'Arcy et al. [251]. 7.1. Small molecule DUB inhibitors As highlighted in this review, DUBs catalyze the removal of ubiquitin moieties from substrates. For the most part the eviction of such ubiquitin moieties results in enhanced protein stability. However, a number of DUBs (USP14, RPN11, UCH-L5) are also physically and functionally associated with the 26S proteasome and act in a timely and substrate specific manner to remove ubiquitin for proper substrate processing, and as such targeted inhibition of proteasome associated DUBs may mimic the effect of bortezomib [252,253]. A small molecule inhibitor of the DUB USP14 was identified through a high through put screen called IU1 (inhibitor of USP14) [254]. This molecule, when exposed to cells, enhanced degradation of several proteasome substrates implicated in neurodegenerative disease, as USP14 is essential for the maintenance of synaptic ubiquitin levels and neuromuscular junction development [255,256]. USP14 inhibition led to quicker 474

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One of the first DUB inhibitors PR-619, is a broad spectrum DUB inhibitor which leads to an overload of ubiquitinated proteins, protein aggregate formation and subsequent inhibition of the UPS. PR-619 also leads to the activation of the autophagy pathway by inducing the transport of lysosomes to the aggregates formed [270,271]. However, due to its broad spectrum activity PR-619 is highly toxic in vivo which has therefore limited its effect in clinical development.

inhibitors compared to the other members of the A, B or E prostaglandin series. PGJ2, Δ12-PGJ2 and 15Δ-PGJ2 are sequentially metabolized products of PGD2 [278–280]. PGs such as Δ12-PGJ2 and Δ7PGA1 methyl ester have antitumour and antiviral activities and are easily taken up by cells and accumulate in the nuclei via covalent interactions that ultimately lead to growth inhibition. α, β- unsaturated ketones are highly susceptible to nucleophilic compounds with thiols as they are particularly strong nucleophiles that PGs react with enzymatically or non-enzymatically. Specifically, these cyclopentone PGs interact with the thiol nucleophiles at the β position within the cyclopentenone ring. Aside from acting as Michael acceptors another possible mechanism through which these prostaglandins can lead to UPS inhibition is through protein carbonylation and oxidative stress induced by cyclopentone PGs which eventually leads to the decrease in activities of S6 ATPase and 26 proteasome [281]. This oxidative stress arises when reactive oxygen species (ROS) are generated and can have potential damaging effects on cellular components. Interestingly, UCHL1 is one such protein which is oxidized in cells treated with 15Δ-PGJ2. Δ12-PGJ2 and 15Δ-PGJ2 are potent cyclopentone PGs and have been reported to inhibit UCH-L3 and UCH-L1 respectively [282,283]. Additionally, cyclopentone PGs induced unfolding and aggregation of UCH-L1, thus inhibiting its enzyme activity [284]. UCH-L1 is abundant in the brain with its expression particularly localized to the Lewy bodies or the neurofibrillary tangles. Furthermore, UCH-L1 has been documented to be involved in a ubiquitin-dependent proteolytic pathway and has been implicated in the pathogenesis of Parkinson's disease. A single thiol group on Cys152 of UCH-L1, irrespective of the presence of 5 other cysteine groups in UCH-L1, reacts with the α, β- unsaturated carbonyl center in the cyclopentenone rings of PG leading to the formation of a covalent adduct destroying its native structure and eventually its DUB activity [284]. Notably, 15Δ-PGJ2 also inactivates p53 function by its binding to Cys277 reducing significantly its binding and transcriptional activity. As discussed the majority of the DUBs are cysteine proteases which are highly reactive nucleophiles and cyclopentone PGs have Micheal acceptors attributing to the inhibitory effect of this set of compounds towards DUBs.

7.2. Michael acceptors The formation of covalent bonds is an important criterion for the design of anticancer molecules. Most antineoplastic agents bind directly to their substrates. However, most these agents lack target selectivity. Michael acceptors, on the other hand can be structurally modified to react selectively with specific nucleophiles. The Michael reaction comprises both Michael acceptors and Michael donors, and comprises of a nucleophilic addition of a carbanion or other types of nucleophiles. The α, β- unsaturated compounds (ketone) undergoing Michael addition is called the Michael acceptor and the highly reactive nucleophiles (cysteines) are referred to as Michael donors and together their product is referred to as the Michael adduct. The 1,4 conjugate reaction of α, βunsaturated carbonyl compounds and nucleophiles is referred to as the Michael reaction. In case of DUB inhibitors, the addition of the Michael acceptor traps the catalytic cysteine of the DUB active site rendering the targeted DUBs inactive. WP1130 (Degrasyn) is a small molecule, selective deubiquitinase inhibitor of both USP and UCH subclasses. WP1130 is derived from a compound with a Janus-activated kinase 2 (JAK2) inhibitor activity. WP1130 inhibits DUBs such as USP5, USP9X, USP14, UCH-L1 and UCH37, all of which are known to regulate survival protein stability. WP1130 also suppresses BCR/ABL, is a JAK2 transducer and activator of transcription factor STAT. The addition of WP1130 leads to rapid accumulation of polyubiquitinated K48/K63-linked proteins into juxtanuclear aggresomes, independent of 20S proteasome activity. Furthermore, WP1130 inhibits tumour activated DUBs and results in the downregulation of anti-apoptotic proteins and upregulation proapoptotic proteins such as MCL-1 and p53 [272–274]. Preliminary data has also indicated that it may be clinically relevant as WP1130 in combination with bortezomib had anti-tumour activity in mantle cell lymphoma animal models [275]. Eeyarestatin-1(Eer1) is an inhibitor of the endoplasmic reticulum associated protein degradation (ERAD) pathway [276]. Proteins that are terminally misfolded, are recognized by chaperones on the endoplasmic reticulum and are transported to depots for ubiquitination and proteasomal degradation through the ERAD pathway. Eeyarestatin1 binds directly to p97 ATPase and acts by blocking the degradation of misfolded proteins associated with the p97-associated deubiquitinating complex in cells preventing ataxin-3 mediated deubiquitination of substrates [277]. Notably, eeyarestatin-1 has exhibited anti-cancer properties similar to that of bortezomib.

7.4. Chalcone inhibitors Chalcone or chalconids is an aromatic ketone and an enone that forms the central core for a plethora of critical biological compounds. Chalcone compounds such as G5, b-AP15 and RA-9 have been described to inhibit intrinsic deubiquitinase activity. Chalcones contain cross conjugated α,β-unsaturated ketones and accessible β-carbons and inhibit DUB activity but are unrelated to PGs. Chalcone DUB inhibitors can be highly specific such as b-AP15 or highly broad spectrum like G5. b-AP15 and its analogue VLX1570 are other proteasome associated DUB inhibitors involved in the specific inhibition of USP14 and UCHL5. b-AP15 was identified in cell based screens for compounds enhancing cellular apoptosis independent of either cathepsin D or p53 function [285,286]. Dual inhibition of UCH-L5 and USP14 using RNA interference not only led to the accumulation of proteasomal substrates but led to loss of cell viability [287,288]. Furthermore, b-AP15 has been reported to show anti-neoplastic properties in animal models, multiple myeloma and solid tumours [287,289]. Interestingly, using gene expression and connectivity MAP analysis showed that b-AP15 induced a similar gene expression profile to that of the J series prostaglandin 15ΔPGJ2 [289]. Two small molecules namely G5 and F6 were identified in a small chemical library screen that was used to identify alternative pathways of caspase activation. The two molecules were capable of activating an apoptosome-independent apoptotic pathway by targeting the UPS and inhibiting ubiquitin isopeptidase activity [290,291]. Both inhibitors upregulate the BH3-only protein NOXA, and the inhibitor of apoptosis antagonist SMAC [290]. NOXA has been shown to play acritical role in the induction of mitochondrial fragmentation and caspase activation

7.3. Cyclopentone prostaglandins Cyclopentone prostaglandins are a subset of prostaglandins (PGs) or eicosanoids that induce the accumulation of polyubiquitinated proteins in cells and correspondingly inhibit DUB activity [278]. These eicosanoids are characterized by their electrophilic nature and their distinctive cross conjugated α, β-unsaturated ketone which act as Michael acceptors. Both A series and J series prostaglandins are capable of inactivating wildtype p53 by impairing the conformation and phosphorylation of p53 resulting in the downregulation of p53 transcriptional activity [278]. However, the J series of prostaglandins contain a exocyclic α, β-unsaturated ketone, a unique structural determinant that confers a pro-apoptotic effect through the inhibition of ubiquitin isopeptidase activity regardless of p53 mediated transcriptional suppression [278]. Additionally, prostaglandins of the J series are more potent 475

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inhibitory activity. Curcumin is a diarylheptanoid, belonging to the family of cucuminoids, a natural phenol, bestowing the yellow colour of turmeric. Furthermore, curcumin is a homodimer of feruloylmethane, containing a methoxy and a hydroxyl pharmacophore. Additionally, curcumin is an extensively studied phytochemical with anti-cancer, anti-microbial, anti-inflammatory and anti-oxidant properties to name a few. Studies also show that the anti-tumour property of curcumin arises from the presence of two α,β-unsaturated ketone moieties. Curcumin was observed to induce the accumulation of polyubiquitinated proteins at high concentrations, through a shotgun proteomic approach [300]. This group also showed that curcumin downregulated some of the proteins associated with cellular assembly, organization, biosynthesis and glycolysis. Notably, the accumulation of polyubiquitinated oncogenes such as NF-κB and cyclin D1 potentially indicates the mechanism of action of this intriguing compound. AC17, a 4-arylidene curcumin analog, has been reported to inhibit the deubiquitinase activity of 19S ribosomal particles. Though AC17 is a synthesized product, it acts as an inhibitory kB kinase inhibitor (IκK) and leads to a rapid accumulation of ubiquitinated proteins without inhibiting the proteasome proteolytic activities. It acts differently from its parent compound curcumin which is a proteasome proteolytic inhibitor by serving as an irreversible deubiquitinase inhibitor of 19S ribosomal particles, leading to the inhibition of the NF-κB pathway and reactivation of the proapoptotic protein p53. Treatment with AC17 has been demonstrated to suppress tumour growth in lung xenograft model [301].

[291]. F6 in particular was reported by another group to be associated with the inhibition of USP2 and USP7 and the inhibition of the SENP2 deSUMOylase [292]. Some other known partially selective DUB inhibitors, which happen to fall in the chalcone family are AM146, RA-9, RA-14 and RAMB1 [293,294]. These DUB inhibitors induce the rapid accumulation of polyubiquitinated substrates depleting the pool of free ubiquitin without affecting the 20S proteasome. The functionality of these novel small molecule inhibitors is based on the presence of a α,β-unsaturated carbonyl group in the inhibitor making it susceptible to the nucleophilic attack by the sulfhydryl moieties on the cysteines present in the active sites of DUBs. These chalcone DUB inhibitors directly suppresses the DUB activity of UCH-L1, UCH-L3, USP2, USP5 and USP8 which are particularly known to play key roles in the turnover and the stability of critical regulators of cell survival and proliferation. Additionally, these compounds down regulate cyclin D while concomitantly upregulating p53, p27 and p16 leading to arrest in S-G2/M phase of the cell cycle. Furthermore, these inhibitors, abrogate anchorage independent growth and promote the onset of apoptosis in ovarian, breast and cervical cancer cell lines, displaying no significant alterations in primary human cells [293,294]. 7.5. Natural compounds exhibiting DUB inhibitory activity Many compounds affect the ubiquitin proteasome system without the direct inhibition of the proteasome activity, chiefly brought about by compounds falling under the umbrella of DUB inhibitors. Interestingly, a number of natural products exist exhibiting DUB inhibitory activities. Gambogic acid, a xanthanoid with cytotoxic properties, is isolated from the resin of Garcinia hanburyi. Gambogic acid has an α,β-unsaturated ketone moiety which directs growth inhibition and the induction of apoptosis in cancer cells. Gambogic acid has been used in traditional Chinese medicine for ages and has been shown to have cytotoxic activity on several cancer cells [295]. Gambogic acid displays cytotoxic activity by inducing changes in gene expression profiles and affects the ubiquitin proteasome system. Gambogic acid also lead to enhanced polyubiquitination potentially through the inhibition of chymotrypsin activity associated with the 20s proteasome leading to the induction of apoptosis and cell death. Interestingly, the gene signature profiles of cells exposed to gambogic acid show similar profiles as cells exposed to UPS inhibitors like PGJ2, celastrol and with aferinA marking its potential to inhibit DUB activity [296]. Betulinic acid is a naturally occurring pentacyclic triterpenoid, that has been isolated from many diverse plants like white birch (Betula pubescens), ber tree (Zizyphus mauritina), carpenters herb (Prunella vulgaris), rosemary and tropical carnivorous plants such as Triphyophyllu peltatum and Ancistrocladus heyneanus. Betulinic acid has a variety of biological and medicinal properties including antiviral, antibacterial, anti-inflammatory, antihelmintic and antioxidant properties. Additionally, betulinic acid has been reported to show significant cytotoxic effect on several cell lines and even human melanoma xenograft animal models [297]. A 20% betulinic acid ointment has been used in the treatment of atypical moles (Dysplastic nevi) [298]. Interestingly, the anticancer property of betulinic acid is carried out by inducing apoptotic cell death in cancer cells by triggering apoptosis through the inhibition of transmembrane potential in isolated mitochondria [299]. Betulinic acid inhibits multiple DUBs, resulting in the overall accumulation of polyubiquitinated proteins and resulting in the decreased levels of oncoptoteins [299]. A similar effect was also observed in TRAMP transgenic mice model of prostate cancer, wherein betulinic acid inhibited primary tumours by increasing apoptosis and decreasing angiogenesis through a marked decrease in androgen receptor and cyclin D1 protein levels [299]. Curcumin, a principal component of turmeric (Curcuma longa), a prominent spice ingredient used in most south Asian dishes has DUB

7.6. Ubiquitin variant inhibitors As highlighted in this review, ubiquitination of substrates by E3 ligases occurs through the formation of an isopeptide bond between the C-terminal carboxyl group of ubiquitin and the lysine of the substrate, a process which is reversed through the catalytic properties of DUBs. Despite their low sequence similarity, USP catalytic domains share a common fold that includes a large structurally conserved ubiquitin binding site for the targeted ubiquitin. Ubiquitin binds with low affinity to these ubiquitin binding domains (UBDs) a evolutionary consequence which permits ubiquitin to bind to all UBDs equally. Since ubiquitin binds in general with low affinity to USPs but through a large contact area specific alterations within the ubiquitin molecule itself would result in potential increased binding affinity of the altered ubiquitin to the ubiquitin binding domain of the USPs. Furthermore, as each USP displays a relatively sequence specific UBD any ubiquitin variant should be USP specific. Tight binding of these ubiquitin variants should therefore act as competitive inhibitors of catalytic activity by blocking the recognition of endogenous ubiquitinated substrates. Using this as a backdrop, Sidhu and colleagues generated combinatorial, phage-displayed libraries of ubiquitin variants and effectively identified inhibitors targeting USP8, USP21, USP2a, USP7, USP10 and OTUB1 [302,303]. In all of the cases each of the ubiquitin variants bound to their respective ubiquitin binding domains with a greater efficiency then wild type ubiquitin and more importantly did not cross react with any of the other USPs tested. Importantly, ubiquitin variants targeting OTUB1 selectively inhibited OTUB1 mediated cleavage of K48 di-ubiquitin. Although, ubiquitin variant inhibitors cannot be used in patients due to their relative size 8 kDa ubiquitin variant technology does allow potent inhibition of targeted USPs and can be utilized for future target validation and drug discovery for all UBD proteins. 8. Concluding remarks Since its identification over 35 years ago our understanding of ubiquitin and the enzymes that manipulate its behavior has improved dramatically. We now know that ubiquitination plays a central role in virtually all biological processes including a number pathways involved 476

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