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Reversible protein ubiquitination is a crucial mechanism regulating the progression through the eukaryotic cell cycle. Ubiquitin-dependent signaling is terminated by specific deubiquitinating enzymes (DUBs), which now are known to be integral components of the core cell cycle machinery and cell cycle checkpoints. The importance of DUBs for cell cycle control is underscored by their frequent misregulation in cancer. Here, we discuss the role of deubiquitinating enzymes in controlling proliferation.
The complexity of multicellular organisms depends on carefully orchestrated developmental programs that ensure that cells divide and differentiate at the right time and place. In eukaryotes, cell division is driven by oscillations in the activities of cyclin-dependent kinases (CDKs), which are coupled to the development of the organism by growth- and differentiation factor signaling. This core cell cycle machinery is controlled by checkpoint signaling networks, which continuously scrutinize cells for aberrations and initiate necessary repair responses. The dynamic interplay between core machinery and checkpoints protects multicellular organisms from unscheduled proliferation and cancer.
During the last two decades, ubiquitination has been identified as a crucial regulator of both the core cell cycle machinery and cell cycle checkpoints. Ubiquitinated proteins are often targeted for degradation by the 26S proteasome, the irreversible nature of which is at the heart of the unidirectional progression through the cell cycle program [1, 2]. Reversible ubiquitination, however, can also trigger endocytosis of growth factor receptors or modulate the activity of kinases and transcription factors, thereby empowering cells to interpret signals from their environment or checkpoints. It is not surprising that the misregulation of any of these processes can have dire consequences for cell division in multicellular organisms.
Ubiquitination is a covalent modification, which involves the formation of an isopeptide bond between the carboxy-terminus of ubiquitin and the ε-amino group of a lysine residue within the acceptor protein . This reaction is carried out by an enzymatic cascade involving at least three different activities (E1, ubiquitin activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase). Substrates can be decorated with a single ubiquitin moiety (monoubiquitination), or with ubiquitin chains (multiubiquitination). Depending on the lysine residue of ubiquitin, which is used for chain formation, chains differ in structure and function. K48-linked ubiquitin chains target proteins for degradation by the 26S proteasome, whereas K63-linked ubiquitin chains fulfill mostly non-proteolytic roles in DNA repair or other signaling processes.
Similar to other covalent modifications, such as phosphorylation or methylation, ubiquitination is reversible. The human genome harbors approximately 100 deubiquitinating enzymes (DUBs), which cleave ubiquitin off substrates and thereby terminate ubiquitin-dependent signaling . Reminiscent of the role of phosphatases in signal transduction, DUBs have only lately been recognized as essential and specific components of the ubiquitin pathway. A flurry of recent papers has pointed to pivotal DUB functions in cell cycle control and has tightly linked the misregulation of DUBs to tumorigenesis. Here, we will review the function of DUBs in cell cycle control, and when appropriate discuss their relevance for cancer and drug development.
The majority of human DUBs are cysteine-proteases that based on homologies between their catalytic domains can be clustered into four subfamilies: ubiquitin-specific proteases (Usp), ubiquitin carboxy-terminal hydrolases (UCH), ovarian tumor-like proteases (OTU), and Machado-Jakob-Disease proteases (MJD). They all contain an active-site cysteine residue, which is part of a catalytic triad comprised of cysteine, histidine, and aspartate [4-7]. Although DUBs of the different subfamilies have surprisingly distinct three-dimensional structures, they share a conserved spatial arrangement of the residues of the catalytic triad. In addition to cysteine proteases, one class of DUB-metalloenzymes has been described [8, 9, 18]. These enzymes contain a JAMM-domain coordinating a catalytically important Zn2+-ion. The human genome harbors 58 Usps, 4 UCHs, 14 OUT-, 5 MJD-, and 14 JAMM domain-DUBs . Interestingly, deubiquitination can also be carried out in human cells by viral proteins following a viral infection [10, 11]. These viral DUBs have been identified mainly by innovative functional proteomic approaches , and do not share much sequence or structural similarity with human DUBs .
Most DUBs use a negatively charged cleft on the surface of their catalytic domain to recognize the extended carboxy-terminus of ubiquitin [4,5]. In addition, they often interact with large surfaces close to the hydrophobic patch of ubiquitin. The binding of ubiquitin can trigger major structural rearrangements of the catalytic DUB-domain, resulting in the optimal orientation of catalytic residues towards the scissile isopeptide bond . Many DUBs contain additional ubiquitin-binding motifs, such as UBA, ZnF-UBP, or UIM domains, which potentially endow DUBs with specificity towards ubiquitin chains of defined length or linkage [3, 15]. DUBs can also recognize motifs within the ubiquitinated proteins using additional domains, which in some cases are connected with the catalytic domain by flexible linkers [16, 17]. The simultaneous recognition of motifs within substrates and ubiquitin likely increases the affinity and specificity of DUBs towards their substrates.
As with most regulators of cell cycle progression, the activity of DUBs is tightly controlled (for an example see Figure 1). Several DUBs are synthesized and degraded in a cell-cycle dependent manner. Their expression levels peak at cell cycle stages controlled by their activity, such as the S phase DUB Usp1, the G2/M DUB Cyld, or the mitotic DUB Usp44 [19, 20, 76]. Even if expressed, DUBs often require the incorporation into larger protein complexes to be fully functional. This is certainly the case with yeast Ubp6, human Usp14, and UCH37, which are activated upon interaction with non-catalytic subunits of the 26S proteasome [21-24]. Recent findings with recombinant Usp44, the activation of which requires incubation with cell extracts , and with Usp1, which is activated upon binding to the WD40-repeat containing protein UAF1 , suggest that this mode of regulation occurs more frequently than originally thought. Besides mediating activation, binding partners also help localize DUBs to specific sites, such as early endosomes or the cytoskeleton, or channel them into specific pathways . Some DUBs, such as Usp8, Usp44, or Cyld are likely activated by cell cycle-dependent phosphorylation [19, 20, 26]. By contrast, inactivation of DUBs can occur by proteasomal degradation, dissociation of binding partners, or inhibitory cleavage of the catalytic domain as originally first shown for Usp1 . The inhibitory cleavage of Usp1 requires its own catalytic activity and amino acids within its catalytic domain, which are homologous to the carboxy-terminus of ubiquitin. DUB-activity and specificity, therefore, are controlled by an astonishing variety of mechanisms, attesting to the importance of proper DUB activity for cellular regulation.
Several studies have now demonstrated the importance of deubiquitinating enzymes for proliferation. Similar to ubiquitin ligases, DUBs participate in cell cycle control at almost every level. They have been proposed to function in such different processes as ubiquitin homeostasis, transcriptional control, or checkpoint regulation. Since we are only beginning to decipher mechanisms of DUB-dependent cell cycle control, novel functions of these pivotal enzymes are likely to be discovered in the near future. Our current knowledge of selected DUBs, however, already allows us to distill general features of DUB-dependent cell cycle control, as described in the following paragraphs.
Ubiquitin is synthesized as a linear fusion to other ubiquitin moieties or to ribosomal proteins [28 and references therein]. Its activation depends on the disassembly of these polyproteins by DUBs, such as isopeptidase T. Strikingly, even though ubiquitin targets other proteins for proteasomal or lysosomal degradation, it itself is relatively stable with a half life of several hours [29, 30]. This is also due to the activity of DUBs, which cleave ubiquitin off substrates prior to their translocation into the proteasome or further targeting to the lysosome. A failure of these DUBs to recycle ubiquitin leads to depletion of intracellular ubiquitin and delays cell cycle progression [30, 31]. The effects of DUB inhibition are exacerbated under stress conditions, when increased ubiquitination and degradation serve to remove misfolded and potentially deleterious proteins from the cellular pool . A failure of DUBs to cleave ubiquitin off proteins that are already engaged with the 26S proteasome impairs proteasome function and, consequently, cell cycle progression . Such DUBs that indirectly control proliferation by maintaining ubiquitin levels or proteasome activity may be referred to as housekeeping DUBs. As cancer cells continuously experience cellular stress, these enzymes represent potential targets for therapeutic intervention.
DUBs control cell cycle progression more directly by regulating the stability or activity of ubiquitin ligases. Although it might sound surprising, DUBs are often found in stable complexes with ubiquitin ligases, such as Mdm2 or Brca1, the misregulation of which are known to cause aberrant proliferation and cancer. Among several known examples, the co-regulation of the DUB Usp7/Hausp and its binding partner, the ubiquitin ligase Mdm2, is best understood.
Usp7/Hausp was originally isolated as the cellular binding partner of the herpesvirus ICP0 protein, but soon thereafter, it was shown to associate with the ubiquitin ligase Mdm2 and its substrate p53 [32-35]. Usp7/Hausp interacts with both Mdm2 and p53 through its amino-terminal TRAF-like domain . In the absence of substrates, some RING-finger ubiquitin ligases, including Mdm2, modify themselves and consequently trigger their own proteasomal degradation. This constitutes a negative feedback loop, which effectively ties the levels of ubiquitin ligases to their respective substrates . DUBs are known to be important components of these regulatory networks. In the case of Mdm2, depletion of Usp7/Hausp in cells leads to increased autoubiquitination and premature degradation of Mdm2 even in the presence of p53 [34, 35]. The resulting drop in Mdm2 levels strongly impairs the ubiquitination of the Mdm2-target p53, which in turn can accumulate and induce a cell cycle arrest in G1 or G2. Thus, Usp7/Hausp exerts cell cycle control by shielding a critical ubiquitin ligase from unwanted degradation.
Such intimate relations between ubiquitin ligases and deubiquitinating enzymes are surprisingly widespread. The heterodimeric E3 Brca1/Bard1, for example, is found in complexes with two different DUBs, the UCH Bap1 and the JAMM-domain DUB Brcc36 [37, 38]. Brcc36 stimulates the catalytic activity of Brca1/Bard1 and cooperates with Brca1/Bard1 in mediating a G2/M-checkpoint arrest. Brca1/Bard1 can be activated by the non-proteolytic ubiquitination of the ubiquitin ligase itself . It is tempting to speculate that Brcc36 increases Brca1/Bard1-activity by protecting it from non-productive or even inhibitory autoubiquitination at sites required for substrate- or E2-binding. A similar non-proteolytic regulation of E3 activity by DUBs is observed during mitosis, when inhibition of the E3 APC/C is maintained by Usp44 , as described in more detail below.
The most intimate relationship between DUBs and ubiquitin ligases is illustrated by the A20 protein, which affects proliferation and cell survival by regulating the NF-κB pathway . In this case, the same polypeptide chain harbors domains with DUB- (OTU-domain) and E3-activity (Zn-fingers). In vivo, A20 preferentially disassembles K63-linked ubiquitin chains, which are crucial for NF-κB-activation . Following the removal of K63-linked ubiquitin chains from substrates, such as RIP or Nemo, A20 then assembles K48-linked chains on the same substrates and completes their inactivation by targeting them for degradation. The NF-κB pathway and ubiquitination of RIP is under control of at least one other DUB, Cyld, which is mutated in familial cylindromatosis [42-44]. Combining DUBs and ubiquitin ligases within complexes or even within same polypeptide chain creates effective molecular machines permitting dynamic cell cycle regulation.
Many key cell cycle regulators are synthesized at specific cell cycle stages. Their expression is controlled by transcription factors, such as E2F or c-Myc, the activities of which are tightly coupled to cell cycle progression. Transcription factors are often very short-lived proteins and rapidly degraded by the ubiquitin/proteasome system. In fact, an inverse correlation between transcription factor activity and stability has been described . The transcription factor c-Myc, for example, promotes entry of cells into S phase and is turned over with a half-life of 30 minutes following its ubiquitination by the SCFFbw7 or SCFSkp2 ubiquitin ligases [46, 47]. The bulk of c-Myc is degraded after ubiquitination by a nucleolar Fbw7-isoform, Fbw7γ. A prerequisite of c-Myc-degradation is phosphorylation on two sites, T58 and S62. Mutation of these residues or deletion of the Fbw7 tumor suppressor stabilizes c-Myc and results in cancer .
The DUB Usp28 was isolated as a factor required for c-Myc-induced apoptosis, but subsequently shown to be equally important for c-Myc’s proliferative functions . Usp28 is found in ternary complexes with c-Myc and the Fbw7α-isoform, in which it protects c-Myc from SCFFbw7α-dependent ubiquitination and degradation. Usp28 also interferes with the premature ubiquitination of other Fbw7-substrates, including cyclin E. As described below, Usp28 is an important component of DNA damage checkpoints . Interestingly, DNA damage induces the dissociation of Usp28 from c-Myc and triggers c-Myc-degradation, thereby stalling cell cycle progression after damage . Consistent with a function of Usp28 in promoting proliferation, it is overexpressed in colon and breast carcinoma . Moreover, Burkitt’s lymphoma cells exhibit higher levels of deubiquitination activity, which may contribute to increased steady state levels of c-Myc . Incorporating the substrates into ternary complexes with ubiquitin ligases and DUBs in all likelihood facilitates the dynamic regulation of substrate levels in response to stimuli, such as DNA damage. Inhibiting the DUBs within those complexes, however, could be used to tip the balance towards c-Myc-ubiquitination and degradation, thereby interfering with the proliferation of c-Myc-dependent tumors.
Ubiquitination does not only control the stability, but also the activity of transcription factors . The forkhead transcription factor Foxo4, for example, is activated in response to cellular stress by monoubiquitination . Monoubiquitination of Foxo4 triggers its nuclear translocation and potentially increases the affinity of Foxo4 for transcriptional co-activators or polymerases, which often have ubiquitin-binding domains [54, 55]. As a consequence of its activation, Foxo4 stimulates the transcription of the CDK-inhibitor p27, which results in cell cycle arrest. When the cellular conditions have improved and the cell cycle machinery needs to resume, Foxo4 is deubiquitinated by the already described Usp7/Hausp. The deubiquitination of Foxo4 by Usp7/Hausp thus ensures the transient nature of a stress-induced cell cycle arrest .
Cell-cycle specific transcription is also indirectly regulated by DUBs that modulate chromatin structure. The accessibility of chromatin for the transcription apparatus is determined by a highly orchestrated interplay of distinct histone modifications, referred to as the histone code. One of these modifications is the transient ubiquitination of histones, which in fact provided the first example of ubiquitination in cells [56, 57]. The ubiquitination of histone H2A by the Bmi1/RING1A heterodimeric ubiquitin ligase is an important regulatory event in HOX gene expression and X-chromosome inactivation . H2A is deubiquitinated upon progression through mitosis , which potentially facilitates histone phosphorylation and chromosome segregation. The mitotic deubiquitination of H2A within intact nucleosomes is catalyzed by Usp16/Ubp-M . Loss of Usp16/Ubp-M increases the amount of ubiquitinated H2A and, consequently, impairs progression of cells through mitosis. Additional DUBs, including the S phase-specific Usp3 , limit histone ubiquitination at other stages of the cell cycle. Regulation of transcription and chromatin structure are most intimately connected with each other in the SAGA transcriptional co-activator complex . A subunit of this complex, Ubp8, deubiquitinates histone H2B, which is required for transcriptional control at several loci. The regulation of chromatin structure and transcription is one of the key mechanisms by which DUBs exert cell cycle control.
The progression through the different stages of the cell cycle program is coordinated with the development of the organism by growth- and differentiation-factor signaling. Both the strength and duration of growth-factor signaling determine whether cells pause in quiescence, progress through the cell cycle, or enter a differentiation program . The signaling is terminated by the endocytosis and lysosomal targeting of ligand-bound growth-factor receptors, which is triggered by their ubiquitination at the plasma membrane [64, 65]. Following the sorting of ubiquitinated receptors to early endosomes, further trafficking to the lysosome depends on consecutive ubiquitin-dependent interactions with ESCRT-complexes. Several DUBs play important roles in regulating the endocytic downregulation of activated growth-factor receptors. Among these, Usp8/UbpY interacts with and is phosphorylated by activated epidermal growth factor receptor (EGFR), but its precise function in EGFR-signaling remains to be established. Some siRNA-studies suggest that Usp8/UbpY-dependent deubiquitination of EGFR promotes further trafficking to the lysosome and EGFR degradation [66, 67]. By contrast, another siRNA-study and a USP8 knockout in mice came to the opposite conclusion that deubiquitination in fact rescues EGFR from degradation and allows its recycling to the plasma membrane [68, 69]. This discrepancy may originate from differences in cell types. Alternatively, it might be a consequence of functions of Usp8/UbpY at additional steps of endocytosis, when Usp8/UbpY is known to protect the critical endocytic regulators Hrs and STAM from proteasomal degradation. Consistent with a function in EGFR- and cell cycle regulation, Usp8/UbpY is expressed in a cell cycle-dependent manner . It is absent from quiescent cells, that did not experience growth factor signaling for prolonged periods of time. A potential function of Usp8/UbpY in cell cycle control is supported by an oncogenic fusion between Usp8/UbpY and the PI-3K subunit p85β, which was isolated from a leukemia patient .
Several other DUBs, including yeast Doa4, Drosophila Faf, and human AMSH, regulate endocytosis and protein trafficking to lysosomes [72-74]. The Drosophila melanogaster protein fat facets (Faf) was in fact one of the first DUBs for which a specific cellular function had been described. Faf binds to the epsin homolog Liquid facets (Lqf). Together, Faf and Lqf control the endocytosis of the Notch ligand Delta, and loss of faf or lqf activity results in perturbed Notch signaling and defective eye development. In addition to its functions in endocytosis, the human Faf homolog, Usp9, affects progression through mitosis by deubiquitinating the chromosomal passenger protein survivin . By controlling the endocytosis and lysosomal trafficking of activated growth- and differentiation factor receptors, DUBs form a critical link between proliferation and cell cycle exit.
It is vital for multicellular organisms to prevent mistakes in proliferation. This is the task of checkpoint signaling networks that sense intracellular damage or incomplete cell cycle stages, and as a consequence, reversibly stall cell cycle progression. Following the repair of damage or completion of a cell cycle stage, the checkpoints are silenced and the cell cycle machinery resumes. Two well studied examples are the DNA damage checkpoint and the mitotic spindle checkpoint, both of which are intimately controlled by deubiquitination and impaired in a wide range of cancers.
The DNA damage checkpoint senses different kinds of DNA damage, such as DNA crosslinks or breaks, leading to activation of kinase cascades and cell cycle arrest in G1 or G2. A key component of the DNA damage checkpoint is the transcription factor p53. Its regulation by opposing activities, Mdm2-dependent ubiquitination/degradation and Usp7/Hausp-dependent deubiquitination/stabilization, was described before. Similar to Usp7/Hausp, Usp28 protects crucial DNA damage checkpoint components from unscheduled degradation . Usp28 was originally isolated as a binding partner of the checkpoint mediators 53BP1, claspin, and Mdc1. The depletion of Usp28 by siRNA or expression of catalytically inactive Usp28 in a cell culture model triggered the premature degradation of its binding partners, but also of kinase specificity factors and an effector kinase, Chk2. This attenuates DNA damage signaling and interferes with p53-dependent apoptosis. As mentioned before, Usp28 also protects the transcription factor c-Myc from SCFFbw7-dependent ubiquitination, but dissociates from c-Myc upon DNA damage . By controlling the turnover of checkpoint components and a proliferative transcription factor, Usp28 can elegantly co-regulate checkpoint activation and cell cycle progression. While Usp28 protects proteins from unwanted degradation, Usp1 regulates checkpoints non-proteolytically by counteracting the ubiquitination of FANCD2 and PCNA [27,76]. FANCD2 is transiently monoubiquitinated in S phase, which is required for normal cell cycle progression. Monoubiquitination also targets FANCD2 to sites of DNA breaks, where it interacts with recombination enzymes and participates in DNA repair. Similarly, monoubiquitination of PCNA occurs in response to DNA damage or stalled replication forks and recruits DNA-repair polymerases . The Usp1/UAF1-complex directly binds to modified FANCD2 and PCNA and catalyzes their deubiquitination . In the event of DNA damage, Usp1 is inactivated by transcriptional repression, proteasomal degradation, and autocleavage. This triggers monoubiquitination of FANCD2 or PCNA and allows cells to initiate necessary DNA repair. The ubiquitination of FANCD2 is misregulated in patients with Fanconi Anemia, underscoring its importance for cell cycle regulation and maintaining genomic stability.
Upon entering mitosis, condensed chromosomes have to achieve bipolar attachment to the mitotic spindle, or otherwise cell division would result in aneuploid progeny. The genomic integrity of dividing cells is ensured by the spindle checkpoint, which is activated by mis- or unattached chromosomes . As a consequence, the spindle checkpoint inhibits the critical ubiquitin ligase APC/C, which arrests cells at metaphase and gives them the necessary time to complete chromosome attachment. Once this is achieved, the spindle checkpoint is rapidly turned off and sister chromatid separation is initiated. Intriguingly, spindle checkpoint silencing is triggered by the APC/C-dependent multiubiquitination of the substrate-targeting factor Cdc20 and the resulting dissociation of its inhibitor Mad2 [79, 80]. The DUB Usp44 counteracts APC/C-activation by deubiquitinating Cdc20, thereby preventing premature Mad2-dissociation and spindle checkpoint silencing [20; Figure 2]. Reducing the levels of Usp44 results in an almost complete loss of spindle checkpoint function. These phenotypes can be rescued by co-depleting APC/C-subunits or the APC/C-specific E2 UbcH10, which strongly suggests that Usp44 reinforces the spindle checkpoint by directly opposing APC/C-dependent ubiquitination.
Following the silencing of the spindle checkpoint, the fully activated APC/C orchestrates progression through mitosis by targeting several key mitotic regulators for sequential degradation . This “substrate ordering” of the APC/C is also controlled by DUB activity, although the molecular nature of the respective enzymes remains unknown . In this case, deubiquitination delays the assembly of ubiquitin chains on substrates, which bind the APC/C weakly and consequently are degraded late in mitosis. Deubiquitination, therefore, turns out to be a major regulator of the APC/C and progression through mitosis.
The surge in recent studies on the reversible nature of ubiquitination has brought deubiquitination into the limelight of cell cycle regulation. DUBs also function in cell survival and development, and thus, it is not surprising that they are frequently mutated or misregulated in cancer. Our increasing understanding of their catalytic activity, regulation, and substrate specificity will empower us to exploit DUB inhibition as a therapeutic strategy against cancer. Indeed, initial studies support the idea that DUB inhibition is of therapeutic value . Continuing our efforts to dissect DUB function and regulation will not only deepen our understanding of the complexities of cell cycle regulation, but it will almost certainly increase our chances of developing specific and effective anti-proliferative drugs.
We thank Lingyan Jin, Adam Williamson, and Julia Schaletzky for many fruitful discussions. We are also grateful to Julia Schaletzky for critically reading the manuscript. Our work is supported by a PEW Scholar Award and an NIH Director’s New Innovator Award.
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