APC/CCdh1-dependent proteolysis of USP1 regulates the response to UV-mediated DNA damage

Targeted protein destruction of critical cellular regulators during the G1 phase of the cell cycle is achieved by anaphase-promoting complex/cyclosome^Cdh1 (APC/C^Cdh1), a multisubunit E3 ubiquitin ligase. Cells lacking Cdh1 have been shown to accumulate deoxyribonucleic acid (DNA) damage, suggesting that it may play a previously unrecognized role in maintaining genomic stability. The ubiquitin-specific protease 1 (USP1) is a known critical regulator of DNA repair and genomic stability. In this paper, we report that USP1 was degraded in G1 via APC/C^Cdh1. USP1 levels were kept low in G1 to provide a permissive condition for inducing proliferating cell nuclear antigen (PCNA) monoubiquitination in response to ultraviolet (UV) damage before DNA replication. Importantly, expression of a USP1 mutant that cannot be degraded via APC/C^Cdh1 inhibited PCNA monoubiquitination during G1, likely compromising the recruitment of trans-lesion synthesis polymerase to UV repair sites. Thus, we propose a role for APC/C^Cdh1 in modulating the status of PCNA monoubiquitination and UV DNA repair before S phase entry.     

APC/CCdh1 controls CtIP stability during the cell cycle and in response to DNA damage

Human cells have evolved elaborate mechanisms for responding to DNA damage to maintain genome stability and prevent carcinogenesis. For instance, the cell cycle can be arrested at different stages to allow time for DNA repair. The APC/C^Cdh1 ubiquitin ligase mainly regulates mitotic exit but is also implicated in the DNA damage‐induced G₂ arrest. However, it is currently unknown whether APC/C^Cdh1 also contributes to DNA repair. Here, we show that Cdh1 depletion causes increased levels of genomic instability and enhanced sensitivity to DNA‐damaging agents. Using an integrated proteomics and bioinformatics approach, we identify CtIP, a DNA‐end resection factor, as a novel APC/C^Cdh1 target. CtIP interacts with Cdh1 through a conserved KEN box, mutation of which impedes ubiquitylation and downregulation of CtIP both during G₁ and after DNA damage in G₂. Finally, we find that abrogating the CtIP–Cdh1 interaction results in delayed CtIP clearance from DNA damage foci, increased DNA‐end resection, and reduced homologous recombination efficiency. Combined, our results highlight the impact of APC/C^Cdh1 on the maintenance of genome integrity and show that this is, at least partially, achieved by controlling CtIP stability in a cell cycle‐ and DNA damage‐dependent manner.     

Skp1-Cul1-F-box Ubiquitin Ligase (SCFβTrCP)-mediated Destruction of the Ubiquitin-specific Protease USP37 during G2-phase Promotes Mitotic Entry

Ubiquitin-mediated proteolysis is a key regulatory process in cell cycle progression. The Skp1-Cul1-F-box (SCF) and anaphase-promoting complex (APC) ubiquitin ligases target numerous components of the cell cycle machinery for destruction. Throughout the cell cycle, these ligases cooperate to maintain precise levels of key regulatory proteins, and indirectly, each other. Recently, we have identified the deubiquitinase USP37 as a regulator of the cell cycle. USP37 expression is cell cycle-regulated, being expressed in late G₁ and ubiquitinated by APC^Cdh1 in early G₁. Here we report that in addition to destruction at G₁, a major fraction of USP37 is degraded at the G₂/M transition, prior to APC substrates and similar to SCF^βTrCP substrates. Consistent with this hypothesis, USP37 interacts with components of the SCF in a βTrCP-dependent manner. Interaction with βTrCP and subsequent degradation is phosphorylation-dependent and is mediated by the Polo-like kinase (Plk1). USP37 is stabilized in G₂ by depletion of βTrCP as well as chemical or genetic manipulation of Plk1. Similarly, mutation of the phospho-sites abolishes βTrCP binding and renders USP37 resistant to Plk1 activity. Expression of this mutant hinders the G₂/M transition. Our data demonstrate that tight regulation of USP37 levels is required for proper cell cycle progression.     

Phosphorylation and dephosphorylation regulate APC/CCdh1 substrate degradation

The Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase activated by its G1 specific adaptor protein Cdh1 is a major regulator of the cell cycle. The APC/C^Cdh1 mediates degradation of dozens of proteins, however, the kinetics and requirements for their degradation are largely unknown. We demonstrate that overexpression of the constitutive active CDH1^m11 mutant that is not inhibited by phosphorylation results in mitotic exit in the absence of the FEAR and MEN pathways, and DNA re-replication in the absence of Cdc7 activity. This mode of mitotic exit also reveals additional requirements for APC/C^Cdh1 substrate degradation, which for some substrates such as Pds1 or Clb5 is dephosphorylation, but for others such as Cdc5 is phosphorylation.     

CAPNS1 Regulates USP1 Stability and Maintenance of Genome Integrity

Calpains regulate a wide spectrum of biological functions, including migration, adhesion, apoptosis, secretion, and autophagy, through the modulating cleavage of specific substrates. Ubiquitous microcalpain (μ-calpain) and millicalpain (m-calpain) are heterodimers composed of catalytic subunits encoded, respectively, by CAPN1 and CAPN2 and a regulatory subunit encoded by CAPNS1. Here we show that calpain is required for the stability of the deubiquitinating enzyme USP1 in several cell lines. USP1 modulates DNA replication polymerase choice and repair by deubiquitinating PCNA. The ubiquitinated form of the USP1 substrate PCNA is stabilized in CAPNS1-depleted U2OS cells and mouse embryonic fibroblasts (MEFs), favoring polymerase-η loading on chromatin and increased mutagenesis. USP1 degradation directed by the cell cycle regulator APC/C^cdh1, which marks USP1 for destruction in the G₁ phase, is upregulated in CAPNS1-depleted cells. USP1 stability can be rescued upon forced expression of calpain-activated Cdk5/p25, previously reported as a cdh1 repressor. These data suggest that calpain stabilizes USP1 by activating Cdk5, which in turn inhibits cdh1 and, consequently, USP1 degradation. Altogether these findings point to a connection between the calpain system and the ubiquitin pathway in the regulation of DNA damage response and place calpain at the interface between cell cycle modulation and DNA repair.     

Anaphase-promoting complex/cyclosome controls the stability of TPX2 during mitotic exit

TPX2, a microtubule-associated protein, is required downstream of Ran-GTP to induce spindle assembly. TPX2 activity appears to be tightly regulated during the cell cycle, and we report here one molecular mechanism for this regulation. We found that TPX2 protein levels are cell cycle regulated, peaking in mitosis and declining sharply during mitotic exit. TPX2 is degraded in mitotic extracts, as well as in HeLa cells exiting from mitosis. This instability depends, both in vitro and in vivo, on the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. In a reconstituted system, TPX2 is efficiently ubiquitinated by APC/C that has been activated by Cdh1. Two discrete elements in TPX2 are required for recognition by APC/C^Cdh1: a KEN box and a novel element in amino acids 1 to 86. Interestingly, the latter element, which has no known APC/C recognition motifs, is required for the ubiquitination of TPX2 by APC/C^Cdh1 in vitro and for its degradation in vivo. We conclude that APC/C^Cdh1 controls the stability of TPX2, thereby ensuring accurate regulation of the spindle assembly in the cell cycle.     

The ubiquitin proteasome system — Implications for cell cycle control and the targeted treatment of cancer

Two families of E3 ubiquitin ligases are prominent in cell cycle regulation and mediate the timely and precise ubiquitin–proteasome-dependent degradation of key cell cycle proteins: the SCF (Skp1/Cul1/F-box protein) complex and the APC/C (anaphase promoting complex or cyclosome). While certain SCF ligases drive cell cycle progression throughout the cell cycle, APC/C (in complex with either of two substrate recruiting proteins: Cdc20 and Cdh1) orchestrates exit from mitosis (APC/C^Cdc20) and establishes a stable G1 phase (APC/C^Cdh1). Upon DNA damage or perturbation of the normal cell cycle, both ligases are involved in checkpoint activation. Mechanistic insight into these processes has significantly improved over the last ten years, largely due to a better understanding of APC/C and the functional characterization of multiple F-box proteins, the variable substrate recruiting components of SCF ligases. Here, we review the role of SCF- and APC/C-mediated ubiquitylation in the normal and perturbed cell cycle and discuss potential clinical implications of SCF and APC/C functions. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

APC/CCdc20 targets E2F1 for degradation in prometaphase

The mechanisms that control E2F-1 activity are complex. We previously showed that Chk1 and Chk2 are required for E2F1 stabilization and p73 target gene induction following DNA damage. To gain further insight into the processes regulating E2F1 protein stability, we focused our investigation on the mechanisms responsible for regulating E2F1 turnover. Here we show that E2F1 is a substrate of the anaphase-promoting complex or cyclosome (APC/C), a ubiquitin ligase that plays an important role in cell cycle progression. Ectopic expression of the APC/C activators Cdh1 and Cdc20 reduced the levels of co-expressed E2F-1 protein. Co-expression of DP1 with E2F1 blocked APC/C-induced E2F1 degradation, suggesting that the E2F1/DP1 heterodimer is protected from APC/C regulation. Following Cdc20 knockdown, E2F1 levels increased and remained stable in extracts over a time course, indicating that APC/C^Cdc20 is a primary regulator of E2F1 stability in vivo. Moreover, cell synchronization experiments showed that siRNA directed against Cdc20 induced an accumulation of E2F1 protein in prometaphase cells. These data suggest that APC/C^Cdc20 specifically targets E2F1 for degradation in early mitosis and reveal a novel mechanism for limiting free E2F1 levels in cells, failure of which may compromise cell survival and/or homeostasis.Key words: cell cycle, ubiquitination, E2F1, APC/C, Cdc20, Cdh1     

Damaged DNA-binding Protein 1 (DDB1) Interacts with Cdh1 and Modulates the Function of APC/CCdh1

APC/C^Cdh1 plays a key role in mitotic exit and has essential targets in the G₁ phase; however, these mechanisms are poorly understood. In this report, we provide evidence that damaged DNA-binding protein 1 (DDB1) is capable of binding the WD40 domains of Cdh1, but not of Cdc20, through its BPA and BPC domains. Moreover, cells lacking DDB1 exhibit markedly elevated levels of the protein substrates of APC/C^Cdh1. Depletion of DDB1 in mitotic cells significantly delays mitotic exit, which demonstrates that the interaction between DDB1 and Cdh1 plays a critical role in regulating APC/C^Cdh1 activity. However, cells depleted of Cdh1 demonstrated no change in the UV-induced degradation of Cdt1, the main function of DDB1 as an E3 ligase. Strikingly, the APC/C^Cdh1 substrate levels are normal in cell knockdowns of Cul4A and Cul4B, which, along with DDB1, form an E3 ligase complex. This finding indicates that DDB1 modulates the function of APC/C^Cdh1 in a manner independent on the Cul4-DDB1 complex. Our results suggest that DDB1 may functionally regulate mitotic exit by modulating APC/C^Cdh1 activity. This study reveals that there may be cross-talk among DDB1, Cdh1, and Skp2 in the control of cell cycle division.     

Degradation of the Human Mitotic Checkpoint Kinase Mps1 Is Cell Cycle-regulated by APC-cCdc20 and APC-cCdh1 Ubiquitin Ligases

Mps1 is a dual specificity protein kinase with key roles in regulating the spindle assembly checkpoint and chromosome-microtubule attachments. Consistent with these mitotic functions, Mps1 protein levels fluctuate during the cell cycle, peaking at early mitosis and abruptly declining during mitotic exit and progression into the G₁ phase. Although evidence in budding yeast indicates that Mps1 is targeted for degradation at anaphase by the anaphase-promoting complex (APC)-c^Cdc20 complex, little is known about the regulatory mechanisms that govern Mps1 protein levels in human cells. Here, we provide evidence for the ubiquitin ligase/proteosome pathway in regulating human Mps1 levels during late mitosis through G₁ phase. First, we showed that treatment of HEK 293T cells with the proteosome inhibitor MG132 resulted in an increase in both the polyubiquitination and the accumulation of Mps1 protein levels. Next, Mps1 was shown to co-precipitate with APC and its activators Cdc20 and Cdh1 in a cell cycle-dependent manner. Consistent with this, overexpression of Cdc20 or Cdh1 led to a marked reduction of endogenous Mps1 levels during anaphase or G₁ phase, respectively. In contrast, depletion of Cdc20 or Cdh1 by RNAi treatment both led to the stabilization of Mps1 protein during mitosis or G₁ phase, respectively. Finally, we identified a single D-box motif in human Mps1 that is required for its ubiquitination and degradation. Failure to appropriately degrade Mps1 is sufficient to trigger centrosome amplification and mitotic abnormalities in human cells. Thus, our results suggest that the sequential actions of the APC-c^Cdc20 and APC-c^Cdh1 ubiquitin ligases regulate the clearance of Mps1 levels and are critical for Mps1 functions during the cell cycle in human cells.     

New origin firing is inhibited by APC/CCdh1 activation in S-phase after severe replication stress

Defects in DNA replication and repair are known to promote genomic instability, a hallmark of cancer cells. Thus, eukaryotic cells have developed complex mechanisms to ensure accurate duplication of their genomes. While DNA damage response has been extensively studied in tumour cells, the pathways implicated in the response to replication stress are less well understood especially in non-transformed cells. Here we show that in non-transformed cells, APC/C^Cdh1 is activated upon severe replication stress. Activation of APC/C^Cdh1 prevents new origin firing and induces permanent arrest in S-phase. Moreover, Rad51-mediated homologous recombination is also impaired under these conditions. APC/C^Cdh1 activation in S-phase occurs after replication forks have been processed into double strand breaks. Remarkably, this activation, which correlates with decreased Emi1 levels, is not prevented by ATR/ATM inhibition, but it is abrogated in cells depleted of p53 or p21. Importantly, we found that the lack of APC/C^Cdh1 activity correlated with an increase in genomic instability. Taken together, our results define a new APC/C^Cdh1 function that prevents cell cycle resumption after prolonged replication stress by inhibiting origin firing, which may act as an additional mechanism in safeguarding genome integrity.     

APC/CCdc20 targets E2F1 for degradation in prometaphase

The mechanisms that control E2F-1 activity are complex. We previously showed that Chk1 and Chk2 are required for E2F1 stabilization and p73 target gene induction following DNA damage. To gain further insight into the processes regulating E2F1 protein stability, we focused our investigation on the mechanisms responsible for regulating E2F1 turnover. Here we show that E2F1 is a substrate of the anaphase promoting complex or cyclosome (APC/C), a ubiquitin ligase that plays an important role in cell cycle progression. Ectopic expression of the APC/C activators Cdh1 and Cdc20 reduced the levels of co-expressed E2F-1 protein. Co-expression of DP1 with E2F1 blocked APC/C-induced E2F1 degradation, suggesting that the E2F1/DP1 heterodimer is protected from APC/C regulation. Following Cdc20 knockdown, E2F1 levels increased and remained stable in extracts over a time course, indicating that APC/C^Cdc20 is a primary regulator of E2F1 stability in vivo. Moreover, cell synchronization experiments showed that siRNA directed against Cdc20 induced an accumulation of E2F1 protein in prometaphase cells. These data suggest that APC/C^Cdc20 specifically targets E2F1 for degradation in early mitosis and reveal a novel mechanism for limiting free E2F1 levels in cells, failure of which may compromise cell survival and/or homeostasis.     

The Anaphase-Promoting Complex/Cyclosome Activator Cdh1 Modulates Rho GTPase by Targeting p190 RhoGAP for Degradation

Cdh1 is an activator of the anaphase-promoting complex/cyclosome and contributes to mitotic exit and G₁ maintenance by targeting cell cycle proteins for degradation. However, Cdh1 is expressed and active in postmitotic or quiescent cells, suggesting that it has functions other than cell cycle control. Here, we found that homozygous Cdh1 gene-trapped (Cdh1^GT/GT) mouse embryonic fibroblasts (MEFs) and Cdh1-depleted HeLa cells reduced stress fiber formation significantly. The GTP-bound active Rho protein was apparently decreased in the Cdh1-depleted cells. The p190 protein, a major GTPase-activating protein for Rho, accumulated both in Cdh1^GT/GT MEFs and in Cdh1-knockdown HeLa cells. Cdh1 formed a physical complex with p190 and stimulated the efficient ubiquitination of p190, both in in vitro and in vivo. The motility of Cdh1-depleted HeLa cells was impaired; however, codepletion of p190 rescued the migration activity of these cells. Moreover, Cdh1^GT/GT embryos exhibited phenotypes similar to those observed for Rho-associated kinase I and II knockout mice: eyelid closure delay and disruptive architecture with frequent thrombus formation in the placental labyrinth layer, respectively. Furthermore, the p190 protein accumulated in the Cdh1^GT/GT embryonic tissues. Our data revealed a novel function for Cdh1 as a regulator of Rho and provided insights into the role of Cdh1 in cell cytoskeleton organization and cell motility.The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit complex that functions as an E3 ubiquitin ligase for various cell cycle proteins (19, 46). Proteins ubiquitinated by APC/C are recognized and degraded by the 26S proteasome to ensure proper cell cycle progression. APC/C activity is strictly dependent on coactivator proteins that interact with APC/C during specific phases of the cell cycle. Cdh1 (also known as Fzr, Hct1, or Srw) is one of the coactivators that maintain APC/C activity from anaphase of mitosis until the end of the G₁ phase of the cell cycle (43, 53).The role of Cdh1 (APC/C^Cdh1) on cell-cycle progression has been well studied; however, several studies have shed light into another aspect of Cdh1''s function. For example, expression of Cdh1 is not restricted to cycling cells; APC/C^Cdh1 is also present and active in quiescent cultured cells (9). Furthermore, immunohistochemical analysis has shown that Cdh1 is expressed in a wide variety of tissues that are predominantly composed of postmitotic cells, such as neurons, where APC/C^Cdh1 has a high cyclin B ubiquitination activity (1, 16). It has been reported that APC/C^Cdh1 promotes axonal growth and patterning (20) and is required for neuronal survival (1). These results highlight the importance of the APC/C activator Cdh1 in neurons. However, Cdh1 has also been shown to participate in the differentiation of tissues such as the muscle (25). Given that Cdh1 is ubiquitously expressed in organs containing quiescent cells, there might be additional roles for Cdh1.Rho GTPase proteins play a central role in the regulation of cell shape, polarity, and locomotion via their effects on actin polymerization, actomyosin contractility, cell adhesion, and microtubule dynamics (13). Small G proteins, which include Rho, act as molecular switches that cycle between an inactive GDP-bound state and an active GTP-bound state. The latter form of Rho proteins interacts with and activates downstream effector proteins. The activity of Rho GTPases is controlled by three class of key regulators: (i) guanine nucleotide exchange factors (GEFs), which catalyze the exchange of GDP to GTP for their activation (41); (ii) GTPase activating proteins (GAPs), which stimulate the intrinsic GTPase activity for their inactivation (8); and (iii) guanine nucleotide dissociation inhibitors (GDIs), which interact with GDP-bound Rho GTPases and sequester them in the cytoplasm to inhibit the exchange of GDP to GTP (33). In addition to these canonical regulations, recent studies indicate that the ubiquitination pathway is also involved in the modulation of Rho GTPase activity. Smurf1, which is a HECT domain E3 ubiquitin ligase, controls the local levels of RhoA at the cell periphery by targeting it for degradation (40, 55). Therefore, the regulatory mechanisms of Rho GTPase activity seem to be more complex than previously thought. It thus remains to be clarified whether other ubiquitin ligases also play a role in Rho signaling by targeting its components directly or indirectly.In this study, we found that the APC/C activator Cdh1 modulated actin organization. Mouse embryonic fibroblasts (MEFs) derived from a homozygous Cdh1 gene-trapped ([GT] Cdh1^GT/GT) mouse model displayed decreased numbers of stress fibers and focal adhesions (FAs). Consistent with these phenotypes, Rho activity was apparently reduced in Cdh1-deficient cells. Cdh1 regulated Rho activity via the targeting of p190 for degradation. We also found that Cdh1 knockdown cells showed decreased motility, which was rescued by codepletion of p190. Furthermore, phenotypic similarities between Cdh1^GT/GT embryos and ROCK (also known as Rho-kinase, which is the important Rho downstream effector of actin cytoskeleton formation) knockout (KO) mice (44, 49) support our notion that Cdh1 plays a role in the Rho/ROCK signaling axis. Collectively, our findings suggest an alternative role for Cdh1 other than cell cycle regulation and reveal Cdh1 as a new regulator of Rho.