ATM-mediated NuSAP phosphorylation induces mitotic arrest

NuSAP is a microtubule-associated protein that plays an important role in spindle assembly. NuSAP deficiency in mice leads to early embryonic lethality. Spindle assembly in NuSAP-deficient cells is highly inefficient and chromosomes remain dispersed in the mitotic cytoplasm. ATM is a key kinase that phosphorylates a series of substrates to mediate G1/S control. However, the role of ATM at the G2/M phase is not well understood. Here we demonstrate that ectopic expression of NuSAP lead to mitotic arrest observably dependent on the kinase activity of ATM. When endogenous ATM was depleted or its kinase activity was inhibited, NuSAP could not cause mitotic arrest. We further show ATM interacts with NuSAP and phosphorylates NuSAP on Ser124. The phosphorylation and interaction occur specifically at G2/M-phase. Collectively, our work has uncovered an ATM-dependent checkpoint pathway that prevents mitotic progression by targeting a microtubule-associated protein, NuSAP.     

Phosphorylation of Mcl‐1 by CDK1–cyclin B1 initiates its Cdc20‐dependent destruction during mitotic arrest

The balance between cell cycle progression and apoptosis is important for both surveillance against genomic defects and responses to drugs that arrest the cell cycle. In this report, we show that the level of the human anti‐apoptotic protein Mcl‐1 is regulated during the cell cycle and peaks at mitosis. Mcl‐1 is phosphorylated at two sites in mitosis, Ser64 and Thr92. Phosphorylation of Thr92 by cyclin‐dependent kinase 1 (CDK1)–cyclin B1 initiates degradation of Mcl‐1 in cells arrested in mitosis by microtubule poisons. Mcl‐1 destruction during mitotic arrest requires proteasome activity and is dependent on Cdc20/Fizzy, which mediates recognition of mitotic substrates by the anaphase‐promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Stabilisation of Mcl‐1 during mitotic arrest by mutation of either Thr92 or a D‐box destruction motif inhibits the induction of apoptosis by microtubule poisons. Thus, phosphorylation of Mcl‐1 by CDK1–cyclin B1 and its APC/C^Cdc20‐mediated destruction initiates apoptosis if a cell fails to resolve mitosis. Regulation of apoptosis, therefore, is linked intrinsically to progression through mitosis and is governed by a temporal mechanism that distinguishes between normal mitosis and prolonged mitotic arrest.     

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.     

Involvement of the telomeric protein Pin2/TRF1 in the regulation of the mitotic spindle

Pin2/TRF1 was independently identified as a telomeric DNA-binding protein (TRF1) that regulates telomere length, and as a protein (Pin2) that can bind the mitotic kinase NIMA and suppress its lethal phenotype. We have previously demonstrated that Pin2/TRF1 levels are cell cycle-regulated and its overexpression induces mitotic arrest and then apoptosis. This Pin2/TRF1 activity can be potentiated by microtubule-disrupting agents, but suppressed by phosphorylation of Pin2/TRF1 by ATM; this negative regulation is critical in mediating for many, but not all, ATM-dependent phenotypes. Interestingly, Pin2/TRF1 specifically localizes to mitotic spindles in mitotic cells and affects the microtubule polymerization in vitro. These results suggest a role of Pin2/TRF1 in mitosis. However, nothing is known about whether Pin2/TRF1 affects the spindle function in mitotic progression. Here we characterized a new Pin2/TRF1-interacting protein, EB1, that was originally identified in our yeast two-hybrid screen. Pin2/TRF1 bound EB1 both in vitro and in vivo and they also co-localize at the mitotic spindle in cells. Furthermore, EB1 inhibits the ability of Pin2/TRF1 to promote microtubule polymerization in vitro. Given that EB1 is a microtubule plus end-binding protein, these results further confirm a specific interaction between Pin2/TRF1 and the mitotic spindle. More importantly, we have shown that inhibition of Pin2/TRF1 in ataxia-telangiectasia cells is able to fully restore their mitotic spindle defect in response to microtubule disruption, demonstrating for the first time a functional involvement of Pin2/TRF1 in mitotic spindle regulation.     

NuSAP,a novel microtubule-associated protein involved in mitotic spindle organization

Here, we report on the identification of nucleolar spindle-associated protein (NuSAP), a novel 55-kD vertebrate protein with selective expression in proliferating cells. Its mRNA and protein levels peak at the transition of G2 to mitosis and abruptly decline after cell division. Microscopic analysis of both fixed and live mammalian cells showed that NuSAP is primarily nucleolar in interphase, and localizes prominently to central spindle microtubules during mitosis. Direct interaction of NuSAP with microtubules was demonstrated in vitro. Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain. In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis. In addition, many NuSAP-depleted interphase cells had deformed nuclei. Both overexpression and knockdown of NuSAP impaired cell proliferation. These results suggest a crucial role for NuSAP in spindle microtubule organization.     

Phosphorylation of NuSAP by Cdk1 regulates its interaction with microtubules in mitosis

Cell division in eukaryotes depends on a fine control of the dynamic changes of microtubules. Nucleolar and spindle-associated protein (NuSAP) is a microtubule-binding and -bundling protein essential for the integrity of the anaphase spindle and cell division. NuSAP contains two consensus cdk phosphorylation sites in its microtubule-binding domain. Here we show NuSAP is phosphorylated by cdk1 in early mitosis. This phosphorylation inhibits the binding of NuSAP to microtubules. During metaphase-to anaphase transition, NuSAP is dephosphorylated to promote spindle midzone formation and cell cycle progression. Expression of cdk1 phosphorylation-null mutant causes extensive bundling of microtubules in the prometaphase spindle. Our results suggest that phosphorylation and dephosphorylation of NuSAP during progression of mitosis regulate spindle organization through modulation of the dynamics of microtubules.     

Structural activation of Mad2 in the mitotic spindle checkpoint: the two-state Mad2 model versus the Mad2 template model

The inheritance of a normal assortment of chromosomes during each cell division relies on a cell-cycle surveillance system called the mitotic spindle checkpoint. The existence of sister chromatids that do not achieve proper bipolar attachment to the mitotic spindle in a cell activates this checkpoint, which inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C) and delays the onset of anaphase. The mitotic arrest deficiency 2 (Mad2) spindle checkpoint protein inhibits APC/C through binding to its mitotic-specific activator, Cdc20. Binding of Mad2 to Cdc20 involves a large conformational change of Mad2 and requires the Mad1-Mad2 interaction in vivo. Two related but distinct models of Mad1-assisted activation of Mad2, the "two-state Mad2" and the "Mad2 template" models, have been proposed. I review the recent structural, biochemical, and cell biological data on Mad2, discuss the differences between the two models, and propose experiments that test their key principles.     

Stabilization of P/CAF,as a ubiquitin ligase toward MDM2, suppresses mitotic cell death through p53-p21 activation in HCT116 cells with SIRT2 suppression

We previously reported that the suppression of SIRT2, an NAD + -dependent protein deacetylases, induces p53 accumulation via degradation of p300 and the subsequent MDM2 degradation, eventually leading to apoptosis in HeLa cells. The present study identified a novel pathway of p53 accumulation by SIRT2 suppression in HCT116(p53+/+) cells in which SIRT2 suppression led to escape from mitotic cell death caused by spindle assembly checkpoint activation induced by microtubule inhibitors such as nocodazole but not apoptosis or G1 or G2 arrest. We found that SIRT2 interacts with P/CAF, a histone acetyltransferase, which also acts as a ubiquitin ligase against MDM2. SIRT2 suppression led to an increase of P/CAF acetylation and its stabilization followed by a decrease in MDM2 and activation of the p53-p21 pathway. Depression of mitotic cell death in HCT116(p53+/+) cells with SIRT2 suppression was released by suppression of P/CAF or p21. Thus, the P/CAF-MDM2-p53-p21 axis enables the escape from mitotic cell death and confers resistance to nocodazole in HCT116(p53+/+) cells with SIRT2 suppression. As SIRT2 has attracted attention as a potential target for cancer therapeutics for p53 regulation, the present study provides a molecular basis for the efficacy of SIRT2 for future cancer therapy based on p53 regulation. These findings also suggest an undesirable function of the SIRT2 suppression associated with activation of the p53-p21 pathway in the suppression of mitotic cell death caused by spindle assembly checkpoint activation.     

Defective in mitotic arrest 1/ring finger 8 is a checkpoint protein that antagonizes the human mitotic exit network

A molecular pathway homologous to the S. cerevisiae mitotic exit network (MEN) and S. pombe septation initiation network has recently been described in higher eukaryotes and involves the tumor suppressor kinase LATS1 and its subunit MOB1A. The yeast MEN/septation initiation network pathways are regulated by the ubiquitin ligase defective in mitotic arrest 1 (Dma1p), a checkpoint protein that helps maintain prometaphase arrest when cells are exposed to microtubule poisons. We identified here the RING domain protein ring finger 8 (RNF8) as the human orthologue of the yeast protein Dma1p. Like its yeast counterparts, human DMA1/RNF8 localized at the midbody and its depletion by siRNA compromised mitotic arrest of nocodazole-treated cells in a manner dependent on the MEN. Depletion of MAD2, a spindle checkpoint protein, also compromised mitotic arrest, but in a MEN-independent manner. Thus, two distinct checkpoint pathways maintain mitotic arrest in cells exposed to microtubule poisons.     

The Arabidopsis anaphase-promoting complex or cyclosome: molecular and genetic characterization of the APC2 subunit

In yeast and animals, the anaphase-promoting complex or cyclosome (APC/C) is an essential ubiquitin protein ligase that regulates mitotic progression and exit by controlling the stability of cell cycle regulatory proteins, such as securin and the mitotic cyclins. In plants, the function, regulation, and substrates of the APC/C are poorly understood. To gain more insight into the roles of the plant APC/C, we characterized at the molecular level one of its subunits, APC2, which is encoded by a single-copy gene in Arabidopsis. We show that the Arabidopsis gene is able to partially complement a budding yeast apc2 ts mutant. By yeast two-hybrid assays, we demonstrate an interaction of APC2 with two other APC/C subunits: APC11 and APC8/CDC23. A reverse-genetic approach identified Arabidopsis plants carrying T-DNA insertions in the APC2 gene. apc2 null mutants are impaired in female megagametogenesis and accumulate a cyclin-beta-glucuronidase reporter protein but do not display metaphase arrest, as observed in other systems. The APC2 gene is expressed in various plant organs and does not seem to be cell cycle regulated. Finally, we report intriguing differences in APC2 protein subcellular localization compared with that in other systems. Our observations support a conserved function of the APC/C in plants but a different mode of regulation.     

State of the APC/C: Organization,function, and structure

The ubiquitin-proteasome protein degradation system is involved in many essential cellular processes including cell cycle regulation, cell differentiation, and the unfolded protein response. The anaphase-promoting complex/cyclosome (APC/C), an evolutionarily conserved E3 ubiquitin ligase, was discovered 15 years ago because of its pivotal role in cyclin degradation and mitotic progression. Since then, we have learned that the APC/C is a very large, complex E3 ligase composed of 13 subunits, yielding a molecular machine of approximately 1 MDa. The intricate regulation of the APC/C is mediated by the Cdc20 family of activators, pseudosubstrate inhibitors, protein kinases and phosphatases and the spindle assembly checkpoint. The large size, complexity, and dynamic nature of the APC/C represent significant obstacles toward high-resolution structural techniques; however, over the last decade, there have been a number of lower resolution APC/C structures determined using single particle electron microscopy. These structures, when combined with data generated from numerous genetic and biochemical studies, have begun to shed light on how APC/C activity is regulated. Here, we discuss the most recent developments in the APC/C field concerning structure, substrate recognition, and catalysis.     

Anaphase-promoting complex/cyclosome controls HEC1 stability

Objective: Chromosome segregation during mitosis requires a physically large proteinaceous structure called the kinetochore to generate attachments between chromosomal DNA and spindle microtubules. It is essential for kinetochore components to be carefully regulated to guarantee successful cell division. Depletion, mutation or dysregulation of kinetochore proteins results in mitotic arrest and/or cell death. HEC1 (high expression in cancer) has been reported to be a kinetochore protein, depletion of which, by RNA interference, results in catastrophic mitotic exit. Materials and methods and results: To investigate how HEC1 protein is controlled post‐translation, we analysed the role of anaphase‐promoting complex/cyclosome (APC/C)‐Cdh1 in degradation of HEC1 protein. In this study, we show that HEC1 is an unstable protein and can be targeted by endogenous ubiquitin‐proteasome system in HEK293T cells. Results of RNA interference and in vivo ubiquitination assay indicated that HEC1 could be ubiquitinated and degraded by APC/C‐hCdh1 E3 ligase. The evolutionally conserved D‐box at the C‐terminus functioned as the degron of HEC1, destruction of which resulted in resistance to degradation mediated by APC/C‐Cdh1. Overexpression of non‐degradable HEC1 (D‐box destroyed) induced accumulation of cyclin B protein in vivo and triggered mitotic arrest. Conclusion: APC/C‐Cdh1 controls stability of HEC1, ensuring normal cell cycle progression.     

State of the APC/C: organization, function, and structure

The ubiquitin-proteasome protein degradation system is involved in many essential cellular processes including cell cycle regulation, cell differentiation, and the unfolded protein response. The anaphase-promoting complex/cyclosome (APC/C), an evolutionarily conserved E3 ubiquitin ligase, was discovered 15 years ago because of its pivotal role in cyclin degradation and mitotic progression. Since then, we have learned that the APC/C is a very large, complex E3 ligase composed of 13 subunits, yielding a molecular machine of approximately 1 MDa. The intricate regulation of the APC/C is mediated by the Cdc20 family of activators, pseudosubstrate inhibitors, protein kinases and phosphatases and the spindle assembly checkpoint. The large size, complexity, and dynamic nature of the APC/C represent significant obstacles toward high-resolution structural techniques; however, over the last decade, there have been a number of lower resolution APC/C structures determined using single particle electron microscopy. These structures, when combined with data generated from numerous genetic and biochemical studies, have begun to shed light on how APC/C activity is regulated. Here, we discuss the most recent developments in the APC/C field concerning structure, substrate recognition, and catalysis.     

Regulation of Chk1

The execution of the mitotic program with high fidelity is dependent upon precise spatiotemporal regulation of posttranslational protein modifications. For example, the timely polyubiquitination of critical mitotic regulators by Anaphase Promoting Complex/Cyclosome (APC/C) is essential for the metaphase to anaphase transition and mitotic exit. The spindle assembly checkpoint prevents unscheduled activity of APC/C-Cdc20 in early mitosis, allowing bipolar attachment of kinetochores to mitotic spindle and facilitating equal segregation of sister chromatids. The critical effector of the spindle checkpoint, Mitotic arrest deficient 2 (Mad2), is recruited to unattached kinetochores forming a complex with other regulatory proteins to efficiently and cooperatively inhibit APC/C-Cdc20. A weakened and/or dysfunctional spindle checkpoint has been linked to the development of genomic instability in both cell culture and animal models, and evidence suggests that aberrant regulation of the spindle checkpoint plays a critical role in human carcinogenesis. Recent studies have illuminated a network of both degradative and non-degradative ubiquitination events that regulate the metaphase to anaphase transition and mitotic exit. Within this context, our recent work showed that the HECT (Homologous to E6-AP C-terminus)-family E3 ligase Smurf2 (Smad specific ubiquitin regulatory factor 2), known as a negative regulator of transforming growth factor-beta (TGF-β) signaling, is required for a functional spindle checkpoint by promoting the functional localization and stability of Mad2. Here we discuss putative models explaining the role of Smurf2 as a new regulator in the spindle checkpoint. The dynamic mitotic localization of Smurf2 to the centrosome and other critical mitotic structures provides implications about mitotic checkpoint control dependent on various ubiquitination events. Finally, deregulated Smurf2 activity may contribute to carcinogenesis by perturbed mitotic control.     

The spindle and kinetochore–associated (Ska) complex enhances binding of the anaphase-promoting complex/cyclosome (APC/C) to chromosomes and promotes mitotic exit

The spindle and kinetochore–associated (Ska) protein complex is a heterotrimeric complex required for timely anaphase onset. The major phenotypes seen after small interfering RNA–mediated depletion of Ska are transient alignment defects followed by metaphase arrest that ultimately results in cohesion fatigue. We find that cells depleted of Ska3 arrest at metaphase with only partial degradation of cyclin B1 and securin. In cells arrested with microtubule drugs, Ska3-depleted cells exhibit slower mitotic exit when the spindle checkpoint is silenced by inhibition of the checkpoint kinase, Mps1, or when cells are forced to exit mitosis downstream of checkpoint silencing by inactivation of Cdk1. These results suggest that in addition to a role in fostering kinetochore–microtubule attachment and chromosome alignment, the Ska complex has functions in promoting anaphase onset. We find that both Ska3 and microtubules promote chromosome association of the anaphase-promoting complex/cyclosome (APC/C). Chromosome-bound APC/C shows significantly stronger ubiquitylation activity than cytoplasmic APC/C. Forced localization of Ska complex to kinetochores, independent of microtubules, results in enhanced accumulation of APC/C on chromosomes and accelerated cyclin B1 degradation during induced mitotic exit. We propose that a Ska-microtubule-kinetochore association promotes APC/C localization to chromosomes, thereby enhancing anaphase onset and mitotic exit.     

Two distinct pathways for inhibiting pds1 ubiquitination in response to DNA damage

The presence of DNA damage activates a conserved cellular response known as the DNA damage checkpoint pathway. This pathway induces a cell cycle arrest that persists until the damage is repaired. Consequently, the failure to arrest in response to DNA damage is associated with genomic instability. In budding yeast, activation of the DNA damage checkpoint pathway leads to a mitotic cell cycle arrest. Following the detection of DNA damage, the checkpoint signal is transduced via the Mec1 kinase, which in turn activates two kinases, Rad53 and Chk1 that act in parallel pathways to bring about the cell cycle arrest. The downstream target of Rad53 is unknown. The target of Chk1 is Pds1, an inhibitor of anaphase initiation whose degradation is a prerequisite for mitotic progression. Pds1 degradation is dependent on its ubiquitination by the anaphase-promoting complex/cyclosome ubiquitin ligase, acting in conjunction with the Cdc20 protein (APC/CCdc20). Previous studies showed that the Rad53 and Chk1 pathways independently lead to Pds1 stabilization but the mechanism for this was unknown. In the present study we show that both the Chk1 and the Rad53 pathways inhibit the APC/CCdc20-dependent ubiquitination of Pds1 but they affect different steps of the process: the Rad53 pathway inhibits the Pds1-Cdc20 interaction whereas Chk1-dependent phosphorylation of Pds1 inhibits the ubiquitination reaction itself. Finally, we show that once the DNA damage is repaired, Pds1 dephosphorylation is involved in the recovery from the checkpoint induced cell cycle arrest.     

The role of stathmin in the regulation of the cell cycle

Stathmin is the founding member of a family of proteins that play critically important roles in the regulation of the microtubule cytoskeleton. Stathmin regulates microtubule dynamics by promoting depolymerization of microtubules and/or preventing polymerization of tubulin heterodimers. Upon entry into mitosis, microtubules polymerize to form the mitotic spindle, a cellular structure that is essential for accurate chromosome segregation and cell division. The microtubule-depolymerizing activity of stathmin is switched off at the onset of mitosis by phosphorylation to allow microtubule polymerization and assembly of the mitotic spindle. Phosphorylated stathmin has to be reactivated by dephosphorylation before cells exit mitosis and enter a new interphase. Interfering with stathmin function by forced expression or inhibition of expression results in reduced cellular proliferation and accumulation of cells in the G2/M phases of the cell cycle. Forced expression of stathmin leads to abnormalities in or a total lack of mitotic spindle assembly and arrest of cells in the early stages of mitosis. On the other hand, inhibition of stathmin expression leads to accumulation of cells in the G2/M phases and is associated with severe mitotic spindle abnormalities and difficulty in the exit from mitosis. Thus, stathmin is critically important not only for the formation of a normal mitotic spindle upon entry into mitosis but also for the regulation of the function of the mitotic spindle in the later stages of mitosis and for the timely exit from mitosis. In this review, we summarize the early studies that led to the identification of the important mitotic function of stathmin and discuss the present understanding of its role in the regulation of microtubules dynamics during cell-cycle progression. We also describe briefly other less mature avenues of investigation which suggest that stathmin may participate in other important biological functions and speculate about the future directions that research in this rapidly developing field may take.     

KEN-box-dependent degradation of the Bub1 spindle checkpoint kinase by the anaphase-promoting complex/cyclosome

The spindle checkpoint is a cell cycle surveillance mechanism that ensures the fidelity of chromosome segregation during mitosis and meiosis. Bub1 is a protein serine-threonine kinase that plays multiple roles in chromosome segregation and the spindle checkpoint. In response to misaligned chromosomes, Bub1 directly inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C) by phosphorylating its activator Cdc20. The protein level and the kinase activity of Bub1 are regulated during the cell cycle; they peak in mitosis and are low in G1/S phase. Here we show that Bub1 is degraded during mitotic exit and that degradation of Bub1 is mediated by APC/C in complex with its activator Cdh1 (APC/C(Cdh1)). Overexpression of Cdh1 reduces the protein levels of ectopically expressed Bub1, whereas depletion of Cdh1 by RNA interference increases the level of the endogenous Bub1 protein. Bub1 is ubiquitinated by immunopurified APC/C(Cdh1) in vitro. We further identify two KEN-box motifs on Bub1 that are required for its degradation in vivo and ubiquitination in vitro. A Bub1 mutant protein with both KEN-boxes mutated is stable in cells but fails to elicit a cell cycle phenotype, indicating that degradation of Bub1 by APC/C(Cdh1) is not required for mitotic exit. Nevertheless, our study clearly demonstrates that Bub1, an APC/C inhibitor, is also an APC/C substrate. The antagonistic relationship between Bub1 and APC/C may help to prevent the premature accumulation of Bub1 during G1.     

Basic mechanism of eukaryotic chromosome segregation

We now have firm evidence that the basic mechanism of chromosome segregation is similar among diverse eukaryotes as the same genes are employed. Even in prokaryotes, the very basic feature of chromosome segregation has similarities to that of eukaryotes. Many aspects of chromosome segregation are closely related to a cell cycle control that includes stage-specific protein modification and proteolysis. Destruction of mitotic cyclin and securin leads to mitotic exit and separase activation, respectively. Key players in chromosome segregation are SMC-containing cohesin and condensin, DNA topoisomerase II, APC/C ubiquitin ligase, securin-separase complex, aurora passengers, and kinetochore microtubule destabilizers or regulators. In addition, the formation of mitotic kinetochore and spindle apparatus is absolutely essential. The roles of principal players in basic chromosome segregation are discussed: most players have interphase as well as mitotic functions. A view on how the centromere/kinetochore is formed is described.