Keep it focused: PRMT6 drives the localization of RCC1 to chromosomes to facilitate mitosis, cell proliferation, and tumorigenesis.

Huang et al. (2021) identified a mechanism acting through the arginine methyltransferase PRMT6 that stabilizes the interaction of RCC1 with chromatin, promoting cell proliferation and tumorigenicity. Targeting this mechanism might enhance the treatment of tumors such as glioblastoma.

Timed degradation of Mcl-1 controls mitotic cell death.

Mitotic arrest can result in cell death through the process of apoptosis. We have shown by live-cell imaging that the ubiquitin-proteasome dependent proteolysis of the apoptotic regulator Mcl-1 under the control of the anaphase-promoting complex or cyclosome (APC/C) provides a timing mechanism that distinguishes prolonged mitotic arrest from normal mitosis.

Atypical APC/C-dependent degradation of Mcl-1 provides an apoptotic timer during mitotic arrest.

The initiation of apoptosis in response to the disruption of mitosis provides surveillance against chromosome instability. Here, we show that proteolytic destruction of the key regulator Mcl-1 during an extended mitosis requires the anaphase-promoting complex or cyclosome (APC/C) and is independent of another ubiquitin E3 ligase, SCFFbw7 Using live-cell imaging, we show that the loss of Mcl-1 during mitosis is dependent on a D box motif found in other APC/C substrates, while an isoleucine-arginine (IR) C-terminal tail regulates the manner in which Mcl-1 engages with the APC/C, converting Mcl-1 from a Cdc20-dependent and checkpoint-controlled substrate to one that is degraded independently of checkpoint strength. This mechanism ensures a relatively slow but steady rate of Mcl-1 degradation during mitosis and avoids its catastrophic destruction when the mitotic checkpoint is satisfied, providing an apoptotic timer that can distinguish a prolonged mitotic delay from normal mitosis. Importantly, we also show that inhibition of Cdc20 promotes mitotic cell death more effectively than loss of APC/C activity through differential effects on Mcl-1 degradation, providing an improved strategy to kill cancer cells.

USP9X Limits Mitotic Checkpoint Complex Turnover to Strengthen the Spindle Assembly Checkpoint and Guard against Chromosomal Instability.

Faithful chromosome segregation during mitosis depends on the spindle assembly checkpoint (SAC), which delays progression through mitosis until every chromosome has stably attached to spindle microtubules via the kinetochore. We show here that the deubiquitinase USP9X strengthens the SAC by antagonizing the turnover of the mitotic checkpoint complex produced at unattached kinetochores. USP9X thereby opposes activation of anaphase-promoting complex/cyclosome (APC/C) and specifically inhibits the mitotic degradation of SAC-controlled APC/C substrates. We demonstrate that depletion or loss of USP9X reduces the effectiveness of the SAC, elevates chromosome segregation defects, and enhances chromosomal instability (CIN). These findings provide a rationale to explain why loss of USP9X could be either pro- or anti-tumorigenic depending on the existing level of CIN.

Phosphorylation of XIAP by CDK1-cyclin-B1 controls mitotic cell death.

Regulation of cell death is crucial for the response of cancer cells to drug treatments that cause arrest in mitosis, and is likely to be important for protection against chromosome instability in normal cells. Prolonged mitotic arrest can result in cell death by activation of caspases and the induction of apoptosis. Here, we show that X-linked inhibitor of apoptosis (XIAP) plays a key role in the control of mitotic cell death. Ablation of XIAP expression sensitises cells to prolonged mitotic arrest caused by a microtubule poison. XIAP is stable during mitotic arrest, but its function is controlled through phosphorylation by the mitotic kinase CDK1-cyclin-B1 at S40. Mutation of S40 to a phosphomimetic residue (S40D) inhibits binding to activated effector caspases and abolishes the anti-apoptotic function of XIAP, whereas a non-phosphorylatable mutant (S40A) blocks apoptosis. By performing live-cell imaging, we show that phosphorylation of XIAP reduces the threshold for the onset of cell death in mitosis. This work illustrates that mitotic cell death is a form of apoptosis linked to the progression of mitosis through control by CDK1-cyclin-B1.

Prolonged mitotic arrest induces a caspase-dependent DNA damage response at telomeres that determines cell survival.

A delay in the completion of metaphase induces a stress response that inhibits further cell proliferation or induces apoptosis. This response is thought to protect against genomic instability and is important for the effects of anti-mitotic cancer drugs. Here, we show that mitotic arrest induces a caspase-dependent DNA damage response (DDR) at telomeres in non-apoptotic cells. This pathway is under the control of Mcl-1 and other Bcl-2 family proteins and requires caspase-9, caspase-3/7 and the endonuclease CAD/DFF40. The gradual caspase-dependent loss of the shelterin complex protein TRF2 from telomeres promotes a DDR that involves DNA-dependent protein kinase (DNA-PK). Suppression of mitotic telomere damage by enhanced expression of TRF2, or the inhibition of either caspase-3/7 or DNA-PK during mitotic arrest, promotes subsequent cell survival. Thus, we demonstrate that mitotic stress is characterised by the sub-apoptotic activation of a classical caspase pathway, which promotes telomere deprotection, activates DNA damage signalling, and determines cell fate in response to a prolonged delay in mitosis.

Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins.

Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.

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.

Microtubule assembly by the Apc protein is regulated by importin-beta--RanGTP.

Mutations in the tumour suppressor Adenomatous polyposis coli (Apc) initiate most sporadic colorectal cancers. Apc is implicated in regulating microtubule (MT) dynamics in interphase and mitosis. However, little is known about the underlying mechanism or regulation of this Apc function. We identified importin-beta as a binding partner of Apc that regulates its effect on MTs. Apc binds importin-beta in vitro and in Xenopus egg extracts, and RanGTP inhibits this interaction. The armadillo-like repeat domain of importin-beta binds to the middle of Apc, where it can compete with beta-catenin. In addition, two independent sites in the C terminus of Apc bind the N-terminal region of importin-beta. Binding to importin-beta reduces the ability of Apc to assemble and bundle MTs in vitro and to promote assembly of microtubule asters in Xenopus egg extracts, but does not affect the binding of Apc to MTs or to EB1. Depletion of Apc decreases the formation of cold-stable spindles in Xenopus egg extracts. Importantly, the ability of purified Apc to rescue this phenotype was reduced when it was constitutively bound to importin-beta. Thus, importin-beta binds to Apc and negatively regulates the MT-assembly and spindle-promoting activity of Apc in a Ran-regulatable manner.

Apoptosis and autophagy: Regulation of caspase-9 by phosphorylation.

Cell death by the process of apoptosis plays important roles in development, tissue homeostasis, diseases and drug responses. The cysteine aspartyl protease caspase-9 plays a central role in the mitochondrial or intrinsic apoptotic pathway that is engaged in response to many apoptotic stimuli. Caspase-9 is activated in a large multimeric complex, the apoptosome, which is formed with apoptotic peptidase activating factor 1 (Apaf-1) in response to the release of cytochrome c from mitochondria. Once activated, caspase-9 cleaves and activates the effector caspases 3 and 7 to bring about apoptosis. This pathway is tightly regulated at multiple steps, including apoptosome formation and caspase-9 activation. Recent work has shown that caspase-9 is the direct target for regulatory phosphorylation by multiple protein kinases activated in response to extracellular growth/survival factors, osmotic stress or during mitosis. Here, we review these advances and discuss the possible roles of caspase-9 phosphorylation in the regulation of apoptosis during development and in pathological states, including cancer.

p38alpha- and DYRK1A-dependent phosphorylation of caspase-9 at an inhibitory site in response to hyperosmotic stress.

The cysteine aspartyl protease caspase-9 is a critical component of the intrinsic apoptotic pathway. Activation of caspase-9 is inhibited by phosphorylation at Thr125, which is catalysed by the mitogen-activated protein kinases (MAPKs) ERK1/2 in response to growth factors, by the cyclin-dependent protein kinase CDK1-cyclin B1 during mitosis, and at a basal level by the dual-specificity tyrosine-phosphorylation regulated protein kinase DYRK1A. Here we show that inhibitory phosphorylation of caspase-9 at Thr125 is induced in mammalian cells by hyperosmotic stress. This response does not require ERK1/2 or ERK5, but it is diminished by ablation of DYRK1A expression by siRNA or chemical inhibition of DYRK1A by harmine. Phosphorylation of Thr125 in response to hyperosmotic stress is also reduced by chemical inhibition of p38 MAPK and is abolished in p38 alpha(-/-) mouse embryonic fibroblasts. These results show that both DYRK1A and p38 alpha play roles in the inhibitory phosphorylation of caspase-9 following hyperosmotic stress and suggest a functional interaction between these protein kinases. Phosphorylation of caspase-9 at Thr125 may restrain apoptosis during the acute response to hyperosmotic stress.

hnRNP-U is a specific DNA-dependent protein kinase substrate phosphorylated in response to DNA double-strand breaks.

Cellular responses to DNA damage are orchestrated by the large phosphoinositol-3-kinase related kinases ATM, ATR and DNA-PK. We have developed a cell-free system to dissect the biochemical mechanisms of these kinases. Using this system, we identify heterogeneous nuclear ribonucleoprotein U (hnRNP-U), also termed scaffold attachment factor A (SAF-A), as a specific substrate for DNA-PK. We show that hnRNP-U is phosphorylated at Ser59 by DNA-PK in vitro and in cells in response to DNA double-strand breaks. Phosphorylation of hnRNP-U suggests novel functions for DNA-PK in the response to DNA damage.

Cell-cycle control in the face of damage--a matter of life or death.

Cells respond to DNA damage or defects in the mitotic spindle by activating checkpoints that arrest the cell cycle. Alternatively, damaged cells can undergo cell death by the process of apoptosis. The correct balance between these pathways is important for the maintenance of genomic integrity while preventing unnecessary cell death. Although the molecular mechanisms of the cell cycle and apoptosis have been elucidated, the links between them have not been clear. Recent work, however, indicates that common components directly link the regulation of apoptosis with cell-cycle checkpoints operating during interphase, whereas in mitosis, the control of apoptosis is directly coupled to the cell-cycle machinery. These findings shed new light on how the balance between cell-cycle progression and cell death is controlled.

DYRK1A phosphorylates caspase 9 at an inhibitory site and is potently inhibited in human cells by harmine.

DYRK1A is a member of the dual-specificity tyrosine-phosphorylation-regulated protein kinase family and is implicated in Down's syndrome. Here, we identify the cysteine aspartyl protease caspase 9, a critical component of the intrinsic apoptotic pathway, as a substrate of DYRK1A. Depletion of DYRK1A from human cells by short interfering RNA inhibits the basal phosphorylation of caspase 9 at an inhibitory site, Thr125. DYRK1A-dependent phosphorylation of Thr125 is also blocked by harmine, confirming the use of this beta-carboline alkaloid as a potent inhibitor of DYRK1A in cells. We show that harmine not only inhibits the protein-serine/threonine kinase activity of mature DYRK1A, but also its autophosphorylation on tyrosine during translation, indicating that harmine prevents formation of the active enzyme. When co-expressed in cells, DYRK1A interacts with caspase 9, strongly induces Thr125 phosphorylation and inhibits caspase 9 auto-processing. Phosphorylation of caspase 9 by DYRK1A involves co-localization to the nucleus. These results indicate that DYRK1A sets a threshold for the activation of caspase 9 through basal inhibitory phosphorylation of this protease. Regulation of apoptosis through inhibitory phosphorylation of caspase 9 may play a role in the function of DYRK1A during development and in pathogenesis.

A mechanism coupling cell division and the control of apoptosis.

Our recent results demonstrate that caspase activation is regulated during the cell cycle, establishing a direct link between the regulation of apoptosis and cell division (Allan and Clarke, 2007). We show that phosphorylation of caspase-9 is critical for the balance between these processes, restraining the initiation of apoptosis during mitosis. This mechanism is likely to be important in determining sensitivity to anti-cancer drugs that target mitotic cells. We propose that regulation of the phosphorylation of caspase-9 during prolonged mitotic arrest may provide a timing mechanism that initiates apoptosis and destroys an aberrant cell if mitosis is not successfully resolved. This mechanism may play an important role in anti-cancer surveillance and might be exploited to improve cell killing by anti-cancer drugs that target mitotic cells.

Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis.

Cyclin B1 is a key regulatory protein controlling cell cycle progression in vertebrates. Cyclin B1 binds CDK1, a cyclin-dependent kinase catalytic subunit, forming a complex that orchestrates mitosis through phosphorylation of key proteins. Cyclin B1 regulates both the activation of CDK1 and its subcellular localization, which may be critical for substrate selection. Here, we demonstrate that cyclin B1 is concentrated on the outer plate of the kinetochore during prometaphase. This localization requires the cyclin box region of the protein. Cyclin B1 is displaced from individual kinetochores to the spindle poles by microtubule attachment to the kinetochores, and this displacement is dependent on the dynein/dynactin complex. Depletion of cyclin B1 by vector-based siRNA causes inefficient attachment between kinetochores and microtubules, and chromosome alignment defects, and delays the onset of anaphase. We conclude that cyclin B1 accumulates at kinetochores during prometaphase, where it contributes to the correct attachment of microtubules to kinetochores and efficient alignment of the chromosomes, most likely through localized phosphorylation of specific substrates by cyclin B1-CDK1. Cyclin B1 is then transported from each kinetochore as microtubule attachment is completed, and this relocalization may redirect the activity of cyclin B1-CDK1 and contribute to inactivation of the spindle assembly checkpoint.

A mitotic role for BRCA1/BARD1 in tumor suppression?

The tumor-suppressor protein BRCA1 is thought to act by preserving genomic integrity. In this issue of Cell, Joukov et al. demonstrate that the BRCA1/BARD1 heterodimer participates in mitotic spindle assembly, a process conducted by the GTPase Ran. Loss of this mitotic function might contribute to tumorigenesis.

Regulation of Claspin degradation by the ubiquitin-proteosome pathway during the cell cycle and in response to ATR-dependent checkpoint activation.

Claspin is involved in ATR-dependent activation of Chk1 during DNA replication and in response to DNA damage. We show that degradation of Claspin by the ubiquitin-proteosome pathway is regulated during the cell cycle. Claspin is stabilized in S-phase but is abruptly degraded in mitosis and is absent from early G(1) cells in which the phosphorylation of Chk1 by ATR is abrogated. In response to hydroxyurea, UV or aphidicolin, Claspin is phosphorylated in the Chk1-binding domain and its protein levels are increased in an ATR-dependent manner. Thus, the Chk1 pathway is regulated through both phosphorylation of Claspin and its controlled degradation.

Cell biology: Ran, mitosis and the cancer connection.

The small GTPase Ran has been shown to regulate HURP, a protein that interacts with several mitotic spindle assembly factors. This discovery sheds new light on the role of Ran in the fidelity of mitosis and in cancer.