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.

Phosphorylation of importin-α1 by CDK1-cyclin B1 controls mitotic spindle assembly.

Importin-α serves as an adaptor linking importin-ß to proteins carrying a nuclear localization sequence (NLS). During interphase, this interaction enables nuclear protein import, while in mitosis it regulates spindle assembly factors (SAFs) and controls microtubule nucleation, stabilization and spindle function. Here, we show that human importin-α1 is regulated during the cell cycle and is phosphorylated at two sites (threonine 9 and serine 62) during mitosis by the major mitotic protein kinase CDK1-cyclin B. Mutational analysis indicates that the mitotic phosphorylation of importin-α1 inhibits its binding to importin-ß and promotes the release of TPX2 and KIFC1, which are then targeted like importin-ß to the spindle. Loss of importin-α1 or expression of a non-phosphorylated mutant of importin-α1 results in the formation of shortened spindles with reduced microtubule density and induces a prolonged metaphase, whereas phosphorylation-mimicking mutants are functional in mitosis. We propose that phosphorylation of importin-α1 is a general mechanism for the spatial and temporal control of mitotic spindle assembly by CDK1-cyclin B1 that acts through the release of SAFs such as TPX2 and KIFC1 from inhibitory complexes that restrict spindle assembly.

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 Crm1 by CDK1-cyclin-B promotes Ran-dependent mitotic spindle assembly.

Mitotic spindle assembly in animal cells is orchestrated by a chromosome-dependent pathway that directs microtubule stabilization. RanGTP generated at chromosomes releases spindle assembly factors from inhibitory complexes with importins, the nuclear transport factors that facilitate protein import into the nucleus during interphase. In addition, the nuclear export factor Crm1 has been proposed to act as a mitotic effector of RanGTP through the localized assembly of protein complexes on the mitotic spindle, notably at centrosomes and kinetochores. It has been unclear, however, how the functions of nuclear transport factors are controlled during mitosis. Here, we report that human Crm1 is phosphorylated at serine 391 in mitosis by CDK1-cyclin-B (i.e. the CDK1 and cyclin B complex). Expression of Crm1 with serine 391 mutated to either non-phosphorylated or phosphorylation-mimicking residues indicates that phosphorylation directs the localization of Crm1 to the mitotic spindle and facilitates spindle assembly, microtubule stabilization and chromosome alignment. We find that phosphorylation of Crm1 at serine 391 enhances its RanGTP-dependent interaction with RanGAP1-RanBP2 and promotes their recruitment to the mitotic spindle. These results show that phosphorylation of Crm1 controls its molecular interactions, localization and function during mitosis, uncovering a new mechanism for the control of mitotic spindle assembly by CDK1-cyclin-B. We propose that nuclear transport factors are controlled during mitosis through the selection of specific molecular interactions by protein phosphorylation.

Chromatin-bound NLS proteins recruit membrane vesicles and nucleoporins for nuclear envelope assembly via importin-α/ß.

The mechanism for nuclear envelope (NE) assembly is not fully understood. Importin-ß and the small GTPase Ran have been implicated in the spatial regulation of NE assembly process. Here we report that chromatin-bound NLS (nuclear localization sequence) proteins provide docking sites for the NE precursor membrane vesicles and nucleoporins via importin-α and -ß during NE assembly in Xenopus egg extracts. We show that along with the fast recruitment of the abundant NLS proteins such as nucleoplasmin and histones to the demembranated sperm chromatin in the extracts, importin-α binds the chromatin NLS proteins rapidly. Meanwhile, importin-ß binds cytoplasmic NE precursor membrane vesicles and nucleoporins. Through interacting with importin-α on the chromatin NLS proteins, importin-ß targets the membrane vesicles and nucleoporins to the chromatin surface. Once encountering Ran-GTP on the chromatin generated by RCC1, importin-ß preferentially binds Ran-GTP and releases the membrane vesicles and nucleoporins for NE assembly. NE assembly is disrupted by blocking the interaction between importin-α and NLS proteins with excess soluble NLS proteins or by depletion of importin-ß from the extract. Our findings reveal a novel molecular mechanism for NE assembly in Xenopus egg extracts.

Clathrin recruits phosphorylated TACC3 to spindle poles for bipolar spindle assembly and chromosome alignment.

Transforming acidic coiled-coil-containing protein 3 (TACC3) has been implicated in mitotic spindle assembly, although the mechanisms involved are largely unknown. Here we identify that clathrin heavy chain (CHC) binds specifically to phosphorylated TACC3 and recruits it to spindle poles for proper spindle assembly and chromosome alignment. Phosphorylation of Xenopus TACC3 at serine 620 (S620) and S626, but not S33, is required for its binding with CHC. Knockdown of CHC by RNA interference (RNAi) abolishes the targeting of TACC3 to spindle poles and results in abnormal spindle assembly and chromosome misalignment, similar to the defects caused by TACC3 knockdown. Furthermore, the binding of CHC with phosphorylated TACC3 is inhibited by importin ß and this inhibition is reversed by the presence of the GTP-binding nuclear protein Ran in the GTP-bound state. Together, these results indicate that the recruitment of phosphorylated TACC3 to spindle poles by CHC ensures proper spindle assembly and chromosome alignment, and is regulated by Ran.

The methylated N-terminal tail of RCC1 is required for stabilisation of its interaction with chromatin by Ran in live cells.

BACKGROUND: Regulator of chromosome condensation 1 (RCC1) is the guanine nucleotide exchange factor for Ran GTPase. Localised generation of Ran-GTP by RCC1 on chromatin is critical for nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Both the N-terminal tail of RCC1 and its association with Ran are important for its interaction with chromatin in cells. In vitro, the association of Ran with RCC1 induces a conformational change in the N-terminal tail that promotes its interaction with DNA. RESULTS: We have investigated the mechanism of the dynamic interaction of the alpha isoform of human RCC1 (RCC1alpha) with chromatin in live cells using fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) fusions. We show that the N-terminal tail stabilises the interaction of RCC1alpha with chromatin and this function can be partially replaced by another lysine-rich nuclear localisation signal. Removal of the tail prevents the interaction of RCC1alpha with chromatin from being stabilised by RanT24N, a mutant that binds stably to RCC1alpha. The interaction of RCC1alpha with chromatin is destabilised by mutation of lysine 4 (K4Q), which abolishes alpha-N-terminal methylation, and this interaction is no longer stabilised by RanT24N. However, alpha-N-terminal methylation of RCC1alpha is not regulated by the binding of RanT24N. Conversely, the association of Ran with precipitated RCC1alpha does not require the N-terminal tail of RCC1alpha or its methylation. The mobility of RCC1alpha on chromatin is increased by mutation of aspartate 182 (D182A), which inhibits guanine-nucleotide exchange activity, but RCC1alphaD182A can still bind nucleotide-free Ran and its interaction with chromatin is stabilised by RanT24N. CONCLUSIONS: These results show that the stabilisation of the dynamic interaction of RCC1alpha with chromatin by Ran in live cells requires the N-terminal tail of RCC1alpha. alpha-N-methylation is not regulated by formation of the binary complex with Ran, but it promotes chromatin binding through the tail. This work supports a model in which the association of RCC1alpha with chromatin is promoted by a conformational change in the alpha-N-terminal methylated tail that is induced allosterically in the binary complex with Ran.

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.

Dynamic localisation of Ran GTPase during the cell cycle.

BACKGROUND: Ran GTPase has multiple functions during the cell division cycle, including nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. The activity of Ran is determined by both its guanine nucleotide-bound state and its subcellular localization. RESULTS: Here, we have characterised the localisation and mobility of Ran coupled to green fluorescent protein (GFP) during the cell cycle in live human cells. Ran-GFP is nuclear during interphase and is dispersed throughout the cell during mitosis. GFP-RanQ69L, a mutant locked in the GTP-bound state, is less highly concentrated in the nucleus and associates with nuclear pore complexes within the nuclear envelope. During mitosis, GFP-RanQ69L is excluded from chromosomes and localizes to the spindle. By contrast, GFP-RanT24N, a mutant with low affinity for nucleotides, interacts relatively stably with chromatin throughout the cell cycle and is highly concentrated on mitotic chromosomes. CONCLUSION: These results show that Ran interacts dynamically with chromatin, nuclear pore complexes and the mitotic spindle during the cell cycle. These interactions are dependent on the nucleotide-bound state of the protein. Our data indicate that Ran-GTP generated at chromatin is highly mobile and interacts dynamically with distal structures that are involved in nuclear transport and mitotic spindle assembly.

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.