The Cdc20-binding Phe Box of the Spindle Checkpoint Protein BubR1 Maintains the Mitotic Checkpoint Complex During Mitosis

The spindle checkpoint ensures accurate chromosome segregation by monitoring kinetochore-microtubule attachment. Unattached or tensionless kinetochores activate the checkpoint and enhance the production of the mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20. MCC is a critical checkpoint inhibitor of the anaphase-promoting complex/cyclosome, a ubiquitin ligase required for anaphase onset. The N-terminal region of BubR1 binds to both Cdc20 and Mad2, thus nucleating MCC formation. The middle region of human BubR1 (BubR1M) also interacts with Cdc20, but the nature and function of this interaction are not understood. Here we identify two critical motifs within BubR1M that contribute to Cdc20 binding and anaphase-promoting complex/cyclosome inhibition: a destruction box (D box) and a phenylalanine-containing motif termed the Phe box. A BubR1 mutant lacking these motifs is defective in MCC maintenance in mitotic human cells but is capable of supporting spindle-checkpoint function. Thus, the BubR1M-Cdc20 interaction indirectly contributes to MCC homeostasis. Its apparent dispensability in the spindle checkpoint might be due to functional duality or redundant, competing mechanisms.     

Phosphorylation of Mixed Lineage Leukemia 5 by Cdc2 Affects Its Cellular Distribution and Is Required for Mitotic Entry

The human mixed lineage leukemia-5 (MLL5) gene is frequently deleted in myeloid malignancies. Emerging evidence suggests that MLL5 has important functions in adult hematopoiesis and the chromatin regulatory network, and it participates in regulating the cell cycle machinery. Here, we demonstrate that MLL5 is tightly regulated through phosphorylation on its central domain at the G₂/M phase of the cell cycle. Upon entry into mitosis, the phosphorylated MLL5 delocalizes from condensed chromosomes, whereas after mitotic exit, MLL5 becomes dephosphorylated and re-associates with the relaxed chromatin. We further identify that the mitotic phosphorylation and subcellular localization of MLL5 are dependent on Cdc2 kinase activity, and Thr-912 is the Cdc2-targeting site. Overexpression of the Cdc2-targeting MLL5 fragment obstructs mitotic entry by competitive inhibition of the phosphorylation of endogenous MLL5. In addition, G₂ phase arrest caused by depletion of endogenous MLL5 can be compensated by exogenously overexpressed full-length MLL5 but not the phosphodomain deletion or MLL5-T912A mutant. Our data provide evidence that MLL5 is a novel cellular target of Cdc2, and the phosphorylation of MLL5 may have an indispensable role in the mitotic progression.     

Phosphorylation of Microtubule-binding Protein Hec1 by Mitotic Kinase Aurora B Specifies Spindle Checkpoint Kinase Mps1 Signaling at the Kinetochore

The spindle assembly checkpoint (SAC) is a quality control device to ensure accurate chromosome attachment to spindle microtubule for equal segregation of sister chromatid. Aurora B is essential for SAC function by sensing chromosome bi-orientation via spatial regulation of kinetochore substrates. However, it has remained elusive as to how Aurora B couples kinetochore-microtubule attachment to SAC signaling. Here, we show that Hec1 interacts with Mps1 and specifies its kinetochore localization via its calponin homology (CH) domain and N-terminal 80 amino acids. Interestingly, phosphorylation of the Hec1 by Aurora B weakens its interaction with microtubules but promotes Hec1 binding to Mps1. Significantly, the temporal regulation of Hec1 phosphorylation orchestrates kinetochore-microtubule attachment and Mps1 loading to the kinetochore. Persistent expression of phosphomimetic Hec1 mutant induces a hyperactivation of SAC, suggesting that phosphorylation-elicited Hec1 conformational change is used as a switch to orchestrate SAC activation to concurrent destabilization of aberrant kinetochore attachment. Taken together, these results define a novel role for Aurora B-Hec1-Mps1 signaling axis in governing accurate chromosome segregation in mitosis.     

Global analysis of Cdc14 phosphatase reveals diverse roles in mitotic processes

Cdc14 phosphatase regulates multiple events during anaphase and is essential for mitotic exit in budding yeast. Cdc14 is regulated in both a spatial and temporal manner. It is sequestered in the nucleolus for most of the cell cycle by the nucleolar protein Net1 and is released into the nucleus and cytoplasm during anaphase. To identify novel binding partners of Cdc14, we used affinity purification of Cdc14 and mass spectrometric analysis of interacting proteins from strains in which Cdc14 localization or catalytic activity was altered. To alter Cdc14 localization, we used a strain deleted for NET1, which causes full release of Cdc14 from the nucleolus. To alter Cdc14 activity, we generated mutations in the active site of Cdc14 (C283S or D253A), which allow binding of substrates, but not dephosphorylation, by Cdc14. Using this strategy, we identified new interactors of Cdc14, including multiple proteins involved in mitotic events. A subset of these proteins displayed increased affinity for catalytically inactive mutants of Cdc14 compared with the wild-type version, suggesting they are likely substrates of Cdc14. We have also shown that several of the novel Cdc14-interacting proteins, including Kar9 (a protein that orients the mitotic spindle) and Bni1 and Bnr1 (formins that nucleate actin cables and may be important for actomyosin ring contraction) are specifically dephosphorylated by Cdc14 in vitro and in vivo. Our findings suggest the dephosphorylation of the formins may be important for their observed localization change during exit from mitosis and indicate that Cdc14 targets proteins involved in wide-ranging mitotic events.     

Cellular abundance of Mps1 and the role of its carboxyl terminal tail in substrate recruitment

Mps1 is a protein kinase that regulates normal mitotic progression and the spindle checkpoint in response to spindle damage. The levels of Mps1 are relatively low in cells during interphase but elevated in mitosis or upon activation of the spindle checkpoint, although the dynamic range of Mps1 expression and the Mps1 catalytic mechanism have not been carefully characterized. Our recent structural studies of the Mps1 kinase domain revealed that the carboxyl-terminal tail region of Mps1 is unstructured, raising the question of whether this region has any functional role in Mps1 catalysis. Here we first determined the cellular abundance of Mps1 during cell cycle progression and found that Mps1 levels vary between 60,000 per cell in early G(1) and 110,000 per cell during mitosis. We studied phosphorylation of a number of Mps1 substrates in vitro and in culture cells. Unexpectedly, we found that the unstructured carboxyl-terminal region of Mps1 plays an essential role in substrate recruitment. Kinetics studies using the purified recombinant wild type and mutant kinases indicate that the carboxyl-terminal tail is largely dispensable for autophosphorylation of Mps1 but critical for trans-phosphorylation of substrates in vitro and in cultured cells. Mps1 mutant without the unstructured tail region is defective in mediating spindle assembly checkpoint activation. Our results underscore the importance of the unstructured tail region of Mps1 in kinase activation.     

Phosphorylation of Cyclin-dependent Kinase 5 (Cdk5) at Tyr-15 Is Inhibited by Cdk5 Activators and Does Not Contribute to the Activation of Cdk5

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. In contrast to other Cdks that promote cell proliferation, Cdk5 plays a role in regulating various neuronal functions, including neuronal migration, synaptic activity, and neuron death. Cdks responsible for cell proliferation need phosphorylation in the activation loop for activation in addition to binding a regulatory subunit cyclin. Cdk5, however, is activated only by binding to its activator, p35 or p39. Furthermore, in contrast to Cdk1 and Cdk2, which are inhibited by phosphorylation at Tyr-15, the kinase activity of Cdk5 is reported to be stimulated when phosphorylated at Tyr-15 by Src family kinases or receptor-type tyrosine kinases. We investigated the activation mechanism of Cdk5 by phosphorylation at Tyr-15. Unexpectedly, however, it was found that Tyr-15 phosphorylation occurred only on monomeric Cdk5, and the coexpression of activators, p35/p25, p39, or Cyclin I, inhibited the phosphorylation. In neuron cultures, too, the activation of Fyn tyrosine kinase did not increase Tyr-15 phosphorylation of Cdk5. Further, phospho-Cdk5 at Tyr-15 was not detected in the p35-bound Cdk5. In contrast, expression of active Fyn increased p35 in neurons. These results indicate that phosphorylation at Tyr-15 is not an activation mechanism of Cdk5 but, rather, indicate that tyrosine kinases could activate Cdk5 by increasing the protein amount of p35. These results call for reinvestigation of how Cdk5 is regulated downstream of Src family kinases or receptor tyrosine kinases in neurons, which is an important signaling cascade in a variety of neuronal activities.     

Positive and Negative Regulation of Vertebrate Separase by Cdk1-Cyclin B1 May Explain Why Securin Is Dispensable

Sister chromatid cohesion is established during replication by entrapment of both dsDNAs within the cohesin ring complex. It is dissolved in anaphase when separase, a giant cysteine endopeptidase, cleaves the Scc1/Rad21 subunit of cohesin, thereby triggering chromosome segregation. Separase is held inactive by association with securin until this anaphase inhibitor is destroyed at the metaphase-to-anaphase transition by ubiquitin-dependent degradation. The relevant ubiquitin ligase, the anaphase-promoting complex/cyclosome, also targets cyclin B1, thereby causing inactivation of Cdk1 and mitotic exit. Although separase is essential, securin knock-out mice are surprisingly viable and fertile. Capitalizing on our previous finding that Cdk1-cyclin B1 can also bind and inhibit separase, we investigated whether this kinase might be suitable to maintain faithful timing and execution of anaphase in the absence of securin. We found that, similar to securin, Cdk1-cyclin B1 regulates separase in both a positive and negative manner. Although securin associates with nascent separase to co-translationally assist proper folding, Cdk1-cyclin B1 acts on native state separase. Upon entry into mitosis, Cdk1-cyclin B1-dependent phosphorylation of Ser-1126 renders separase prone to inactivation by aggregation/precipitation. Stable association of Cdk1-cyclin B1 with phosphorylated separase counteracts this tendency and stabilizes separase in an inhibited yet activatable state. These opposing effects are suited to prevent premature cleavage of cohesin in early mitosis while ensuring timely activation of separase by anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. Coupling sister chromatid separation with subsequent exit from mitosis by this simplified mode might have been the common scheme of mitotic control prior to the evolution of securin.     

Cdc25 Phosphatases Are Required for Timely Assembly of CDK1-Cyclin B at the G2/M Transition

Progression through mitosis requires the coordinated regulation of Cdk1 kinase activity. Activation of Cdk1 is a multistep process comprising binding of Cdk1 to cyclin B, relocation of cyclin-kinase complexes to the nucleus, activating phosphorylation of Cdk1 on Thr¹⁶¹ by the Cdk-activating kinase (CAK; Cdk7 in metazoans), and removal of inhibitory Thr¹⁴ and Tyr¹⁵ phosphorylations. This dephosphorylation is catalyzed by the dual specific Cdc25 phosphatases, which occur in three isoforms in mammalian cells, Cdc25A, -B, and -C. We find that expression of Cdc25A leads to an accelerated G₂/M phase transition. In Cdc25A-overexpressing cells, Cdk1 exhibits high kinase activity despite being phosphorylated on Tyr¹⁵. In addition, Tyr¹⁵-phosphorylated Cdk1 binds more cyclin B in Cdc25A-overexpressing cells compared with control cells. Consistent with this observation, we demonstrate that in human transformed cells, Cdc25A and Cdc25B, but not Cdc25C phosphatases have an effect on timing and efficiency of cyclin-kinase complex formation. Overexpression of Cdc25A or Cdc25B promotes earlier assembly and activation of Cdk1-cyclin B complexes, whereas repression of these phosphatases by short hairpin RNA has a reverse effect, leading to a substantial decrease in amounts of cyclin B-bound Cdk1 in G₂ and mitosis. Importantly, we find that Cdc25A overexpression leads to an activation of Cdk7 and increase in Thr¹⁶¹ phosphorylation of Cdk1. In conclusion, our data suggest that complex assembly and dephosphorylation of Cdk1 at G₂/M is tightly coupled and regulated by Cdc25 phosphatases.     

Mitotic Regulation of SEPT9 Protein by Cyclin-dependent Kinase 1 (Cdk1) and Pin1 Protein Is Important for the Completion of Cytokinesis

Precise cell division is essential for multicellular development, and defects in this process have been linked to cancer. Septins are a family of proteins that are required for mammalian cell division, but their function and mode of regulation during this process are poorly understood. Here, we demonstrate that cyclin-dependent kinase 1 (Cdk1) phosphorylates septin 9 (SEPT9) upon mitotic entry, and this phosphorylation controls association with the proline isomerase, Pin1. Both SEPT9 and Pin1 are critical for mediating the final separation of daughter cells. Expression of mutant SEPT9 that is defective in Pin1 binding was unable to rescue cytokinesis defects caused by SEPT9 depletion but rather induced dominant-negative defects in cytokinesis. However, unlike SEPT9 depletion, Pin1 was not required for the accumulation of the exocyst complex at the midbody. These results suggest that SEPT9 plays multiple roles in abscission, one of which is regulated by the action of Cdk1 and Pin1.     

Regulation of a Dynamic Interaction between Two Microtubule-binding Proteins,EB1 and TIP150, by the Mitotic p300/CBP-associated Factor (PCAF) Orchestrates Kinetochore Microtubule Plasticity and Chromosome Stability during Mitosis

The microtubule cytoskeleton network orchestrates cellular dynamics and chromosome stability in mitosis. Although tubulin acetylation is essential for cellular plasticity, it has remained elusive how kinetochore microtubule plus-end dynamics are regulated by p300/CBP-associated factor (PCAF) acetylation in mitosis. Here, we demonstrate that the plus-end tracking protein, TIP150, regulates dynamic kinetochore-microtubule attachments by promoting the stability of spindle microtubule plus-ends. Suppression of TIP150 by siRNA results in metaphase alignment delays and perturbations in chromosome biorientation. TIP150 is a tetramer that binds an end-binding protein (EB1) dimer through the C-terminal domains, and overexpression of the C-terminal TIP150 or disruption of the TIP150-EB1 interface by a membrane-permeable peptide perturbs chromosome segregation. Acetylation of EB1-PCAF regulates the TIP150 interaction, and persistent acetylation perturbs EB1-TIP150 interaction and accurate metaphase alignment, resulting in spindle checkpoint activation. Suppression of the mitotic checkpoint serine/threonine protein kinase, BubR1, overrides mitotic arrest induced by impaired EB1-TIP150 interaction, but cells exhibit whole chromosome aneuploidy. Thus, the results identify a mechanism by which the TIP150-EB1 interaction governs kinetochore microtubule plus-end plasticity and establish that the temporal control of the TIP150-EB1 interaction by PCAF acetylation ensures chromosome stability in mitosis.     

Plk1 Protein Phosphorylates Phosphatase and Tensin Homolog (PTEN) and Regulates Its Mitotic Activity during the Cell Cycle

PTEN is a well known tumor suppressor through the negative regulation of the PI3K signaling pathway. Here we report that PTEN plays an important role in regulating mitotic timing, which is associated with increased PTEN phosphorylation in the C-terminal tail and its localization to chromatin. Pulldown analysis revealed that Plk1 physically interacted with PTEN. Biochemical studies showed that Plk1 phosphorylates PTEN in vitro in a concentration-dependent manner and that the phosphorylation was inhibited by Bi2635, a Plk1-specific inhibitor. Deletional and mutational analyses identified that Plk1 phosphorylated Ser-380, Thr-382, and Thr-383, but not Ser-385, a cluster of residues known to affect the PTEN stability. Interestingly, a combination of molecular and genetic analyses revealed that only Ser-380 was significantly phosphorylated in vivo and that Plk1 regulated the phosphorylation, which was associated with the accumulation of PTEN on chromatin. Moreover, expression of phospho-deficient mutant, but not wild-type PTEN, caused enhanced mitotic exit. Taken together, our studies identify Plk1 as an important regulator of PTEN during the cell cycle.     

Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G₂/M transition. We find that activation of Aurora A first occurs at centrosomes at late G₂ and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.     

ATP-dependent Pre-replicative Complex Assembly Is Facilitated by Adk1p in Budding Yeast

Pre-replicative complex (pre-RC) assembly is a critical part of the mechanism that controls the initiation of DNA replication, and ATP binding and hydrolysis by multiple pre-RC proteins are essential for pre-RC assembly and activation. Here, we demonstrate that Adk1p (adenylate kinase 1 protein) plays an important role in pre-RC assembly in Saccharomyces cerevisiae. Isolated from a genetic screen, adk1^G20S cells with a mutation within the nucleotide-binding site were defective in replication initiation. adk1Δ cells were viable at 25 °C but not at 37°C. Flow cytometry indicated that both the adk1-td (temperature-inducible degron) and adk1^G20S mutants were defective in S phase entry. Furthermore, Adk1p bound to chromatin throughout the cell cycle and physically interacted with Orc3p, whereas the Adk1^G20S protein had a reduced ability to bind chromatin and Orc3p without affecting the cellular ATP level. In addition, Adk1p associated with replication origins by ChIP assay. Finally, Adk1-td protein depletion prevented pre-RC assembly during the M-to-G₁ transition. We suggest that Adk1p regulates ATP metabolism on pre-RC proteins to promote pre-RC assembly and activation.     

Mitotic Phosphorylation of Cdc25B Ser321 Disrupts 14-3-3 Binding to the High Affinity Ser323 Site

Cdc25B is a key regulator of entry into mitosis, and its activity and localization are regulated by binding of the 14-3-3 dimer. There are three 14-3-3 binding sites on Cdc25B, with Ser³²³ being the highest affinity binding and is highly homologous to the Ser²¹⁶ 14-3-3 binding site on Cdc25C. Loss of 14-3-3 binding to Ser³²³ increases cyclin/Cdk substrate access to the catalytic site, thereby increasing its activity. It also affects the localization of Cdc25B. Thus, phosphorylation and 14-3-3 binding to this site is essential for down-regulating Cdc25B activity, blocking its mitosis promoting function. The question of how this inhibitory signal is relieved to allow Cdc25B activation and entry into mitosis is yet to be resolved. Here, we show that Ser³²³ phosphorylation is maintained into mitosis, but phosphorylation of Ser³²¹ disrupts 14-3-3 binding to Ser³²³, mimicking the effect of inhibiting Ser³²³ phosphorylation on both Cdc25B activity and localization. The unphosphorylated Ser³²¹ appears to have a role in stabilizing 14-3-3 binding to Ser³²³, and loss of the Ser hydroxyl group appears to be sufficient to significantly reduce 14-3-3 binding. A consequence of loss of 14-3-3 binding is dephosphorylation of Ser³²³. Ser³²¹ is phosphorylated in mitosis by Cdk1. The mitotic phosphorylation of Ser³²¹ acts to maintain full activation of Cdc25B by disrupting 14-3-3 binding to Ser³²³ and enhancing the dephosphorylation of Ser³²³ to block 14-3-3 binding to this site.     

Monopolar Spindle 1 (MPS1) Kinase Promotes Production of Closed MAD2 (C-MAD2) Conformer and Assembly of the Mitotic Checkpoint Complex

MPS1 kinase is an essential component of the spindle assembly checkpoint (SAC), but its functioning mechanisms are not fully understood. We have shown recently that direct interaction between BUBR1 and MAD2 is critical for assembly and function of the human mitotic checkpoint complex (MCC), the SAC effector. Here we report that inhibition of MPS1 kinase activity by reversine disrupts BUBR1-MAD2 as well as CDC20-MAD2 interactions, causing premature activation of the anaphase-promoting complex/cyclosome. The effect of MPS1 inhibition is likely due to reduction of closed MAD2 (C-MAD2), as expressing a MAD2 mutant (MAD2^L13A) that is locked in the C conformation rescued the checkpoint defects. In the presence of reversine, exogenous C-MAD2 does not localize to unattached kinetochores but is still incorporated into the MCC. Contrary to a previous report, we found that sustained MPS1 activity is required for maintaining both the MAD1·C-MAD2 complex and open MAD2 (O-MAD2) at unattached kinetochores to facilitate C-MAD2 production. Additionally, mitotic phosphorylation of BUBR1 is also affected by MPS1 inhibition but seems dispensable for MCC assembly. Our results support the notion that MPS1 kinase promotes C-MAD2 production and subsequent MCC assembly to activate the SAC.     

Phosphorylation Regulates Kinase and Microtubule Binding Activities of the Budding Yeast Chromosomal Passenger Complex in Vitro

The chromosomal passenger complex (CPC) is a key regulator of mitosis in eukaryotes. It comprises four essential and conserved proteins known in mammals/yeasts as Aurora B/Ipl1, INCENP/Sli15, Survivin/Bir1, and Borealin/Nbl1. These subunits act together in a highly controlled fashion. Regulation of Aurora B/Ipl1 kinase activity and localization is critical for CPC function. Although regulation of CPC localization and kinase activity in vivo has been investigated elsewhere, studies on the complete, four-subunit CPC and its basic biochemical properties are only beginning. Here we describe the biochemical characterization of purified and complete Saccharomyces cerevisiae four-subunit CPC. We determined the affinity of the CPC for microtubules and demonstrated that the binding of CPC to microtubules is primarily electrostatic in nature and depends on the acidic C-terminal tail (E-hook) of tubulin. Moreover, phosphorylation of INCENP/Sli15 on its microtubule binding region also negatively regulates CPC affinity for microtubules. Furthermore, we show that phosphorylation of INCENP/Sli15 is required for activation of the kinase Aurora B/Ipl1 and can occur in trans. Although phosphorylation of INCENP/Sli15 is essential for activation, we determined that a version of the CPC lacking the INCENP/Sli15 microtubule binding region (residues Glu-91 to Ile-631) is able to form an intact complex that retains microtubule binding activity. Thus, we conclude that this INCENP/Sli15 linker domain plays a largely regulatory function and is not essential for complex formation or microtubule binding.