Spindle checkpoint silencing requires association of PP1 to both Spc7 and kinesin-8 motors

The spindle checkpoint is the prime cell-cycle control mechanism that ensures sister chromatids are bioriented before anaphase takes place. Aurora B kinase, the catalytic subunit of the chromosome passenger complex, both destabilizes kinetochore attachments that do not generate tension and simultaneously maintains the spindle checkpoint signal. However, it is unclear how the checkpoint is silenced following chromosome biorientation. We demonstrate that association of type 1 phosphatase (PP1(Dis2)) with both the N terminus of Spc7 and the nonmotor domains of the Klp5-Klp6 (kinesin-8) complex is necessary to counteract Aurora B kinase to efficiently silence the spindle checkpoint. The role of Klp5 and Klp6 in checkpoint silencing is specific to this class of kinesin and independent of their motor activities. These data demonstrate that at least two distinct pools of PP1, one kinetochore associated and the other motor associated, are needed to silence the spindle checkpoint.     

PP2A-B56 opposes Mps1 phosphorylation of Knl1 and thereby promotes spindle assembly checkpoint silencing

The spindle assembly checkpoint (SAC) monitors correct attachment of chromosomes to microtubules, an important safeguard mechanism ensuring faithful chromosome segregation in eukaryotic cells. How the SAC signal is turned off once all the chromosomes have successfully attached to the spindle remains an unresolved question. Mps1 phosphorylation of Knl1 results in recruitment of the SAC proteins Bub1, Bub3, and BubR1 to the kinetochore and production of the wait-anaphase signal. SAC silencing is therefore expected to involve a phosphatase opposing Mps1. Here we demonstrate in vivo and in vitro that BubR1-associated PP2A-B56 is a key phosphatase for the removal of the Mps1-mediated Knl1 phosphorylations necessary for Bub1/BubR1 recruitment in mammalian cells. SAC silencing is thus promoted by a negative feedback loop involving the Mps1-dependent recruitment of a phosphatase opposing Mps1. Our findings extend the previously reported role for BubR1-associated PP2A-B56 in opposing Aurora B and suggest that BubR1-bound PP2A-B56 integrates kinetochore surveillance and silencing of the SAC.     

Spindle assembly checkpoint: the third decade

The spindle assembly checkpoint controls cell cycle progression during mitosis, synchronizing it with the attachment of chromosomes to spindle microtubules. After the discovery of the mitotic arrest deficient (MAD) and budding uninhibited by benzymidazole (BUB) genes as crucial checkpoint components in 1991, the second decade of checkpoint studies (2001-2010) witnessed crucial advances in the elucidation of the mechanism through which the checkpoint effector, the mitotic checkpoint complex, targets the anaphase-promoting complex (APC/C) to prevent progression into anaphase. Concomitantly, the discovery that the Ndc80 complex and other components of the microtubule-binding interface of kinetochores are essential for the checkpoint response finally asserted that kinetochores are crucial for the checkpoint response. Nevertheless, the relationship between kinetochores and checkpoint control remains poorly understood. Crucial advances in this area in the third decade of checkpoint studies (2011-2020) are likely to be brought about by the characterization of the mechanism of kinetochore recruitment, activation and inactivation of checkpoint proteins, which remains elusive for the majority of checkpoint components. Here, we take a molecular view on the main challenges hampering this task.     

Moving right along: how PP1 helps clear the checkpoint

Spindle checkpoint silencing is crucial for cell-cycle progression, but mechanisms underlying this process remain mysterious. Two papers, one in this issue of Developmental Cell (Meadows et?al., 2011) and one in Current Biology (Rosenberg et?al., 2011), begin to show how phosphatase PP1-gamma connects chromosome-microtubule attachment with anaphase entry.     

Role for the silencing protein Dot1 in meiotic checkpoint control

During the meiotic cell cycle, a surveillance mechanism called the "pachytene checkpoint" ensures proper chromosome segregation by preventing meiotic progression when recombination and chromosome synapsis are defective. The silencing protein Dot1 (also known as Pch1) is required for checkpoint-mediated pachytene arrest of the zip1 and dmc1 mutants of Saccharomyces cerevisiae. In the absence of DOT1, the zip1 and dmc1 mutants inappropriately progress through meiosis, generating inviable meiotic products. Other components of the pachytene checkpoint include the nucleolar protein Pch2 and the heterochromatin component Sir2. In dot1, disruption of the checkpoint correlates with the loss of concentration of Pch2 and Sir2 in the nucleolus. In addition to its checkpoint function, Dot1 blocks the repair of meiotic double-strand breaks by a Rad54-dependent pathway of recombination between sister chromatids. In vegetative cells, mutation of DOT1 results in delocalization of Sir3 from telomeres, accounting for the impaired telomeric silencing in dot1.     

Spindle checkpoint requires Mad1-bound and Mad1-free Mad2

The spindle checkpoint prevents anaphase from occurring until all chromosomes have attached properly to the mitotic spindle. The checkpoint components Mad1 and Mad2 associate with unattached kinetochores and are probably involved in triggering the checkpoint. We now demonstrate that in Xenopus egg extracts Mad1 and Mad2 form a stable complex, whereas a fraction of Mad2 molecules is not bound to Mad1. The checkpoint establishment and maintenance are lost upon titrating out free Mad2 with an excess of Mad1 or a truncated Mad1 (amino acids 326-718, Mad1C) that contains the Mad2-binding region. Mad1N (amino acids 1-445) that binds kinetochores, but not Mad2, reduces Mad1 and Mad2 at kinetochores and abolishes checkpoint maintenance. Furthermore, the association between Mad2 and Cdc20, the activator for the anaphase-promoting complex, is enhanced under checkpoint-active condition compared with that at metaphase. Immunodepletion analysis shows that the Mad1-free Mad2 protein is unable to bind Cdc20, consistent with the model that kinetochore localization of Mad2 facilitates the formation of Mad2-Cdc20 complex. This study demonstrates that the ratio between Mad1 and Mad2 is critical for maintaining a pool of Mad1-free Mad2 that is necessary for the spindle checkpoint. We propose that Mad2 may become activated and dissociated from Mad1 at kinetochores and is replenished by the pool of Mad1-free Mad2.     

Polo-like kinase-1 regulates kinetochore-microtubule dynamics and spindle checkpoint silencing

Polo-like kinase-1 (Plk1) is a highly conserved kinase with multiple mitotic functions. Plk1 localizes to prometaphase kinetochores and is reduced at metaphase kinetochores, similar to many checkpoint signaling proteins, but Plk1 is not required for spindle checkpoint function. Plk1 is also implicated in stabilizing kinetochore-microtubule attachments, but these attachments are most stable when kinetochore Plk1 levels are low at metaphase. Therefore, it is unclear how Plk1 function at kinetochores can be understood in the context of its dynamic localization. In this paper, we show that Plk1 activity suppresses kinetochore-microtubule dynamics to stabilize initial attachments in prometaphase, and Plk1 removal from kinetochores is necessary to maintain dynamic microtubules in metaphase. Constitutively targeting Plk1 to kinetochores maintained high activity at metaphase, leading to reduced interkinetochore tension and intrakinetochore stretch, a checkpoint-dependent mitotic arrest, and accumulation of microtubule attachment errors. Together, our data show that Plk1 dynamics at kinetochores control two critical mitotic processes: initially establishing correct kinetochore-microtubule attachments and subsequently silencing the spindle checkpoint.     

Resisting arrest: recovery from checkpoint arrest through dephosphorylation of Chk1 by PP1

The G2 DNA damage checkpoint prevents mitotic entry in the presence of damaged DNA, and thus is essential for cells to replicate with stable genetic inheritance. Whilst significant progress has been made in the past 10 years on the mechanism of checkpoint activation, little attention has been paid to how the DNA damage checkpoint is switched off to allow cell cycle re-entry. Insight into the mechanism of cell cycle re-entry was recently provided by our finding that the Schizosaccharomyces pombe type 1 phosphatase (PP1) Dis2 dephosphorylates the checkpoint effector kinase Chk1. This occurs on a site phosphorylated by the ATR homologue Rad3 in response to DNA damage, and results in Chk1 inactivation and checkpoint release. Here we discuss the implications of this finding on DNA damage checkpoint signaling, and speculate on models for checkpoint maintenance and release.     

KNL1/Spc105 recruits PP1 to silence the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) delays anaphase onset until kinetochores accomplish bioriented microtubule attachments [1]. Although several centromeric and kinetochore kinases, including Aurora B, regulate kinetochore-microtubule attachment and/or SAC activation [2-4], the molecular mechanism that translates bioriented attachment into SAC silencing remains unclear [5]. Employing a method to rapidly induce exact gene replacement in budding yeast [6], we show here that the binding of protein phosphatase 1?(PP1/Glc7) to the evolutionarily conserved RVSF motif of the kinetochore protein Spc105 (KNL1/Blinkin/CASC5) is essential for viability by silencing the SAC, while it plays an auxiliary nonessential role for physical chromosome segregation. Although Aurora B may inhibit this binding, persistent PP1-Spc105 interaction does not affect chromosome segregation and is insufficient to silence the SAC in the absence of microtubules, indicating that dynamic regulation of this?interaction is dispensable. However, the amount of PP1 targeted to kinetochores must be finely tuned, because recruitment of either no or one extra copy of PP1 to Spc105 is detrimental, illustrating the vital impact of targeting an exiguous fraction of PP1 to the kinetochore. We propose that the PP1-Spc105 interaction enables local regulation of dynamic phosphorylation and dephosphorylation at the kinetochore to couple microtubule attachment and SAC silencing.     

Spindle assembly checkpoint,aneuploidy and tumorigenesis

Comment on: Li M, et al. J Cell Biol 2009; 185:983-94.     

Microtubule binding by KNL-1 contributes to spindle checkpoint silencing at the kinetochore

Accurate chromosome segregation requires coordination between microtubule attachment and spindle checkpoint signaling at the kinetochore. The kinetochore-localized KMN (KNL-1/Mis12 complex/Ndc80 complex) network, which mediates microtubule attachment and scaffolds checkpoint signaling, harbors two distinct microtubule-binding activities: the load-bearing activity of the Ndc80 complex and a less well-understood activity in KNL-1. In this paper, we show that KNL-1 microtubule-binding and -bundling activity resides in its extreme N terminus. Selective perturbation of KNL-1 microtubule binding in Caenorhabditis elegans embryos revealed that this activity is dispensable for both load-bearing attachment formation and checkpoint activation but plays a role in checkpoint silencing at the kinetochore. Perturbation of both microtubule binding and protein phosphatase 1 docking at the KNL-1 N terminus additively affected checkpoint silencing, indicating that, despite their proximity in KNL-1, these two activities make independent contributions. We propose that microtubule binding by KNL-1 functions in checkpoint silencing by sensing microtubules attached to kinetochores and relaying their presence to eliminate generation of the checkpoint signal.     

Activating and silencing the mitotic checkpoint through CENP-E-dependent activation/inactivation of BubR1

The mitotic checkpoint prevents advance to anaphase prior to successful attachment of every centromere/kinetochore to mitotic spindle microtubules. Using purified components and Xenopus egg extracts, the kinetochore-associated microtubule motor CENP-E is now shown to be the activator of the essential checkpoint kinase BubR1. Since kinase activity and the checkpoint are silenced following CENP-E-dependent microtubule attachment in extracts or binding of CENP-E antibodies that do not disrupt CENP-E association with BubR1, CENP-E mediates silencing of BubR1 signaling. Checkpoint signaling requires the normal level of BubR1 containing a functional Mad3 domain implicated in Cdc20 binding, but only a small fraction need be kinase competent. This supports bifunctional roles for BubR1 in the checkpoint: an enzymatic one requiring CENP-E-dependent activation of its kinase activity at kinetochores and a stoichiometric one as a direct inhibitor of Cdc20.     

Recovery from DNA damage checkpoint arrest by PP1-mediated inhibition of Chk1

The G2 DNA damage checkpoint delays mitotic entry via the upregulation of Wee1 kinase and the downregulation of Cdc25 phosphatase by Chk1 kinase, and resultant inhibitory phosphorylation of Cdc2. While checkpoint activation is well understood, little is known about how the checkpoint is switched off to allow cell cycle re-entry. To identify proteins required for checkpoint release, we screened for genes in Schizosaccharomyces pombe that, when overexpressed, result in precocious mitotic entry in the presence of DNA damage. We show that overexpression of the type I protein phosphatase Dis2 sensitises S. pombe cells to DNA damage, causing aberrant mitoses. Dis2 abrogates Chk1 phosphorylation and activation in vivo, and dephosphorylates Chk1 and a phospho-S345 Chk1 peptide in vitro. dis2Delta cells have a prolonged chk1-dependent arrest and a compromised ability to downregulate Chk1 activity for checkpoint release. These effects are specific for the DNA damage checkpoint, because Dis2 has no effect on the chk1-independent response to stalled replication forks. We propose that inactivation of Chk1 by Dis2 allows mitotic entry following repair of DNA damage in the G2-phase.