Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle checkpoint

The spindle assembly checkpoint (SAC) is the major surveillance system that ensures that sister chromatids do not separate until all chromosomes are correctly bioriented during mitosis. Components of the checkpoint include Mad1, Mad2, Mad3 (BubR1), Bub3, and the kinases Bub1, Mph1 (Mps1), and Aurora B. Checkpoint proteins are recruited to kinetochores when individual kinetochores are not bound to spindle microtubules or not under tension. Kinetochore association of Mad2 causes it to undergo a conformational change, which promotes its association to Mad3 and Cdc20 to form the mitotic checkpoint complex (MCC). The MCC inhibits the anaphase-promoting complex/cyclosome (APC/C) until the checkpoint is satisfied. SAC silencing derepresses Cdc20-APC/C activity. This triggers the polyubiquitination of securin and cyclin, which promotes the dissolution of sister chromatid cohesion and mitotic progression. We, and others, recently showed that association of PP1 to the Spc7/Spc105/KNL1 family of kinetochore proteins is necessary to stabilize microtubule-kinetochore attachments and silence the SAC. We now report that phosphorylation of the conserved MELT motifs in Spc7 by Mph1 (Mps1) recruits Bub1 and Bub3 to the kinetochore and that this is required to maintain the SAC signal.     

KNL1: bringing order to the kinetochore

KNL1 is an evolutionarily conserved kinetochore-associated protein essential for accurate chromosome segregation in eukaryotic cells. This large scaffold protein, predicted to be almost entirely unstructured, is involved in diverse mitotic processes including kinetochore assembly, chromosome congression, and mitotic checkpoint signaling. How this kinetochore “hub” coordinates protein–protein interactions spatially and temporally during mitosis to orchestrate these processes is an area of active investigation. Here we summarize the current understanding of KNL1 and discuss possible mechanisms by which this protein actively contributes to multiple aspects of mitotic progression.     

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.     

The hairpin region of Ndc80 is important for the kinetochore recruitment of Mph1/MPS1 in fission yeast

The establishment of proper kinetochore-microtubule attachments facilitates faithful chromosome segregation. Incorrect attachments activate the spindle assembly checkpoint (SAC), which blocks anaphase onset via recruitment of a cohort of SAC components (Mph1/MPS1, Mad1, Mad2, Mad3/BubR1, Bub1 and Bub3) to kinetochores. KNL1, a component of the outer kinetochore KMN network (KNL1/Mis12 complex/Ndc80 complex), acts as a platform for Bub1 and Bub3 localization upon its phosphorylation by Mph1/MPS1. The Ndc80 protein, a major microtubule-binding site, is critical for MPS1 localization to the kinetochores in mammalian cells. Here we characterized the newly isolated mutant ndc80-AK01 in fission yeast, which contains a single point mutation within the hairpin region. This hairpin connects the preceding calponin-homology domain with the coiled-coil region. ndc80-AK01 was hypersensitive to microtubule depolymerizing reagents with no apparent growth defects without drugs. Subsequent analyses indicated that ndc80-AK01 is defective in SAC signaling, as mutant cells proceeded into lethal cell division in the absence of microtubules. Under mitotic arrest conditions, all SAC components (Ark1/Aurora B, Mph1, Bub1, Bub3, Mad3, Mad2 and Mad1) did not localize to the kinetochore. Further genetic analyses indicated that the Ndc80 hairpin region might act as a platform for the kinetochore recruitment of Mph1, which is one of the most upstream SAC components in the hierarchy. Intriguingly, artificial tethering of Mph1 to the kinetochore fully restored checkpoint signaling in ndc80-AK01 cells, further substantiating the notion that Ndc80 is a kinetochore platform for Mph1. The hairpin region of Ndc80, therefore, plays a critical role in kinetochore recruitment of Mph1.     

Regulated targeting of protein phosphatase 1 to the outer kinetochore by KNL1 opposes Aurora B kinase

Regulated interactions between kinetochores and spindle microtubules are essential to maintain genomic stability during chromosome segregation. The Aurora B kinase phosphorylates kinetochore substrates to destabilize kinetochore–microtubule interactions and eliminate incorrect attachments. These substrates must be dephosphorylated to stabilize correct attachments, but how opposing kinase and phosphatase activities are coordinated at the kinetochore is unknown. Here, we demonstrate that a conserved motif in the kinetochore protein KNL1 directly interacts with and targets protein phosphatase 1 (PP1) to the outer kinetochore. PP1 recruitment by KNL1 is required to dephosphorylate Aurora B substrates at kinetochores and stabilize microtubule attachments. PP1 levels at kinetochores are regulated and inversely proportional to local Aurora B activity. Indeed, we demonstrate that phosphorylation of KNL1 by Aurora B disrupts the KNL1–PP1 interaction. In total, our results support a positive feedback mechanism by which Aurora B activity at kinetochores not only targets substrates directly, but also prevents localization of the opposing phosphatase.     

KNL1 and the CENP-H/I/K complex coordinately direct kinetochore assembly in vertebrates

Chromosome segregation during mitosis requires the assembly of a large proteinaceous structure termed the kinetochore. In Caenorhabditis elegans, KNL-1 is required to target multiple outer kinetochore proteins. Here, we demonstrate that the vertebrate KNL1 counterpart is essential for chromosome segregation and is required to localize a subset of outer kinetochore proteins. However, unlike in C. elegans, depletion of vertebrate KNL1 does not abolish kinetochore localization of the microtubule-binding Ndc80 complex. Instead, we show that KNL1 and CENP-K, a subunit of a constitutively centromere-associated complex that is missing from C. elegans, coordinately direct Ndc80 complex localization. Simultaneously reducing both hKNL1 and CENP-K function abolishes all aspects of kinetochore assembly downstream of centromeric chromatin and causes catastrophic chromosome segregation defects. These findings explain discrepancies in kinetochore assembly pathways between different organisms and reveal a surprising plasticity in the assembly mechanism of an essential cell division organelle.     

Structure of a central component of the yeast kinetochore: the Spc24p/Spc25p globular domain

The Ndc80 complex, a kinetochore component conserved from yeast to humans, is essential for proper chromosome alignment and segregation during mitosis. It is an approximately 570 A long, rod-shaped assembly of four proteins--Ndc80p (Hec1), Nuf2p, Spc24p, and Spc25p--with globular regions at either end of a central shaft. The complex bridges from the centromere-proximal inner kinetochore layer at its Spc24/Spc25 globular end to the microtubule binding outer kinetochore layer at its Ndc80/Nuf2 globular end. We report the atomic structures of the Spc24/Spc25 globular domain, determined both by X-ray crystallography at 1.9 A resolution and by NMR. Spc24 and Spc25 fold tightly together into a single globular entity with pseudo-2-fold symmetry. Conserved residues line a common hydrophobic core and the bottom of a cleft, indicating that the functional orthologs from other eukaryotes will have the same structure and suggesting a docking site for components of the inner kinetochore.     

Yeast Mps1p phosphorylates the spindle pole component Spc110p in the N-terminal domain

The yeast spindle pole body (SPB) component Spc110p (Nuf1p) undergoes specific serine/threonine phosphorylation as the mitotic spindle apparatus forms, and this phosphorylation persists until cells enter anaphase. We demonstrate that the dual-specificity kinase Mps1p is essential for the mitosis-specific phosphorylation of Spc110p in vivo and that Mps1p phosphorylates Spc110p in vitro. Phosphopeptides generated by proteolytic cleavage were identified and sequenced by mass spectrometry. Ser(60), Thr(64), and Thr(68) are the major sites in Spc110p phosphorylated by Mps1p in vitro, and alanine substitution at these sites abolishes the mitosis-specific isoform in vivo. This is the first time that phosphorylation sites of an SPB component have been determined, and these are the first sites of Mps1p phosphorylation identified. Alanine substitution for any one of these phosphorylated residues, in conjunction with an alanine substitution at residue Ser(36), is lethal in combination with alleles of SPC97, which encodes a component of the Tub4p complex. Consistent with a specific dysfunction for the alanine substitution mutations, simultaneous mutation of all four serine/threonine residues to aspartate does not confer any defect. Sites of Mps1p phosphorylation and Ser(36) are located within the N-terminal globular domain of Spc110p, which resides at the inner plaque of the SPB and binds the Tub4p complex.     

Bub1 is a fission yeast kinetochore scaffold protein, and is sufficient to recruit other spindle checkpoint proteins to ectopic sites on chromosomes

The spindle checkpoint delays anaphase onset until all chromosomes have attached in a bi-polar manner to the mitotic spindle. Mad and Bub proteins are recruited to unattached kinetochores, and generate diffusible anaphase inhibitors. Checkpoint models propose that Mad1 and Bub1 act as stable kinetochore-bound scaffolds, to enhance recruitment of Mad2 and Mad3/BubR1, but this remains untested for Bub1. Here, fission yeast FRAP experiments confirm that Bub1 stably binds kinetochores, and by tethering Bub1 to telomeres we demonstrate that it is sufficient to recruit anaphase inhibitors in a kinase-independent manner. We propose that the major checkpoint role for Bub1 is as a signalling scaffold.     

DNA damage activates the SAC in an ATM/ATR-dependent manner, independently of the kinetochore

The DNA damage checkpoint and the spindle assembly checkpoint (SAC) are two important regulatory mechanisms that respond to different lesions. The DNA damage checkpoint detects DNA damage, initiates protein kinase cascades, and inhibits the cell cycle. The SAC relies on kinetochore-dependent assembly of protein complexes to inhibit mitosis when chromosomes are detached from the spindle. The two checkpoints are thought to function independently. Here we show that yeast cells lacking the DNA damage checkpoint arrest prior to anaphase in response to low doses of the DNA damaging agent methyl methane sulfonate (MMS). The arrest requires the SAC proteins Mad1, Mad2, Mad3, Bub1, and Bub3 and works through Cdc20 and Pds1 but unlike the normal SAC, does not require a functional kinetochore. Mec1 (ATR) and Tel1 (ATM) are also required, independently of Chk1 and Rad53, suggesting that Mec1 and Tel1 inhibit anaphase in response to DNA damage by utilizing SAC proteins. Our results demonstrate cross-talk between the two checkpoints and suggest that assembling inhibitory complexes of SAC proteins at unattached kinetochores is not obligatory for their inhibitory activity. Furthermore, our results suggest that there are novel, important targets of ATM and ATR for cell cycle regulation.     

Neurabins recruit protein phosphatase-1 and inhibitor-2 to the actin cytoskeleton

Inhibitor-2 (I-2) bound protein phosphatase-1 (PP1) and several PP1-binding proteins from rat brain extracts, including the actin-binding proteins, neurabin I and neurabin II. Neurabins from rat brain lysates were sedimented by I-2 and its structural homologue, I-4. The central domain of both neurabins bound PP1 and I-2, and mutation of a conserved PP1-binding motif abolished neurabin binding to both proteins. Microcystin-LR, a PP1 inhibitor, also attenuated I-2 binding to neurabins. Immunoprecipitation of neurabin I established its association with PP1 and I-2 in HEK293T cells and suggested that PP1 mediated I-2 binding to neurabins. The C terminus of I-2, although not required for PP1 binding, facilitated PP1 recruitment by neurabins, which also targeted I-2 to polymerized F-actin. Mutations that attenuated PP1 binding to I-2 and neurabin I suggested distinct and overlapping sites for these two proteins on the PP1 catalytic subunit. Immunocytochemistry in epithelial cells and cultured hippocampal neurons showed that endogenous neurabin II and I-2 colocalized at actin-rich structures, consistent with the ability of neurabins to target the PP1.I-2 complex to actin cytoskeleton and regulate cell morphology.     

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.     

Pho85 phosphorylates the Glc7 protein phosphatase regulator Glc8 in vivo

The budding yeast Glc7 serine/threonine protein phosphatase-1 is regulated by Glc8, the yeast ortholog of mammalian phosphatase inhibitor-2. In this work, we demonstrated that similarly to inhibitor-2, Glc8 function is regulated by phosphorylation. The cyclin-dependent protein kinase, Pho85, in conjunction with the related cyclins Pcl6 and Pcl7 comprise the major Glc8 kinase in vivo and in vitro. Several glc7 mutations are dependent on the presence of Glc8 for viability. For example, glc7 alleles R121K, R142H, and R198D are lethal in combination with a glc8 deletion. We found that glc7-R121K is lethal in combination with a pho85 deletion. This finding indicates that Pho85 is the sole Glc8 kinase in vivo. Furthermore, glc7-R121K is also lethal when combined with deletions of pcl6, plc7, pcl8, and pcl10, indicating that these related cyclins redundantly activate Pho85 for Glc8 phosphorylation in vivo. In vitro kinase assays and genetic results indicate that Pho85 cyclins Pcl6 and Pcl7 comprise the predominant Glc8 kinase.     

TORC1-regulated protein kinase Npr1 phosphorylates Orm to stimulate complex sphingolipid synthesis

The evolutionarily conserved Orm1 and Orm2 proteins mediate sphingolipid homeostasis. However, the homologous Orm proteins and the signaling pathways modulating their phosphorylation and function are incompletely characterized. Here we demonstrate that inhibition of nutrient-sensitive target of rapamycin complex 1 (TORC1) stimulates Orm phosphorylation and synthesis of complex sphingolipids in Saccharomyces cerevisiae. TORC1 inhibition activates the kinase Npr1 that directly phosphorylates and activates the Orm proteins. Npr1-phosphorylated Orm1 and Orm2 stimulate de novo synthesis of complex sphingolipids downstream of serine palmitoyltransferase. Complex sphingolipids in turn stimulate plasma membrane localization and activity of the nutrient scavenging general amino acid permease 1. Thus activation of Orm and complex sphingolipid synthesis upon TORC1 inhibition is a physiological response to starvation.     

An Mtw1 complex promotes kinetochore biorientation that is monitored by the Ipl1/Aurora protein kinase

Chromosome segregation depends on kinetochore biorientation so that sister kinetochores attach to microtubules from opposite poles and come under tension. The budding yeast Ipl1/Aurora protein kinase allows the absence of tension to activate the spindle checkpoint. We found that checkpoint activation in the mtw1-1 kinetochore mutant requires Ipl1p, suggesting that Mtw1p promotes tension. We isolated mtw1-1 dosage suppressors and identified Dsn1, a kinetochore protein that immunoprecipitates with the Mif2/CENP-C and Cse4/CENP-A proteins, as well as the Mtw1, Nnf1, and Nsl1 kinetochore proteins. mtw1 and dsn1 mutant strains exhibit similar phenotypes, suggesting that Mtw1p and Dsn1p act together. Although mtw1 mutant cells contained unattached chromosomes, attachment was restored by impairing Ipl1p function. These results suggest that mtw1 mutant kinetochores are competent to bind microtubules but Ipl1p generates unattached chromosomes. We therefore propose that an Mtw1 complex is required for kinetochore biorientation that is monitored by the Ipl1p kinase.     

Scm3 is essential to recruit the histone h3 variant cse4 to centromeres and to maintain a functional kinetochore

The kinetochore is a complex multiprotein structure located at centromeres that is essential for proper chromosome segregation. Budding-yeast Cse4 is an essential evolutionarily conserved histone H3 variant recruited to the centromere by an unknown mechanism. We have identified Scm3, an inner kinetochore protein that immunopurifies with Cse4. Scm3 is essential for viability and localizes to all centromeres. Construction of a conditional SCM3 allele reveals that depletion results in metaphase arrest, with duplicated spindle poles, short spindles, and unequal DNA distribution. The metaphase arrest is mediated by the mitotic spindle checkpoint being dependent on Mad1 and the Aurora kinase B homolog Ipl1. Scm3 interacts with both Ndc10 and Cse4 and is essential to establish centromeric chromatin after DNA replication. In addition, Scm3 is required to maintain kinetochore function throughout the cell cycle. We propose a model in which Ndc10/Scm3 binds to centromeric DNA, which is in turn essential for targeting Cse4 to centromeres.     

The kinetochore protein Bub1 participates in the DNA damage response

The DNA damage response (DDR) and the spindle assembly checkpoint (SAC) are two critical mechanisms by which mammalian cells maintain genome stability. There is a growing body of evidence that DDR elements and SAC components crosstalk. Here we report that Bub1 (budding uninhibited by benzimidazoles 1), one of the critical kinetochore proteins essential for SAC, is required for optimal DDRs. We found that knocking-down Bub1 resulted in prolonged H2AX foci and comet tail formation as well as hypersensitivity in response to ionizing radiation (IR). Further, we found that Bub1-mediated Histone H2A Threonine 121 phosphorylation was induced after IR in an ATM-dependent manner. We demonstrated that ATM phosphorylated Bub1 on serine 314 in response to DNA damage in vivo. Finally, we showed that ATM-mediated Bub1 serine 314 phosphorylation was required for IR-induced Bub1 activation and for the optimal DDR. Together, we elucidate the molecular mechanism of DNA damage-induced Bub1 activation and highlight a critical role of Bub1 in DDR.