CELL CYCLE 2016, VOL. 15, NO. 10, 1313–1314 http://dx.doi.org/10.1080/15384101.2016.1159888
CELL CYCLE NEWS & VIEWS
Mph1/MPS1 checks in at the kinetochore America Hervas-Aguilar and Jonathan B.A. Millar Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Gibbet Hill, Coventry, UK ARTICLE HISTORY Received 21 February 2016; Accepted 26 February 2016
The kinetochore is the major attachment site between chromosomes and microtubules during mitosis but also acts as the platform for the action of a surveillance system, known as the spindle assembly checkpoint (SAC), which ensures sister chromatids do not separate until all chromosomes are correctly bioriented1. Components of the SAC include the Mad1, Mad2, BubR1 (Mad3) and Bub3 proteins and the Bub1, Mps1 (Mph1) and Aurora-B kinases. When individual kinetochores are not bound to spindle microtubules checkpoint proteins are recruited to the unattached kinetochore and the SAC is activated. Kinetochore association of Mad2 induces a conformational change which allows it to bind Mad3 and Cdc20 to form a potent inhibitor, the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex/cyclosome (APC/C), a specialized E3 ubiquitin ligase. When the checkpoint is satisfied the spindle checkpoint signal is silenced and the Cdc20-APC/C is activated. This triggers the poly-ubiquitination of securin and cyclin, which allows the dissolution of sister chromatid cohesion and mitotic progression1. This is a crucial step during the cell cycle as it ensures accurate segregation of sister chromatids to daughter cells. Genetic and cell biological experiments have established that the recruitment of spindle checkpoint proteins to kinetochores occurs in a hierarchical manner, with the Aurora B and Mps1 kinases as the most upstream elements. Once at the kinetochore Mps1 phosphorylates the threonine residues in multiple repetitive motifs (MELT) in the KNL1 kinetochore protein (also known as BLINKIN, Spc7 and Spc105) to create a binding site for the Bub3:Bub1:BubR1 complex2. Mps1 further phosphorylates the central, non-catalytic region of Bub1 to promote the association of Mad1:Mad2 complex to Bub13. More recently, attention has been focused on how Mps1 interacts with the kinetochores to control these downstream checkpoint signaling events. Crucial to this is the microtubule-binding NDC80 (nuclear division cycle 80) complex (NDC80-C), which consists of HEC1 (highly expressed in cancer 1), NUF2 (nuclear filament–related 2), SPC24 and SPC254. Two recent papers in Science have reported that, in human cells, microtubules compete with Mps1 for binding to NDC80-C to regulate checkpoint signaling5,6. More particularly, the human Mps1 kinase contains 2 domains, an N-terminal extension (NTE) and a middle region
(MR), which interact with the microtubule-binding calponin homology (CH) domains of HEC1 and NUF2, respectively. The NTE and MR regions are separated by a conserved tetratricopeptide repeat domain (TPR). Kinetochore interaction of an MPS1 variant lacking the MR domain is sensitive to inhibition of Aurora B kinase5,6, however it is not clear how this is controlled as interaction of the MR domain with the CH domain of NUF2, but not interaction of the NTE with the CH domain of HEC1, is stimulated by Aurora B mediated phosphorylation of the HEC1 tail. Nevertheless, together these results indicate that interaction of human MPS1 with the kinetochore is regulated both by microtubules and Aurora B kinase, either directly or indirectly. Curiously, the N-terminal region of fission yeast Mps1 kinase (known as Mph1) is shorter than that of human MPS1 and lacks recognizable NTE, TPR and MR domains. Moreover, the N-terminal 150 amino acids of Mph1 are dispensable for kinetochore association. To probe the role of the Ndc80 in checkpoint signaling, Toda and colleagues subjected HEC1 (Ndc80) to random mutagenesis, re-integrated mutants into the genome and selected for cells that were viable, and thus could sustain microtubule interaction with the kinetochore, but sensitive to thiabendazole, a microtubule depolymerizing, a phenotype that is associated with mutants that are defective in spindle checkpoint signaling7. One particular mutant, ndc80AK01, was selected for further study. The authors demonstrate that spindle checkpoint signaling was defective and Mph1, and all other downstream spindle checkpoint proteins including Bub3, Bub1, Mad1 and Mad2, fail to bind the kinetochore in the ndc80-AK01 cells. Importantly, association of these latter proteins and checkpoint signaling were restored when Mph1 was artificially tethered to the kinetochore, indicating that the prime defect of ndc80-AK01 cells is the failure of Mph1 to associate with the Ndc80-AK01 protein. Surprisingly, this mutant protein harbours a leucine to proline amino acid substitution in a hairpin region of Ndc80, which is located immediately Cterminal to the CH domain. There are a number of potential explanations for this observation. Firstly, the binding site of Mph1 to the NDC80-C in yeast may be distinct to the binding site for MPS1 in human cells or, secondly, that the hairpin region provides an additional binding site that is cryptic in human MPS1 or, thirdly, that the structural integrity of the
CONTACT Jonathan B.A. Millar [email protected] Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Gibbet Hill, Coventry CV4 7AL, UK. News and Views to: Chmielewska AE, et al. The hairpin region of Ndc80 is important for the kinetochore recruitment of Mph1/MPS1 in fission yeast. Cell Cycle 2016; 15(5):740-7; PMID: 26900649; http://dx.doi.org/10.1080/15384101.2016.1148842 © 2016 Taylor & Francis
1314
A. HERVAS-AGUILAR AND J. B. A. MILLAR
hairpin region is required for the structural integrity of the CH domain of Ndc80 to which Mph1 binds. However, the latter scenario seems somewhat unlikely given the absence of the NTE and MR regions in Mph1. Importantly, association of Mph1 to kinetochores is also regulated by Aurora B kinase in fission yeast. This suggests that we have much more to learn about how the spindle checkpoint is activated and inactivated at kinetochores and, more particularly, how Mph1/MPS1 activity is controlled by this dynamic association.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
References [1] London N, Biggins S. Nat Rev Mol Cell Biol 2014; 15:736-47; PMID:25303117; http://dx.doi.org/10.1038/nrm3888 [2] Primorac I, et al. Elife 2013; 2:e01030; PMID:24066227; http://dx.doi. org/10.7554/eLife.01030 [3] London N, Biggins S. Genes Dev 2014; 28:140-52; PMID:24402315; http://dx.doi.org/10.1101/gad.233700.113 [4] Martin-Lluesma S, et al. Science 2002; 297:2267-70; PMID:12351790; http://dx.doi.org/10.1126/science.1075596 [5] Hiruma Y, et al. Science 2015; 348:1264-7; PMID:26068855; http://dx. doi.org/10.1126/science.aaa4055 [6] Ji Z, et al. Science 2015; 348:1260-4; PMID:26068854; http://dx.doi. org/10.1126/science.aaa4029 [7] Chmielewska AE, et al. Cell Cycle 2016 ; 15(5):740-7; PMID:26900649; http://dx.doi.org/10.1080/15384101.2016.1148842
CELL CYCLE NEWS & VIEWS
Mph1/MPS1 checks in at the kinetochore America Hervas-Aguilar and Jonathan B.A. Millar Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Gibbet Hill, Coventry, UK ARTICLE HISTORY Received 21 February 2016; Accepted 26 February 2016
The kinetochore is the major attachment site between chromosomes and microtubules during mitosis but also acts as the platform for the action of a surveillance system, known as the spindle assembly checkpoint (SAC), which ensures sister chromatids do not separate until all chromosomes are correctly bioriented1. Components of the SAC include the Mad1, Mad2, BubR1 (Mad3) and Bub3 proteins and the Bub1, Mps1 (Mph1) and Aurora-B kinases. When individual kinetochores are not bound to spindle microtubules checkpoint proteins are recruited to the unattached kinetochore and the SAC is activated. Kinetochore association of Mad2 induces a conformational change which allows it to bind Mad3 and Cdc20 to form a potent inhibitor, the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex/cyclosome (APC/C), a specialized E3 ubiquitin ligase. When the checkpoint is satisfied the spindle checkpoint signal is silenced and the Cdc20-APC/C is activated. This triggers the poly-ubiquitination of securin and cyclin, which allows the dissolution of sister chromatid cohesion and mitotic progression1. This is a crucial step during the cell cycle as it ensures accurate segregation of sister chromatids to daughter cells. Genetic and cell biological experiments have established that the recruitment of spindle checkpoint proteins to kinetochores occurs in a hierarchical manner, with the Aurora B and Mps1 kinases as the most upstream elements. Once at the kinetochore Mps1 phosphorylates the threonine residues in multiple repetitive motifs (MELT) in the KNL1 kinetochore protein (also known as BLINKIN, Spc7 and Spc105) to create a binding site for the Bub3:Bub1:BubR1 complex2. Mps1 further phosphorylates the central, non-catalytic region of Bub1 to promote the association of Mad1:Mad2 complex to Bub13. More recently, attention has been focused on how Mps1 interacts with the kinetochores to control these downstream checkpoint signaling events. Crucial to this is the microtubule-binding NDC80 (nuclear division cycle 80) complex (NDC80-C), which consists of HEC1 (highly expressed in cancer 1), NUF2 (nuclear filament–related 2), SPC24 and SPC254. Two recent papers in Science have reported that, in human cells, microtubules compete with Mps1 for binding to NDC80-C to regulate checkpoint signaling5,6. More particularly, the human Mps1 kinase contains 2 domains, an N-terminal extension (NTE) and a middle region
(MR), which interact with the microtubule-binding calponin homology (CH) domains of HEC1 and NUF2, respectively. The NTE and MR regions are separated by a conserved tetratricopeptide repeat domain (TPR). Kinetochore interaction of an MPS1 variant lacking the MR domain is sensitive to inhibition of Aurora B kinase5,6, however it is not clear how this is controlled as interaction of the MR domain with the CH domain of NUF2, but not interaction of the NTE with the CH domain of HEC1, is stimulated by Aurora B mediated phosphorylation of the HEC1 tail. Nevertheless, together these results indicate that interaction of human MPS1 with the kinetochore is regulated both by microtubules and Aurora B kinase, either directly or indirectly. Curiously, the N-terminal region of fission yeast Mps1 kinase (known as Mph1) is shorter than that of human MPS1 and lacks recognizable NTE, TPR and MR domains. Moreover, the N-terminal 150 amino acids of Mph1 are dispensable for kinetochore association. To probe the role of the Ndc80 in checkpoint signaling, Toda and colleagues subjected HEC1 (Ndc80) to random mutagenesis, re-integrated mutants into the genome and selected for cells that were viable, and thus could sustain microtubule interaction with the kinetochore, but sensitive to thiabendazole, a microtubule depolymerizing, a phenotype that is associated with mutants that are defective in spindle checkpoint signaling7. One particular mutant, ndc80AK01, was selected for further study. The authors demonstrate that spindle checkpoint signaling was defective and Mph1, and all other downstream spindle checkpoint proteins including Bub3, Bub1, Mad1 and Mad2, fail to bind the kinetochore in the ndc80-AK01 cells. Importantly, association of these latter proteins and checkpoint signaling were restored when Mph1 was artificially tethered to the kinetochore, indicating that the prime defect of ndc80-AK01 cells is the failure of Mph1 to associate with the Ndc80-AK01 protein. Surprisingly, this mutant protein harbours a leucine to proline amino acid substitution in a hairpin region of Ndc80, which is located immediately Cterminal to the CH domain. There are a number of potential explanations for this observation. Firstly, the binding site of Mph1 to the NDC80-C in yeast may be distinct to the binding site for MPS1 in human cells or, secondly, that the hairpin region provides an additional binding site that is cryptic in human MPS1 or, thirdly, that the structural integrity of the
CONTACT Jonathan B.A. Millar [email protected] Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Gibbet Hill, Coventry CV4 7AL, UK. News and Views to: Chmielewska AE, et al. The hairpin region of Ndc80 is important for the kinetochore recruitment of Mph1/MPS1 in fission yeast. Cell Cycle 2016; 15(5):740-7; PMID: 26900649; http://dx.doi.org/10.1080/15384101.2016.1148842 © 2016 Taylor & Francis
1314
A. HERVAS-AGUILAR AND J. B. A. MILLAR
hairpin region is required for the structural integrity of the CH domain of Ndc80 to which Mph1 binds. However, the latter scenario seems somewhat unlikely given the absence of the NTE and MR regions in Mph1. Importantly, association of Mph1 to kinetochores is also regulated by Aurora B kinase in fission yeast. This suggests that we have much more to learn about how the spindle checkpoint is activated and inactivated at kinetochores and, more particularly, how Mph1/MPS1 activity is controlled by this dynamic association.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
References [1] London N, Biggins S. Nat Rev Mol Cell Biol 2014; 15:736-47; PMID:25303117; http://dx.doi.org/10.1038/nrm3888 [2] Primorac I, et al. Elife 2013; 2:e01030; PMID:24066227; http://dx.doi. org/10.7554/eLife.01030 [3] London N, Biggins S. Genes Dev 2014; 28:140-52; PMID:24402315; http://dx.doi.org/10.1101/gad.233700.113 [4] Martin-Lluesma S, et al. Science 2002; 297:2267-70; PMID:12351790; http://dx.doi.org/10.1126/science.1075596 [5] Hiruma Y, et al. Science 2015; 348:1264-7; PMID:26068855; http://dx. doi.org/10.1126/science.aaa4055 [6] Ji Z, et al. Science 2015; 348:1260-4; PMID:26068854; http://dx.doi. org/10.1126/science.aaa4029 [7] Chmielewska AE, et al. Cell Cycle 2016 ; 15(5):740-7; PMID:26900649; http://dx.doi.org/10.1080/15384101.2016.1148842
