RESUMO
Maintenance of chromosomal stability relies on coordination between various processes that are critical for proper chromosome segregation in mitosis. Here we show that monopolar spindle 1 (Mps1) kinase, which is essential for the mitotic checkpoint, also controls correction of improper chromosome attachments. We report that Borealin/DasraB, a member of the complex that regulates the Aurora B kinase, is directly phosphorylated by Mps1 on residues that are crucial for Aurora B activity and chromosome alignment. As a result, cells lacking Mps1 kinase activity fail to efficiently align chromosomes due to impaired Aurora B function at centromeres, leaving improper attachments uncorrected. Strikingly, Borealin/DasraB bearing phosphomimetic mutations restores Aurora B activity and alignment in Mps1-depleted cells. Mps1 thus coordinates attachment error correction and checkpoint signaling, two crucial responses to unproductive chromosome attachments.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Cromossomos Humanos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Alelos , Aurora Quinase B , Aurora Quinases , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Ativação Enzimática , Células HeLa , Humanos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mutação , Fosforilação , Plasmídeos , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Fuso Acromático/metabolismo , TransfecçãoRESUMO
Error-free chromosome segregation depends on timely activation of the multi-subunit E3 ubiquitin ligase APC/C. Activation of the APC/C initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for destruction. The APC/C is the principle target of the mitotic checkpoint, which prevents segregation while chromosomes are unattached to spindle microtubules. We now report the identification and characterization of APC16, a conserved subunit of the APC/C. APC16 was found in association with tandem-affinity-purified mitotic checkpoint complex protein complexes. APC16 is a bona fide subunit of human APC/C: it is present in APC/C complexes throughout the cell cycle, the phenotype of APC16-depleted cells copies depletion of other APC/C subunits, and APC16 is important for APC/C activity towards mitotic substrates. APC16 sequence homologues can be identified in metazoans, but not fungi, by four conserved primary sequence stretches. We provide evidence that the C. elegans gene K10D2.4 and the D. rerio gene zgc:110659 are functional equivalents of human APC16. Our findings show that APC/C is composed of previously undescribed subunits, and raise the question of why metazoan APC/C is molecularly different from unicellular APC/C.
Assuntos
Mitose , Complexos Ubiquitina-Proteína Ligase/genética , Ubiquitina-Proteína Ligases/genética , Ciclossomo-Complexo Promotor de Anáfase , Animais , Caenorhabditis elegans , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/genética , Sequência Conservada/genética , Células HeLa , Humanos , Proteínas Mad2 , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/genética , Proteínas Repressoras/metabolismo , Homologia de Sequência de Aminoácidos , Fuso Acromático/metabolismo , Espectrometria de Massas em Tandem , Complexos Ubiquitina-Proteína Ligase/isolamento & purificação , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Aurora Quinase B , Aurora Quinases , Pontos de Checagem do Ciclo Celular , Sequência Conservada , Cristalografia por Raios X , Proteínas do Citoesqueleto , Evolução Molecular , Células HeLa , Humanos , Microtúbulos/metabolismo , Mitose , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Relação Estrutura-AtividadeRESUMO
Genetic mutations in the mitotic regulatory kinase BUBR1 are associated with the cancer-susceptible disorder mosaic variegated aneuploidy (MVA). In patients with biallelic mutations, a missense mutation pairs with a truncating mutation. Here, we show that cell lines derived from MVA patients with biallelic mutations have an impaired mitotic checkpoint, chromosome alignment defects, and low overall BUBR1 abundance. Ectopic expression of BUBR1 restored mitotic checkpoint activity, proving that BUBR1 dysfunction causes chromosome segregation errors in the patients. Combined analysis of patient cells and functional protein replacement shows that all MVA mutations fall in two distinct classes: those that impose specific defects in checkpoint activity or microtubule attachment and those that lower BUBR1 protein abundance. Low protein abundance is the direct result of the absence of transcripts from truncating mutants combined with high protein turnover of missense mutants. In this group of missense mutants, the amino acid change consistently occurs in or near the BUBR1 kinase domain. Our findings provide a molecular explanation for chromosomal instability in patients with biallelic genetic mutations in BUBR1.