Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling.
Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling.
复制标题
DOI：
10.7554/elife.01030
复制
发表时间：
2013-09-24
期刊：
影响因子：
7.7
通讯作者：
Musacchio A
中科院分区：
文献类型：
--
作者：
Primorac I;Weir JR;Chiroli E;Gross F;Hoffmann I;van Gerwen S;Ciliberto A;Musacchio A
在信号网络中，通过磷酸化来调节大分子相互作用是至关重要的。在纺锤体组装检查点(SAC)，它实现了无错误的染色体分离，磷酸化促进了SAC蛋白向无张力动点的招募。SAC激酶Mps1磷酸化动粒亚基Spc105/Knl1上的多个Met-Glu-Leu-Thr(Met-Glu-Leu-Thr，Met-Glu-Leu-Thr，熔融)基序。然后，磷酸化的熔融基序(MELTP)促进下游信号成分的招募。MELTP基序是如何识别的尚不清楚。在这项研究中，我们报告了7叶片β螺旋桨Bub3是MELTP阅读器。它包含一个非常保守的界面，以一种以前未知的结合模式将MELTP序列对接在β螺旋桨的一侧。针对Bub3界面的突变阻止了SAC激酶Bub1的动粒募集。关键的是，它们也会导致检查点缺陷，表明Bub3识别磷酸化的目标是检查点信号所必需的。我们的数据首次提供了关于磷酸化如何促进检查点蛋白招募到动素中枢的详细机制洞察。DOI：http://dx.doi.org/10.7554/eLife.01030.001细胞周期是一个细胞分裂产生两个几乎相同的子细胞的过程。细胞周期的两个关键部分是原始细胞中染色体的复制，以及这些染色体在两个子细胞之间的分离。这些和细胞周期的其他部分都受到严格的监管，以防止错误，错误可能导致癌症和其他疾病。在染色体复制发生后，被称为姐妹染色单体的一模一样的染色体对仍然彼此紧密相连。这些姐妹染色单体排列在细胞中央，由称为微管的蛋白质细丝将它们连接到称为纺锤体的双极结构。为了使细胞正确分裂，每对姐妹染色单体必须连接到纺锤体的相反两极。一个称为纺锤体组件检查点(SAC)的信号网络确保姐妹染色单体有足够的时间正确排列并纠正可能的问题。一旦一切就位，SAC就会释放它的“断裂”，然后微管就会将姐妹染色单体拉离彼此。通过这种方式，每个子细胞都会收到与母细胞相同的染色体。微管并不直接附着在姐妹染色单体上，而是与聚集在每个姐妹染色单体上的称为动点的蛋白质复合体相连。具体地说，每个微管与一个非常大的蛋白质复合体结合，称为KMN网络。Knl1是这个网络的一部分，它招募两个SAC蛋白-Bub1和Bub3-到动粒。众所周知，当SAC活性时，磷酸基团被添加到KnL1，并且KnL1只有在其被磷酸化后才能招募Bub1和Bub3。然而，Knl1、Bub1和Bub3之间相互作用的细节尚不清楚，也不清楚这些相互作用是否对SAC至关重要。现在，Primorac等人。已有研究表明，Bub3通过包含多个熔融基序(其中M、E、L和T都是氨基酸)的区域直接与KN11结合，并且只有当这些“熔融重复序列”被磷酸化时，这种相互作用才会发生。此外，一旦被绑定到Knl1，Bub3就会将Bub1招募到动毛核。这些结果表明，Bub1-Bub3复合体对磷酸化Knl1的识别在纺锤体组装检查点中起着核心作用，这些结果强调了磷酸化作为调节细胞周期中事件时序的一种方式的重要性。DOI：http://dx.doi.org/10.7554/eLife.01030.002
Regulation of macromolecular interactions by phosphorylation is crucial in signaling networks. In the spindle assembly checkpoint (SAC), which enables errorless chromosome segregation, phosphorylation promotes recruitment of SAC proteins to tensionless kinetochores. The SAC kinase Mps1 phosphorylates multiple Met-Glu-Leu-Thr (MELT) motifs on the kinetochore subunit Spc105/Knl1. The phosphorylated MELT motifs (MELTP) then promote recruitment of downstream signaling components. How MELTP motifs are recognized is unclear. In this study, we report that Bub3, a 7-bladed β-propeller, is the MELTP reader. It contains an exceptionally well-conserved interface that docks the MELTP sequence on the side of the β-propeller in a previously unknown binding mode. Mutations targeting the Bub3 interface prevent kinetochore recruitment of the SAC kinase Bub1. Crucially, they also cause a checkpoint defect, showing that recognition of phosphorylated targets by Bub3 is required for checkpoint signaling. Our data provide the first detailed mechanistic insight into how phosphorylation promotes recruitment of checkpoint proteins to kinetochores. DOI: http://dx.doi.org/10.7554/eLife.01030.001 The cell cycle is the process by which a cell divides to produce two near-identical daughter cells. Two crucial parts of the cell cycle are the duplication of the chromosomes in the original cell, and the segregation of these chromosomes between the two daughter cells. These and other parts of the cell cycle are strictly regulated to prevent errors, which can lead to cancer and other diseases. After chromosome duplication has taken place, the pairs of identical chromosomes, known as sister chromatids, remain tightly bound to each other. These sister chromatids line up in the middle of the cell, with protein filaments called microtubules connecting them to a bipolar structure called the spindle. For the cell to divide correctly, the sister chromatids in each pair must be connected to opposite poles of the spindle. A signalling network known as the spindle assembly checkpoint (SAC) ensures that the sister chromatids have enough time to line up correctly and to correct possible problems. Once everything is in place, the SAC releases its ‘break’, and the microtubules then pull the sister chromatids away from each other. This way, each daughter cell receives the same complement of chromosomes that was present in the mother cell. The microtubules are not directly attached to the sister chromatids but to protein complexes called kinetochores that assemble on each sister chromatid. In particular, each microtubule binds to a very large protein complex called the KMN network. Knl1, which is part of this network, recruits two SAC proteins–Bub1 and Bub3–to the kinetochore. It is known that a phosphate group is added to Knl1 when the SAC is active, and that Knl1 can only recruit Bub1 and Bub3 after it has been phosphorylated. However, the details of the interactions between Knl1, Bub1 and Bub3 are not understood, and it is not clear whether these interactions are essential for the SAC. Now Primorac et al. have shown that Bub3 binds directly to Knl1 through a region that contains multiple MELT motifs (where M, E, L and T are all amino acids), and that this interaction only happens if these ‘MELT repeats’ have been phosphorylated. Moreover, once bound to the Knl1, Bub3 then recruits Bub1 to the kinetochore. By showing that the recognition of phosphorylated Knl1 by the Bub1-Bub3 complex has a central role in the spindle assembly checkpoint, these results highlight the importance of phosphorylation as a way of regulating the timing of events during the cell cycle. DOI: http://dx.doi.org/10.7554/eLife.01030.002