Phosphorylation of Sli15 by Ipl1 is important for proper CPC localization and chromosome stability in Saccharomyces cerevisiae.
Phosphorylation of Sli15 by Ipl1 is important for proper CPC localization and chromosome stability in Saccharomyces cerevisiae.
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DOI：
10.1371/journal.pone.0089399
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Stark MJ
中科院分区：
文献类型：
--
作者：
Makrantoni V;Corbishley SJ;Rachidi N;Morrice NA;Robinson DA;Stark MJ
染色体过客复合物（CPC）是真核细胞分裂的关键调节因子，由蛋白激酶Aurora B/Ipl 1及其激活剂（INCENP/Sli 15）和两种另外的蛋白（Survivin/Bir 1和Borealin/Nbl 1）组成。在这里，我们已经确定了多个网站的CPC自磷酸化酵母Sli 15位于其中央微管结合域，并检查其磷酸化的功能意义，通过Ipl 1通过这些网站的突变，无论是不可磷酸化的丙氨酸（sli 15 - 20 A）或酸性残基，以模拟组成性磷酸化（sli 15 - 20 D）。这两个突变体sli 15等位基因赋予染色体不稳定性，但这既不是由Sli 15激活Ipl 1激酶的能力的变化介导的，也不是由染色体双向取向效率降低介导的，这是细胞分裂中需要CPC功能的关键过程。相反，我们发现，模仿组成型磷酸化的Sli 15上的Ipl 1磷酸化位点的CPC在中期的离域，而阻止磷酸化的Sli 15上的Ipl 1网站驱动Sli 15过度本地化的有丝分裂纺锤体在前期后期细胞。与这些结果相一致，直接相互作用的Sli 15与微管在体外大大减少，无论是由Ipl 1磷酸化后，或当Ipl 1依赖的磷酸化位点的组成性磷酸化是模仿天冬氨酸或谷氨酸取代。此外，我们发现，模仿Ipl 1磷酸化的Sli 15干扰的“张力检查点”-CPC依赖的机制，通过该机制，细胞激活纺锤体组装检查点，以延迟后期在没有张力的情况下对kinetochore-microtubule附件。因此，Sli 15的Ipl 1依赖性磷酸化抑制了其与微管在体内和体外的关联，并可能负调节张力检查点机制。
The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here we have identified multiple sites of CPC autophosphorylation on yeast Sli15 that are located within its central microtubule-binding domain and examined the functional significance of their phosphorylation by Ipl1 through mutation of these sites, either to non-phosphorylatable alanine (sli15-20A) or to acidic residues to mimic constitutive phosphorylation (sli15-20D). Both mutant sli15 alleles confer chromosome instability, but this is mediated neither by changes in the capacity of Sli15 to activate Ipl1 kinase nor by decreased efficiency of chromosome biorientation, a key process in cell division that requires CPC function. Instead, we find that mimicking constitutive phosphorylation of Sli15 on the Ipl1 phosphorylation sites causes delocalization of the CPC in metaphase, whereas blocking phosphorylation of Sli15 on the Ipl1 sites drives excessive localization of Sli15 to the mitotic spindle in pre-anaphase cells. Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions. Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the ‘tension checkpoint’ – the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments. Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism.
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