Sivakumar Sushama, Daum John R, Tipton Aaron R, Rankin Susannah, Gorbsky Gary J
Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104.
Mol Biol Cell. 2014 Mar;25(5):594-605. doi: 10.1091/mbc.E13-07-0421. Epub 2014 Jan 8.
The spindle and kinetochore-associated (Ska) protein complex is a heterotrimeric complex required for timely anaphase onset. The major phenotypes seen after small interfering RNA-mediated depletion of Ska are transient alignment defects followed by metaphase arrest that ultimately results in cohesion fatigue. We find that cells depleted of Ska3 arrest at metaphase with only partial degradation of cyclin B1 and securin. In cells arrested with microtubule drugs, Ska3-depleted cells exhibit slower mitotic exit when the spindle checkpoint is silenced by inhibition of the checkpoint kinase, Mps1, or when cells are forced to exit mitosis downstream of checkpoint silencing by inactivation of Cdk1. These results suggest that in addition to a role in fostering kinetochore-microtubule attachment and chromosome alignment, the Ska complex has functions in promoting anaphase onset. We find that both Ska3 and microtubules promote chromosome association of the anaphase-promoting complex/cyclosome (APC/C). Chromosome-bound APC/C shows significantly stronger ubiquitylation activity than cytoplasmic APC/C. Forced localization of Ska complex to kinetochores, independent of microtubules, results in enhanced accumulation of APC/C on chromosomes and accelerated cyclin B1 degradation during induced mitotic exit. We propose that a Ska-microtubule-kinetochore association promotes APC/C localization to chromosomes, thereby enhancing anaphase onset and mitotic exit.
纺锤体和动粒相关(Ska)蛋白复合体是适时进入后期所必需的异源三聚体复合体。在小干扰RNA介导的Ska缺失后观察到的主要表型是短暂的排列缺陷,随后是中期阻滞,最终导致黏连蛋白疲劳。我们发现,缺失Ska3的细胞在中期停滞,细胞周期蛋白B1和分离酶仅部分降解。在用微管药物阻滞的细胞中,当纺锤体检查点通过抑制检查点激酶Mps1而被沉默时,或者当细胞通过Cdk1失活而在检查点沉默下游被迫退出有丝分裂时,缺失Ska3的细胞表现出较慢的有丝分裂退出。这些结果表明,除了在促进动粒-微管附着和染色体排列中发挥作用外,Ska复合体在促进后期起始中也有功能。我们发现,Ska3和微管都促进后期促进复合体/细胞周期体(APC/C)与染色体的结合。与细胞质中的APC/C相比,与染色体结合的APC/C显示出明显更强的泛素化活性。在不依赖微管的情况下,将Ska复合体强制定位于动粒会导致APC/C在染色体上的积累增加,并在诱导的有丝分裂退出过程中加速细胞周期蛋白B1的降解。我们提出,Ska-微管-动粒的结合促进了APC/C定位于染色体,从而增强后期起始和有丝分裂退出。