Sridharan Vinidhra, Park Hyewon, Ryu Hyunju, Azuma Yoshiaki
From the Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045.
From the Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045
J Biol Chem. 2015 Feb 6;290(6):3269-76. doi: 10.1074/jbc.C114.601906. Epub 2015 Jan 6.
Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1-interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated topoisomerase IIα (TopoIIα), a major centromeric small ubiquitin-like modifier (SUMO) substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Further analysis of mitotic chromosomes revealed that PICH localized to the centromere independent of mitotic SUMOylation. Additionally, we found that PICH is modified by SUMO2/3 on mitotic chromosomes and in vitro. PICH SUMOylation is highly dependent on protein inhibitor of activated STAT, PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA binding capability, indicating that SUMOylation might regulate its DNA-dependent ATPase activity. Collectively, our findings suggest a novel SUMO-mediated regulation of the function of PICH at mitotic centromeres.
有丝分裂期的小泛素样修饰蛋白（SUMO）化在真核生物中对染色体的准确分离起着至关重要的作用，尽管其分子机制尚未完全明确。在非洲爪蟾卵提取物实验中，我们发现聚（ADP - 核糖）聚合酶1（PARP1）在有丝分裂期的着丝粒处被SUMO2/3修饰，并且其酶活性可能受SUMO化调控。为了确定有丝分裂期SUMO化的分子机制，我们分析了SUMO化的PARP1特异性结合蛋白。我们鉴定出Polo样激酶1相互作用的检查点解旋酶（PICH）是非洲爪蟾卵提取物中SUMO化PARP1的相互作用伴侣。有趣的是，PICH还与SUMO化的拓扑异构酶IIα（TopoIIα）结合，后者是主要的着丝粒小泛素样修饰蛋白（SUMO）底物。纯化的重组人PICH与SUMO化底物相互作用，表明PICH直接与SUMO相互作用，且这种相互作用在物种间保守。对有丝分裂染色体的进一步分析表明，PICH定位于着丝粒，不依赖于有丝分裂期的SUMO化。此外，我们发现PICH在有丝分裂染色体上以及体外都被SUMO2/3修饰。PICH的SUMO化高度依赖于信号转导和转录激活因子的蛋白抑制剂PIASy，这与其他有丝分裂染色体SUMO底物一致。最后，PICH的SUMO化显著降低了其DNA结合能力，表明SUMO化可能调控其依赖于DNA的ATP酶活性。总的来说，我们的研究结果提示了一种新的SUMO介导的对有丝分裂期着丝粒处PICH功能的调控机制。
