IPL1 REGULATION OF THE ASE1 AND KIP3 PROTEINS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。染色体必须准确地分离到子细胞中，以防止基因组不稳定和非整倍性，这是所有肿瘤的一个标志。染色体分离的一个关键调控因子是Ipl1，它是保守的Aurora蛋白激酶家族的出芽酵母同源物。我们发现Ipl1在着丝点双向定位、纺锤体组装和纺锤体拆卸中起作用。尽管Ipl1在染色体分离相关的多个过程中起着关键作用，但其调控这些功能的关键靶点尚不清楚。我们已经确定了两个优秀的候选蛋白，Ipl1可能调节其在纺锤体组装和纺锤体拆卸中的功能。由于Ase1蛋白的过表达抑制纺锤体组装中的ipl1突变表型，我们突变了Ase1中5个ipl1一致磷酸化位点（Ase1- 5a）。引人注目的是，Ase1-5A突变体与ipl1突变体具有相同的纺锤体组装缺陷。综上所述，这些数据强烈表明Ipl1可能磷酸化Ase1以调节纺锤体组装。同样，我们在Kip3马达蛋白中发现了一个单一的Ipl1共识位点突变，表型上显示了Ipl1突变体在纺锤体拆卸中的缺陷。此外，Ipl1在体外可以磷酸化Ase1和Kip3。由于这些研究表明Ase1和Kip3可能是Ipl1的关键靶点，我们建议在体内确定Ase1和Kip3是否在Ipl1的共识位点磷酸化。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ipl1 regulation of the Ase1 and Kip3 proteins Chris Breed, Chitra Kotwaliwale and Sue Biggins Fred Hutchinson Cancer Research Center Chromosomes must accurately segregate to daughter cells to prevent genomic instability and aneuploidy, a hallmark of all tumors. A key regulator of chromosome segregation is Ipl1, the budding yeast homolog of the conserved Aurora protein kinase family. We have found that Ipl1 has roles in kinetochore biorientation, spindle assembly and spindle disassembly. Despite its critical role in multiple processes related to chromosome segregation, the key targets that Ipl1 regulates for these functions are unknown. We have identified two excellent candidate proteins that Ipl1 may regulate to mediate its functions in spindle assembly and spindle disassembly. Because the overexpression of the Ase1 protein suppresses the ipl1 mutant phenotype in spindle assembly, we mutated the five Ipl1 consensus phosphorylation sites in Ase1 (Ase1-5A). Strikingly, the Ase1-5A mutant has the same spindle assembly defects as an ipl1 mutant. Taken together, these data strongly suggest that Ipl1 may phosphorylate Ase1 to regulate spindle assembly. Similarly, we have found a single Ipl1 consensus site mutation in the Kip3 motor protein phenocopies the ipl1 mutant defect in spindle disassembly. In addition, Ipl1 can phosphorylate Ase1 and Kip3 in vitro. Because these studies suggest that Ase1 and Kip3 may be key targets of Ipl1, we propose to determine whether Ase1 and Kip3 are phosphorylated on the Ipl1 consensus sites in vivo.