IPL1 REGULATION OF THE ASE1 AND KIP3 PROTEINS

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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.