This proposal is directed at understanding the requirements for proper chromosome segregation during mitosis. Using a clever screen to detect mutations which lead to increases in ploidy, the PI previously isolated a conditional mutation in the IPL1 gene which has significant sequence homology with a number of kinases implicated in chromosome segregation in higher eucaryotes (e.g., human Aur1, Drosophila """"""""aurora""""""""). He has done a great deal of work characterizing this (and related) genes which is described in the Progress Report. This previous work motivates the work proposed in the current grant, and thus will be briefly reviewed. He has demonstrated that the IPL1 is an essential gene., and that it is required in each cell cycle. Mutants missegregate chromosomes, but do not arrest thus suggesting they have a defect in a mitotic checkpoint. In order to find possible substrates for the putative kinase, he employed a genetic technique: he screened for mutations which were lethal in the presence of tsipl1 mutation at permissive temp (they couldn't lose the wild type IPL1 gene on a URA3 plasmid). This careful search generated 14 mutations; it was initially impossible to classify them into complementation groups because they exhibited unlinked noncomplemention. However, he characterized their phenotypes with respect to chromosome segregation, etc., and cloned 4 of them. They turned out to be: TUB3 and TUB1 (the alpha subunit of tubulin), CIN8 (one of 2 yeast kinesins), and BUB1 (one subunit of a checkpoint kinase). All of these can be related to chromosome segregation, and seem reasonable candidates for target proteins. He isolated two high copy suppressors of tsipl1: one is a dominant negative fragment of the gene GLC7(PP1) that encodes the catalytic subunit of protein phosphatase1;the second was GLC8, a regulator subunit of PP1. The data obtained is consistent with the model that PP1 acts as an antagonist of IPL1, and that GLC8 can act to activate or inhibit PP1 depending on its concentration. All the data presented, and it includes both biochemical assays and genetic tests, is consistent with this hypothesis. For example, a mutation of GLC8 or O/E of GLC7(PP1) lead to a missegregation phenotype similar to that express in the ipl1 mutant. He proposes that IPL1 phosphorylates a target(s) necessary for proper chromosome segregation, that PP1 inactivates the target, and suggests 3 cogent models which will test their interaction and which are be tested in the proposal. All the experiments in this proposal are aimed at understanding the role of the Ilp1 kinase (and to some extent, the PP1 phosphatase) in chromosome segregation fidelity. He proposes to determine the location of the Ilp1 kinase; he suggests that it may be located on microtubules. Since the protein is present at relatively low abundance, he presents several alternative approaches, including in vitro assays to determine association with microtubules. He proposes experiments to examine the checkpoint in ipl1 mutants; these include experiments to investigate kinetochore separation done with Andrew Murray. Extensive experiments are proposed to understand the relationship of the Ilp1 kinase, alpha-tubulin, Cin8 kinesin, and Bub1/3 kinase (to determine if they are the targets). These include direct measurements of the phosphorylation state of the putative targets in WT and ipl1 cells. The PI suggests three different in vivo approaches (with appropriate controls), as well as a in vitro assay for target proteins that give positive results above using purified Ilp1 kinase. He has already partially purified the Ilp1 kinase. A third approach makes use of an altered 2hybrid system. Since the interaction with the Ilp1 kinase with a substrate is likely to be transitory, he proposes making a mutation which, in other related kinases, maintains protein stability but eliminates kinase activity. (In the case of Human Cdk2 kinase, the mutant binds its substrate but cannot phosphorylate it and release it.) He will combine the mutant ipl1 with gal4DB, and fuse the potential targets with the galAc domain. The experiments will also be done with IPL1. He suggests that should the conserved mutation act as proposed, it might be a dominant negative mutation; O/E suppressors of the dom neg ipl1 mutation might identify target genes. A series of experiments are proposed to investigate the relationship between PP1 and Ilp1 kinase. He will ask whether Ilp1 kinase is dephosphorylated by PP1, and if so, whether that event affects its activity. He will determine if Ilp1 phosphorylates the regulatory subunit of PP1 (encoded by GLC8). The precedent from mammalian cells suggests specific residues that may be targets, and further suggests that these might interfere with activation of GLC8 by other kinases. These experiments include techniques analogous to the ones above, and include both in vitro and in vivo approaches. The PI already has purified PP1 and has partially purified Ilp1 kinase. He will also ask, if Ilp1 kinase acts on certain target proteins, whether PP1 dephosphorylates them. The final work proposed is planned for during the last 2 years of the grant. The PI suggests that the other sli mutants isolated may identify target or regulators of IPL1. An alternative explanation would be that the sli mutations lead to an overproduction of PP1. He suggests experiments to test all three hypotheses, as well as the cloning and characterization of the wild type sli genes.
