This proposal outlines an integrated set of yeast-based technologies to investigate a kinase signaling network. We detail methodology that enables the investigation of phosphorylation-dependent control of protein-protein interaction profiles. While general in scope, the methods applied in this proposal will focus on the yeast Aurora kinase, Ipl1. Overexpression of the Aurora kinase is established in many cancers and small molecule inhibition results in phenotypes such as apoptosis and mitotic catastrophe resulting in tumor regression. Our integrated methods address the fact that phosphorylation affects protein-protein interactions and hence we will contribute an important factor in building a dynamic interactome map. The dynamic behavior of the cell is most evident during mitosis and dependent on a network of protein-protein interactions with evidence accumulating that the Ipl1 kinase is a master regulator. We hypothesize that phosphorylation sites will specifically modulate protein- protein interactions and these will associate with the various mitotic roles of Ipl1. Our analysis of the Ipl1 signaling network will be conducted by: i) identifying substrates and cellular pathways associated with Ipl1 using optimized methods based on the principle of synthetic dosage lethality, ii) determine the effects of Ipl1- initiated phosphorylation on protein-protein interactions by a phosphomutant scanning yeast two-hybrid method, iii) associate the Ipl1 phosphorylation sites and corresponding protein-protein interactions with mitotic pathways and roles of Ipl1, and iv) test our predictions for anti-cancer targets in a pancreatic cancer cell model based on our synthetic dosage lethality and phosphomutant experiments. The association of phosphomodulated interactions with the role of Ipl1 in orchestrating chromosome segregation will interface with biological and structural studies of the kinetochore and spindle. This proposal addresses a bottleneck in proteomics research - identifying the biological consequence and pathways associated with the thousands of phosphorylation events currently being identified. On a therapeutic level, this research investigates how Aurora kinase misregulation can initiate cell cycle defects such as chromosomal instability.
We are investigating the role of the Aurora kinase protein in cell proliferation and division and the implications this has on cancer. Our goal is to develop technology in the baker's yeast that allows us to understand how this protein, and other related proteins, communicates with the cell division machinery. A better understanding of these complicated protein networks will allow us to find therapies and design better drugs that would preferentially target cancerous cells.
