| Protein phosphorylation is a ubiquitous regulatory mechanism for cellular signal propagation, and the complexity of signaling networks presents a challenge to protein kinase substrate identification. Chemical genetic control of kinase function provides a handle for kinase pathway analysis. Here, we apply this approach to three kinases that function in a signaling network that regulates exit from mitosis in the budding yeast, Saccharomyces cerevisiae. These include the mitogen-activating protein kinase, Cdc15, the nuclear Dbf2-related kinase, Dbf2, and the Polo-like kinase, Cdc5. Each kinase was successfully engineered for selective chemical inhibition in vivo. We found that monospecific pharmacological inhibition of Cdc5 delays anaphase nucleus migration into the bud, revealing a novel Cdc5 function. Additionally, chemical genetic, bioinformatic, and yeast proteomic tools were combined for Cdc5 substrate identification. Systematically chosen candidate Cdc5 substrates were examined for loss of phosphorylation upon cellular Cdc5 inhibition. The identified Cdc5 targets include Spc72, a spindle pole body (SPB) component and microtubule anchor required for nuclear positioning. Spc72 binds Cdc5 in a cell cycle specific manner, and in vivo Cdc5 inhibition prevents mitotic Spc72 phosphorylation. Studies in vitro demonstrate direct Spc72 phosphorylation by Cdc5. Finally, we expanded our knowledge of Cdc5 function at the SPB by examining SPB-localized proteins for presence in a Cdc5 complex. In summary, a chemical genetic approach was used to inhibit three protein kinases from diverse families, which led to a greater understanding of Cdc5 cellular function. |
