Grant: Doctoral: Vanier Canada Graduate Scholarships

The human genome encodes ~21,300 proteins, over 95% of which are subjected to reversible phosphorylation at nearly a million sites by at least 568 protein kinases (PK) and about 156 protein phosphatases (PP). Many phosphorylation sites act as on/off switches in proteins to control their diverse functions. Transcription factors (TFs) represent one of the largest families of targets for PK and PP, and may include over 1600 different proteins that bind to DNA. TFs bind immediately upstream of genes to regulate their abilities to produce their protein products. Only a relatively small number of transcription factors have been well studied and shown to be controlled by phosphorylation. Knowledge of the regulation of the vast majority of TFs remains very limited, because it has been extremely difficult to track the activation or inhibition of specific TFs in response to diverse treatments. Our aim in this project will be to develop and apply methods that will permit tracking of TFs in high-throughput, and establishment of their interactions with PK and PP and target genes in regulatory networks by wet lab and predictive bioinformatics approaches. Dr. Steven Pelech's lab has access to purified preparations of over 360 different PK and over 30 PP. The combination of the microarray technology with the subcellular fractionation of treated human cancer cells will be used to monitor TFs locations and phosphorylation states following treatments with diverse factors, mediators, and (PK/PP) inhibitors. I will also use purified preparations of PK and PP to try to phosphorylate and dephosphorylate TFs. Altered phosphorylation of these TFs will be tracked by differential binding of the phosphorylated TFs to the phosphosite-specific antibodies printed on the antibody microarrays. Immunohistochemistry and subcellular fractionation will be applied to confirm that potential PK-TF and PP-TF pairs of interacting proteins reside in the same locations inside of cells.