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Dr. PelloisUnderstanding the post-translational process of protein S-acylation and its function in the context of signal transduction. The primary objective of the proposed research is to contribute to our understanding of the post-translational process of protein S-acylation in the context of the Src family of non-receptor tyrosine kinases (PTKs). Src proteins are involved in many complex signaling pathways. Elevated expression and activity of Src PTKs have been detected in cancer cells and it is widely believed that these proteins contribute to tumour formation, progression to advanced-stage disease, and metastasis. Insight into the function of Src S-acylation has come from recent studies where it was shown that Src PTKs have to be membrane bound to be functional and that S-acylation is required for their proper localization. The challenge now at hand is to determine how the process of S-acylation is regulated and to understand how this regulation is integrated with other cellular events that also influence the activity of Src PTKs. Our initial aims are (1) to monitor in real-time the S-acylation of Src PTKs in live cells and to determine the dynamics of the process, (2) to characterize the regulation of Src proteins by simultaneous detection of their palmitoylation and phosphorylation state, and (3) to identify the enzymes involved in the attachment and cleavage of palmitate to Src proteins. Accordingly, our initial focus will be to develop chemical tools that will include (1) proteins that contain photocleavable lipids that mimic the palmitoyl group in chemical structure and function, (2) a chemical conversion technique to assay protein palmitoylation and phosphorylation on a proteomic scale, (3) photoaffinity probes that are substrates for protein acyl transferases. Our short-term goals will be to validate these tools in both in vitro and in vivo assays. The long-term goal will be to utilize these tools to investigate the biology of the process of S-acylation. |
