PROJECT SUMMARY O-GlcNAc transferase (OGT), an essential human protein, attaches N-acetylglucosamine (GlcNAc) to Ser/Thr residues of proteins in the nucleus and cytoplasm. Dysregulated OGT expression and activity have been linked to insulin resistance, diabetic complications, and cancer, making OGT a possible drug target. Developing approaches to exploit OGT as a target requires understanding its physiological roles and the mechanisms by which it achieves them. Studies to probe OGT's physiological roles have relied heavily on knockdown (KD) or conditional knockout (KO) experiments and the resulting phenotypes have been attributed to loss of O-GlcNAc. However, OGT has a second catalytic activity and also binds cellular proteins that it does not glycosylate; some phenotypes attributed to O-GlcNAc loss may therefore be due to loss of other activities. Previously, the importance of OGT's catalytic and noncatalytic functions could not be assessed because methods to replace endogenous OGT with variants did not exist. We have now established methods to replace OGT and will analyze cells containing specifically altered copies to deconvolute OGT's physiological roles. Growth phenotypes, quantitative proteomics, and biochemistry will be used to address several questions. Is OGT's noncatalytic scaffolding activity a major driver of physiology and what pathways are linked to it? What is the shortest OGT construct that still supports cell survival and what proteins does it glycosylate and bind? How do OGT's substrates and binding partners interact with the TPR domain and are there opportunities to target specific regions of this domain? We will also use a small molecule inhibitor we recently developed in a chemogenomics screen to identify genetic vulnerabilities to loss of O-GlcNAc. In addition to advancing scientific knowledge about one of the most fundamentally important proteins in mammalian biology, the work in this proposal will provide the foundation to guide approaches to exploit OGT as a therapeutic target.