PTP1B, a tyrosine specific protein phosphatase, is a key regulator of insulin signaling. We recently found that PTP1B is regulated by sumoylation. This discovery could have an impact on our understanding of health issues, such as diabetes, obesity, and heart disease. Sumoylation is a post-translational modification, and its dysregulation has been closely linked to pathogenesis of a variety of human disorders by altering the subcellular localization, protein stability, and enzymatic activity of its targeted proteins. To determine the molecular mechanism of PTP1B sumoylation in insulin-induced regulation of glucose metabolism, I will focus on the following 3 specific aims. In aim 1, I will identify the predominant sumoylation sites in PTP1B using peptide microarray as well as mass spectrometry, and further determine whether mutation of these sites affects the enzymatic activity of PTP1B. Subsequently in aim 2, I will examine the relationship between the phosphorylation and the sumoylation in regulation of PTP1B. Specific Ser352Ala mutation, and alanine substitution of acidic patches proximal to the SUMO core consensus sites, (psi)KxE, of PTP1B will be utilized for analysis of phosphorylation-dependent (PDSM) and/or negative-charge-dependent (NDSM) sumoylation in PTP1B, respectively. The effect of mitotic/stress-induced phosphorylation in PTP1B sumoylation will be evaluated by an in vivo sumoylation assay. Finally in aim 3, I hypothesize that PTP1B sumoylation occurs in the proximity of nuclear envelope. Using a combination of confocal microscopy and bimolecular fluorescence complementation techniques, I will determine the correlation between PTP1B sumoylation and its subcellular localization, which will be confirmed biochemically by density gradient centrifugation. PUBLIC HEALTH RELEVANCE Physiological significance of PTP1B sumoylation is accompanied by reduction of its enzymatic activity and its inability to attenuate insulin signaling. With the existence of PTP1B knockout animal model, our long term objective is to use genetic models to further advance our knowledge in regulation of glucose metabolism by SUMO modification of PTP1 B. These findings have potential therapeutic value in reducing the burden of diabetes as well as obesity, and ensure the physical well-being of the public.