Phosphorylation Dependent Regulation of PTP1B by SUMO Modification

描述（申请人提供）：PTP1B是酪氨酸特异性蛋白磷酸酶，是胰岛素信号传导的关键调节因子。我们最近发现PTP1B受sumo化调节。这一发现可能会对我们对糖尿病、肥胖和心脏病等健康问题的理解产生影响。Sumoylation是一种翻译后修饰，其失调通过改变其靶蛋白的亚细胞定位、蛋白质稳定性和酶活性，与多种人类疾病的发病机制密切相关。为了确定PTP1B summoylation在胰岛素诱导的糖代谢调节中的分子机制，我将重点关注以下3个具体目标。在目标1中，我将使用肽芯片和质谱技术确定PTP1B中主要的summoylation位点，并进一步确定这些位点的突变是否会影响PTP1B的酶活性。随后在目标2中，我将研究PTP1B调控中磷酸化和sumo化之间的关系。PTP1B的特异性Ser352Ala突变和靠近SUMO核心共识位点（psi）KxE的酸性斑块的丙氨酸替代将分别用于分析PTP1B的磷酸化依赖性（PDSM）和/或负电荷依赖性（NDSM） sumoylation。有丝分裂/应激诱导磷酸化对PTP1B趋近化的影响将通过体内趋近化实验进行评估。最后，在目标3中，我假设PTP1B酰化发生在核膜附近。利用共聚焦显微镜和双分子荧光互补技术的结合，我将确定PTP1B summoylation与其亚细胞定位之间的相关性，并将通过密度梯度离心进行生化证实。PTP1B sumo化的生理意义伴随着其酶活性的降低和无法减弱胰岛素信号。随着PTP1B敲除动物模型的存在，我们的长期目标是利用遗传模型进一步推进我们对PTP1 b的SUMO修饰调控糖代谢的认识。这些发现在减轻糖尿病和肥胖负担方面具有潜在的治疗价值，并确保公众的身体健康。

DESCRIPTION (provided by applicant): 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.