Phosphorylation Dependent Regulation of PTP1B by SUMO Modification

SUMO 修饰对 PTP1B 的磷酸化依赖性调节

• 批准号: 7587934
• 负责人: Shu-Chin Jenny Yip
• 金额: $ 5.13万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-28 至 2010-04-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7587934
• 关键词: Affect; Alanine; Animal Model; Attenuated; Biological Assay; Charge; Confocal Microscopy; Consensus; Density Gradient Centrifugation; Diabetes Mellitus; Disease; Ensure; Fluorescence; Genetic Models; Health; Heart Diseases; Human; Insulin; Knowledge; Link; Mass Spectrum Analysis; Mitotic; Modification; Molecular; Mutation; Nuclear Envelope; Obesity; PTPN1 gene; Pathogenesis; Peptides; Personal Satisfaction; Phosphorylation; Physiological; Post-Translational Protein Processing; Protein phosphatase; Proteins; Regulation; Site; Stress; Techniques; Therapeutic; Tyrosine; glucose metabolism; in vivo; insulin signaling; knockout animal; public health relevance

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.

描述（由申请人提供）：PTP1B，一种酪氨酸特异性蛋白磷酸酶，是胰岛素信号传导的关键调节剂。我们最近发现 PTP1B 受到 sumoylation 的调节。这一发现可能会影响我们对糖尿病、肥胖和心脏病等健康问题的理解。 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 苏酰化在胰岛素诱导的葡萄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，我将使用肽微阵列和质谱鉴定 PTP1B 中的主要苏酰化位点，并进一步确定这些位点的突变是否影响 PTP1B 的酶活性。随后在目标 2 中，我将研究磷酸化和苏酰化在 PTP1B 调节中的关系。特定的 Ser352Ala 突变和 PTP1B 的 SUMO 核心共有位点 (psi)KxE 附近的酸性斑块的丙氨酸取代将分别用于分析 PTP1B 中的磷酸化依赖性 (PDSM) 和/或负电荷依赖性 (NDSM) sumoylation。有丝分裂/应激诱导的磷酸化对 PTP1B sumoylation 的影响将通过体内 sumoylation 测定进行评估。最后，在目标 3 中，我假设 PTP1B sumoylation 发生在核膜附近。结合共聚焦显微镜和双分子荧光互补技术，我将确定 PTP1B 苏酰化与其亚细胞定位之间的相关性，并通过密度梯度离心进行生化证实。公共健康相关性 PTP1B sumoylation 的生理意义伴随着其酶活性的降低以及其无法减弱胰岛素信号传导。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进行SUMO修饰调节葡萄糖代谢的认识。这些发现对于减轻糖尿病和肥胖症的负担，并确保公众的身体健康具有潜在的治疗价值。