Phosphorylation Dependent Regulation of PTP1B by SUMO Modification

描述(申请人提供)：PTP1B是一种酪氨酸特异性蛋白磷酸酶，是胰岛素信号的关键调节因子。我们最近发现，PTP1B受相思甲基化的调节。这一发现可能会对我们对糖尿病、肥胖症和心脏病等健康问题的理解产生影响。苏莫化是一种翻译后修饰，它的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而与多种人类疾病的发病机制密切相关。为了确定PTP1B苏莫化在胰岛素诱导的糖代谢调节中的分子机制，我将重点关注以下三个特定的目的。在目标1中，我将利用多肽芯片和质谱仪鉴定PTP1B中主要的苏莫化位点，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PTP1B的磷酸化和SUMO化在调节中的关系。特异的Ser352Ala突变和PTP1B相扑核心共识位点(Psi)KxE附近酸性斑块的丙氨酸置换将分别用于分析PTP1B的磷酸化依赖(PDSM)和/或负电荷依赖(NDSM)SUM化。有丝分裂/应激诱导的磷酸化在PTP1B总合作用中的作用将通过体内总合作用试验来评估。最后，在目标3中，我假设PTP1B相扑发生在核膜附近。使用共聚焦显微镜和双分子荧光互补技术相结合，我将确定PTP1B苏莫化与其亚细胞定位的相关性，这将通过密度梯度离心法得到生化证实。与公共健康相关的PTP1B总合作用的生理意义伴随着它的酶活性的降低和它不能减弱胰岛素信号。随着PTP1B基因敲除动物模型的存在，我们的长期目标是利用遗传模型通过PTP1B的相扑修饰来进一步提高我们对糖代谢调节的认识。这些发现对于减轻糖尿病和肥胖的负担以及保障公众的身体健康具有潜在的治疗价值。

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.