Characterization of a dual specificity phosphatase hYVH1

双特异性磷酸酶 hYVH1 的表征

• 批准号: 6584726
• 负责人: PANAYIOTIS O VACRATSIS
• 金额: $ 1.42万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-06 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6584726
• 关键词: Saccharomyces cerevisiae; X ray crystallography; catalyst; cell growth regulation; enzyme activity; enzyme mechanism; enzyme substrate; fungal proteins; mass spectrometry; mutant; phosphopeptides; phosphorylation; postdoctoral investigator; protein protein interaction; protein purification; protein structure; protein tyrosine phosphatase; serine

DESCRIPTION (provided by applicant): Human YVH1 is a 36 kDa dual specificity phosphatase widely expressed in human tissues. Orthologues of YVH1 are conserved throughout higher eukaryotes from yeast to humans. In addition to its phosphatase domain, YVH1 members posses a C-terminal Zn-finger like domain that shows higher sequence identity than the catalytic domain. Although hYVH1 is able to complement the growth defect caused by disruption of the S. cerevisiae YVH1 gene, its physiological role in human cells is completely unknown. Data presented in this proposal demonstrates for the first time that hYVH1 is phosphorylated in vivo on serine residues. Efforts will be made to identify the hYVH1 in vivo phosphorylation sites using mass spectrometry and phosphopeptide mapping and further analyze the role of the individual phosphorylation events in regulating hYVH1 activities. Also an affinity purification-based approach in conjunction with substrate-trapping mutants and mass spectrometry will be employed to search for hYVH1 substrates and binding partners. Finally, large amounts of hYVH1 can be expressed and purified from bacteria cells. Crystallization experiments will be performed with the aim of obtaining an X-ray structure of hYVH1. It is anticipated that the structure of hYVH1 will reveal important insights into catalysis and the function of the zinc-finger binding domain.

描述（由申请人提供）：人YVH1是一种36 kDa的双特异性磷酸酶，广泛表达于人体组织中。YVH1的同源基因在从酵母到人类的高等真核生物中都是保守的。除了磷酸酶结构域外，YVH1成员还具有c端锌指状结构域，该结构域比催化结构域具有更高的序列同一性。虽然hYVH1能够补充酿酒葡萄球菌YVH1基因破坏引起的生长缺陷，但其在人类细胞中的生理作用是完全未知的。本研究的数据首次证明hYVH1在体内的丝氨酸残基上被磷酸化。我们将利用质谱法和磷酸化肽定位技术确定hYVH1在体内的磷酸化位点，并进一步分析个体磷酸化事件在调节hYVH1活性中的作用。此外，基于亲和纯化的方法，结合底物捕获突变体和质谱法，将用于寻找hYVH1底物和结合伙伴。最后，可以从细菌细胞中大量表达和纯化hYVH1。将进行结晶实验，目的是获得hYVH1的x射线结构。预计hYVH1的结构将揭示锌指结合结构域的催化和功能的重要见解。