Characterization of a dual specificity phosphatase hYVH1

描述(申请人提供)：人YVH1是一种在人体组织中广泛表达的36 kDa双特异性磷酸酶。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。除了其磷酸酶结构域，YVH1成员还具有一个C-末端的锌指结构域，其序列同源性高于催化结构域。虽然hYVH1能够弥补酿酒酵母YVH1基因突变导致的生长缺陷，但它在人类细胞中的生理作用完全未知。本提案中提供的数据首次证明了hYVH1在体内丝氨酸残基上被磷酸化。我们将努力利用质谱学和磷酸肽图谱来确定hYVH1在体内的磷酸化位点，并进一步分析单个磷酸化事件在调节hYVH1活性中的作用。此外，还将采用基于亲和纯化的方法，结合底物捕获突变体和质谱学来寻找hYVH1底物和结合伙伴。最后，可以从细菌细胞中表达和纯化大量的hYVH1。将进行结晶实验，目的是获得hYVH1的X射线结构。预计hYVH1的结构将揭示重要的催化作用和锌指结合结构域的功能。

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.