Characterization of a dual specificity phosphatase hYVH1

描述（由申请人提供）：人YVH 1是一种在人体组织中广泛表达的36 kDa双特异性磷酸酶。YVH 1的直系同源物在从酵母到人类的高等真核生物中是保守的。除了其磷酸酶结构域，YVH 1成员还具有C-末端锌指样结构域，其显示出比催化结构域更高的序列同一性。虽然hYVH 1能够弥补由S.酿酒酵母YVH 1基因，其在人类细胞中的生理作用是完全未知的。 本提案中提供的数据首次证明hYVH 1在体内丝氨酸残基上被磷酸化。 将努力确定hYVH 1在体内磷酸化位点，使用质谱和磷酸肽图谱，并进一步分析的作用，个人的磷酸化事件在调节hYVH 1的活动。 此外，结合底物捕获突变体和质谱法的亲和纯化为基础的方法将被用来寻找hYVH 1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hYVH 1。将进行结晶实验，目的是获得hYVH 1的X射线结构。 预计hYVH 1的结构将揭示锌指结合结构域的催化和功能的重要见解。

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.