CLONING AND CHARACTERIZATION OF NEW PROTEIN TYROSINE PHOSPHATASES

利用表达克隆策略，编码人类蛋白质的cDNA. 分离得到酪氨酸磷酸酶。表达蛋白激酶结构域的细菌 角质形成细胞生长因子受体(BEK/成纤维细胞生长因子 受体-2)被噬菌体中的成纤维细胞cDNA文库感染 原核表达载体，并用抗-HBs单抗进行筛选 磷酸酪氨酸抗体。在几个表现出抗原性降低的克隆中 磷酸酪氨酸识别，一种表现出磷酸酶活性的 试管中的激活剂。该4.1kb的cDNA编码了一个推导的蛋白质 185个氨基酸与痘苗病毒有有限的序列相似性 磷酸酶VH1。纯化的重组蛋白经脱磷处理 几种激活的生长因子受体以及丝氨酸- 体外磷酸化酪蛋白。丝氨酸磷酸酶和酪氨酸磷酸酶 一种半胱氨酸的诱变使活性完全丧失。 VH1和VH1相关的人类蛋白(VHR)中的残基保守。这些 特性表明VHR能够调节细胞内事件 通过酪氨酸和丝氨酸的磷酸化来调节。

Using an expression cloning strategy, cDNA encoding a human protein tyrosine-phosphatase was isolated. Bacteria expressing the kinase domain of the keratinocyte growth factor receptor (bek/fibroblast growth factor receptor-2) were infected with a fibroblast cDNA library in a phagemid prokaryotic expression vector and screened with a monoclonal anti- phosphotyrosine antibody. among several clones showing decreased anti- phosphotyrosine recognition, one displayed phosphatase activity toward the kinase in vitro. The 4.1-kilobase cDNA encoded a deduced protein of 185 amino acids with limited sequence similarity to the vaccinia virus phosphatase VH1. The purified recombinant protein dephosphorylated several activated growth factor receptors as well as serine- phosphorylated casein in vitro. Both serine and tyrosine phosphatase activities were completely abolished by mutagenesis of a single cysteine residue conserved in VH1 and the VH1-related human protein (VHR). These properties suggest that VHR is capable of regulating intracellular events mediated by both tyrosine and serine phosphorylation.