CLONING AND CHARACTERIZATION OF NEW PROTEIN TYROSINE PHOSPHATASES

新蛋白酪氨酸磷酸酶的克隆和表征

• 批准号: 3774919
• 负责人: T MIKI
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3774919
• 关键词: caseins; complementary DNA; enzyme activity; enzyme mechanism; fibroblast growth factor; fibroblasts; genetic library; growth factor receptors; human genetic material tag; human tissue; keratinocyte; molecular cloning; monoclonal antibody; phosphoprotein phosphatase; phosphorylation; protein kinase; protein sequence; recombinant proteins; serine; site directed mutagenesis; transfection /expression vector; tyrosine; vaccinia virus

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.

使用表达克隆策略，编码人蛋白质的cDNA 分离酪氨酸磷酸酶。 表达激酶结构域的细菌 角化细胞生长因子受体（BEK/成纤维细胞生长因子 受体-2）用噬菌粒中的成纤维细胞cDNA文库感染 原核表达载体，并用单克隆抗- 磷酸酪氨酸抗体 在几个克隆中， 磷酸酪氨酸识别，一个显示磷酸酶活性， 体外激酶。 4.1-β-淀粉酶cDNA编码一个推导的蛋白质， 与牛痘病毒具有有限序列相似性的185个氨基酸 磷酸酶VH 1。 纯化的重组蛋白脱磷酸化 几种活化的生长因子受体以及丝氨酸- 体外磷酸化酪蛋白。 丝氨酸和酪氨酸磷酸酶 活性完全消除了诱变的一个半胱氨酸 在VH 1和VH 1相关的人蛋白（VHR）中保守的残基。 这些 特性表明VHR能够调节细胞内事件 由酪氨酸和丝氨酸磷酸化介导。