REGULATORY MECHANISMS OF PROTEIN TYROSINE PHOSPHATASES

蛋白质酪氨酸磷酸酶的调控机制

• 批准号: 2100544
• 负责人: HARRY CHARBONNEAU
• 金额: $ 10.33万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2100544
• 关键词: Baculoviridae; Sf9 cell line; conformation; enzyme activity; enzyme induction /repression; enzyme mechanism; enzyme structure; enzyme substrate; fluorescence microscopy; human genetic material tag; immunofluorescence technique; immunoprecipitation; isozymes; laboratory rabbit; mass spectrometry; membrane activity; northern blottings; phosphoprotein phosphatase; phosphorylation; protein engineering; protein sequence; proteolysis; site directed mutagenesis; virus protein; virus virus interaction

The steady state level of protein tyrosine phosphorylation is dependent upon the relative activities of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases. Regulation of PTP activity constitutes an important mechanism for controlling cellular functions which are mediated by protein tyrosine phosphorylation such as growth and development. Despite recent progress in identifying new PTPs, relatively little is known about their substrates, regulation, and cellular functions. The broad objective of these proposed studies is to elucidate the regulatory mechanisms that control the biological activities of PTPs. We will investigate the function and regulation of two nonreceptor enzymes, the T-cell PTP and a distinct baculoviral PTP. The substrate preference and intracellular localization of the T-cell PTP are regulated by its C- terminal domain. Using in vitro mutagenesis, we will determine the location of specific sites within the C-terminal domain that act to regulate substrate specificity. Immunofluorescence will be used to determine the precise intracellular site where the T-cell PTP is localized. Mutagenesis of a hydrophobic, C-terminal segment will be used to determine whether it is responsible for localization of the enzyme. We have identified a PTP (BVP)that is encoded by a baculovirus, Autographa californica. BVP and two cellular homologs comprise a new subfamily of enzymes. We will investigate the function, mechanism of regulation, and substrates of the BVP by analyzing its role in viral function. It is important to determine whether mammalian forms of this subfamily exist and if so, to define their role in controlling tyrosine phosphorylation. We have evidence for the existence of a human homolog and will isolate a cDNA clone encoding it so that it can be expressed and characterized. Aberrant tyrosine phosphorylation resulting from the unregulated activity of oncogenic tyrosine kinases is thought to be an important event leading to cellular transformation. Knowledge of PTP regulation is crucial to understanding how the delicate balance between protein tyrosine kinases and PTPs is maintained during both oncogenesis and normal cell growth. Information gained about the function and regulation of PTPs may lead to the development of methods for manipulating the level of tyrosine phosphorylation that may have application in research and treatment of diseases such as cancer.

蛋白质酪氨酸磷酸化的稳态水平取决于 蛋白质酪氨酸磷酸酶（PTP）和 蛋白酪氨酸激酶。 PTP活动的调节构成 控制介导的细胞功能的重要机制 通过蛋白质酪氨酸磷酸化，例如生长和发育。 尽管最近在识别新的PTP方面取得了进展，但鲜为人知 关于它们的底物，调节和细胞功能。 这些提出的研究的广泛目标是阐明 控制PTP的生物学活性的调节机制。 我们 将研究两种非受体酶的功能和调节， T细胞PTP和独特的细菌病毒PTP。 底物偏好 T细胞PTP的细胞内定位受其c-调节 终端域。 使用体外诱变，我们将确定 C末端域内特定站点的位置 调节底物特异性。 免疫荧光将用于 确定T细胞PTP所定位的精确细胞内部位。 疏水，C末端段的诱变将用于确定 它是否负责酶的定位。 我们已经确定了由杆状病毒签名编码的PTP（BVP） 加州。 BVP和两个细胞同源物包括一个新的亚科 酶。 我们将研究调节的功能，机制和 BVP的底物通过分析其在病毒功能中的作用。 这是 确定是否存在哺乳动物形式，并且 如果是这样，则定义它们在控制酪氨酸磷酸化中的作用。 我们 有证据表明存在人类同源物，并将分离cDNA 克隆编码它，以便可以表达和表征。 不受管制的活性导致异常的酪氨酸磷酸化 肿瘤酪氨酸激酶被认为是一个重要事件 到细胞转化。 对PTP调节的了解对于 了解蛋白质酪氨酸激酶和 在肿瘤发生和正常细胞生长期间，PTP均维持。 获得有关PTP的功能和调节的信息可能会导致 开发操纵酪氨酸水平的方法 磷酸化可能在研究和治疗中应用 癌症等疾病。