PROTEIN TYROSINE DEPHOSPHORYLATION & SIGNAL TRANSDUCTION

蛋白质酪氨酸去磷酸化

• 批准号: 3198457
• 负责人: NICHOLAS K TONKS
• 金额: $ 33.01万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1996-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3198457
• 关键词: HeLa cells; biological signal transduction; cell growth regulation; embryo /fetus tissue /cell culture; enzyme inhibitors; enzyme mechanism; enzyme substrate; gel filtration chromatography; gene expression; human tissue; nucleic acid sequence; phosphorylation; polymerase chain reaction; protein structure function; protein tyrosine kinase; site directed mutagenesis

It is now well established that a the phosphorylation of proteins on tyrosyl residues is an essential element in the control of both normal and neoplastic cell growth. This is a reversible process representing a balance between the activity of kinases and phosphates. Considerable research effort has focused on the kinases while the phosphatases have been relatively neglected. Thus, the broad, long term objectives of this research program are to characterize the structure properties and mode of regulation of the protein tyrosine phosphatases (PTPase), of which very little is known at present. This will provide a necessary and complementary perspective from which to achieve an understanding of the role of protein tyrosine phosphorylation in the control of cell function. In reference to the health-relatedness of the project it is anticipated that information pertaining to the structure and regulation of the PTPases will provide indicators to direct the search for potential biochemical causes of pathological conditions such as diabetes and transformation, and suggest means by which they may be countered. Specific Aim 1 deals with the role of the C terminal segment of a low Mr T Cell-PTPase, in modulating its activity; it also addresses the identification and characterization of proteins that blind to, and appear to regulate the activity of the enzyme following expression in BHK cells. Synthetic peptides derived from sequences in the C terminal segment will be tested as inhibitors of a truncated PTPase expressed in Sf9 cells. Co-immunoprecipitation studies and affinity chromatography over columns constructed using the C terminal segment of the PTPase will be used to identify binding proteins. The phosphorylation of PTPase by PTKs in vivo will be investigated, and coprecipitation with the PTKs will be sought as a means to look for phosphorylation on activity. The role of phosphorylation of seryl residues in the TC-PTPase by p34cdc2 in modulating activity in vitro and in vivo will also be studied. In specific Aim 3, the identity and mode of regulation of the PTPase that dephosphorylates p34cdc2 and commits the cell to mitosis will be ascertained. Protein biochemical approaches will be utilized with particular attention paid to the fundamental step towards understanding the control of the cell cycle. Finally, in specific Aim 4, three new PTPase cDNAs that have been identified in HeLa cells by PCR will be sequenced. The proteins will be expressed and characterized at the enzymatic level using site directed mutagenesis to elucidate the significance of some of the structural features.

现在已经很好地确定蛋白质的磷酸化 酪酶残基是控制正常和控制的重要元素 肿瘤细胞生长。 这是一个可逆过程，代表 激酶和磷酸盐活性之间的平衡。 大量 研究工作集中于激酶，而磷酸酶已经 相对忽略。 因此，这是广泛的长期目标 研究计划将表征结构属性和模式 调节蛋白质酪氨酸磷酸酶（PTPase），其中非常 目前知之甚少。 这将提供必要的 互补的观点可以从中了解 蛋白酪氨酸磷酸化在细胞功能控制中的作用。 关于该项目的健康相关性 与PTPases的结构和调节有关的信息 将提供指标，以指导寻找潜在的生化 病理状况的原因，例如糖尿病和转化，以及 建议将它们反驳的手段。 特定目标1处理 低MR T细胞PTPase的C末端段的作用，在调节中 它的活动；它还解决了 视而不见并似乎调节酶活性的蛋白质 在BHK细胞中表达后面。 衍生自的合成肽 C末端段中的序列将作为A的抑制剂进行测试 在SF9细胞中表达的截短PTPase。 共免疫沉淀研究 和使用C端子构建的列的亲和力色谱法 PTPase的片段将用于识别结合蛋白。 这 将研究PTK在体内对PTPase的磷酸化，并研究 将寻求与PTK的共同沉积作为寻找的一种手段 活性的磷酸化。 雪茄残基磷酸化的作用 在P34CDC2的TC-PTPase中，体外和体内调节活性 也将研究。 在特定目标3中，身份和模式 对p34CDC2的pTPase的调节并提交细胞 将确定有丝分裂。 蛋白质生化方法将是 特别关注的是迈向的基本步骤 了解细胞周期的控制。 最后，在特定的目标4中， 通过PCR在HeLa细胞中鉴定出的三个新的PTPase CDNA将会 被测序。 蛋白质将在 使用位点定向诱变来阐明酶促水平 某些结构特征的意义。