TYROSINE PHOSPHATASES IN GLOMERULAR CELL INJURY

描述（改编自申请人的摘要）： 本项目旨在明确酪氨酸磷酸酶在肾小球疾病中的作用， 细胞损伤 不同的生长因子和细胞因子被分泌或 在患病的肾小球中表达并作用于肾小球细胞，包括 系膜细胞 通过酪氨酸磷酸化的信号转导是一种 介导这些介质的生物学效应的主要途径。 的 细胞中总酪氨酸磷酸含量的水平取决于 蛋白酪氨酸激酶的平衡活性， 蛋白酪氨酸磷酸酶（PTPases）。 他们研究分子的计划 酪氨酸磷酸化/去磷酸化的机制，决定了 系膜细胞的激活或失活状态。 他们的假设是 沿着酪氨酸激酶，表达和/或 肾小球系膜细胞特异性激活以及其他更普遍存在的 酪氨酸磷酸酶负责肾小球系膜细胞损伤， 肾脏，尤其是肾小球损伤。 他们使用了聚合酶链 基于反应的策略，以分离1.5 kb的独特PTB， 试图克隆全长cDNA。 这种磷酸酶将 在大肠杆菌中表达大肠杆菌中使用质粒表达载体在诱导 条件 将纯化PTK并测试其活性， 表征了 抗肽抗体和抗细菌抗体 产生的PTB将被提高并用于免疫沉淀， 随后在系膜细胞中测定活性。 为了理解这个角色 细胞质和核因子，他们将研究克隆的调控 肾小球系膜细胞特异性PTD 3 cDNA以及另外两种细胞质PTD 3 cDNA， PTPases，PTP 1B和PTP 1C。 生长因子和细胞因子在 将研究调节这些磷酸酶的活性。 的cdna 将编码这些PTPases的细胞转染到系膜细胞中， 探索它们激活或停用必要信号的能力， 系膜细胞功能 系膜细胞的初步研究 过表达PTP 1C表明，这种PTP 1C增强了细胞的增殖， 细胞的潜力。 PTPases对信号转导的调节作用 酶如磷脂酶Cg 1、磷脂酰肌醇3激酶、促分裂原 活化的蛋白激酶和其它酪氨酸磷酸化蛋白质将被 探讨了 他们将研究最近发现的一组细胞质 蛋白质进行酪氨酸磷酸化和功能作为转录 某些靶基因的因子。 这些蛋白质被称为信号 转录因子和转录激活因子（STAT）。 它们是酪氨酸 磷酸化，并显示PDGF刺激STAT 1，也诱导酪氨酸 JAK 1的磷酸化。 使用凝胶迁移率变化分析，他们计划 研究系膜细胞中STAT的其他家族成员的活化， 过表达PTPases。 生长因子如PDGF单独或联合 还将研究与选定的相关细胞因子的组合。 效果 将在PTPs中测定PTPases对STAT和JAK活化的影响。 转染的系膜细胞，使用免疫沉淀和免疫印迹。

DESCRIPTION (Adapted from the Applicant's Abstract): The long-term goal of this project is to define the role of tyrosine phosphatases in glomerular cell injury. Different growth factors and cytokines are secreted or expressed in the diseased glomerulus and act on glomerular cells including mesangial cells. Signal transduction through tyrosine phosphorylation is a major pathway that mediates the biologic effects of these mediators. The level of total tyrosine phosphate content in the cell depends upon the balanced activities of protein tyrosine kinases and recently recognized protein tyrosine phosphatases (PTPases). Their plan to study molecular mechanisms of tyrosine phosphorylation/dephosphorylation that determine the activation or deactivation state of mesangial cells. They hypothesis is that, along with tyrosine kinases, alteration in the expression and/or activation of mesangial cell specific as well as other more ubiquitous tyrosine phosphatases are responsible for mesangial cell injury during renal, especially glomerular injury. They have employed a polymerase chain reaction based strategy to isolate 1.5 kb of a unique PTPase and are attempting to clone the full-length cDNA. This phosphatase will be expressed in E. coli using a plasmid expression vector under inducible conditions. PTPase will be purified and its activity will be tested and characterized. Antipeptide antibodies and antibody against bacterially produced PTPase will be raised and used to immunoprecipitate and subsequently to assay activity in mesangial cells. To understand the role of cytoplasmic and nuclear factors, they will study regulation of cloned mesangial cell specific PTPase cDNA as well as two other cytoplasmic PTPases, PTP1B and PTP1C. Role of growth factors and cytokines in regulating the activity of these phosphatases will be studied. cDNAs encoding for these PTPases will be transfected into mesangial cells and explored for their ability to activate or deactivate signals necessary for mesangial cell function. Preliminary studies in mesangial cells overexpressing PTP1C demonstrate that this PTPase enhances the proliferative potential of the cells. Regulatory effects of PTPases on signal transducing enzymes such as phospholipase Cg1, phosphatidylinositol 3 kinase, mitogen activated protein kinase and other tyrosine phosphorylated proteins will be explored. They will study a recently identified group of cytoplasmic proteins that undergo tyrosine phosphorylation and function as transcription factors for certain target genes. These proteins are called signal transducers and activators of transcription (STATs). They are tyrosine phosphorylated and show that PDGF stimulates STAT1 and also induces tyrosine phosphorylation of JAK1. Using a gel mobility shift assay, they plan to study activation of other family members of STATs in mesangial cells that overexpress PTPases. The effect of growth factors such as PDGF alone or in combination with selected relevant cytokines will also be studied. Effect of PTPases on activation of STATs and JAKs will be determined in PTPase transfected mesangial cells using immunoprecipitation and immunoblotting.