The relation between LAR class protein tyrosine phosphates and Semaphorin-Plexin signaling

LAR类蛋白酪氨酸磷酸与Semaphorin-Plexin信号传导的关系

基本信息

批准号：
13670128
负责人：
NAKAMURA Fumio
金额：
$ 2.62万
依托单位：
Yokohama City University, School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670128/
关键词：
Protein tyrosine Phosphatase Semaphorin Plexin Axon guidance Signal Transduction チロシンリン酸化

项目摘要

We have investigated whether LAR class Protein Tyrosine Phosphatase (PTP) is involved in Semaphorin-3A (Sema3A) signaling. In Drosophila, DLAR mutant exhibits 'bypass' phenotype of ISNb motoneuron projection that is observed in Dsemal and DPlexA mutants, suggesting the relation between the PTP and Semaphorin signaling. In mammals, LAR class PTP consists of LAR, PTP delta, and PTP sigma. We expressed a series of truncated mutant of these PTPs in chick dorsal root ganglions and tested Sema3A-induced growth cone collapse. We found that PTP delta mutants attenuated Sema3A signaling. To explore the role of PTP delta in Sema3A signaling and in axon guidance, we performed following experiments:1) Seeking tyrosine phosphorylated proteins interacting with PTP delta and Plexins, 2) Utilizing yeast hybrid system for isolation of PTPdelta substrates, 3) Evaluation of extracellular dornain of PTPdelta, and 4) Screening the genes up-regulated by Sema3A stimulation.1) PTPdelta from mouse embryonic ce … More rebral neurons was co-immunoprecipitated with a 100kDa phosphotyrosine containing protein. The phosphorylation level transiently decreased after Sema3A stimulation. Ectopically expressed Plexin-A4 in rat primary neurons also associated with a 100kDa protein that was rapidly phosphorylated after Sema3A stimulation. The identities of these two 100kDa proteins are currently unknown.2) Among 3X10^7 clones of mouse brain library screened, 47 clones of AIRC were isolated. AIRC is ADE1 and ADE2 enzyme complex that is involved in purine de novo synthesis. GST-pull down assay confirmed the interaction between PTP delta and AIRC.3) A fusion protein of alkaline Phosphatase and PTP delta extracellular domain bound to several specific regions in mouse embryonic brain such as subcortical layer of cerebrum, fascicules retroflex us and oblongata. The protein promoted the outgrowth of embryonic cerebral neurons.4) Using rat brain cDNA arrays, we compared the RNAs from embryonic brain dissociated cultures with or without Sema3A stimulation. We found that PKA regulatory subunit, stannin, and HAR1R-62 were up-regulated by Sema3A. Less

我们研究了 LAR 类蛋白酪氨酸磷酸酶 (PTP) 是否参与 Semaphorin-3A (Sema3A) 信号传导。在果蝇中，DLAR 突变体表现出在 Dsemal 和 DPlexA 突变体中观察到的 ISNb 运动神经元投射的“旁路”表型，这表明了两者之间的关系。 PTP 和信号蛋白信号传导在哺乳动物中，LAR 类 PTP 由 LAR、PTP delta 和 PTP 组成。我们在鸡背根神经节中表达了一系列 PTP 的截短突变体，并测试了 Sema3A 诱导的生长锥塌陷，我们发现 PTP delta 突变体减弱了 Sema3A 信号传导，以探索 PTP delta 在 Sema3A 信号传导和轴突引导中的作用。，我们进行了以下实验：1）寻找与 PTP delta 和 Plexins 相互作用的酪氨酸磷酸化蛋白，2）利用用于分离 PTPdelta 底物的酵母杂交系统，3) 评估 PTPdelta 的细胞外结构，以及 4) 筛选受 Sema3A 刺激上调的基因。1) 来自小鼠胚胎细胞的 PTPdelta 与 100kDa 磷酸酪氨酸共免疫沉淀异位表达的 Plexin-A4 后磷酸化水平短暂降低。在大鼠原代神经元中，也与 Sema3A 刺激后快速磷酸化的 100kDa 蛋白相关。这两种 100kDa 蛋白的身份目前未知。2) 在筛选的 3X10^7 个小鼠脑库克隆中，分离出 47 个 AIRC 克隆。 ADE1 和 ADE2 酶复合物参与嘌呤从头合成，证实了 PTP delta 和 PTP 之间的相互作用。 AIRC.3)碱性磷酸酶和PTP δ胞外结构域的融合蛋白与小鼠胚胎脑的几个特定区域(例如大脑皮层下层、反卷束和扁形)结合。4)使用该蛋白促进胚胎脑神经元的生长。大鼠脑 cDNA 阵列，我们比较了有或没有 Sema3A 刺激的胚胎脑分离培养物的 RNA，我们发现 PKA 调节亚基锡宁，和 HAR1R-62 被 Sema3A Less 上调。