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

• 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

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（Sema 3A）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，表明PTP和Semaphorin信号传导之间的关系。在哺乳动物中，LAR类PTP由LAR、PTP δ和PTP σ组成。我们在鸡背根神经节中表达了这些PTP的一系列截短突变体，并测试了Sema 3A诱导的生长锥塌陷。我们发现PTP δ突变体减弱了Sema 3A信号传导。为了探讨PTP δ在Sema 3A信号转导和轴突导向中的作用，我们进行了以下实验：1）寻找与PTP δ和丛蛋白相互作用的酪氨酸磷酸化蛋白，2）利用酵母杂交系统分离PTP δ底物，3）评估PTP δ的胞外通道，4）筛选受Sema 3A刺激上调的基因。1）从小鼠胚胎细胞中提取PTP δ，并将其与神经元丛蛋白（Plexins）相互作用。2）利用酵母杂交系统分离PTP δ底物，3）鉴定PTP δ胞外通道，4）筛选受Sema 3A刺激上调的基因。 ...更多信息 用含100 kDa磷酸酪氨酸的蛋白质免疫共沉淀脑神经元。Sema 3A刺激后磷酸化水平短暂下降。在大鼠原代神经元中异位表达的丛蛋白A4也与Sema 3A刺激后迅速磷酸化的100 kDa蛋白相关。这两种100 kDa蛋白质的身份目前尚不清楚。2）在筛选的小鼠脑库的3X 10^7个克隆中，分离出47个AIRC克隆。AIRC是参与嘌呤从头合成的ADE 1和ADE 2酶复合物。3）碱性磷酸酶（ALP）与PTP δ胞外区的融合蛋白结合于小鼠胚胎脑的几个特定区域，如大脑皮质下层、卷缩束和长圆束。该蛋白促进胚胎脑神经元的生长。4）使用大鼠脑cDNA阵列，我们比较了来自有或没有Sema 3A刺激的胚胎脑分离培养物的RNA。我们发现PKA调节亚基，stannin和HAR 1 R-62被Sema 3A上调。少

项目成果

Nakamura F, Goshima Y: "Protein Tyrosine Phosphatase in Axon Guidance. (Japanese, Review)"Cell Technology. 20. 873-881 (2001)

Nakamura F、Goshima Y：“轴突指导中的蛋白质酪氨酸磷酸酶。（日语，评论）”细胞技术。

中村史雄, 五嶋良郎: "神経回路形成とチロシンホスファターゼ"細胞工学. 20. 873-881 (2001)

Fumio Nakamura、Yoshio Goshima：“神经回路形成和酪氨酸磷酸酶”细胞工程 20. 873-881 (2001)。

Sasaki Y, Cheng C, Uchida Y et al.: "Fyn and Cdk5 mediate Semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex"Neuron. 35. 907-920 (2002)

Sasaki Y、Cheng C、Uchida Y 等人：“Fyn 和 Cdk5 介导 Semaphorin-3A 信号传导，该信号参与大脑皮层树突方向的调节”神经元。

Suto F, Murakami Y, Nakamura F, Goshima Y, Fujisawa H: "Identification and characterization of a novel mouse plexin. Plexin-A4."Mechanisms of Development. 120. 385-396 (2003)

Suto F、Murakami Y、Nakamura F、Goshima Y、Fujisawa H：“新型小鼠丛蛋白的鉴定和表征。Plexin-A4。”发展机制。

Suto F, Murakami Y, Nakamura F et al.: "Identification and characterization of a novel mouse plexin, plexin-A4"Mechanisms of Development. 120. 385-396 (2003)

Suto F、Murakami Y、Nakamura F 等人：“新型小鼠丛蛋白 plexin-A4 的鉴定和表征”发育机制。