ANALYSIS OF NOVEL PLECKSTRIN HOMOLOGY DOMAIN

新型 PLECKSTRIN 同源结构域分析

• 批准号: 6293563
• 负责人: EDWARD Y SKOLNIK
• 金额: $ 24.38万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-10 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6293563
• 关键词: B lymphocyte; binding proteins; blood proteins; cell membrane; enzyme inhibitors; gene expression; gene targeting; genetically modified animals; immunoprecipitation; laboratory mouse; phosphatidylinositol 3 kinase; phosphatidylinositols; phosphoproteins; protein protein interaction; protein sequence; protein structure function; protein tyrosine phosphatase; tyrosine; vanadium; yeast two hybrid system

Phosphatidylinositol 3-kinase (PI3K) is an important enzyme in signal transduction pathways. PI3K generates the second messengers PI(3,4)P2 and PI(3,4,5)P3, which mediate responses by binding to pleckstrin homology (PH) domains in downstream signaling molecules. This application proposes to identify the roles of two new 3-phosphoinositides (3-PI) binding proteins that the investigators identified in PI3K signaling. Spiel is a PH domain containing protein that has an N-terminal SH2 and a C-terminal PH domain with a tyrosine (tyr) phosphorylation site located between these domains. Spiel likely functions as an adaptor molecule to couple proteins bound to its SH2 domain and/or phospho-tyrosine to 3-PIs in stimulated cells via its PH domain. The investigators plan: (1) To determine the mechanism whereby expression of Spiel (Y139F) in which the tyr phosphorylation site (Y139) is mutated to alanine enhances activation of AKT in response to pervanadate stimulation, and the physiological stimuli that lead to Spiel tyrosine phosphorylation. The investigators will test whether Spiel interacts via pY139 with proteins that negatively regulate signaling in B cells, determine cellular localization of Spiel in stimulated cells and whether Spiel localizes SHIP/SHP/Cbl to plasma membrane (PM), whether Spiel (Y139F) acts pre- or post- generation of Ptdins (3,4,5) P3 to enhance AKT activation and whether overexpression of Spiel (Y139F) enhances activation of signaling molecules upstream of PI3K and AKT activation. In addition, the investigators propose to identify the upstream signaling system that leads to Spiel tyr phosphorylation. (2) The investigators propose to identify: A) tyr phosphorylated proteins that bind the SH2 domain of Spiel and (B) SH2 domain containing proteins that bind pY139 on Spiel. Once proteins are identified, the investigators will test (C) their in vivo relevance to Spiel signaling and (D) their mode of regulation. (E) The investigators hypothesize that PIP7, a second new PH domain containing protein that the investigators identified that binds 3-PIs with high affinity, functions as an adaptor molecule in cells, and the investigators will identify proteins that bind PIP7 and determine their relevance to PIP7 and PI3 kinase signaling as for Spiel. (3) To determine the role for Spiel in development and identify signaling pathways in which Spiel plays an essential function, the investigators will create mice nullizygous for Spiel. The investigators will analyze Spiel -/- mice by determining whether their B and T cell development is normal; whether they have normal serum Ig and humoral responses; and whether the T and B cells isolated from these animals function normally following stimulation. Spiel -/- cell and cell lines will be used to definitely confirm or refute essential roles for Spiel in mediating the functions identified in previous aims.

磷脂酰肌醇3-激酶（PI 3 K）是一种重要的酶， 信号转导途径PI 3 K产生第二信使P1（3，4）P2 和PI（3，4，5）P3，其通过结合普列克底物蛋白同源物（PH）介导应答 下游信号分子的结构域。本申请建议 鉴定两种新的3-磷酸肌醇（3-PI）结合蛋白的作用， 研究人员在PI 3 K信号中发现的。Spiel是一个PH结构域， 一种具有N-末端SH 2和C-末端PH结构域的蛋白质，该结构域带有酪氨酸 (tyr)磷酸化位点位于这些结构域之间。Spiel可能 作为接头分子，将结合到其SH 2结构域的蛋白质偶联 和/或磷酸酪氨酸通过其PH结构域在刺激的细胞中转化为3-PI。的 研究者计划：（1）确定Spiel表达的机制， 其中tyr磷酸化位点（Y139）突变为丙氨酸 增强AKT对过钒酸盐刺激的响应的活化， 导致Spiel酪氨酸磷酸化的生理刺激。的 研究人员将测试Spiel是否通过pY 139与蛋白质相互作用， 负调节B细胞中的信号传导，确定 刺激细胞中的Spiel以及Spiel是否将SHIP/SHP/Cbl定位于血浆 膜（PM），Spiel（Y139 F）是否在Ptdins产生前或产生后起作用 （3，4，5）P3增强AKT激活和Spiel是否过表达 （Y139 F）增强PI 3 K和AKT上游信号分子的活化 activation.此外，调查人员建议确定上游 导致Spiel tyr磷酸化的信号系统。(2)调查人员 建议鉴定：A）结合SH 2结构域的tyr磷酸化蛋白， Spiel和（B）含有SH 2结构域的蛋白质，其结合Spiel上的pY 139。一旦 蛋白质被鉴定，研究人员将测试（C）他们的体内 与Spiel信号传导的相关性和（D）它们的调节模式。(E)的 研究人员假设PIP 7是第二个新的含有PH结构域的蛋白质 研究人员发现，它与3-PI结合的亲和力很高， 作为细胞中的衔接分子，研究人员将确定 结合PIP 7并确定其与PIP 7和PI 3激酶相关性的蛋白质 就像Spiel一样。(3)确定Spiel在开发中的作用， 确定Spiel发挥重要作用的信号通路， 研究人员将创建Spiel的无效合子小鼠。调查人员将 通过确定Spiel -/-小鼠的B和T细胞发育是否 正常;是否具有正常的血清IG和体液反应;以及是否 从这些动物中分离的T和B细胞在下列条件下正常发挥功能 刺激. Spiel -/-细胞和细胞系将用于明确确认 或反驳Spiel在调解中确定的功能中的重要作用， 以前的目标。

项目成果

Genetic analysis of the myotubularin family of phosphatases in Caenorhabditis elegans.

秀丽隐杆线虫磷酸酶肌管蛋白家族的遗传分析。

• DOI: 10.1074/jbc.m303259200
• 发表时间: 2003
• 期刊: The Journal of biological chemistry
• 作者: Xue,Yingzi;Fares,Hanna;Grant,Barth;Li,Zhai;Rose,AnnM;Clark,ScottG;Skolnik,EdwardY
• 通讯作者: Skolnik,EdwardY