SIGNAL TRANSDUCTION PATHWAYS OF POLYOMA MIDDLE T

中型多发性瘤的信号转导途径

• 批准号: 6575617
• 负责人: BRIAN S SCHAFFHAUSEN
• 金额: $ 22.84万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6575617
• 关键词: Polyomavirus; apoptosis; biological signal transduction; cell transformation; enzyme activity; laboratory mouse; laboratory rabbit; mitogen activated protein kinase; neoplastic transformation; nuclear magnetic resonance spectroscopy; oncoproteins; phosphatidylinositols; phosphorylation; protein binding; protein protein interaction; protein sequence; protein structure function; stress proteins; tissue /cell culture; transforming virus; tumor antigens; virus antigen; virus genetics; virus protein

This subproject concerns middle T (MT), the major transfOrming protein of murine polyomavirus. MT causes cell transformation and sends both pro- and anti-apoptotic signals. Since MT targets host regulatory pathways, the information obtained here will be broadly relevant to understanding cell growth regulation. The common N-termini of the polyoma T antigens represent a DnaJ domain. The MT J plays a critical role in transformation by participating in recruitment of protein phosphatase 2A (PP2A). DnaI proteins are involved in protein foldingi/unfolding, translocation and regulation of protein complexes through interactions with DnaK molecular chaperones. For MT, the J domain functions in a novel manner, with recruitment of PP2A being independent of DnaK. Genetic analysis will identify specific J domain residues important for this MT function. Biochemical investigation of the J domain in middle T-PP2A interaction will determine if J interacts directly with PP2A or whether J performs an intramolecular chaperone function. Contributions of the J domain binding of DnaK to MT transformation will be tested in different cellular backgrounds. Cellular mRNAs whose expression are regulated by interactions of DnaKs with middle T will also be identified. MT sends an apoptotic signal. Genetics connect this MT signal to its PP2A binding and to activation of JNK kinases. Experiments will determine the cellular target of MT that causes apoptosis and the mechanisms by which MT activates members of the stress-activated MAPK family. A proline-rich stretch near the Cterminus from residues 332 to 347 is important for both transformation and the apoptotic signal. We will define critical residues in this region. We will also carry out two hybrid analysis to identify the host protein(s) interacting with this region. The major forms of phosphatidylinositol 3-kinase (PI3-K) interact with MT and other signal generators via SH2 domains of the regulatory 85 kDa subunit. 5112 domains, found in more than 100 proteins, represent major connectors of tyrosine phosphorylation signaling. NMR structural analysis will be performed to probe the basis of SH2 specificity and to examine interactions with two novel ligands, doubly tyrosine phosphorylated peptides and phosphatidylinositol 3,4,5 triphosphate (PIP3). NMR will provide leads to be used for genetic experiments to test the importance of SH2 interactions with these novel ligands in signaling via PI3-K.

该次要注射涉及中间t（MT），这是鼠多瘤病毒的主要转化蛋白。 MT导致细胞转化，并发送促凋亡信号。由于MT目标宿主调节途径，因此这里获得的信息将与理解细胞生长调节广泛相关。多瘤T抗原的常见N末端代表DNAJ结构域。 MT J通过参与募集蛋白质磷酸酶2a（PP2A）而在转化中起关键作用。 DNAi蛋白通过与DNAK分子伴侣的相互作用而参与蛋白质折叠，易位和调节蛋白质复合物。对于MT，J域以新颖的方式起作用，PP2A的募集独立于DNAK。遗传分析将确定对此MT功能重要的特定J域残基。中间T-PP2A相互作用中J结构域的生化研究将确定J是否直接与PP2A相互作用，或者J是否执行分子内伴侣蛋白功能。 DNAK对MT转化的J结构域结合的贡献将在不同的细胞背景中进行测试。还将鉴定出DNAKS与中间T的相互作用调节的细胞mRNA。 MT发送凋亡信号。遗传学将此MT信号连接到其PP2A结合和JNK激酶的激活。实验将确定导致凋亡的MT的细胞靶标和MT激活压力激活MAPK家族成员的机制。从残基332到347附近的Cterminus附近的脯氨酸富含脯氨酸的伸展对于转化和凋亡信号都很重要。我们将定义该地区的关键残留物。我们还将进行两项混合分析，以确定与该区域相互作用的宿主蛋白。磷脂酰肌醇3-激酶（PI3-K）的主要形式通过调节85 kDa亚基的SH2结构域与MT和其他信号发生器相互作用。在100多种蛋白质中发现的5112结构域代表酪氨酸磷酸化信号的主要连接器。 NMR结构分析将进行探测SH2特异性的基础，并检查与两种新型配体的相互作用，即双酪氨酸磷酸化肽和磷脂酰肌醇3,4,5三磷酸（PIP3）。 NMR将提供铅用于基因实验，以测试通过PI3-K在信号传导中与这些新型配体相互作用的重要性。