SIGNAL TRANSDUCTION PATHWAYS OF POLYOMA MIDDLE T

中型多发性瘤的信号转导途径

基本信息

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
项目类别：
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

项目摘要

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.

该子项目涉及鼠多瘤病毒的主要转化蛋白中间 T (MT)。 MT 引起细胞转化并发出促凋亡和抗凋亡信号。由于 MT 以宿主调控途径为目标，因此此处获得的信息将与了解细胞生长调控广泛相关。多瘤 T 抗原的共同 N 末端代表 DnaJ 结构域。 MT J 通过参与蛋白磷酸酶 2A (PP2A) 的募集在转化中发挥关键作用。 DnaI 蛋白通过与 DnaK 分子伴侣的相互作用参与蛋白质折叠/解折叠、易位和蛋白质复合物的调节。对于 MT，J 结构域以一种新颖的方式发挥作用，PP2A 的募集独立于 DnaK。遗传分析将识别对该 MT 功能重要的特定 J 结构域残基。对中间 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 J 是否执行分子内伴侣功能。 DnaK 的 J 结构域结合对 MT 转化的贡献将在不同的细胞背景中进行测试。其表达受 DnaK 与中 T 相互作用调节的细胞 mRNA 也将被鉴定。 MT 发出凋亡信号。遗传学将此 MT 信号与其 PP2A 结合以及 JNK 激酶的激活联系起来。实验将确定 MT 导致细胞凋亡的细胞靶点以及 MT 激活应激激活的 MAPK 家族成员的机制。 C 末端附近从残基 332 到 347 的富含脯氨酸的延伸对于转化和凋亡信号都很重要。我们将定义该区域的关键残留物。我们还将进行两次混合分析，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 85 kDa 调节亚基的 SH2 结构域与 MT 和其他信号发生器相互作用。在 100 多种蛋白质中发现了 5112 个结构域，代表酪氨酸磷酸化信号传导的主要连接器。将进行 NMR 结构分析，以探究 SH2 特异性的基础，并检查与两种新型配体（双酪氨酸磷酸化肽和磷脂酰肌醇 3,4,5 三磷酸 (PIP3)）的相互作用。 NMR 将提供用于基因实验的线索，以测试 SH2 与这些新型配体相互作用在 PI3-K 信号传导中的重要性。