SIGNAL TRANSDUCTION PATHWAYS OF POLYOMA MIDDLE T

这个子项目涉及小鼠多瘤病毒的主要转化蛋白MidT(MT)。MT可引起细胞转化，并同时发出促和抗凋亡信号。由于MT的靶标是宿主调控途径，因此这里获得的信息将广泛地与理解细胞生长调控有关。多瘤T抗原的共同N末端代表一个DNAJ结构域。MT J通过参与蛋白磷酸酶2A(PP2A)的募集，在转化过程中起着关键作用。DNAi蛋白通过与DNAK分子伴侣相互作用，参与蛋白质折叠/去折叠、转位和蛋白质复合体的调节。对于MT，J结构域以一种新的方式发挥作用，PP2A的招募独立于DNAK。遗传分析将确定对MT功能重要的特定J结构域残基。对T-PP2A中间相互作用中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下进行测试。还将鉴定其表达受DNAKs与中间T相互作用调控的细胞mRNAs。MT发出细胞凋亡信号。遗传学将这种MT信号与其PP2A结合和JNK激酶的激活联系在一起。实验将确定导致细胞凋亡的MT的细胞靶点，以及MT激活应激激活的MAPK家族成员的机制。在332-347位氨基酸残基附近的富含Pro的片段对于转化和细胞凋亡信号都是重要的。我们将定义这一地区的关键残留物。我们还将进行两个杂交分析，以确定与该区域相互作用的宿主蛋白(S)。磷脂酰肌醇3-激酶(PI3-K)的主要形式是通过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个结构域是酪氨酸磷酸化信号的主要连接点。核磁共振结构分析将被用来探索SH2特异性的基础，并研究与两个新的配体，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。核磁共振将提供线索，用于基因实验，以测试SH2与这些新配体相互作用在PI3-K信号转导中的重要性。

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.