SIGNAL TRANSDUCTION PATHWAYS OF POLYOMA MIDDLE T

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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.