The purpose of this project is to investigate the role(s) of a set of cellular proteins, which coimmunoprecipitate with polyomavirus middle tumor (T) antigen, in normal growth control and in polynmavirus tumorignenesis. Such knowledge of mechanisms involved in normal and aberrant growth is necessary for an informed approach to the prevention and cure of neoplastic diseases. There are three specific aims designed to accomplish this purpose. First, the middle Tassociated proteins (MTAPs) will be characterized in more detail by a variety of biochemical techniques. Complete tryptic peptide mapping will be employed to analyzed their primary structure. The MTAPs will be identified directly in whole cell NP40 lysates analyzed by twodimensional (2D) gel electrophoresis. Variation in the middle T complexes in different subcellular localizations will be analyzed by subcellular fractionation experiments, and by immunofluorescence experiments using antisera which detect different populations of middle T. To get some preliminary information about the composition and approximate size of the complex(es) middle T forms with the various MTAPs NP40 lysates of labeled cells expressing middle T will be analyzed on sucrose gradients, followed by immunoprecipitation of middle T and 2D gel analysis.
The second aim of this project is to generate and characterize a set of monoclonal antibodies direct against the MTAPs. An in vitro immunization system for making monoclonal antibodies will be used as it requires less immunogen and is effective in making antibodies to conserved epitopes. The MTAPs will be purified by elution from an antimiddle T immunoaffinity column, and in some cases by preparative 2D gels or antiphosphotyrosine immunoaffinity purification. Hybridoma supernatants will be screened by a combination of immuniprecipitation and biochemical assay techniques. Initial characterization of the antibodies will be performed by Western blotting.
The third aim of this project is to utilize the monoclonal antibodies to investigate the function of the MTAPs in normal and middle Ttransformed cells. The tyrosine and phospholipid kinase activities associated with the various individual MTAPs, the composition of the complexes formed by them, the state of their biochemical modification, and their subcellular location will be assayed in different cell growth states and in the presence or absence of wildtype or transformationdefective mutant middle T antigens. In addition, antibody microinjection experiments will be performed to determine if the MTAPs are essential for progression of the cell cycle or for middle Tmediated transformation.
