Pallas Lab - Emory University Medical School
4124/4162 Rollins Research Center
The primary purpose of the research in my laboratory is to understand the molecular basis of the control of cell proliferation and of mechanisms by which this control is circumvented in neoplastic cell growth. Our approach has been to investigate the roles of a set of cellular proteins which associate with polyomavirus small and/or middle tumor (T) antigens. Middle T antigen (MT) is capable of transforming a wide variety of cell types to a state of uncontrolled cell proliferation. In some cases MT requires the help of the small T antigen (ST) for full transformation to occur. These proteins appear to exert their effects by associating with and altering the function of cellular proteins that are important players in the control of cell proliferation. The major goals of our work have been to identify and obtain cDNAs for all the associated proteins, to investigate their roles in the control of cell proliferation, and to determine the effect of their interaction with T antigens on these functions.
Most of our recent efforts have focused on one of the proteins that we identified in complex with the T antigens, protein phosphatase 2A (PP2A). Our goal is to understand the roles of PP2A, a multisubunit, multifunctional phosphatase, in normal and T antigen-perturbed cell proliferation. We have been able to raise precipitating antisera directed against the PP2A complex. These antibodies, in combination with mutational analysis, have allowed us to probe the regulation of PP2A subunit assembly and PP2A association with MT. Among the mutants of PP2A that we generated are catalytically inactive mutants. These mutants have been used to study the role of PP2A activity in MT complex assembly and function. In addition to forming complexes with the normal PP2A regulatory subunits, the catalytically inactive mutants complex with several cellular proteins which do not bind active, wild-type PP2A. These proteins represent potential substrates or regulators of PP2A and are being isolated and studied. We have identified the first of these as a PP2A methylesterase, an enzyme which removes a regulatory methyl group from the carboxy-terminus of the PP2A catalytic subunit. The cloning of this methylesterase, the first mammalian protein methylesterase to be cloned, and the production of monoclonal antibodies capable of distinguishing the methylation state of PP2A together have facilitated many new experiments investigating the role of PP2A methylation. Our mutational analyses have also generated a series of mutants that are defective in binding of one or more regulatory subunits and/or in methylation. These mutants are being analyzed in yeast and in mammalian cells to investigate the roles of these different subunits and of methylation in regulating PP2A activity, localization, and the formation of PP2A complexes. To do this we are employing both mammalian and yeast systems and using genetic, biochemical, and cell biological techniques. Finally, we have identified a new family of regulatory subunits that associate with wild-type PP2A. These proteins appear to be scaffolding proteins involved in targeting PP2A to different cellular compartments and are likely involved in integrating PP2A phosphatase activity with the activity of cellular kinases. The study of these targeting subunits is rapidly becoming a major focus of our lab as understanding their roles is key to understanding the regulation of PP2A functions on a molecular level.