Regulation of Protein Dephosphroylation

Mumby, Marc

Abstract

Protein phosphatase 2A (PP2A) is a major form of serine/threonine phosphase in many cell types. Biochemical and genetic evidence have shown that PP2A is involved in a number of cellular signalling pathways, including those controlling cell growth and proliferation. The native enzyme is a heterotrimer composed of a catalytic subunit (C), a scaffold subunit (A), and a regulatory subunit (R). The A and C subunits are ubiquitously expressed and form a common AC core complex. There are multiple families of regulatory subunits that generate a diversity of enzyme forms. Association of different regulatory subunits with the core complex alters enzymatic activity and substrate specificity. Alteration of PP2A activity by protein-protein interactions plays an important role in modulating signal transduction pathways. For example, interaction of DNA tumor virus oncoproteins with the AC core complex is necessary but not sufficient from transformation. Our goals are to identify the repertoire of PP2A regulatory subunits, determine the molecular basis for their interaction with the core enzyme, determine their effects on enzyme activity and specificity, and determine how they control phosphorylation of cellular proteins. Complimentary DNA clones corresponding to different classes and isoforms of PP2A regulatory subunits will be isolated and characterized. The expression and distribution of regulatory subunits in mouse tissues will be determined by Northern analysis, RNase protection, and immunoblotting. Full length cDNAs for the regulatory subunits will be expressed in bacteria or Sf9 cells and assayed for their interaction with PP2A in vitro using reconstitution systems. The in vivo effects of regulatory subunits on the MAP kinase pathway, the AP-l transcription factor, cell proliferation, and the intracellular localization of PP2A will be determined by expressing heterologous subunits in mammalian cells. The structural basis for the control of PP2A activity and specificity by regulatory subunits will be determined. Chemical modification, mutagenesis, and the yeast two-hybrid system will be used to identify sites on the regulatory subunits that interact with the A and C subunits. Novel regulatory subunits and other proteins that interact with PP2A will be identified using the two-hybrid system and affinity chromatography on glutathione S-transferase-A subunit fusion protein columns.