Altering the Holoenzyme Composition and Substrate Specificity of Protein Phosphatase 2A

Abstract

This chapter describes to the process of generating C subunit mutants with altered binding abilities, an introduction and expressing these mutants in mammalian cells, and the characterization in terms of catalytic activity and complex composition. The same methods can be applied for the generation of A subunit mutants with altered binding properties. Most of the intracellular protein phosphatase 2A (PP2A) complexes contain the C subunit and a regulatory A subunit. The heterodimer between the C and A subunit represents the core complex, to which one of several regulatory B-type subunits binds. From the large pool of different B-type subunits, it results in the formation of many heterotrimeric holoenzymes. Thus, the general term PP2A does not stand for a single entity but rather for a number of distinct di- or trimeric PP2A complexes. The chapter discusses detailed protocols routinely used in the lab for expressing C subunit mutants in mammalian cells, and for characterizing the effects of C subunit mutations on both complex formation and phosphatase activity.