Post-translational carboxyl methylation of protein phosphatase 2A: A therapeutic target

Protein phosphatase 2A (PP2A) is one of the major protein serine/thereonine phosphatases in eukaryotes. The holoenzyme contains a catalytic subunit (C), a scaffold subunit (A) and one of a variety of regulatory (B) subunits. Reversible carboxyl-methylation at the C-terminal leucine of PP2A C subunit is an important post-translational modification, which controls the association of B subunits and consequently the specificities and activities of PP2A. Down-regulation of PP2A methylation has been proposed to correlate with some diseases.;This dissertation focuses mainly on the characterization of the kinetic properties of the methylating (MTase) and demethylating (MEase) enzymes of PP2A under different conditions and the identification of factors that can affect PP2A methylation both in vitro and in vivo . The Km values for the substrates of MTase and MEase are determined. The active sites of both enzymes are identified by mutagenesis analysis in collaboration with Shi's Group (Molecular Biology Department, Princeton University). Biochemical assays reveal that some regulatory subunits impose inhibitory effects to the methylation reaction, whereas some other subunit can protect PP2A from demethylation. This suggests that the methylation may be subject to further regulations even with the fact that itself is already a regulatory mechanism for PP2A. Although S-adenosyl-homocysteine (SAH) is proved as a competitive inhibitor of methylation by the kinetic studies, the absolute methylation levels remain unchanged in the mouse hearts with induced high levels of SAH and homocysteine. Instead, the population of the demethylated AC dimer is found increased, accompanied with an elevation of the total PP2A level. Hence, there may be a compensatory mechanism that maintains PP2A methylation level, and the observed increase of demethylated PP2A could result in irregular cellular activities related with cardiovascular diseases. Recognized as a potential therapeutic target, PP2A methylation system has been used to screen small-molecule inhibitors for MEase. Tocopherol is found to inhibit both methylation and demethylation using pure proteins. Structure-activity relationship studies of tocopherol-PP2A system have been performed in close collaboration with Semmelhack's Group (Chemistry Department, Princeton University), and several essential structure moieties on tocopherol for the interaction are identified. All results from the biochemical studies and animal models have provided improved understanding of the PP2A methylation system, and support the concept that it is an important aspect of PP2A regulation.