Isolation and characterization of a dominant-negative protein phosphatase 2A catalytic subunit active site mutant

Reversible protein phosphorylation on serine and threonine residues has been recognized as a major mechanism governing signal transduction and cellular regulation. Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase that has been highly conserved throughout the evolution of eukaryotes. The classical subunit composition of PP2A is a heterotrimeric complex consisting of a catalytic subunit (C), a regulatory subunit (A), and one of several different B regulatory subunits. The C and A subunits are expressed constitutively and ubiquitously as a tightly bound core heterodimer. The A/C complexes associate with different B subunits, which confer substrate specificity. Studies have shown that this enzyme plays a critical role in several diverse processes such as regulation of metabolism, transcription, translation, cell cycle, oncogenic transformation and apoptosis. The work presented here demonstrates the use of a yeast complementation assay to analyze functional complementation of a human protein in S. cerevisiae. Site-directed mutagenesis was used to create two distinct, catalytically impaired C subunit mutants of the human PP2A genes. Expression of the mutant subunit in yeast prevented growth, even in the presence of functional C subunit proteins. This dominant growth defect is consistent with a dominant-interfering mode of action. These mutants were further characterized in a mammalian cell system. The interfering mechanism of the dominant-negative protein enabled questions regarding C subunit regulation to be addressed. This work shows that regulation of the catalytic subunit is a post-transcriptional event that involves a tightly regulated feedback mechanism involving protein turnover. In addition, a slow growth phenotype was observed in the mutant expressing cell lines.