A PP2A active site mutant impedes growth and causes misregulation of native catalytic subunit expression

Activity of protein phosphatase 2A (PP2A) is tightly regulated and performs a diverse repertoire of cellular functions. Previously we isolated a dominant-negative active site mutant of the PP2A catalytic (C) subunit using a yeast complementation assay. We have established stable fibroblastic cell lines expressing epitope-tagged versions of the wild-type and H118N mutant C subunits and have used these cells to investigate mechanisms that regulate PP2A activity. Cells expressing the mutant C subunit exhibit a decreased growth rate and a prolonged G1 cell cycle phase. The mutant protein is enzymatically inactive, but extracts made from cells expressing the HI18NC subunit show normal levels of total PP2A activity in vitro. The H118N mutant shows reduced binding to the regulatory A subunit, but binds normally to the alpha 4 protein, a non-canonical regulator of PP2A. Expression of the H118N mutant interferes with the normal control of C subunit abundance, causing accumulation of the endogenous wild-type protein as well as the mutant transgene product. Our results indicate that the H118N mutant isoform retards C subunit turnover and suggest that PP2A C subunit turnover may be important for normal cell cycle progression.