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

Abstract

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 H118N C 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 alpha4 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.

Identification and Characterization of an Alternatively Spliced Isoform of the Human Protein Phosphatase 2Aα Catalytic Subunit

“…Most probably, we observed a combination of these two factors, which raises an interesting question: could these effects be separated by generating a PP2Ac mutant, which is enzymatically inactive but still is incorporated into heterotrimeric complexes? In a recent work, a PP2Ac active site mutant (H118N) was found to completely lose its interaction with the PR65/A subunit, whereas the interaction with ␣4 remained unchanged (33). This suggests that the regulation of PP2Ac by ␣4 might be so robust that any modification that results in a defective enzyme is readily detected and sequestered in an inactive form.…”