title: 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.
authors.affiliation: Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island 02912, USA.
authors.forename: Donna L
authors.initials: DL
authors.lastname: Lizotte
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Blakeslee
authors.forename: Albert
authors.initials: A
authors.lastname: Siryaporn
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Heath
authors.forename: Alison
authors.initials: A
authors.lastname: DeLong
mesh_terms: D020134:Catalytic Domain; D002453:Cell Cycle; D002460:Cell Line; D049109:Cell Proliferation; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D009154:Mutation; D054648:Protein Phosphatase 2 / Q000096:biosynthesis* / Q000235:genetics; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics