A PP2A active site mutant impedes growth and causes misregulation of native catalytic subunit expression |
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Authors: | Lizotte Donna L Blakeslee Joshua J Siryaporn Albert Heath Jeffrey T DeLong Alison |
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Affiliation: | Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island 02912, USA. |
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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. |
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Keywords: | protein phosphatase 2A catalytic site mutant G1 phase delay regulated protein turnover |
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