Functional Expression of Human PP2Ac in Yeast Permits the Identification of Novel C-terminal and Dominant-negative Mutant Forms

The protein phosphatase 2A (PP2A) holoenzyme is structurally conserved among eukaryotes. This reflects a conservation of function in vivo because the human catalytic subunit (PP2Ac) functionally replaced the endogenous PP2Ac of Saccharomyces cerevisiae and bound the yeast regulatory PR65/A subunit (...

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Bibliographic Details
Published in:The Journal of biological chemistry 1999-08, Vol.274 (34), p.24038-24046
Main Authors: Evans, David R.H., Myles, Timothy, Hofsteenge, Jan, Hemmings, Brian A.
Format: Article
Language:English
Subjects:
Alleles
Catalytic Domain
Computer Simulation
Humans
Leucine
Mutation
Phosphoprotein Phosphatases - chemistry
Phosphoprotein Phosphatases - physiology
Protein Phosphatase 2
Saccharomyces cerevisiae
Saccharomyces cerevisiae - enzymology
Structure-Activity Relationship
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Summary:The protein phosphatase 2A (PP2A) holoenzyme is structurally conserved among eukaryotes. This reflects a conservation of function in vivo because the human catalytic subunit (PP2Ac) functionally replaced the endogenous PP2Ac of Saccharomyces cerevisiae and bound the yeast regulatory PR65/A subunit (Tpd3p) forming a dimer. Yeast was employed as a novel system for mutagenesis and functional analysis of human PP2Ac, revealing that the invariant C-terminal leucine residue, a site of regulatory methylation, is apparently dispensable for protein function. However, truncated forms of human PP2Ac lacking larger portions of the C terminus exerted a dominant interfering effect, as did several mutant forms containing a substitution mutation. Computer modeling of PP2Ac structure revealed that interfering amino acid substitutions clustered to the active site, and consistently, the PP2Ac-L199P mutant protein was catalytically impaired despite binding Tpd3p. Thus, interfering forms of PP2Ac titrate regulatory subunits and/or substrates into non-productive complexes and will serve as useful tools for studying PP2A function in mammalian cells. The transgenic approach employed here, involving a simple screen for interfering mutants, may be applicable generally to the analysis of structure-function relationships within protein phosphatases and other conserved proteins and demonstrates further the utility of yeast for analyzing gene function.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.274.34.24038