Functional expression of human PP2Ac in yeast permits the identification of novel C-terminal and dominant-negative mutant forms
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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.
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The Cα mutant is catalytically inactive but binds the A subunit normally, and likely interferes with PP2A function by titrating B subunits.
“This Cα mutant is catalytically inactive, while showing normal A subunit binding, and thus presumably interferes with PP2A function through titration of B subunits [52].”
From Physiologic functions of PP2A: Lessons from genetically modified mice (2019) · cites this paper as [52]
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- Authors
- Evans, D. R. H., Myles, T., Hofsteenge, J., & Hemmings, B. A.
- Affiliations
- Friedrich Miescher Institute (CH)
- Corresponding author
- Hemmings, Brian A.
- ORCID
- Evans, David R.H.
- Venue
- Journal of Biological Chemistry
- Publisher
- Elsevier BV
- Published
- 1999-08
- Volume
- 274
- Issue
- 34
- Pages
- 24038-24046
- ISSN
- 0021-9258 (ISSN-L)
- OpenAlex
- W2062228248
- Cited by
- 65
- References
- 43
- Citations by year
- 2026: 1 · 2024: 2 · 2022: 1 · 2021: 1 · 2019: 1 · 2018: 3 · 2017: 3 · 2015: 1 · 2013: 1 · 2012: 5
- FWCI
- 1.84
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Journal of Biological Chemistry (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- National Cancer Institute
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Tyrosine Phosphatases
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
- Keywords
- protein phosphatase 2A, Saccharomyces cerevisiae, dominant-negative mutant, C-terminal truncation
- MeSH terms
- Alleles, Computer Simulation, Humans, Leucine, Mutation, Phosphoprotein Phosphatases, Saccharomyces cerevisiae, Structure-Activity Relationship, Catalytic Domain, Protein Phosphatase 2
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