Cited by 65Protein Tyrosine PhosphatasesProtein Kinase Regulation and GTPase SignalingFungal and yeast genetics research

Functional expression of human PP2Ac in yeast permits the identification of novel C-terminal and dominant-negative mutant forms

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Evans, David R.H., Myles, Timothy, Hofsteenge, Jan, Hemmings, Brian A.·1999·Journal of Biological Chemistry

Abstract

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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Crossref · 10.1074/jbc.274.34.24038

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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].”

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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.
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
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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