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Analysis by in vitro mutagenesis of PP2A alpha okadaic acid responsive sequences

S Kaneko1, H Shima, T Amagasa

  • 1Carcinogenesis Division, National Cancer Center Research Institute, Tokyo, Japan.

Biochemical and Biophysical Research Communications
|September 14, 1995
PubMed

Insights

Mutations in protein phosphatase 2A alpha (PP2A alpha) near amino acid 269 confer resistance to okadaic acid. Specific amino acid changes, including cysteine at 269 and surrounding residues, are crucial for PP2A

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Protein phosphatase 2A alpha (PP2A alpha) is a key enzyme in cellular signaling.
  • Okadaic acid is a potent inhibitor of PP2A, widely used to study its function.
  • Previous studies identified a mutation conferring okadaic acid resistance in PP2A alpha.

Purpose of the Study:

  • To investigate the specific amino acid residues in PP2A alpha responsible for okadaic acid sensitivity.
  • To analyze the okadaic acid responsive sequences within PP2A alpha around codon 269.

Main Methods:

  • Site-directed mutagenesis was used to introduce specific amino acid substitutions in PP2A alpha cDNA.
  • Recombinant mutant PP2A alpha proteins were expressed and purified.
  • In vitro phosphatase assays were performed to determine IC50 values for okadaic acid inhibition.

Main Results:

  • Recombinant PP2A alpha mutants Y265F, C266G, Y267G, and C269Y exhibited significant resistance to okadaic acid.
  • The IC50 values for these mutants ranged from 10 to 24 nM, compared to 0.24 nM for wild-type PP2A alpha.
  • These findings indicate that residues Y265, C266, Y267, and C269 are critical for okadaic acid sensitivity.

Conclusions:

  • The sensitivity of PP2A alpha to okadaic acid is dependent on multiple amino acid residues, not solely cysteine at position 269.
  • Amino acids Y265, C266, and Y267, in addition to C269, play essential roles in mediating the inhibitory effect of okadaic acid on PP2A alpha.

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