Analysis by in vitro mutagenesis of PP2A alpha okadaic acid responsive sequences.
It has been shown that the protein serine/threonine phosphatase type 2A alpha catalytic subunit (PP2A alpha) has a mutation consisting of a glycine substitution for cysteine at 269 in the okadaic acid resistant variant of CHO cells, and that the mutant protein is resistant to okadaic acid (Shima, H. et al. Proc. Natl. Acad. Sci. USA 91, 9267-9271, 1994). In this study we analyzed okadaic acid responsive sequences in PP2A alpha by introducing a mutation at around codon 269 of the cDNA strand. The recombinant mutant PP2A alpha proteins Y265F, C266G, Y267G and C269Y were resistant to okadaic acid, with IC50s of 10, 24, 20 and 10 nM, respectively, as opposed to the recombinant wild PP2A alpha protein which had an IC50 of 0.24 nM. This observation suggests that not only cysteine at 269, but also other YCY amino acids, between 265-267, are necessary for the high sensitivity of PP2A to okadaic acid.
Kaneko
S
S
Carcinogenesis Division, National Cancer Center Research Institute, Tokyo, Japan.
Shima
H
H
Amagasa
T
T
Takagi
M
M
Sugimura
T
T
Nagao
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004988:Ethers, Cyclic / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D014162:Transfection
D017931:DNA Primers; D004988:Ethers, Cyclic; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases