Characterization of the PP2A alpha gene mutation in okadaic acid-resistant variants of CHO-K1 cells.
Okadaic acid (OA)-resistant variants of Chinese hamster ovary cells, clones CHO/OAR6-6 and CHO/OAR2-3, were isolated from a CHO-K1 culture. These variant cells were 17- to 26-fold more resistant to OA than the parental cells. The phosphorylase phosphatase activity of the variant cell extracts was 2- to 4-fold more resistant to OA than that of the parental cells in the presence of inhibitor 2, a specific inhibitor of type 1 protein serine/threonine phosphatase (PP1). Nucleotide sequencing of PP2A alpha (an isotype of PP2A catalytic subunit) cDNA demonstrated that both variants have a T-->G transversion at the first base of codon 269 (805 nt), which results in substitution of glycine for cysteine. We expressed in COS-1 cells a mutant PP2A alpha tagged with the influenza hemagglutinin epitope. The recombinant mutant PP2A alpha protein immunoprecipitated with an anti-influenza hemagglutinin antibody was more resistant than the wild type to OA, their IC50 values being 0.65 nM and 0.15 nM, and their IC80 values being 4.0 nM and 0.45 nM, respectively. The cysteine at residue 269 present only in highly OA-sensitive protein serine/threonine phosphatase catalytic subunit isozymes, PP2A alpha, PP2A beta, and PPX, is suggested to be involved in the binding of OA. CHO/OAR6-6 and CHO/OAR2-3 cells also overexpressed the P-glycoprotein, and the efflux of OA was more rapid. It is suggested that the PP2A alpha mutation in cooperation with a high level of P-glycoprotein makes the CHO-K1 variants highly resistant to OA.
Shima
H
H
Carcinogenesis Division, National Cancer Center Research Institute, Tokyo, Japan.
Tohda
H
H
Aonuma
S
S
Nakayasu
M
M
DePaoli-Roach
A A
AA
Sugimura
T
T
Nagao
M
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002999:Clone Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D004351:Drug Resistance / Q000235:genetics*; D004988:Ethers, Cyclic / Q000494:pharmacology*; D014644:Genetic Variation; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D017386:Sequence Homology, Amino Acid
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D017931:DNA Primers; D004988:Ethers, Cyclic; D007527:Isoenzymes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases