Characterization of the PP2Aα Gene Mutation in Okadaic Acid- Resistant Variants of CHO-K1 Cells

• Published in: Proceedings of the National Academy of Sciences - PNAS 1994-09, Vol.91 (20), p.9267-9271
• Main Authors: Shima, Hiroshi, Tohda, Hiroko, Aonuma, Shizu, Nakayasu, Michie, DePaoli-Roach, Anna A., Sugimura, Takashi, Nagao, Minako
• Format: Article
• Language: English
• Subjects: 3T3 cells; Amino Acid Sequence; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1 - biosynthesis; Base Sequence; Cell lines; CHO Cells; Clone Cells; Cloning, Molecular; Codons; Complementary DNA; COS cells; Cricetinae; DNA; DNA Primers; Drug Resistance - genetics; Ethers, Cyclic - pharmacology; Genetic mutation; Genetic Variation; Inhibitory concentration 50; Isoenzymes - antagonists & inhibitors; Isoenzymes - genetics; Macromolecular Substances; Molecular Sequence Data; Okadaic Acid; Phosphatases; Phosphoprotein Phosphatases - antagonists & inhibitors; Phosphoprotein Phosphatases - genetics; Point Mutation; Polymerase Chain Reaction; Polymorphism, Genetic; Rats; Recombinant Proteins - antagonists & inhibitors; Sequence Homology, Amino Acid; Sequencing
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.20.9267

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 PP2Aα (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 PP2Aα tagged with the influenza hemagglutinin epitope. The recombinant mutant PP2Aα protein immunoprecipitated with an anti-influenza hemagglutinin antibody was more resistant than the wild type to OA, their IC50values being 0.65 nM and 0.15 nM, and their IC80values 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, PP2Aα, PP2Aβ, 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 PP2Aα mutation in cooperation with a high level of P-glycoprotein makes the CHO-K1 variants highly resistant to OA.