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, c lones 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 p arental cells in the presence of inhibitor 2, a specific inhibitor of type 1 protein serine/threonine phosphatase (PP1). Nucleotide sequenci ng of PP2A alpha (an isotype of PP2A catalytic subunit) cDNA demonstra ted that both variants have a T --> G transversion at the first base o f codon 269 (805 nt), which results in substitution of glycine for cys teine. We expressed in COS-1 cells a mutant PP2A alpha tagged with the influenza he magglutinin epitope. The recombinant mutant PP2A alpha p rotein immunoprecipitated with an anti-influenza hemagglutinin antibod y 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-se nsitive protein serine/threonine phosphatase catalytic subunit isozyme s, 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 t hat the PP2A alpha mutation in cooperation with a high level of P-glyc oprotein makes the CHO-K1 variants highly resistant to OA.