ANALYSIS BY IN-VITRO MUTAGENESIS OF PP2A-ALPHA OKADAIC ACID RESPONSIVE SEQUENCES

It has been shown that the protein serine/threonine phosphatase type 2 A 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 oka daic 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 or the cDNA s trand. The recombinant mutant PP2A alpha proteins Y265F, C266G, Y267G and C269Y were resistant to okadaic acid, with IC(50)s of 10, 24, 20 a nd 10 nM, respectively, as opposed to the recombinant wild PP2A alpha protein which had an IC50 of 0.24 nM. This observation suggests that n ot only cysteine at 269, but also other YCY amino acids, between 265-2 67, are necessary for the high sensitivity of PP2A to okadaic acid. (C ) 1995 Academic Press, Inc.