A PHOSPHATASE-ACTIVITY IN XENOPUS OOCYTE EXTRACTS PREFERENTIALLY DEPHOSPHORYLATES THE MPM-2 EPITOPE

MPM-2 antigens are a large family of mitotic phosphoproteins that cont ain similar phosphoepitopes recognized by the anti-phosphoepitope anti body MPM-2 (MPM-2, epitopes), These proteins are phosphorylated during M phase induction and dephosphorylated from the onset of anaphase thr ough interphase. Since biochemical characterization of the MPM-2 epito pe phosphatase requires a specific assay for its activity, we tested d ifferent methods for measurement of the MPM-2 epitope phosphatase acti vity in crude cell lysates, First, an ELISA-based assay was designed t hat measured the phosphatase-induced reduction of the MPM-2 reactivity in crude M phase cell lysates, Using this assay to follow the phospha tase activity during sequential chromatography of Xenopus oocyte extra cts, one predominant peak of phosphatase activity was detected which w as separated from the majority of PP1 and PP2A activities, This phosph atase activity dephosphorylated the MPM-2 epitope on multiple MPM-2 an tigens, The second method measured dephosphorylation of cdc25, a known MPM-2 antigen, Two major peaks of cdc25 dephosphorylating activities were detected during the sequential chromatography, one that copurifie d with the major peak of MPM-2 epitope phosphatase activity, and the o ther with the major peak of PP2A activity, Finally, we examined whethe r GST-MPM2, a fusion protein between glutathione S-transferase and a 1 9-residue peptide that contained two representative MPM-2 epitope sequ ences, could be dephosphorylated efficiently and specifically by the m ajor MPM-2 epitope phosphatase activity in Xenopus oocyte extracts, Ne ither the crude extract nor the partially purified MPM-2 epitope phosp hatase activity efficiently dephosphorylated the MPM-2 epitope on GST- MPM2. These results demonstrate that the ELISA-based assay preferentia lly detects the MPM-2 epitope phosphatase activity in crude cell lysat es which may represent a physiological MPM-2 epitope phosphatase, (C) 1998 Federation of European Biochemical Societies.