TY - JOUR
T1 - A phosphatase activity in Xenopus oocyte extracts preferentially dephosphorylates the MPM-2 epitope
AU - Che, Shaoli
AU - Wu, Weiguo
AU - Nelman-Gonzalez, Mayra
AU - Stukenberg, Todd
AU - Clark, Richard
AU - Kuang, Jian
N1 - Funding Information:
This work was supported by grants from the National Cancer Institute (R-29 GM48457-02) and the National Science Foundation (MCB-9405699) to Dr. J. Kuang, and by a postdoctoral fellowship provided to W. Wu by the Pharmacology Department, UT Medical School. We thank Drs. A.R. Nebreda and T. Hunt for kindly providing Escherichia coli TG-1 cells harboring the expression vector GEX-KG-cdc25. We also thank Dr. T. Lee for her critiques of the manuscript, Cheryl Ashorn for her technical assistance, and Josephine Neicheril for her administrative assistance.
PY - 1998/3/13
Y1 - 1998/3/13
N2 - MPM-2 antigens are a large family of mitotic phosphoproteins that contain similar phosphoepitopes recognized by the anti-phosphoepitope antibody MPM-2 (MPM-2 epitopes). These proteins are phosphorylated during M phase induction and dephosphorylated from the onset of anaphase through interphase. Since biochemical characterization of the MPM-2 epitope phosphatase requires a specific assay for its activity, we tested different methods for measurement of the MPM-2 epitope phosphatase activity in crude cell lysates. First, an ELISA-based assay was designed that measured the phosphatase-induced reduction of the MPM-2 reactivity in crude M phase cell lysates. Using this assay to follow the phosphatase activity during sequential chromatography of Xenopus oocyte extracts, one predominant peak of phosphatase activity was detected which was separated from the majority of PP1 and PP2A activities. This phosphatase activity dephosphorylated the MPM-2 epitope on multiple MPM-2 antigens. 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 copurified with the major peak of MPM-2 epitope phosphatase activity, and the other with the major peak of PP2A activity. Finally, we examined whether GST-MPM2, a fusion protein between glutathione S-transferase and a 19-residue peptide that contained two representative MPM-2 epitope sequences, could be dephosphorylated efficiently and specifically by the major MPM-2 epitope phosphatase activity in Xenopus oocyte extracts. Neither the crude extract nor the partially purified MPM-2 epitope phosphatase activity efficiently dephosphorylated the MPM-2 epitope on GST-MPM2. These results demonstrate that the ELISA-based assay preferentially detects the MPM-2 epitope phosphatase activity in crude cell lysates which may represent a physiological MPM-2 epitope phosphatase.
AB - MPM-2 antigens are a large family of mitotic phosphoproteins that contain similar phosphoepitopes recognized by the anti-phosphoepitope antibody MPM-2 (MPM-2 epitopes). These proteins are phosphorylated during M phase induction and dephosphorylated from the onset of anaphase through interphase. Since biochemical characterization of the MPM-2 epitope phosphatase requires a specific assay for its activity, we tested different methods for measurement of the MPM-2 epitope phosphatase activity in crude cell lysates. First, an ELISA-based assay was designed that measured the phosphatase-induced reduction of the MPM-2 reactivity in crude M phase cell lysates. Using this assay to follow the phosphatase activity during sequential chromatography of Xenopus oocyte extracts, one predominant peak of phosphatase activity was detected which was separated from the majority of PP1 and PP2A activities. This phosphatase activity dephosphorylated the MPM-2 epitope on multiple MPM-2 antigens. 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 copurified with the major peak of MPM-2 epitope phosphatase activity, and the other with the major peak of PP2A activity. Finally, we examined whether GST-MPM2, a fusion protein between glutathione S-transferase and a 19-residue peptide that contained two representative MPM-2 epitope sequences, could be dephosphorylated efficiently and specifically by the major MPM-2 epitope phosphatase activity in Xenopus oocyte extracts. Neither the crude extract nor the partially purified MPM-2 epitope phosphatase activity efficiently dephosphorylated the MPM-2 epitope on GST-MPM2. These results demonstrate that the ELISA-based assay preferentially detects the MPM-2 epitope phosphatase activity in crude cell lysates which may represent a physiological MPM-2 epitope phosphatase.
KW - Dephosphorylation
KW - MPM-2 antigen
KW - Phosphatase activity
KW - Xenopus oocyte extract
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U2 - 10.1016/S0014-5793(98)00158-6
DO - 10.1016/S0014-5793(98)00158-6
M3 - Article
C2 - 9539156
AN - SCOPUS:0032513068
SN - 0014-5793
VL - 424
SP - 225
EP - 233
JO - FEBS Letters
JF - FEBS Letters
IS - 3
ER -