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At least two kinases phosphorylate the MPM-2 epitope during Xenopus oocyte maturation
1Department of Clinical Investigations, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Abstract:
MPM-2 antigens, a discrete set of phosphoproteins that contain similar phosphoepitopes (the MPM-2 epitope), are associated with various mitotically important structures. The central mitotic regulator cdc2 kinase has been proposed to induce M-phase by phosphorylating many proteins which might include the MPM-2 antigens. To clarify the relationship of cdc2 kinase and the MPM-2 antigens, we developed an in vitro assay that enabled us to specifically detect the kinases that phosphorylate the MPM-2 epitope (ME kinases) in crude cell extracts. Two different ME kinase activities were identified in unfertilized Xenopus eggs, neither of which was cdc2 kinase, but both appeared to be activated by the introduction of cdc2 kinase into oocytes or oocyte extract. The two ME kinases differed in molecular size, substrate specificity, peptide components, and MPM-2 reactivity. The larger one, ME kinase-H, phosphorylated several MPM-2 antigens, while the smaller one, ME kinase-L, phosphorylated mainly one. We purified ME kinase-L to near homogeneity by sequential chromatography and showed that it has the characteristics of the 42-kD microtubule-associated protein (MAP) kinase. Our results support the previous finding that MAP kinase is activated during Xenopus oocyte maturation and suggest that MAP kinase may contribute to oocyte maturation induction by phosphorylating one subtype of MPM-2 epitope.
Insights
Researchers identified two novel kinases that phosphorylate MPM-2 antigens in Xenopus eggs. Neither kinase was cdc2 kinase, but MAP kinase was identified as one of the key MPM-2 phosphorylating enzymes involved in oocyte maturation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- MPM-2 antigens are phosphoproteins crucial for mitotic structures.
- cdc2 kinase is a proposed inducer of M-phase through protein phosphorylation.
Purpose of the Study:
- To clarify the relationship between cdc2 kinase and MPM-2 antigens.
- To identify kinases that phosphorylate the MPM-2 epitope (ME kinases) in cell extracts.
Main Methods:
- Developed an in vitro assay to detect ME kinases in crude cell extracts.
- Purified ME kinase-L using sequential chromatography.
- Characterized ME kinase-L's properties and substrate specificity.
Main Results:
- Identified two distinct ME kinase activities in unfertilized Xenopus eggs.
- Neither ME kinase was cdc2 kinase, but both were activated by cdc2 kinase introduction.
- Purified ME kinase-L exhibited characteristics of 42-kD microtubule-associated protein (MAP) kinase.
- MAP kinase was found to phosphorylate a specific MPM-2 antigen subtype.
Conclusions:
- MAP kinase is activated during Xenopus oocyte maturation.
- MAP kinase likely contributes to oocyte maturation by phosphorylating MPM-2 epitopes.
- cdc2 kinase may indirectly regulate MPM-2 phosphorylation through ME kinases.