PURIFICATION AND CHARACTERIZATION OF A MITOGEN-ACTIVATED PROTEIN-KINASE TYROSINE PHOSPHATASE FROM XENOPUS EGGS

The mitogen-activated protein (MAP) kinases are serine-threonine prote in kinases that are activated by tyrosine and threonine phosphorylatio n by the dual specificity protein kinase MEK (MAP kinase/ERK kinase). The present report describes the purification to near homogeneity and characterization of a protein tyrosine phosphatase from Xenopus laevis eggs that dephosphorylates MAP kinase phosphorylated by MEK. Bacteria lly expressed Xenopus MAP kinase phosphorylated by purified Xenopus ME K was used as substrate throughout the purification. The purification procedure included anion-exchange, cation-exchange, gel filtration, he parin-Sepharose, and chromatography on a column of thiophosphorylated MAP kinase-Sepharose, resulting in a >3000-fold purification. Upon ana lysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a protein of 47 kDa correlated with activity. The phosphatase showed abs olute specificity toward phosphotyrosine and no activity toward phosph othreonyl-phosphoseryl residues of MAP kinase. The pH optimum of the e nzyme was 7.0 with a K(m) of 9.0 muM for phosphorylated MAP kinase. Th e phosphatase was inhibited by ammonium molybdate (IC50, 2 muM), vanad ate (IC50, 250 muM), millimolar concentrations of MnCl2, ZnCl2 and p-n itrophenylphosphate but not by okadaic acid or microcystin. This tyros ine phosphatase may be involved in deactivating MAP kinase in vivo.