Purification and characterization of a mitogen-activated protein kinase tyrosine phosphatase from Xenopus eggs.
The mitogen-activated protein (MAP) kinases are serine-threonine protein kinases that are activated by tyrosine and threonine phosphorylation 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. Bacterially expressed Xenopus MAP kinase phosphorylated by purified Xenopus MEK was used as substrate throughout the purification. The purification procedure included anion-exchange, cation-exchange, gel filtration, heparin-Sepharose, and chromatography on a column of thiophosphorylated MAP kinase-Sepharose, resulting in a > 3000-fold purification. Upon analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a protein of 47 kDa correlated with activity. The phosphatase showed absolute specificity toward phosphotyrosine and no activity toward phosphothreonyl-phosphoseryl residues of MAP kinase. The pH optimum of the enzyme was 7.0 with a Km of 9.0 microM for phosphorylated MAP kinase. The phosphatase was inhibited by ammonium molybdate (IC50, 2 microM), vanadate (IC50, 250 microM), millimolar concentrations of MnCl2, ZnCl2 and p-nitrophenylphosphate but not by okadaic acid or microcystin. This tyrosine phosphatase may be involved in deactivating MAP kinase in vivo.
Sarcevic
B
B
Howard Hughes Medical Institute, University of Colorado School of Medicine, Denver 80262.
Erikson
E
E
Maller
J L
JL
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D009865:Oocytes / Q000201:enzymology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D014982:Xenopus laevis
D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases