Abstract
Preparation of milligram amounts of [32P]p42mapk, phosphorylated at Tyr185 or diphosphorylated at Tyr185/Thr183, for use as specific protein phosphatase substrates is described. Tyr- but not Thr-phosphorylated p42mapk, accumulates when ATP is limiting. Furthermore, Tyr185-phosphorylated p42mapk exhibits an apparent 10-fold decrease in apparent Km (46.6 +/- 6.6 nM) for MAP kinase kinase compared to that for the dephospho form (approximately 476 nM). We conclude that Tyr185 precedes Thr183 phosphorylation, and that this is prerequisite, dramatically increasing the affinity of p42mapk for MAP kinase kinase.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Autoradiography
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Chromatography, Ion Exchange
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Electrophoresis, Polyacrylamide Gel
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Enzyme Activation
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Kinetics
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase Kinases
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Muscles / enzymology
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Phosphorylation
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Protein Kinases / metabolism*
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Protein Serine-Threonine Kinases
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Protein-Tyrosine Kinases
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Rabbits
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Recombinant Proteins / metabolism
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Substrate Specificity
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Threonine / metabolism
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Tyrosine / metabolism
Substances
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Recombinant Proteins
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Threonine
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Tyrosine
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Protein Kinases
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Protein-Tyrosine Kinases
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Protein Serine-Threonine Kinases
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase Kinases