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Ordered phosphorylation of p42mapk by MAP kinase kinase
1Department of Pharmacology, University of Virginia, Charlottesville 22908.
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.
Insights
This study details the preparation of phosphorylated p42 mitogen-activated protein kinase (MAPK) for phosphatase assays. Tyr185 phosphorylation of p42MAPK precedes Thr183 phosphorylation, enhancing its affinity for MAP kinase kinase.
Area of Science:
- Molecular Biology
- Enzymology
- Signal Transduction
Background:
- p42 mitogen-activated protein kinase (MAPK) is a key signaling molecule.
- The phosphorylation status of p42MAPK regulates its activity and interactions.
- Understanding the sequential phosphorylation events is crucial for deciphering MAPK pathway function.
Purpose of the Study:
- To describe the preparation of milligram quantities of [32P]p42MAPK phosphorylated at specific sites (Tyr185 or Tyr185/Thr183).
- To investigate the substrate properties of differentially phosphorylated p42MAPK for protein phosphatases.
- To elucidate the order and functional significance of Tyr185 and Thr183 phosphorylation in p42MAPK activation.
Main Methods:
- Radiolabeling of p42MAPK with [32P]ATP.
- Controlled phosphorylation of p42MAPK using limiting ATP conditions to favor Tyr185 phosphorylation.
- Enzyme kinetic assays to determine the Michaelis constant (Km) for MAP kinase kinase (MAPKK) with different p42MAPK phosphorylation states.
Main Results:
- Milligram amounts of [32P]p42MAPK, phosphorylated at Tyr185 or diphosphorylated at Tyr185/Thr183, were successfully prepared.
- Tyr185-phosphorylated p42MAPK accumulated under conditions of limiting ATP, while Thr183 phosphorylation required further activation.
- Tyr185-phosphorylated p42MAPK showed a significantly lower apparent Km (46.6 nM) for MAPKK compared to the dephospho form (476 nM), indicating a 10-fold increase in affinity.
Conclusions:
- The phosphorylation of Tyr185 on p42MAPK precedes and is a prerequisite for Thr183 phosphorylation.
- This sequential phosphorylation dramatically enhances the affinity of p42MAPK for its upstream activator, MAPKK.
- The findings provide critical insights into the regulatory mechanism of MAPK pathway activation and substrate recruitment.