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Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1
D R Alessi1, Y Saito, D G Campbell
1Department of Biochemistry, University of Dundee, Scotland.
Abstract:
Many growth factors whose receptors are protein tyrosine kinases stimulate the MAP kinase pathway by activating first the GTP-binding protein Ras and then the protein kinase p74raf-1. p74raf-1 phosphorylates and activates MAP kinase kinase (MAPKK). To understand the mechanism of activation of MAPKK, we have identified Ser217 and Ser221 of MAPKK1 as the sites phosphorylated by p74raf-1. This represents the first characterization of sites phosphorylated by this proto-oncogene product. Ser217 and Ser221 lie in a region of the catalytic domain where the activating phosphorylation sites of several other protein kinases are located. Among MAPKK family members, this region is the most conserved, suggesting that all members of the family are activated by the phosphorylation of these sites. A 'kinase-dead' MAPKK1 mutant was phosphorylated at the same residues as the wild-type enzyme, establishing that both sites are phosphorylated directly by p74raf-1, and not by autophosphorylation. Only the diphosphorylated form of MAPKK1 (phosphorylated at both Ser217 and Ser221) was detected, even when the stoichiometry of phosphorylation by p74raf-1 was low, indicating that phosphorylation of one of these sites is rate limiting, phosphorylation of the second then occurring extremely rapidly. Ser217 and Ser221 were both phosphorylated in vivo within minutes when PC12 cells were stimulated with nerve growth factor. Analysis of MAPKK1 mutants in which either Ser217 or Ser221 were changed to glutamic acid, and the finding that inactivation of maximally activated MAPKK1 required the dephosphorylation of both serines, shows that phosphorylation of either residue is sufficient for maximal activation.
Insights
Researchers identified specific phosphorylation sites (Ser217 and Ser221) on MAP kinase kinase (MAPKK1) that are crucial for its activation by the p74raf-1 protein kinase. This discovery clarifies a key step in the MAP kinase pathway signaling cascade.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- The MAP kinase pathway is a critical signaling cascade regulated by growth factors and protein tyrosine kinases.
- Activation of this pathway involves sequential signaling through Ras and p74raf-1, leading to MAP kinase kinase (MAPKK) activation.
Purpose of the Study:
- To elucidate the precise mechanism by which p74raf-1 activates MAPKK.
- To identify the specific amino acid residues on MAPKK1 phosphorylated by p74raf-1.
Main Methods:
- Identification of phosphorylation sites on MAPKK1 using a 'kinase-dead' mutant and wild-type enzyme.
- In vitro phosphorylation assays with p74raf-1 and MAPKK1.
- In vivo phosphorylation analysis in PC12 cells stimulated with nerve growth factor.
- Site-directed mutagenesis of MAPKK1 (Ser217 and Ser221 to glutamic acid).
Main Results:
- Serine 217 (Ser217) and Serine 221 (Ser221) of MAPKK1 were identified as the direct phosphorylation sites by p74raf-1.
- Phosphorylation occurs rapidly, with diphosphorylation (both sites) being the predominant form, suggesting a rate-limiting first phosphorylation.
- Both Ser217 and Ser221 are phosphorylated in vivo upon nerve growth factor stimulation.
- Phosphorylation of either Ser217 or Ser221 is sufficient for maximal MAPKK1 activation.
Conclusions:
- The direct phosphorylation of Ser217 and Ser221 by p74raf-1 is essential for MAPKK1 activation.
- These conserved residues represent key regulatory sites across the MAPKK family.
- Understanding these phosphorylation events provides insight into signal transduction pathways.