Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1
| Autor: | G. Sithanandam, A. Ashworth, C.J. Marshall, David G. Campbell, Y. Saito, U. Rapp, Philip Cohen, Sally A. Cowley, Dario R. Alessi |
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| Jazyk: | angličtina |
| Rok vydání: | 1994 |
| Předmět: |
inorganic chemicals
Recombinant Fusion Proteins DNA Mutational Analysis Molecular Sequence Data macromolecular substances Biology Mitogen-activated protein kinase kinase SH2 domain PC12 Cells environment and public health General Biochemistry Genetics and Molecular Biology MAP2K7 Species Specificity Proto-Oncogene Proteins Serine Animals Protein phosphorylation Amino Acid Sequence Nerve Growth Factors Phosphorylation Protein kinase A Molecular Biology MAPK14 Mitogen-Activated Protein Kinase Kinases General Immunology and Microbiology Sequence Homology Amino Acid General Neuroscience Autophosphorylation Peptide Fragments Enzyme Activation Isoenzymes Proto-Oncogene Proteins c-raf enzymes and coenzymes (carbohydrates) Biochemistry bacteria Protein Kinases Research Article |
| Zdroj: | Europe PubMed Central Scopus-Elsevier |
| ISSN: | 1460-2075 0261-4189 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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