Reconstitution of the Raf-1-MEK-ERK Signal Transduction Pathway In Vitro
| Autor: | S. G. Macdonald, J. Driller, Frank McCormick, R Clark, Craig M. Crews, Lelia Wu, R. L. Erikson |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
MAPK/ERK pathway
MAP Kinase Kinase 1 Mitogen-activated protein kinase kinase Biology Moths Protein Serine-Threonine Kinases Transfection Polymerase Chain Reaction Cell Line Proto-Oncogene Proteins p21(ras) Proto-Oncogene Proteins Animals Humans Cloning Molecular Phosphorylation Protein kinase A Molecular Biology Protein kinase C Mitogen-Activated Protein Kinase Kinases Kinase Cell Biology Protein-Tyrosine Kinases Molecular biology Recombinant Proteins Proto-Oncogene Proteins c-raf Genes src Genes ras Mutagenesis Site-Directed Baculoviridae Proto-oncogene tyrosine-protein kinase Src Research Article Signal Transduction |
| Zdroj: | Molecular and Cellular Biology. 13:6615-6620 |
| ISSN: | 1098-5549 |
| DOI: | 10.1128/mcb.13.11.6615-6620.1993 |
| Popis: | Raf-1 is a serine/threonine kinase which is essential in cell growth and differentiation. Tyrosine kinase oncogenes and receptors and p21ras can activate Raf-1, and recent studies have suggested that Raf-1 functions upstream of MEK (MAP/ERK kinase), which phosphorylates and activates ERK. To determine whether or not Raf-1 directly activates MEK, we developed an in vitro assay with purified recombinant proteins. Epitope-tagged versions of Raf-1 and MEK and kinase-inactive mutants of each protein were expressed in Sf9 cells, and ERK1 was purified as a glutathione S-transferase fusion protein from bacteria. Raf-1 purified from Sf9 cells which had been coinfected with v-src or v-ras was able to phosphorylate kinase-active and kinase-inactive MEK. A kinase-inactive version of Raf-1 purified from cells that had been coinfected with v-src or v-ras was not able to phosphorylate MEK. Raf-1 phosphorylation of MEK activated it, as judged by its ability to stimulate the phosphorylation of myelin basic protein by glutathione S-transferase-ERK1. We conclude that MEK is a direct substrate of Raf-1 and that the activation of MEK by Raf-1 is due to phosphorylation by Raf-1, which is sufficient for MEK activation. We also tested the ability of protein kinase C to activate Raf-1 and found that, although protein kinase C phosphorylation of Raf-1 was able to stimulate its autokinase activity, it did not stimulate its ability to phosphorylate MEK. |
| Databáze: | OpenAIRE |
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