Reconstitution of the Raf-1-MEK-ERK signal transduction pathway in vitro.
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.
Macdonald
S G
SG
Onyx Pharmaceuticals, Richmond, California 94806.
Crews
C M
CM
Wu
L
L
Driller
J
J
Clark
R
R
Erikson
R L
RL
McCormick
F
F
D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D003001:Cloning, Molecular; D011905:Genes, ras; D016391:Genes, src; D006801:Humans; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D009036:Moths; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)