Evidence that extracellular signal-regulated kinases are the insulin-activated Raf-1 kinase kinases

• Published in: The Journal of biological chemistry 1992-01, Vol.267 (2), p.1088-1092
• Main Authors: Lee, R M, Cobb, M H, Blackshear, P J
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Analytical, structural and metabolic biochemistry; Animals; Autoradiography; Biological and medical sciences; Blotting, Western; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzymes and enzyme inhibitors; extracellular signal-regulated kinase; Fundamental and applied biological sciences. Psychology; Insulin - pharmacology; Kinetics; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 6; Mitogen-Activated Protein Kinases; Peptide Mapping; Phosphorylation; Protein Kinases - metabolism; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-raf; Raf-1; Transferases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)48399-8

The Raf-1 proto-oncogene protein kinase can be phosphorylated and activated after stimulation of cells with insulin and a variety of other growth factors and mitogens. We recently presented evidence that insulin and certain other growth factors activated one or more Raf-1 kinase kinase activities (Lee, R.M., Rapp, U. R., and Blackshear, P.J. (1991) J. Biol. Chem. 266, 10351-10357). In the present study, four peaks of Raf-1 kinase kinase activity were identified after anion-exchange chromatography of cell lysates, and two of these were activated by insulin. Further chromatographic characterization of these two peaks of insulin-activated kinase activity indicated that they contained three apparently distinct kinase activities. Two of these activities comigrated with immunoreactive extracellular signal-regulated kinases (ERK) 1 and 2 (mitogen-activated protein kinase) through three different chromatographic separations. Both ERK1 and ERK2 phosphorylated Raf-1 with reasonably high affinity (Km for ERK1 = 90 nM; Km for ERK2 = 120 nM), and produced similar, complex phosphopeptide maps; both kinases also phosphorylated myelin basic protein. The third kinase activity also phosphorylated Raf-1 and myelin basic protein but did not comigrate exactly with either immunoreactive ERK1 or ERK2. We conclude that two and possibly three insulin-activated Raf-1 kinase kinases are members of the ERK family.