Evidence that extracellular signal-regulated kinases are the insulin-activated Raf-1 kinase kinases.
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.
Lee
R M
RM
Howard Hughes Medical Institute Laboratories, Duke University Medical Center, Durham, North Carolina 27710.
Cobb
M H
MH
Blackshear
P J
PJ
D016475:3T3 Cells; D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1; D048053:Mitogen-Activated Protein Kinase 6; D020928:Mitogen-Activated Protein Kinases; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf
D007328:Insulin; D011518:Proto-Oncogene Proteins; D011494:Protein Kinases; D019908:Proto-Oncogene Proteins c-raf; D019950:Mitogen-Activated Protein Kinase 1; D048053:Mitogen-Activated Protein Kinase 6; D020928:Mitogen-Activated Protein Kinases