Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase by G protein and tyrosine kinase oncoproteins.
Mitogen-activated protein kinases (MAPKs) are rapidly phosphorylated and activated in response to a variety of extracellular stimuli in many different cell types. The kinases that activate MAPK, the MAPK/ERK Kinases (MEKs), are also activated by phosphorylation. We have studied the influence of specific oncogenes on the regulation of MEK activity in NIH3T3 and Rat1a fibroblasts. We show that a similar MEK activity phosphorylates and activates MAPK in both growth factor-stimulated (epidermal growth factor and thrombin) and oncogene (gip2, v-src, and v-raf)-transfected cells. Gip2 and v-Src activated MEK-1 in transfected Rat 1a cells, whereas v-Raf activated MEK-1 in transfected NIH3T3 cells. These cell-selective differences in MEK activation parallel constitutive MAPK activation in these cell lines. Stable expression of the v-ras oncogene resulted in little constitutive MEK activation in either cell line, even though both were highly transformed. The growth factor and oncoprotein regulated MEK activity co-fractionated by Mono S chromatography with the 45-kDa MEK-1 protein. We further demonstrate in NIH3T3 and Rat 1a cells that Raf-1 is activated, as measured by its ability to phosphorylate MEK-1, in response to epidermal growth factor but not thrombin. Thus, the regulatory network of protein kinases that activate MAPK converges at MEK but diverges with the kinases that phosphorylate and activate MEK.
Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
A M
AM
Gardner
R R
RR
Vaillancourt
G L
GL
Johnson
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002460:Cell Line; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D015513:Oncogene Proteins / Q000096:biosynthesis / Q000378:metabolism*; D009857:Oncogenes; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D015513:Oncogene Proteins; D011506:Proteins; D004815:Epidermal Growth Factor; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins