Activation of mitogen-activated protein kinase in BC3H1 myocytes by fluoroaluminate.
Treatment of BC3H1 myocytes or 3T3-L1 fibroblasts with fluoroaluminate (AlF4-), a direct activator of G proteins, increased the tyrosine phosphorylation of a 42-kDa cytosolic protein. AlF4- induced a parallel increase in protein kinase activity toward myelin basic protein (MBP) in partially purified cell extracts. To test whether AlF4- was activating the 42-kDa MAP (mitogen-activated protein) kinase, extracts from AlF4--treated cells were taken through the chromatographic steps routinely used to purify MAP kinase from growth factor-stimulated cells. Following phenyl-Superose chromatography, a peak of MBP kinase activity eluted at a position characteristic of MAP kinase. Immunoblotting of the active fractions with anti-phosphotyrosine antibodies revealed a single reactive protein band of Mr 42,000. Stimulation of MAP kinase by AlF4- was rapid, peaking within 15 min and persisting for at least 1 h. In contrast, the activation of MAP kinase by insulin was transient, characteristic of its activation by growth factors in other cell types. Although concentrations of sodium fluoride greater than 1 mM also activated MAP kinase, this effect was shown to be dependent upon the simultaneous presence of aluminum ions in the medium. Activation of MAP kinase by AlF4- was not affected by either cellular depletion of protein kinase C or pretreatment of cells with pertussis toxin. Potential sites of action of AlF4- are discussed. These findings suggest that activation of a G protein(s) in intact cells can initiate events that result in tyrosine phosphorylation and activation of MAP kinase.
Anderson
N G
NG
Department of Internal Medicine, University of Virginia, Charlottesville 22908.
Kilgour
E
E
Sturgill
T W
TW
D000535:Aluminum / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002460:Cell Line; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005461:Fluorine / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D051379:Mice; D008934:Mitogens / Q000494:pharmacology*; D004676:Myelin Basic Protein / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012969:Sodium Fluoride / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism
D008934:Mitogens; D004676:Myelin Basic Protein; C053629:fluoroaluminum; D005461:Fluorine; D014443:Tyrosine; D012969:Sodium Fluoride; D000535:Aluminum; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins