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Activation of mitogen-activated protein kinase in BC3H1 myocytes by fluoroaluminate
N G Anderson1, E Kilgour, T W Sturgill
1Department of Internal Medicine, University of Virginia, Charlottesville 22908.
Abstract:
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.
Insights
Fluoroaluminate (AlF4-) activates G proteins, leading to rapid tyrosine phosphorylation and activation of mitogen-activated protein (MAP) kinase in cells. This G protein activation pathway for MAP kinase differs from growth factor signaling.
Area of Science:
- Cellular signaling pathways
- Signal transduction mechanisms
- G protein-coupled receptor (GPCR) research
Background:
- G proteins are key regulators of cellular signaling.
- Mitogen-activated protein (MAP) kinase is a crucial signaling molecule involved in cell growth and differentiation.
- Understanding upstream activators of MAP kinase is essential for deciphering cellular responses.
Purpose of the Study:
- To investigate the effect of fluoroaluminate (AlF4-), a G protein activator, on MAP kinase activity.
- To determine if AlF4- activates the 42-kDa MAP kinase.
- To elucidate the signaling pathway initiated by G protein activation leading to MAP kinase activation.
Main Methods:
- Treatment of BC3H1 myocytes and 3T3-L1 fibroblasts with AlF4-.
- Assay of tyrosine phosphorylation and myelin basic protein (MBP) kinase activity in cell extracts.
- Chromatographic purification and immunoblotting with anti-phosphotyrosine antibodies to identify activated MAP kinase.
Main Results:
- AlF4- treatment rapidly increased tyrosine phosphorylation and MBP kinase activity, characteristic of MAP kinase activation.
- A 42-kDa protein, identified as MAP kinase, showed increased tyrosine phosphorylation and activity.
- AlF4- activation of MAP kinase was rapid, sustained, and independent of protein kinase C or pertussis toxin-sensitive G proteins.
Conclusions:
- Direct activation of G proteins by AlF4- can lead to the activation of MAP kinase.
- This pathway represents a novel mechanism for MAP kinase activation, distinct from typical growth factor signaling.
- Findings suggest G protein activation initiates downstream events culminating in MAP kinase tyrosine phosphorylation and activation.
