MeHg Exposure Causes Activation of Fyn, Phosphorylation of c-Cbl, and Ubiquitylation of PDGFRα Leading to Reductions in Receptor Level

<div><p>(A) O-2A/OPCs exposed to 30 nM MeHg exhibited higher levels of Fyn kinase activity, as detected by analysis of immunoprecipitated Fyn from these cells using the Universal Tyrosine Kinase Assay Kit (Takara), as described in Materials and Methods. Values (mean ± SD) are expressed as the percent of controls, which were defined from basal Fyn kinase activity without any stimulation. All bars with increased levels of Fyn activity differ from control values at <i>p</i> < 0.001. Increased Fyn activity was blocked by pre-treatment of cells with NAC or with the src-family kinase inhibitor PP1, but not by pre-treatment with NH₄Cl (an inhibitor of lysosomal function).</p> <p>(B) As for (A), Increased c-Cbl phosphorylation (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050035#s4" target="_blank">Materials and Methods</a> for immunoprecipitation assay) associated with MeHg exposure was blocked by co-exposure of cells to NAC or PP1, but was not blocked when cells were exposed to NH₄Cl.</p> <p>(C) Exposure to MeHg was associated with a marked increase in the levels of ubiquitylation of PDGFRα, with this increase being apparent even though receptor levels were themselves reduced by toxicant exposure. NH₄Cl treatment was associated with rescue of levels of total PDGFRα, and therefore was associated with a still more marked increase in the amount of ubquitylated receptor detected. As predicted by the hypothesis that both receptor ubiquitylation and receptor loss are due to activation of Fyn, co-exposure of cells to PP1 rescued receptor levels and greatly reduced the extent of receptor ubiquitylation.</p> <p>The plus symbol (+) indicates exposure of the cells to the indicated substance. IB = immunoblot; IP = immunoprecipitation.</p>