1. PKCζ is tyrosine phosphorylated by EGF and contributes to EGF-induced activation of ERK in Mef cells.
PKCζ is tyrosine phosphorylated by EGF and contributes to EGF-induced activation of ERK in Mef cells. A, WT Mef cells expressing PKCζ (see C) were serum starved and stimulated with increasing concentrations of EGF for 5 minutes, lysed, immunoprecipitated with anti-PKCζ antibody, probed with anti-phosphotyrosine antibody (4G10), and reprobed with anti-PKCζ antibody. Shown is one of six independent experiments with similar results. B, dose-response curve of PKCζ tyrosine phosphorylation in Mef cells treated with EGF. Points, mean from six separate experiments, normalized to the PKCζ reblot; bars, SE. C, serum-starved WT or PKCζ−/− Mef cells were stimulated with different concentrations of EGF for 5 minutes. Aliquots of the cell lysates (60 μg protein each) were analyzed for activation of ERK by Western blotting with phosphosite-specific anti-pERK (Tyr204) antibody. Equal loading was controlled by measuring the contents of β-actin and PKCα per sample using the respective antibodies. For control, equal aliquots of these samples were run for Western blotting with mixed ERK1 and ERK2 antibodies. Representative blots of six independent experiments. D, the quantified data from C of site-specific phosphorylation (activation) of ERK in response to EGF have been normalized to actin and are expressed as percentage related to the respective basal values. Insets, expression levels of PKCζ in the respective Mef cells. Columns, means; bars, SD. *, P < 0.05, significantly higher compared with PKCζ−/− Mef cells.
Christina Valkova et al. Mol Cancer Res 2010;8:
©2010 by American Association for Cancer Research