Fig. 1

EGF induces transient Ras activation and Sos phosphorylation. a Transient Ras activation in HeLa cells. Serum-starved HeLa cells were challenged with 10 ng/ml EGF and Ras activation was determined via Ras-GTP affinity pulldowns. EGFR and Erk phosphorylation were determined using phosphosite-selective antibodies. A quantification of the Ras-GTP kinetics is shown on the right. RBD: Coomassie stain of Ras binding domain used for collecting Ras-GTP. b MEF cells challenged with EGF were processed for Ras and Erk activity assays as in (a). c EGF induces a mobility shift in Sos. HeLa cells were treated with inhibitors for MEK (10 μM U0126), Erk (50 μM FR108204) or Rsk (10 μM BI-D1870) prior to stimulation with EGF. Extracts were processed via western blotting using the indicated antibodies. Asterisk marks an unspecific doublet band. d Minimal Ras model describing Ras deactivation as induced by Ras-GTP-dependent feedback inhibition of Sos. R-GEF: receptor-GEF complex. See experimental section for details. e Simulations of Ras activation/deactivation using the model from (d) in a background of absent, low or high basal GAP activity. f Biochemical analysis of Ras-GTP levels following manipulation of Ras-GAP levels. The indicated Ras-GAP species were knocked down by siRNA (siNF1, siRASA1, siDAB2IP) or transiently overexpressed in HeLa cells (GFP-NF1: GFP-neurofibromin fusion construct; HA-RASA1: HA-tagged RASA1; asterisks mark overexpressed polypeptides). 5 min EGF stimulation is shown as positive control