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Fig. 2 | Cell Communication and Signaling

Fig. 2

From: Feedback activation of neurofibromin terminates growth factor-induced Ras activation

Fig. 2

EGF induces transient Ras-GTP accumulation but sustained up-regulation of nucleotide exchange. a Specificity of the Ras nucleotide exchange assay in permeabilized cells. Serum-starved HeLa cells were permeabilized or mock permeabilized by omitting SLO in the presence of [α-32P]GTP. A 100fold molar excess of unlabeled GTP was included where indicated. Cell extracts prepared at the indicated time points were subjected to Ras-IPs or mock IPs lacking the Y13-259 Ras-antibody. Precipitates were washed and associated radioactivity evaluated by cerenkow counting. b Biochemical assay of time-dependent EGF-induced Ras and Erk activation performed in the absence or presence of the permeabilizing agent SLO. SLO was added simultaneously with EGF. c Nucleotide exchange assay in permeabilized HeLa cells before and 5 min or 20 min after EGF administration. Nucleotides associated to Ras-IPs were additionally eluted from Ras and separated via thin layer chromatography (TLC, on the right). %GTP/(GDP + GTP) values were determined by densitometry and plotted under the panel. Of note, initial values start off high and level off only at later time points. This pattern is owed to the different time required for single Ras proteins versus the whole Ras population to achieve steady-state nucleotide turnover. d Same experiment as in C performed in MEF cells. e Quantification of nucleotides bound to Ras-IPs. On the left, the amount of GDP + GTP bound to Ras at the 6 min assay point (as recorded in (c)) was plotted as the fold increase of radioactivity bound to Ras in EGF-stimulated versus unstimulated cells. On the right, the amount of GTP/(GDP + GTP) in the same assay points was plotted as % GTP/(GDP + GTP). Shown are means ± S.E.M. for three independent experiments. f [α-32P]GTP associated to total cellular protein from untreated or EGF-challenged permeabilized cells determined by a filter binding assay. Shown here is the means ± S.E.M. for three independent experiments. g GppNHp but not GTP promotes strong Ras activation in permeabilized cells at late time points of EGF stimulation. HeLa cells were permeabilized for the indicated time frames in the presence of GTP or GppNHp before (no stim.), 5 min or 20 min after EGF stimulation. Reactions were stopped by cell lysis and cell extracts were subjected to biochemical analysis of Ras and Erk activation

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