The reaction stoichiometry and kinetic constants of the EGFR network model are given in Supplementary Tables S1-S3.

Hek293 cells were pretreated with MEK inhibitor U0126 (5 μM) or with ERK inhibitor 5-Iodotubericidin (IT-5) (5 μM) for 30 minutes, stimulated with 20 nM EGF. (A) Basal and EGF-induced serine-phosphorylation of Akt (S473) was detected in whole-cell lysates pretreated with indicated inhibitors for 5 minutes (upper panel) or with IT-5 inhibitor (lower panel). (B) Gab1 was detected in phosphotyrosine protein immunoprecipitates.

Simulated time-course of membrane-bound SOS (A) and membrane-bound RasGAP (B) in the presence (dashed line) and absence of wortmannin (WM). Membrane-bound forms SOS and RasGAP involve a number of complexes that contain EGFR or membrane-associated GAB1 and are listed in the Supplementary material. Stimulation with 20 nM EGF.

GAB1 protein abundance was 300 nM in control and 100 nM in GAB1-depleted cells (dashed line). Stimulation with 20 nM (A) and 0.5 nM (B) EGF.

Time-course of ERK activation in GAB1 mutants predicted by the computational model. (A) Gab1Δp85, deletion of the p85 PI3K binding sites (dashed line); (B) Gab1ΔSHP2, deletion of the SHP2-binding sites (dashed line).

After preincubation at 37° C for 30 min in the presence or absence 200 nM wortmannin (WM), Hek293 cells were stimulated with 20 nM (A, B, C, E) or 0.05 nM EGF (D). Whole-cell lysates were prepared at the indicated times and analyzed by immunoblotting for (A) serine-phosphorylated Akt (S473), (B, D) threonine/tyrosine-phosphorylated Erk (T202/Y204), or (C) tyrosine-phosphorylated EGFR (Y1173). Membrane was stripped, reprobed for total level of each indicated protein and used as a loading control and for quantitation of phosphorylated Erk (plots B, and D) in the presence (□) or absence of WM (◊) (expressed in arbitrary units as a fraction of the total Erk). Data shown (plots B, and D) are the mean ± SEM from three independent experiments. (E). RasGTP was measured by affinity precipitation with Raf-1 Ras binding domain agarose beads and detected with anti-Ras antibody. Anti-Ras antibodies were also used to determine total Ras level in appropriate lysates (Materials and Methods).

(A) The time-course of Gab1 tyrosine phosphorylation in response to 20 nM EGF in the presence or absence of wortmannin (WM) (200 nM). Cell lysates were prepared at the indicated times as mentioned above and analyzed by immunoblotting for phosphorylation of SHP2 binding site on Gab1 (Y627) (top line) and total Gab1 level (bottom line). All tyrosine phosphorylated proteins were immunoprecipitated from whole-cell lysates (see Materials and Methods) and immunoblotted with anti-Gab1 antibodies (middle line). (B) Computer simulations of the time-course of Gab1 tyrosine phosphorylation in the presence (dashed line) or absence of WM (in the presence of WM, the PI3K catalytic constant is assumed equal to zero). (C) WM induces partial SHP2 dissociation from Gab1 as revealed in SHP2 immunoprecipitates from WM-treated cells probed with anti-Gab1 antibodies. (D) Computer-simulated kinetics of the SHP2-GAB1 association in the presence (dashed line) or absence of WM.

The calculated time-course of the RasGTP fraction (A) and the threonine/tyrosine phosphorylated ERK fraction following stimulation with 20 nM (B) or 1 nM (C) EGF in the absence and presence (dashed line) of wortmannin (WM).

(A) Simulated kinetics of MEK phosphorylation in control cells (solid line), GAB1 suppression (dashed line, [GAB1]=100 nM), or the presence of wortmannin (WM, dashed-dotted), 20 nM EGF. (B). Validation of specific endogenous Gab1 knockdown by siRNA in Hek293 cells. Hek293 cells were transfected with 50 nM Gab1 siRNA, 50 nM Non-Targeting (NT) siRNA or left untreated for 72 hours as described in Materials and Methods. Lysates of quiescent or EGF stimulated (20 nM) cells (5 minutes) were probed with anti-Gab1 antibodies to estimate level of Gab1 suppression. Probing for SHP2 and α-tubulin protein levels was used as specificity and loading control. (C) Time course of MEK phosphorylation in Hek293 cells treated with 20 nM EGF. Cells were either transfected with 50 nM Gab1 siRNA for 72 hours (middle three lines) or 200 nM wortmannin for 30 minutes (bottom three lines) or left untreated (top three lines) before stimulation with EGF. Whole-cell lysates were immunoblotted with anti-phospho-Mek 1/2. Equal Gab1 suppression (~70%) and sample loading were determined with anti-Gab1 and anti-GAPDH antibodies, respectively. Experimental curves of MEK activation were determined in four independent experiments and plotted as the mean ± SEM. Wortmannin (Δ), GAB1 siRNA (□), Control (○). (D) Computer simulated and (E) experimental time course of AKT phosphorylation in Hek293 cells treated with 0.5 nM EGF. Cells were depleted of Gab1 as described above or left untreated before stimulation with ligand. Whole-cell lysates were immunoblotted with anti-phospho-Akt (S473). Membranes were stripped and reprobed with anti-Akt antibodies to show that Gab1 suppression did not affect Akt protein level. Additionally, equal Gab1 suppression (~85%) and sample loading were determined with anti-Gab1 and anti-Grb2 antibodies respectively.

(A) The EGFR concentration was increased by a factor of 10 to 1080 nM. The time-course of EGF (20 nM) induced ERK activation in the absence (1) and the presence (2) of wortmannin (WM). (B) 12 hours starved A431 cells were pretreated with 200nM wortmannin (Δ) or left untreated (○) for 30 minutes before stimulation with 20 nM EGF at indicated time points. Data shown are the mean ± SEM from six independent experiments.