Computational modeling of the dynamics of the MAP kinase cascade activated by surface and internalized EGF receptors

Supplementary Figure 1. (pdf 17K)
Kinetic scheme of the EGF receptor-induced MAP kinase cascade induced by an Shc-dependent and an Shc-independent pathway. Each component is identified by a specific number (blue). Blue numbers in brackets specify the components after internalization. The arrows represent the reaction rates specified in Supplementary Table 1 and characterized by green numbers v1-v62. The second green number identifies reaction rates after internalization.

Supplementary Figure 2. (pdf 3K)
Biochemical reaction schemes of internalization by coated pit-dependent and -independent pathways. The internalization steps modeled for the compounds in Figure 1 are illustrated separately in this figure to avoid confusing additional arrows. (A) Receptor internalization in the Shc pathway initiated by the association of a coated-pit protein (Prot) to the components identified by the blue numbers (identical to those in Fig. 1). After dissociation of Prot, the components are identified by the same numbers as the blue numbers in brackets of Figure 1. v115, v118, v121, v124 are equivalent to k₄. Prot-associated components 91, 92, 93, 94 are not depicted in Figure 1. (B) Receptor internalization in the Grb2 pathway initiated by the coated-pit protein (Prot) identified according to the same principle as in (A). v106, v109, and v112 are equivalent to k₄ and v107, v110, v113 are identical to k₅. Prot-associated components 7, 88, 89, 90 are not depicted in Figure 1. (C) Internalization in both pathways by a (Prot)-independent step. v102, v103, v104, v108, v111, v114, v117, v120, v123, are the same as k6.

Supplementary Figure 3. (pdf 157K)
Time course of ERK-1/2 phosphorylation and c-fos activation in HeLa cells after EGF stimulation. (A) Kinetics of ERK activation. HeLa cells were serum-starved for 24 h and then incubated with different concentrations of EGF for times indicated. Protein samples were blotted onto nitrocellulose membranes and probed for the phosphorylated forms of ERK-1/2 as described in the main text Experimental Protocol. Vinculin was used as an internal standard to normalize protein levels. The different panels show western blots of the time-dependent ERK-1/2 (p42/p44) phosphorylation after stimulation with decreasing amounts of EGF, as indicated. For all concentrations, the value for ERK-1/2 activation at 50 ng/ml EGF after 3 min was used as internal standard for maximal ERK phosphorylation. These experiments were done with the same passage of HeLa cells within 1 day, and were repeated four times with similar results. (B) Induction of c-fos expression after EGF treatment. HeLa cells were serum-starved for 24 h and then incubated with different concentrations of EGF for 1 h as indicated. Immunocytochemistry was done using anti-c-fos rabbit polyclonal antibody as described in the main text Experimental Protocol. Levels of c-fos protein were evaluated by counting the percentage of c-fos-labeled nuclei of duplicate populations of 500 cells for each EGF concentration indicated. Each experiment was repeated twice with similar results.

Supplementary Figure 4. (pdf 8K)
Curves from Figure 2A (green line) and F (blue line) for 0.5 ng/ml EGF calculated as percentage of total are combined.

Supplementary Figure 5. (pdf 11K)
Graphic representation of variability of association (A) and dissociation (B) rates using fixed initial conditions as shown in Supplementary Table 2. Thin horizontal lines represent the values used in the current model; vertical lines between thick bars show the possible variability of parameters on a logarithmic scale leading to the same solution.

Supplementary Figure 6. (pdf 16K)
Parameter variation for two different sets of initial conditions. A second model was calculated assuming underestimated initial conditions, leading to the same solution as in the original model. Compared to the molecule numbers shown in Supplementary Table 2, initial conditions were reduced as follows: Shc, 1/10; SOS, 1/3; Raf, 1/22; MEK, 1/500; ERK, 1/500; phosphatase 1 and 2, ¼; and phosphatase 3, 1/140. Circles indicate the parameters of the presented model from Supplementary Table 1. Crosses within the circles indicate no change of the parameter. Vertical lines between circles and crosses indicate the change of association rates (A) and dissociation rates (B) necessary to obtain the same solution.

Supplementary Figure 7. (pdf 243K)
Determination of concentrations of signaling proteins as described in the main text Experimental Protocol.

Supplementary Table 1 (xls 32K)

Supplementary Table 2 (pdf 59K)

Supplementary Results (pdf 65K)

Top