Figure 1.
FcεRI and LAT dephosphorylation in the hapten-inhibition experiment of Peirce and Metzger [40].
The figure is modified from Fig. 4B in Peirce and Metzger [40]. Square symbols represent FcεRI and the circles represent LAT. The experiment is detailed in Peirce and Metzger [40] and in Materials and Methods of this study. RBL cells were first stimulated with a polyvalent antigen to induce receptor crosslinking and signaling (before time zero in the figure), and then excess monovalent hapten was added (at time zero) to break up the receptor crosslinking. Subsequently, amounts of phosphorylated FcεRI and LAT were assayed at different time points. The figure shows the fraction of phosphorylated FcεRI and LAT remaining after hapten addition.
Figure 2.
Binding and phosphorylation reactions in the model that occur both in raft and in nonraft compartments.
1) Constitutive association of Lyn unique domain with the β subunit of the receptor. 2) Binding and cross-linking of IgE-FcεRI complexes by a multivalent ligand; 3) Transphosphorylation of the β and γ ITAMs by constitutively associated Lyn. 4) Recruitment of Lyn through its SH2 domain to the phosphorlated β ITAM and recruitment of Syk through its two SH2 domains to the doubly phosphorylated γ ITAM. 5) Michaelis-Menton interaction of a receptor aggregate containing Syk with LAT resulting in a phosphorylated LAT. 6) Binding of Grb2 to phosphorylated. Not shown are the reactions that dephosphorylate unprotected tyrosines.
Figure 3.
Predicted effects of lipid raft protection on protein phosphorylation.
Ligand dose-dependent phosphorylation of (A) FcεRI β, (B) FcεRI γ, (C) Syk, and (D) LAT in the model is shown for different levels of lipid raft protection. Phosphorylation is represented as the fraction of each protein in the phosphorylated state. Parameter which represents the level of raft protection (Materials and Methods), has the following interpretations:
represents absolute protection (proteins do not dephosphorylate inside lipid rafts);
represents no protection (proteins dephosphorylate at equal rates inside and outside lipid rafts); and
represents partial protections (proteins dephosphorylate at slower rates inside lipid rafts than outside lipid rafts). In simulations, default values (Table S1) are used for parameters not shown in the figure.
Figure 4.
LAT dephosphorylation in the simulated hapten-inhibition experiment.
Lines represent the simulation results, and symbols represent the experimental data of Peirce and Metzger [40]. Different lines in panels A and B represent different levels of raft protection as indicated by the parameter Experimental data and the line at
in panel A are re-plotted in linear scale in panel C. Experimental data and the line at
in panel B are re-plotted in linear scale in panel D. The top and the bottom panels represent different values of the receptor-Syk dissociation constant in the model:
The value taken from Faeder et al. [41], is used in panels A and C, and
is used in panels B and D. In simulations, steady-state receptor crossliking is induced with a bivalent ligand concentration of 1 nM, and then monovalent hapten is added at 100 µM (Materials and Methods). Time zero indicates the point when hapten is added. In simulations, default values (Table S1) are used for parameters not shown in the figure.
Figure 5.
FcεRI β, FcεRI γ and Syk dephosphorylation in the hapten-inhibition experiment.
Lines represent the simulation results, and symbols represent the experimental data of Mao and Metzger [42]. In each panel, different lines represent different levels of lipid raft protections as indicated by parameter The left panels (A, B, C) represent a receptor-Syk dissociation constant of 0.13 s−1 [41], and the right panels (D, E, F) represent a receptor-Syk dissociation constant of
s−1. In simulations, default values (Table S1) are used for parameters not shown in the figure.
Figure 6.
Effects of lipid raft lifetime increase on protein dephosphorylation.
The hapten inhibition experiment is simulated with a mean raft lifetime s (instead of 10 s, the default value in the model). With this change in
the lumped parameter
(Eq (1)) is adjusted to keep the partition coefficients of proteins
fixed. For other parameters, default values (Table S1) are used. Dephosphorylation kinetics of FcεRI β and γ are shown in panel A and B, respectively. Dephosphorylation kinetics of Syk and LAT are shown in panel C and D, respectively.
Figure 7.
Effects of raft lifetime decrease on dephosphorylation kinetics.
The hapten inhibition experiment is simulated with a mean raft lifetime s (instead of 10 s, the default value in the model). With this change in
the lumped parameter
(Eq (1)) is adjusted to keep the partition coefficients of proteins
fixed. For other parameters, default values (Table S1) are used. Dephosphorylation kinetics of FcεRI β and γ are shown in panel A and B, respectively. Dephosphorylation kinetics of Syk and LAT are shown in panel C and D, respectively.
Figure 8.
Effects of Lyn palmitoylation mutation on protein phosphorylation.
Simulation results are compared with experimental data of Kovarova et al. [29]. In the figure, WT represents wild type Lyn, and CA represents palmitoylation-mutated Lyn. Letter M within parentheses represent model predictions and E represents experimental data. Simulation results presented are for a fixed level of raft protection corresponding to . Default values are used for other parameters, as listed in Table S1.