Results: 5

1.
Figure 2

Figure 2. From: Linear combinations of docking affinities explain quantitative differences in RTK signaling.

Correlations between docking affinities and relative phosphorylation levels for SH2 and PTB domain-containing proteins. (A) Correlation coefficients, r, were determined for the eight SH2 or PTB domain-containing proteins for which both docking affinities and relative phosphorylation levels were measured. Red bars show correlations using docking affinities (sum of KA values); green bars show correlations using only the number of docking sites with KD⩽2 μM. The dotted line indicates a correlation coefficient of 0.9. (B) P-values for the correlations shown in (A). The dotted line indicates a P-value of 0.05. P-values less than 0.05 are marked with an asterisk.

Andrew Gordus, et al. Mol Syst Biol. 2009;5:235-235.
2.
Figure 3

Figure 3. From: Linear combinations of docking affinities explain quantitative differences in RTK signaling.

Linear combinations of docking affinities can predict upstream, but not downstream, signaling events. PLSR models were tested using leave-one-out cross-validation. (A) Predicted relative phosphorylation levels were plotted as a function of observed relative phosphorylation levels for each signaling event. Representative plots are shown for two upstream events (Stat3 pY705 and CrkL pY207) and two downstream events (Stat3 pS727 and SEK1/MKK4 pS257/pT261). The cross-validated residual, Q2, and the P-value are shown for each model. Plots for the other 20 models are provided in Supplementary Figure S6. (B) Q2 and P-values for all 24 PLSR models. Q2 values below zero were set to zero for display purposes. (C) Q2 and P-values for PLSR models built using: (1) the number of docking sites and the docking affinities (ΣKA); (2) only the number of docking sites (no. of sites); and (3) only the four variables with the highest VIP scores (reduced). Red circles represent upstream signaling events and blue circles represent downstream signaling events.

Andrew Gordus, et al. Mol Syst Biol. 2009;5:235-235.
3.
Figure 4

Figure 4. From: Linear combinations of docking affinities explain quantitative differences in RTK signaling.

Contribution of SH2 and PTB domains in predicting the relative phosphorylation levels of upstream signaling proteins. (A) Heat map showing the variable importance in the projection (VIP) for each SH2 or PTB domain-containing protein in each PLSR model of upstream signaling events. The average across all 10 models is shown to the right. (B) Bar graph showing the coefficients for PI3K, CPI3K, in the six statistically significant PLSR models. (C) Bar graph showing the sum of PI3K-docking affinities for each RTK. (D, E) Relative phosphorylation levels for (D) Src pY416 and (E) Stat3 pY705 across the six cell lines, with and without PI3K inhibitor LY294002 (100 μM). Bar graphs for the other eight upstream signaling events are provided in Supplementary Figure 7. Source data is available for this figure at www.nature.com/msb

Andrew Gordus, et al. Mol Syst Biol. 2009;5:235-235.
4.
Figure 5

Figure 5. From: Linear combinations of docking affinities explain quantitative differences in RTK signaling.

Bias for PI3K- and Shc1-binding sites in human RTKs. (A) Heat map showing the bias for each of the 20 amino acids at each position relative to sites of tyrosine phosphorylation in human RTKs. (B) Histogram of the observed/expected frequencies in (A). The line is a log-normal fit to the data. The red bars indicate significant deviations (P<0.05) and reflect biases (red squares in (A)). The biases for Cys at positions −5 and +6 and for Trp at position +7 are likely due to the conservation of structurally important residues at these locations relative to the conserved phosphotyrosine residue in the activation loop of the kinase domain. The biases for Asn at position −3 and Pro at position −2 match the consensus recognition sequence for the PTB domain of Shc1 (NPXpY). The bias for Met at position +3 matches the consensus recognition sequence for the SH2 domains of PI3K (pYXXM) and we frequently observe Asp at position +2 in phosphopeptides that are recognized by these domains. (C) Same as for (A), but using sites of tyrosine phosphorylation on all human proteins excluding RTKs. (D) Same as for (B), but using sites of tyrosine phosphorylation on all human proteins excluding RTKs.

Andrew Gordus, et al. Mol Syst Biol. 2009;5:235-235.
5.
Figure 1

Figure 1. From: Linear combinations of docking affinities explain quantitative differences in RTK signaling.

Measurement of the intrinsic differences among six receptor tyrosine kinases. (A) The full-length coding regions for six RTKs were introduced into Flp-In-293 cells to generate stable cell lines. Each cell line was serum-starved for 24 h and stimulated for 5 min with a saturating concentration of the indicated growth factor. (B) Cell lysates were analyzed by quantitative immunoblotting to determine the relative levels of 24 phosphorylation sites on 23 signaling proteins across the six cell lines. Representative results are shown for four phosphorylation sites. Error bars indicate the range of biological duplicates. The other 20 bar graphs are provided in Supplementary Figure S2. (C) Heat map illustrating the relative levels of the 24 phosphorylation sites across the six cell lines. The columns of this matrix, Y, constitute relative phosphorylation vectors for each signaling event. (D) Protein microarrays comprising almost every human SH2 and PTB domain were printed in individual wells of 96-well microtiter plates and probed with eight concentrations of each phosphopeptide, ranging from 10 nM to 5 μM. Phosphopeptides were derived from established sites of tyrosine phosphorylation on the six RTKs. For each domain–peptide interaction, a saturation-binding curve was obtained and the observed fluorescence, Fobs, was fit to equation (1) to obtain an equilibrium dissociation constant, KD. (E) KD values were converted to KA values (KD=1/KA) and each phosphopeptide was represented as a vector of KA values. (F) Each receptor vector was defined as the sum of its constituent phosphopeptide vectors. The receptor-docking affinity matrix, X, comprises the six receptor vectors. Source data is available for this figure at www.nature.com/msb.

Andrew Gordus, et al. Mol Syst Biol. 2009;5:235-235.

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