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. 2015 Oct;1853(10 Pt A):2560-9.
doi: 10.1016/j.bbamcr.2015.06.002. Epub 2015 Jun 12.

Regions outside of conserved PxxPxR motifs drive the high affinity interaction of GRB2 with SH3 domain ligands

Rebekah R Bartelt  1 , Jonathan Light  1 , Aldo Vacaflores  2 , Alayna Butcher  1 , Madhana Pandian  1 , Piers Nash  3 , Jon C D Houtman  4
Affiliations

Regions outside of conserved PxxPxR motifs drive the high affinity interaction of GRB2 with SH3 domain ligands

Rebekah R Bartelt et al. Biochim Biophys Acta. 2015 Oct.

Abstract

SH3 domains are evolutionarily conserved protein interaction domains that control nearly all cellular processes in eukaryotes. The current model is that most SH3 domains bind discreet PxxPxR motifs with weak affinity and relatively low selectivity. However, the interactions of full-length SH3 domain-containing proteins with ligands are highly specific and have much stronger affinity. This suggests that regions outside of PxxPxR motifs drive these interactions. In this study, we observed that PxxPxR motifs were required for the binding of the adaptor protein GRB2 to short peptides from its ligand SOS1. Surprisingly, PxxPxR motifs from the proline rich region of SOS1 or CBL were neither necessary nor sufficient for the in vitro or in vivo interaction with full-length GRB2. Together, our findings show that regions outside of the consensus PxxPxR sites drive the high affinity association of GRB2 with SH3 domain ligands, suggesting that the binding mechanism for this and other SH3 domain interactions may be more complex than originally thought.

Keywords: CBL; GRB2; SH3 domains; SOS1; Signal transduction.

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Figures

Figure 1
Figure 1. PxxPxR motifs are required for binding of full-length GRB2 to SOS1 peptides
Full-length human GRB2 was incubated with a peptide array derived from the PRR of SOS1. The binding of GRB2 to the array was assessed by immunoblotting with an anti-GRB2 antibody. Peptides containing PxxPxR motifs are described below the blot.
Figure 2
Figure 2. SOS1 peptide with a PxxPxR motif does not significantly inhibit the interaction of GRB2 with the complete proline rich region of SOS1
A) Full-length GRB2 was pre-incubated with a SOS1 site 1 peptide. The ability of the peptide to inhibit the binding of GRB2 to the complete proline rich region of SOS1 was then assessed via ITC. Each point on the curve is an average ± 95% confidence interval of 2 or 3 replicates. The curves were fit using the Boltzmann sigmoidal equation in GraphPad Prism. B) The values for affinity, change in enthalpy (ΔH) and stoichiometry from three independent experiments are shown. The statistical analysis was performed using GraphPad Prism via ANOVA analysis and p values for the comparison of the no peptide control to 2:1 or 5:1 molar ratio of peptide are shown.
Figure 3
Figure 3. The consensus PxxPxR motifs of SOS1 are not required for the high affinity interaction with GRB2
A) The sequence for SOS1 wild-type, SOS1 Δ1234Z, SOS1 ΔSite AB and SOS1 scrambled are shown. B) The binding of GRB2 to the complete proline rich regions of SOS1 were assessed by ITC. The values for affinity, ΔH and stoichiometry were determined using the Origin ITC analysis package. The individual values for affinity, ΔH and stoichiometry were plotted with the average ± 95% confidence interval using GraphPad Prism. The statistical analysis was performed using GraphPad Prism via ANOVA analysis and the p values < 0.50 are shown.
Figure 4
Figure 4. Regions outside of the consensus PxxPxR motifs are required for the high affinity interaction of GRB2 with SOS1
A) Raw ITC tracings for the interaction of GRB2 with SOS1 WT, SOS1 Δ1234Z and SOS1 scrambled. Each trace is representative of at least 4 separate injections. B) The hydrodynamic radius of GRB2, SOS1 WT, SOS1 Δ1234Z or SOS1 scrambled, both alone and in combination, was determined using DLS. The individual values, with the mean ± 95% confidence intervals were plotted using GraphPad Prism. Statistical analysis was performed using GraphPad Prism via ANOVA analysis and the p values are shown.
Figure 5
Figure 5. The consensus PxxPxR motifs of CBL are not required for the high affinity interaction with GRB2
A) The sequence for CBL wild-type and CBLΔ123 are shown. B) The binding of GRB2 to the complete proline rich regions of CBL were assessed by ITC. The values for affinity, ΔH and stoichiometry were determined using the Origin ITC analysis package. The individual values for affinity, ΔH and stoichiometry were plotted with the average ± 95% confidence interval using GraphPad Prism. The statistical analysis was performed using GraphPad Prism via ANOVA analysis and the p values < 0.50 are shown.
Figure 6
Figure 6. SOS1 wild-type and SOS1 Δ1234Z are recruited to the plasma membrane upon TCR activation in human T cells
The expression of YFP fusion proteins in Jurkat E6.1 T cells stably expressing SOS1 wild-type PRR-YFP (WT-YFP), SOS1 Δ1234Z PRR-YFP (Δ1234Z-YFP), SOS1 scrambled PRR-YFP (Scram-YFP) or YFP alone (YFP) was assessed by A) flow cytometry or B) immunoblotting. C) Jurkat E6.1 T cells stably expressing SOS1 wild-type PRRYFP (WT-YFP), SOS1 Δ1234Z PRR-YFP (Δ1234Z-YFP) or YFP alone (YFP) were activated using coverslips coated with a stimulatory anti-TCR antibody. The YFP fluorescence and LAT phosphorylation occurring at the membrane was imaged using TIRF microscopy. LAT phosphorylation was assessed by immunofluorescence using a phospho-specific LAT Y226 antibody. The total YFP fluorescence in the cell was imaged by EPI fluorescence. D) The fluorescence in the TIRF and EPI fluorescence channel was quantified for individual cells using NIH Image. The individual values and the mean ± 95% confidence intervals were plotted using GraphPad Prism. The statistical analysis was performed using GraphPad Prism via ANOVA analysis and the p value for each comparison is shown.
Figure 7
Figure 7. Comparison of the current and revised model for the interaction of GRB2 with SH3 domain ligands
The current model suggests that PxxPxR motifs are critical for the interaction of GRB2 with SH3 domain ligands. These interactions are low affinity and have variable ligand specificity driven by an unknown mechanism, making the poor clinical targets. Our revised model indicates that the interaction of GRB2 with SH3 domain ligands is driven by multiple contacts outside of the PxxPxR motifs. These interactions are high affinity and have ligand specificity based on sequence differences; thus, these interactions could potentially be targeted for clinical intervention.
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