Dynalogo: an interactive sequence logo with dynamic thresholding of matched quantitative proteomic data.

SUMMARY: Current web-based sequence logo analyses for studying domain-peptide interactions are often conducted only on high affinity binders due to conservative data thresholding. We have developed Dynalogo, a combination of threshold varying tool and sequence logo generator written in the R statistical programming language, which allows on-the-fly visualization of binding specificity over a wide range of affinity interactions. Hence researchers can easily explore their dataset without the constraint of an arbitrary threshold. After importing quantitative data files, there are various data filtering and visualizing features available. Using a threshold control, users can easily track the dynamic change of enrichment and depletion of amino acid characters in the sequence logo panel. The built-in export function allows downloading filtered data and graphical outputs for further analyses. Dynalogo is optimized for analysis of modular domain-peptide binding experiments but the platform offers a broader application including quantitative proteomics. AVAILABILITY AND IMPLEMENTATION: Dynalogo application, user manual and sample data files are available at https://dynalogo.cam.uchc.edu. The source code is available at https://github.com/lafontaine-uchc/dynalogo. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Src homology 2 domains enhance tyrosine phosphorylation in vivo by protecting binding sites in their target proteins from dephosphorylation.

Phosphotyrosine (pTyr)-dependent signaling is critical for many cellular processes. It is highly dynamic, as signal output depends not only on phosphorylation and dephosphorylation rates but also on the rates of binding and dissociation of effectors containing phosphotyrosine-dependent binding modules such as Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains. Previous in vitro studies suggested that binding of SH2 and PTB domains can enhance protein phosphorylation by protecting the sites bound by these domains from phosphatase-mediated dephosphorylation. To test whether this occurs in vivo, we used the binding of growth factor receptor bound 2 (GRB2) to phosphorylated epidermal growth factor receptor (EGFR) as a model system. We analyzed the effects of SH2 domain overexpression on protein tyrosine phosphorylation by quantitative Western and far-Western blotting, mass spectrometry, and computational modeling. We found that SH2 overexpression results in a significant, dose-dependent increase in EGFR tyrosine phosphorylation, particularly of sites corresponding to the binding specificity of the overexpressed SH2 domain. Computational models using experimentally determined EGFR phosphorylation and dephosphorylation rates, and pTyr-EGFR and GRB2 concentrations, recapitulated the experimental findings. Surprisingly, both modeling and biochemical analyses suggested that SH2 domain overexpression does not result in a major decrease in the number of unbound phosphorylated SH2 domain-binding sites. Our results suggest that signaling via SH2 domain binding is buffered over a relatively wide range of effector concentrations and that SH2 domain proteins with overlapping binding specificities are unlikely to compete with one another for phosphosites in vivo.