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. 2018 Jul 31;115(31):E7303-E7312.
doi: 10.1073/pnas.1803598115. Epub 2018 Jul 16.

Deep mutational analysis reveals functional trade-offs in the sequences of EGFR autophosphorylation sites

Aaron J Cantor  1   2   3 , Neel H Shah  1   2   3 , John Kuriyan  4   2   3   5   6
Affiliations

Deep mutational analysis reveals functional trade-offs in the sequences of EGFR autophosphorylation sites

Aaron J Cantor et al. Proc Natl Acad Sci U S A. .

Abstract

Upon activation, the epidermal growth factor receptor (EGFR) phosphorylates tyrosine residues in its cytoplasmic tail, which triggers the binding of Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains and initiates downstream signaling. The sequences flanking the tyrosine residues (referred to as "phosphosites") must be compatible with phosphorylation by the EGFR kinase domain and the recruitment of adapter proteins, while minimizing phosphorylation that would reduce the fidelity of signal transmission. To understand how phosphosite sequences encode these functions within a small set of residues, we carried out high-throughput mutational analysis of three phosphosite sequences in the EGFR tail. We used bacterial surface display of peptides coupled with deep sequencing to monitor phosphorylation efficiency and the binding of the SH2 and PTB domains of the adapter proteins Grb2 and Shc1, respectively. We found that the sequences of phosphosites in the EGFR tail are restricted to a subset of the range of sequences that can be phosphorylated efficiently by EGFR. Although efficient phosphorylation by EGFR can occur with either acidic or large hydrophobic residues at the -1 position with respect to the tyrosine, hydrophobic residues are generally excluded from this position in tail sequences. The mutational data suggest that this restriction results in weaker binding to adapter proteins but also disfavors phosphorylation by the cytoplasmic tyrosine kinases c-Src and c-Abl. Our results show how EGFR-family phosphosites achieve a trade-off between minimizing off-pathway phosphorylation and maintaining the ability to recruit the diverse complement of effectors required for downstream pathway activation.

Keywords: EGFR; SH2; deep mutational scanning; signaling; specificity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Overview of EGFR signal transduction at the membrane and a bacterial surface display scheme to analyze the specificity of tyrosine kinases and phosphotyrosine-binding proteins. (A) Illustration of membrane-proximal EGFR signaling components. Autophosphorylation of the tyrosine phosphosites in the C-terminal cytoplasmic tail (red circles) by the activated kinase domain produces binding sites for many downstream effectors, a subset of which are depicted. These effectors go on to activate second-messenger pathways, also depicted. Grb2, growth factor receptor-bound protein 2; MAPK, mitogen-activated protein kinases; PI3K, phosphoinositide 3-kinase regulatory subunit; PKC, protein kinase C; Plcγ1, phospholipase C-gamma-1; Shc1, SH2 domain-containing-transforming protein C1. (B) Workflow for determining phosphosite specificity profiles of tyrosine kinases and phosphotyrosine-binding proteins by bacterial surface display coupled with FACS and deep sequencing. Phosphotyrosine on the surface of the cells is detected either by immunostaining with an anti-phosphotyrosine antibody or, for binding profiles, with either a tandem SH2 or PTB construct fused to GFP. The frequency of each peptide-coding sequence in the highly phosphorylated population, or enrichment, and thus the relative efficiency of phosphorylation or binding for each peptide, is determined by counting the number of sequencing reads for each peptide in the sorted and unsorted populations.
Fig. 2.
Fig. 2.
Comparison of intrinsic EGFR and c-Src substrate specificity with EGFR-family phosphosite sequences. (A) Histogram of peptide read frequency ratios from EGFR phosphorylation of a library of human phosphosites obtained by bacterial surface display and deep sequencing. The distribution of ratios of read frequencies for input and sorted samples are plotted from two replicate experiments. (B) Read-frequency ratios for two replicate Human-pTyr library phosphorylation experiments plotted against each other. Peptides with ratios above the 75th percentile in both replicates (gray box) were counted as highly phosphorylated in C. (C) Phospho-pLogo of highly phosphorylated peptides for EGFR in the bacterial surface display experiment. The height of each letter corresponds to the negative log-odds ratio of binomial probabilities of finding a given amino acid residue at a particular sequence position at higher versus lower frequencies than the expected positional frequency for all peptides in the library. Higher values indicate an enrichment of a residue versus the background distribution. Red lines indicate the log-odds ratio values for a significance level of 0.05, as defined in ref. . (D) Sequence-pLogo of EGFR-family C-terminal tail tyrosines. Sequence segments surrounding tyrosine were extracted from the regions C-terminal to the kinase domain for metazoan EGFR-family protein sequences. The positional amino acid frequency in these segments was compared with the frequency in metazoan intracellular and transmembrane proteins and was plotted as a pLogo. (E and F) Phospho-pLogo of highly phosphorylated sequences from c-Src phosphorylation (E) and c-Abl phosphorylation (F) of the Human-pTyr library (raw data are from ref. 32). Sequences above the 75th percentile in three replicates are included in the highly phosphorylated set.
Fig. 3.
Fig. 3.
Effect of single amino acid substitutions on the phosphorylation of three EGFR phosphosite peptides by EGFR. (A) Sequences of three human EGFR C-terminal tail phosphosites. (B) Heat maps showing the effect of all single amino acid substitutions (except tyrosine and cysteine) on the phosphorylation level of three EGFR phosphosite peptides relative to the wild-type peptide upon phosphorylation by EGFR, measured by bacterial surface display and deep sequencing. Squares for each substitution x at each wild-type position i are colored as log-twofold enrichment relative to wild type (∆Exi), calculated from read frequency ratios of sorted and input samples. Wild-type residue squares (∆Ewti = 0 by definition) are indicated by gray squares. The ∆E scales for each peptide, displayed in the top right corner of each heat map, are not directly comparable because different optimized cell-sorting parameters were used for each peptide. Red and blue colors indicate variants that were phosphorylated more or less, respectively, than the wild-type sequence. Row and column mean ∆E values are displayed separately. Data are the variantwise mean of at least two replicates. (C) Enrichment values for the −2 column (∆Ex−2) for each peptide. Error bars indicate the SEM.
Fig. 4.
Fig. 4.
Comparison of c-Src and EGFR specificity with respect to EGFR substrates. (A) Relative phosphorylation of EGFR-family phosphosites and reported cytoplasmic EGFR substrates by c-Src versus EGFR. Log-twofold enrichment values relative to a non–tyrosine-containing control peptide were calculated from peptide read frequencies in sorted and input samples. These enrichment values (denoted ∆E*) were corrected by the relative expression level measured for each peptide by cell sorting and deep sequencing. ∆E* values are not comparable on the same scale between kinases. The mean of three replicates with 95% CIs for each kinase is plotted. (B) Venn diagram showing membership of peptides in the top quartile of ∆E* values for each kinase. (C) Specific activities measured for c-Src and EGFR by an NADH-coupled assay against selected peptides at 0.5 mM peptide. Three EGFR C-terminal tail phosphosites and one c-Src substrate, noted below each set of bars, were measured. Error bars indicate the 95% CI of the mean. (D) Heat map showing the effect of single amino acid substitutions on the phosphorylation level of the EGFR Tyr-1086 phosphosite relative to wild type upon phosphorylation by c-Src, as measured by bacterial surface display and deep sequencing. ∆Exi is displayed as a heat map, as described in Fig. 2.
Fig. 5.
Fig. 5.
Effect of single amino acid substitutions on the binding of the Grb2 SH2 domain and Shc1 PTB domain to two EGFR phosphosites. Log-twofold changes in read frequency ratios relative to wild-type (∆Exi) were determined by cell sorting after labeling phosphorylated bacteria displaying peptides with tandem copies of the Shc1 PTB domain (A) or the Grb2 SH2 domain (B) fused to GFP. ∆Exi values for single amino acid substitutions are displayed as heat maps, as described in Fig. 2.
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