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

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) andPhosphotyrosine 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. In order 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 while maintaining the ability to recruit the diverse complement of effectors required for downstream pathway activation.Cantor et al.

MotifAnalyzer‐PDZ: A computational program to investigate the evolution of PDZ‐binding target specificity

“…Kinase domains typically recognize and phosphorylate serine, threonine, or tyrosine residues in target proteins, and although some kinases are promiscuous, with motifs limited to a small number of amino acid residues, others are more specific, with up to 10 amino acid positions indicating a favorable phosphorylation site . For example, if we look for motif‐satisfying sequences that match the Src motif (E/D‐X‐I/L/V‐ Y ‐E/G‐X‐L/F, where X is any amino acid and the phosphorylated tyrosine is in bold ) in the entire human and choanoflagellate proteomes, we find 165 sequences in H. sapiens and 64 in M. brevicollis that contain all motif‐satisfying residues (Table S2).…”

A two-component protein condensate of EGFR and Grb2 regulates Ras activation at the membrane

“…To maintain the EGFR TAIL in its active, phosphorylated state, a separate Src-family kinase protein, Hck, is tethered to the membrane (also through His tag chemistry) and ATP is provided in solution. Src kinases do not normally phosphorylate the EGFR TAIL efficiently (19), but in this system the pre-incubation with Hck suffices to ensure robust phosphorylation of the tail. A similar strategy was used previously to maintain LAT in its phosphorylated state for condensation phase transition studies (5, 30, 42).…”

“…There is an obvious conceptual connection between EGFR and the LAT signaling system in T cells. The ~200 residue long cytoplasmic tail of EGFR resembles LAT in that both are intrinsically-disordered and contain multiple sites of tyrosine phosphorylation that recruit adaptor proteins upon receptor activation (19). Phosphorylation at tyrosine residues Y1068, Y1086, Y1148, and Y1173 in the EGFR tail creates sites to which Grb2 can bind via its SH2 domain.…”

Design and evaluation of engineered protein biosensors for live-cell imaging of EGFR phosphorylation

“…A biosensor with low binding affinity for its target phosphorylation site is particularly relevant in the context of EGFR. The amino acid composition of residues surrounding the phosphosites in EGFR has likely evolved to avoid off-pathway phosphorylation by intracellular kinases, a consequence of which is weaker binding to adapter proteins (36).…”