Deep mutational analysis reveals functional trade-offs in the sequences of EGFR autophosphorylation sites
| Autor: | Neel H. Shah, John Kuriyan, Aaron J. Cantor |
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| Rok vydání: | 2018 |
| Předmět: |
inorganic chemicals
Phosphotyrosine binding 0301 basic medicine Proteome Protein Conformation EGFR DNA Mutational Analysis specificity macromolecular substances environment and public health Biochemistry 03 medical and health sciences 0302 clinical medicine deep mutational scanning Humans Phosphorylation Tyrosine 030304 developmental biology 0303 health sciences Multidisciplinary Epidermal Growth Factor biology Chemistry Autophosphorylation Signal transducing adaptor protein Biological Sciences Cell biology ErbB Receptors SH2 enzymes and coenzymes (carbohydrates) 030104 developmental biology PNAS Plus Protein kinase domain 030220 oncology & carcinogenesis biology.protein bacteria Generic health relevance GRB2 signaling Tyrosine kinase Receptor Signal Transduction Proto-oncogene tyrosine-protein kinase Src |
| Zdroj: | Cantor, AJ; Shah, NH; & Kuriyan, J. (2018). Deep mutational analysis reveals functional trade-offs in the sequences of EGFR autophosphorylation sites. Proceedings of the National Academy of Sciences of the United States of America, 115(31), E7303-E7312. doi: 10.1073/pnas.1803598115. UC Berkeley: Retrieved from: http://www.escholarship.org/uc/item/7nb1t9db Proceedings of the National Academy of Sciences of the United States of America, vol 115, iss 31 Proceedings of the National Academy of Sciences of the United States of America |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | Significance Phosphorylation of tyrosine residues in the cytoplasmic tail of the epidermal growth factor receptor (EGFR) by its kinase domain propagates a rich variety of information downstream of growth factor binding. The amino acid sequences surrounding each phosphorylation site encode the extent of phosphorylation as well as the extent of binding by multiple effector proteins. By profiling the kinase activity of EGFR alongside the binding specificities of an SH2 domain and a PTB domain for thousands of defined phosphorylation site sequences, we discovered that the sequences surrounding the phosphorylation sites in EGFR are not optimal and that discrimination against phosphorylation by cytoplasmic tyrosine kinases such as c-Src and c-Abl is likely to have shaped the evolution of these sequences. 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. |
| Databáze: | OpenAIRE |
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