Abstract:The inhibition of tyrosine kinases is a successful approach for the treatment of cancers and the discovery of kinase inhibitor drugs is the focus of numerous academic and pharmaceutical laboratories. With this goal in mind, several strategies have been developed to measure kinase activity and to screen novel tyrosine kinase inhibitors. Nevertheless, a general non-radioactive and inexpensive approach, easy to implement and adapt to a range of applications, is still missing. Herein, using Bcr-Abl tyrosine kinase… Show more
“…This assay utilizes a previously developed scaffold for bacterial surface-display of peptides, the enhanced circularly permuted OmpX (eCPX) protein (Rice and Daugherty, 2008). This scaffold has been used to measure phosphorylation levels of tyrosine-containing peptides on the surface of E. coli cells upon addition of a tyrosine kinase to the cell suspension, followed by detection using a pan-phosphotyrosine antibody and flow cytometry (Henriques et al, 2013). We expanded this technique by applying it to libraries of genetically encoded peptides and coupling it to fluorescence-activated cell sorting (FACS), followed by deep sequencing (Figure 2).…”
Section: Resultsmentioning
confidence: 99%
“…10.7554/eLife.20105.004Figure 2.A high-throughput assay for tyrosine kinase specificity.Top left panel: E. coli cells are transformed with plasmids encoding a library of peptide variants fused to the bacterial surface-display scaffold, eCPX (Rice and Daugherty, 2008). Top right panel: Expression of the peptide-scaffold fusions is induced to permit surface-display of the peptides, then the peptides on the extracellular surface of the cells are phosphorylated by the addition of a tyrosine kinase to the cell suspension (Henriques et al, 2013). Bottom right panel: Phosphorylated cells are labeled with a fluorescent pan-phosphotyrosine antibody, and cells with a high fluorescence signal are isolated by fluorescence-activated cell sorting.…”
Section: Resultsmentioning
confidence: 99%
“…Top right panel: Expression of the peptide-scaffold fusions is induced to permit surface-display of the peptides, then the peptides on the extracellular surface of the cells are phosphorylated by the addition of a tyrosine kinase to the cell suspension (Henriques et al, 2013). Bottom right panel: Phosphorylated cells are labeled with a fluorescent pan-phosphotyrosine antibody, and cells with a high fluorescence signal are isolated by fluorescence-activated cell sorting.…”
Section: Resultsmentioning
confidence: 99%
“…Bacteria bearing the surface-displayed peptide libraries were prepared using previously described protocols with slight modifications (Henriques et al, 2013; Rice and Daugherty, 2008). Typically, 100 ng of library DNA was used to transform 100 μL of electrocompetent E. coli MC1061 cells.…”
The sequence of events that initiates T cell signaling is dictated by the specificities and order of activation of the tyrosine kinases that signal downstream of the T cell receptor. Using a platform that combines exhaustive point-mutagenesis of peptide substrates, bacterial surface-display, cell sorting, and deep sequencing, we have defined the specificities of the first two kinases in this pathway, Lck and ZAP-70, for the T cell receptor ζ chain and the scaffold proteins LAT and SLP-76. We find that ZAP-70 selects its substrates by utilizing an electrostatic mechanism that excludes substrates with positively-charged residues and favors LAT and SLP-76 phosphosites that are surrounded by negatively-charged residues. This mechanism prevents ZAP-70 from phosphorylating its own activation loop, thereby enforcing its strict dependence on Lck for activation. The sequence features in ZAP-70, LAT, and SLP-76 that underlie electrostatic selectivity likely contribute to the specific response of T cells to foreign antigens.DOI:
http://dx.doi.org/10.7554/eLife.20105.001
“…This assay utilizes a previously developed scaffold for bacterial surface-display of peptides, the enhanced circularly permuted OmpX (eCPX) protein (Rice and Daugherty, 2008). This scaffold has been used to measure phosphorylation levels of tyrosine-containing peptides on the surface of E. coli cells upon addition of a tyrosine kinase to the cell suspension, followed by detection using a pan-phosphotyrosine antibody and flow cytometry (Henriques et al, 2013). We expanded this technique by applying it to libraries of genetically encoded peptides and coupling it to fluorescence-activated cell sorting (FACS), followed by deep sequencing (Figure 2).…”
Section: Resultsmentioning
confidence: 99%
“…10.7554/eLife.20105.004Figure 2.A high-throughput assay for tyrosine kinase specificity.Top left panel: E. coli cells are transformed with plasmids encoding a library of peptide variants fused to the bacterial surface-display scaffold, eCPX (Rice and Daugherty, 2008). Top right panel: Expression of the peptide-scaffold fusions is induced to permit surface-display of the peptides, then the peptides on the extracellular surface of the cells are phosphorylated by the addition of a tyrosine kinase to the cell suspension (Henriques et al, 2013). Bottom right panel: Phosphorylated cells are labeled with a fluorescent pan-phosphotyrosine antibody, and cells with a high fluorescence signal are isolated by fluorescence-activated cell sorting.…”
Section: Resultsmentioning
confidence: 99%
“…Top right panel: Expression of the peptide-scaffold fusions is induced to permit surface-display of the peptides, then the peptides on the extracellular surface of the cells are phosphorylated by the addition of a tyrosine kinase to the cell suspension (Henriques et al, 2013). Bottom right panel: Phosphorylated cells are labeled with a fluorescent pan-phosphotyrosine antibody, and cells with a high fluorescence signal are isolated by fluorescence-activated cell sorting.…”
Section: Resultsmentioning
confidence: 99%
“…Bacteria bearing the surface-displayed peptide libraries were prepared using previously described protocols with slight modifications (Henriques et al, 2013; Rice and Daugherty, 2008). Typically, 100 ng of library DNA was used to transform 100 μL of electrocompetent E. coli MC1061 cells.…”
The sequence of events that initiates T cell signaling is dictated by the specificities and order of activation of the tyrosine kinases that signal downstream of the T cell receptor. Using a platform that combines exhaustive point-mutagenesis of peptide substrates, bacterial surface-display, cell sorting, and deep sequencing, we have defined the specificities of the first two kinases in this pathway, Lck and ZAP-70, for the T cell receptor ζ chain and the scaffold proteins LAT and SLP-76. We find that ZAP-70 selects its substrates by utilizing an electrostatic mechanism that excludes substrates with positively-charged residues and favors LAT and SLP-76 phosphosites that are surrounded by negatively-charged residues. This mechanism prevents ZAP-70 from phosphorylating its own activation loop, thereby enforcing its strict dependence on Lck for activation. The sequence features in ZAP-70, LAT, and SLP-76 that underlie electrostatic selectivity likely contribute to the specific response of T cells to foreign antigens.DOI:
http://dx.doi.org/10.7554/eLife.20105.001
“…This approach assesses the sufficiency of particular types of residues at specific sites to confer phosphorylation by the kinase of interest. To gain an understanding of EGFR kinase specificity in the context of defined sequences, we developed a high-throughput assay, based on bacterial surface-display (35,36), fluorescence-activated cell sorting, and deep sequencing, to screen for efficiently phosphorylated tyrosine phosphosites (31) (Figure 1B). This multiplexed bacterial surface-display kinase assay has been used recently to characterize tyrosine kinase specificity in the T-cell receptor signaling pathway (31), and to analyze tyrosine kinase specificity on substrates spanning the human proteome (32).…”
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.
Microbes employ cell membranes for reducing exogenous stresses. Polyamino acid display on microbial cell surfaces and their effects on microbial chemical stress tolerance were examined. Growth analysis revealed that displays of polyarginine, polyaspartate and polytryptophan substantially enhanced tolerance of Escherichia coli to NaCl. A titration assay indicated that polyarginine and polyaspartate altered cell surface charges, implying tolerance enhancement via ion atmosphere and/or ionic bond network formations for electrostatic ion repulsion. The enhancement by polytryptophan may have arisen from surface hydrophobicity increase for hydrophobic ion exclusion, because of a strong correlation between hydrophobic characters of amino acids and their effects on tolerance enhancement. The display also enhanced tolerance to other salts and/or alcohols in E. coli and to NaCl in Saccharomyces cerevisiae. Thus polyamino acid display has the potential as an approach for conferring chemical stress tolerance on various microbes.
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