Abstract
The specificity of tyrosine kinases is attributed predominantly to localization effects dictated by non-catalytic domains. We developed a method to profile the specificities of tyrosine kinases by combining bacterial surface-display of peptide libraries with next-generation sequencing. Using this, we showed that the tyrosine kinase ZAP-70, which is critical for T cell signaling, discriminates substrates through an electrostatic selection mechanism encoded within its catalytic domain (Shah et al., 2016). Here, we expand this high-throughput platform to analyze the intrinsic specificity of any tyrosine kinase domain against thousands of peptides derived from human tyrosine phosphorylation sites. Using this approach, we find a difference in the electrostatic recognition of substrates between the closely related Src-family kinases Lck and c-Src. This divergence likely reflects the specialization of Lck to act in concert with ZAP-70 in T cell signaling. These results point to the importance of direct recognition at the kinase active site in fine-tuning specificity.
Highlights
The ~30 cytoplasmic tyrosine kinases are characterized by the presence of a tyrosine kinase domain and one or more modular binding domains, such as SH2 and SH3 domains (Robinson et al, 2000)
We have shown recently that there is an important role for direct substrate selection by the kinase active sites of two cytoplasmic tyrosine kinases, the Src-family kinase Lck and the Syk-family kinase ZAP-70, in the response of T cells to antigen recognition (Shah et al, 2016)
We purchased peptide-coding oligonucleotides generated by massively parallel on-chip DNA synthesis (Twist Bioscience), and we cloned these coding sequences into plasmids containing a bacterial surface-display scaffold gene (Rice and Daugherty, 2008)
Summary
The ~30 cytoplasmic tyrosine kinases are characterized by the presence of a tyrosine kinase domain and one or more modular binding domains, such as SH2 and SH3 domains (Robinson et al, 2000). To analyze the differential specificities of Lck and ZAP-70, we had adapted a high-throughput platform to simultaneously measure the efficiency of phosphorylation by a tyrosine kinase of hundreds of genetically encoded peptides in bacterial surface-display libraries (Figure 1B) (Shah et al, 2016). Prior studies employing peptide library screens with degenerate sequences have shown that all eight of the human Src-family kinases have similar substrate sequence preferences (Songyang et al, 1995; Schmitz et al, 1996; Deng et al, 2014) They prefer a large aliphatic residue immediately upstream of the target tyrosine residue in their substrates, a feature that distinguishes their specificity from that of ZAP-70, which prefers an aspartic acid residue at this position (Shah et al, 2016). Our approach provides a tool that is deployed to interrogate the proteome-wide specificity of any tyrosine kinase, by leveraging the speed and accuracy of next-generation sequencing
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