Investigating the Role of Tyrosine Phosphorylation on SH2 Domain Binding

Introduction: Src homology 2 (SH2) domains are structurally conserved protein domains that are involved in tyrosine kinase signaling cascades due to their ability to recognize phosphotyrosine (pTyr) containing peptides. These domains play a critical role in cell signaling and mediating protein-protein interactions through the regulation of cellular processes such as cell growth and differentiation, and dysregulated SH2 domain signaling has been seen in many different forms of cancer. We have previously discovered that SH2 domains are heavily tyrosine phosphorylated, but little research has been conducted on the impact of tyrosine phosphorylation on these domains.
This work uses a mixed experimental and computational approach to study the role of tyrosine phosphorylation on SH2 domains. Using our previously developed protein domain analysis pipeline, we are able to gain insight into the role of post-translational modifications (PTMs) on SH2 domains through the overlay of PTMs with ligand binding interfaces. Guided by the hypotheses generated from this analysis, we explore the impact of tyrosine phosphorylation on SH2 domains in vitro, focusing on the changes to ligand binding specificity and affinity. One SH2 domain of interest is PTPN11, a tandem SH2 containing phosphatase that has been implicated in several diseases. While pY62 within the N-SH2 domain has been shown to alter PTPN11 signaling, the mechanism behind this is still understudied. This work will also expand to additional SH2 domains with different pTyr sites across the binding interface, including Lyn-SH2 Y194 as a positive control for binding changes based on prior literature [1].

Materials and

Methods: A common method used to study tyrosine phosphorylation is through the use of phosphomimics, where a Y is mutated to E or D. Although these amino acids are the closest in structural similarity to pTyr, these are ultimately poor substitutes for phosphorylation because they do not accurately reflect the size and charge change that occurs upon modification. To avoid this issue, this work utilizes our lab’s engineered kinase toolkit, a process that involves the coexpression of a substrate and a kinase in E. coli, with the kinase being targeted to the substrate through an interaction between an SH3 domain linked to the kinase domain and a polyproline sequence linked to the substrate. After these proteins are coexpressed, the substrate and kinase are induced individually, and the resulting native phosphoprotein is purified using affinity purification. When off-target phosphorylation is a concern, the site of interest is mutated to a phenylalanine, and the effects resulting from the inability of a specific site to be phosphorylated can be measured.

Results, Conclusions, and Discussions: Through the use of qualitative and quantitative assays, we have been able to characterize the impact of site-specific tyrosine phosphorylation on SH2 domain binding specificity and affinity. Using a far-western approach to detect protein-protein interactions, we were able to globally detect differences in binding specificity patterns upon modification of specific sites within our SH2 domains of interest, accounting for off-target effects from additional tyrosine sites within the respective domains through phenylalanine mutations. Further, using biolayer interferometry with known canonical binding partners of the respective SH2 domains, we are able to quantify the affinity changes resulting from site-specific phosphorylation. Successful recapitulation of the trends from the Lyn Y194 study provides confidence in the validity of our assays.

Acknowledgements (Optional): Alekhya Kandoor, Swathi Shekharan, and Kristen Naegle, Ph.D.
NIH T32