Abstract
Tyrosine phosphorylation is a post-translational modification that is critical for the regulation of many
cellular processes, including cell growth, differentiation, and signal transduction. The addition of a
phosphate group to tyrosine residues on proteins can alter their activity, localization, and interactions
with other proteins. Recent studies have revealed that thousands of phosphotyrosine sites occur within
structured protein domains, suggesting that phosphorylation may directly regulate domain function. Among
these domains, Src homology 2 (SH2) domains are of particular interest. SH2 domains are functional
units characterized by their ability to bind to phosphotyrosine-containing proteins, giving them a central
role in phosphotyrosine-mediated signaling. They also represent one of the most heavily phosphorylated
protein domains, with over 184 unique phosphotyrosine sites identified across 120 SH2 domains in the
human proteome. Despite this, the functional consequences of phosphorylation on SH2 domains remain
largely unexplored. This dissertation aims to address this gap in knowledge by investigating the role of
tyrosine phosphorylation in regulating SH2 domain function, using an integrated approach that combines
computational and experimental methods.
First, we utilize CoDIAC, a structural analysis pipeline for SH2 domains, to identify conserved
phosphotyrosine sites and generate hypotheses regarding their functional roles. From this analysis, we
were able to identify several regions within the SH2 domain binding interface containing highly conserved
phosphotyrosine sites, and using structural information, we were able to generate hypotheses regarding the
potential functional consequences of phosphorylation at these sites.
Second, we explore the use of phosphomimic mutations to study the effect of site-specific tyrosine
phosphorylation on SH2 domain binding to phosphopeptides. We develop a dot blot overlay assay to
screen for differences in binding between wild-type and phosphomimic mutations. Although phosphomimic
mutations have their limitations, we were able to identify specific phosphosites that significantly alter SH2
domain binding to phosphopeptides, and others that do not.
Lastly, we investigate native phosphorylation of SH2 domains using SiSa-KiT, our lab’s synthetic toolkit
designed to enable the native phosphorylation of proteins through co-expression with kinases in E. coli.
We designed a system where we isolated a phosphotyrosine site of interest on an SH2 domain to study the
effect of native phosphorylation on SH2 domain binding. We made several advances, such as successfully
using IMAC to enrich for phosphorylated SH2 domains and utilizing a new, tyrosine-free tag to replace
the GST tag for the detection of SH2-ligand interactions in a dot blot overlay assay. However, we found
that the number of mutations introduced ultimately reduced the binding capability of the SH2 domain,
thus preventing us from drawing conclusions regarding the effect of native phosphorylation on SH2 domain
binding.
Overall, this dissertation aims to provide a better understanding of the role of tyrosine phosphorylation in regulating SH2 domain function. By combining computational and experimental approaches, we hope to shed light on the functional consequences of phosphorylation on SH2 domains and provide insights into the
broader role of tyrosine phosphorylation in cellular signaling.
