Abstract
Tyrosine phosphorylation is a critical regulator of cell signaling, especially within signaling pathways that control cell growth, survival, proliferation, motility, and differentiation. Dysregulation within these pathways is common within the context of cancer, and there is a growing focus on developing drug treatments that target tyrosine kinases and their downstream effectors. However, proper development of these drugs depends on a comprehensive understanding not only of how these proteins interact within the cell, but also how pathways interconnect and ultimately allow the cell to utilize alternative mechanisms of action and develop resistance to the treatment plans. Unfortunately, of the 46,000 phosphotyrosine (pTyr) sites within the human proteome, only about 5% have a known function, leaving us with significant gaps in our understanding of these protein-protein interactions and hindering our ability to effectively control cell behavior. One limitation in our ability to explore pTyr function is an over-reliance on phospho-specific antibodies that leads to us over-studying the same small subset of sites in every research study and potentially missing more critical sites elsewhere. Mass spectrometry helps us explore these overlooked sites, but the rarity of tyrosine phosphorylation makes it easy for the pTyr peptides to get lost in the background of the cell sample. Some improvements have been made within the phosphoproteomics field, but the high cost of reagents makes it inaccessible to most labs. Additionally, studying specific protein-protein interactions in a more in-depth manner relies on the ability to produce phosphorylated protein, but it is challenging to achieve high incorporation efficiency of the phosphate group, especially in the context of proteins containing multiple pTyr sites. In this dissertation, we utilize a newly developed tool from our lab, SISA-KiT, to explore multivalency in tandem SH2 protein interactions with biphosphorylated proteins as well as develop a new tool that will allow for the production of cheaper, multiplexed phosphorylated standards to aid in phosphoproteomics experiments.
In Aim 1, we utilize an ODE model originally developed by Dr. Casim Sarkar to study receptor-ligand interactions to explore the role of avidity in tandem SH2 protein binding events. We gain insight into the conditions necessary to optimize avidity, develop theories on the advantages of tandem SH2 linker structures, and utilize the model to predict preferred binding partners of complex systems. SISA-KiT allows us to produce biphosphorylated protein, which we use in BLI studies to measure the bivalent binding affinity of these proteins and experimentally validate the findings of the model.
In Aim 2, we develop a phosphorylated QconCAT, or PhosCAT, polypeptide composed by concatenating pTyr-containing tryptic peptides from various proteins of interest. SISA-KiT is utilized to drive phosphorylation across this protein with the goal of digesting the protein into individual phosphopeptide standards to use as spike-ins for mass spectrometry experiments. We optimize the PhosCAT design and workflow to achieve the highest degree of phosphorylation and showcase the customizability of this technique which can be adapted for studying a wide range of phosphorylation events. We also introduce a novel application of the PhosCAT protein to study phosphatase specificity and activity.
Overall, this dissertation provides new approaches to improving our understanding of the role of tyrosine phosphorylation in regulating signaling dynamics within a cell. These techniques can improve our ability to treat debilitating illnesses and can easily be adapted to explore the impact of other post-translational modifications.
