Biomedical Sciences ETDs

Publication Date

Summer 6-21-2018

Abstract

Signaling through the Epidermal Growth Factor Receptor (EGFR) plays an important role in both physiological and cancer-related processes. In this work, single-molecule microscopy measurements and computational modeling were closely integrated to better understand the mechanisms that regulate EGFR signaling. Technical improvements were made over the previously described Single-Molecule Pull-down (SiMPull) assay to facilitate direct detection of the phosphorylation state of thousands of individual receptors, and thereby estimate both the fraction of receptors phosphorylated at specific tyrosine residues and the frequency of multisite phosphorylation. These improvements enabled the first direct detection of multisite phosphorylation on full-length Epidermal Growth Factor Receptor (EGFR), and revealed that the extent of phosphorylation varied by tyrosine residue (biased phosphorylation). To help in understanding the underlying processes giving rise to these observations, a rule-based model for EGFR signaling was developed. The model suggested that biased phosphorylation could be explained by variations in adaptor protein abundances. This prediction arises from the structure of the model, in which a phospho-site that is bound by an adaptor protein is sterically protected from the action of phosphatases. Testing model predictions confirmed that overexpression of the adaptor protein Grb2 leads to phosphorylation levels enhanced specifically at a site where this protein binds. Finally, this model was extended to explore the possible mechanisms leading to differential signaling induced by EGFR ligands. Model results suggest that ligand-dependent differences in dimer lifetimes lead to differential multisite phosphorylation and ubiquitination, which in turn could influence signaling kinetics and cellular outcomes.

Keywords

cell signaling, phosphorylation, PTMs, systems biology, quantitative biology, mathematical modeling

Document Type

Dissertation

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Diane S. Lidke, PhD

Second Committee Member

William S. Hlavacek

Third Committee Member

Bridget S. Wilson

Fourth Committee Member

Angela Wandinger-Ness

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