Date of Award

2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cellular, Molecular and Biomedical Sciences

First Advisor

Albert van der Vliet

Abstract

The proto-oncogene tyrosine kinase Src is a ubiquitously expressed non-receptor tyrosine kinase involved in critical cellular functions including wound healing, migration, survival, and proliferation. Accordingly, aberrant Src signaling is also significantly involved in many diseases. Src is most well known as an oncogenic protein, however, there are emerging roles for Src signaling in various tissues involved in inflammatory diseases. One such tissue is the respiratory epithelium, which comprises the first line of defense against inhaled microbial, particulate, and chemical insults and is a critical nexus of innate immune signaling. Recent work has detailed the involvement of Src in many innate injury and wound healing responses within the airway epithelium, specifically with respect to its simultaneous Cys oxidation and activation by the NADPH oxidase family member dual oxidase 1 (DUOX1). To date, however, there has been little investigation into the specifics of this Cys oxidation, the structural alterations that promote oxidative Src activation, or the exact role of Src with respect to the secretion of innate immune cytokines following airway epithelial insult.

Src is classically known to be regulated by conformational changes depending on the phosphorylation of its critical regulatory tyrosine residues. However, Src activation is also known to be regulated by reactive oxidation species (ROS)-mediated Cys oxidation. While redox regulation of Src has been observed for some time, no Cys residues have been directly implicated outside of mutational studies with no exploration of Src conformational changes. Utilizing quantitative mass spectrometry, we identified two Cys residues, C185 and C277, that were predominantly oxidized in Src when compared to the other Cys. Additionally, we confirmed their importance for Src activity using both kinase assays with recombinant C to A mutants, as well as cell lines expressing C to A mutant Src. Finally, utilizing molecular dynamics simulations, we established a role for the oxidation of these two Cys residues in conformational changes associated with promoting kinase activation, suggesting a possible mechanistic explanation for Src activation by ROS.

DUOX1 has previously been identified as a critical mediator of innate epithelial responses to allergens, inducing acute secretion of the epithelial cytokine interleukin-33 (IL-33) and downstream type 2 responses involving the epidermal growth factor receptor (EGFR). However, the importance of Src has not been evaluated in this pathway. Through pharmacologic inhibition and genetic knockout/silencing strategies, we have determined that Src is involved in epithelial IL-33 secretion and downstream type 2 responses following allergen challenge in vivo and in vitro. Interestingly, we also observed that IL-33 was capable of inducing its own secretion in an unexpected acute “autoamplification” pathway where blocking IL-33 signaling inhibited IL-33 secretion following allergen challenge. Similarly, DUOX1 and Src signaling were involved in downstream secretion of the type 2 cytokines IL-5 and IL-13 following direct IL-33 challenge. Finally, following 4 day in vivo IL-33 treatment, we observed Src- and DUOX1-mediated IL-33 secretion, as well as IL-5 and IL-13 secretion, in mouse airways. The secretion of these cytokines was accompanied by DUOX1-dependent alterations of mouse lung function.

In total, this dissertation provides further insight into the redox regulation of the Src kinase and identifies a role for Src in innate epithelial IL-33 signaling and autoamplification.

Language

en

Number of Pages

295 p.

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