Search

Most cellular processes are modulated by protein-protein interactions, which serve as the basis for intracellular signaling cascades. Perturbations in these cascades lead to abnormal signaling events and ultimately result in diseases like cancer. Specialized phosphotyrosine circuitry translates extracellular signals into cellular responses through altered phosphorylation states of tyrosine residues using receptor tyrosine kinases, phosphatases, as well as phosphotyrosine binding domains found in adaptor proteins. The Shc (Src homology 2 domain-containing) family of cytosolic adaptors consists of four evolutionarily related proteins that share structural homology yet possess distinct functional roles. The more recently identified and least characterized member, ShcD, is of clinical relevance on account of its overexpression in malignant gliomas. However, its exact role remains speculative and therefore warrants further inquiry. The purpose of this project, accordingly, is to identify and better understand the mechanisms through which ShcD interacts with other signaling proteins to modulate pathways that likely contribute to glioma occurrence and/or progression. Using a comprehensive, dual-pronged proteomic and transcriptomic approach, I demonstrate that ShcD associates with receptor Tie2 to synergistically promote invasion in glioma cells. Additionally, I show that the ShcD signaling repertoire is not only limited to receptor tyrosine kinases, but it also associates with the phosphatase Shp2 and suppresses Erk activation downstream of neurotrophic receptor TrkB. Lastly, this project examines the regulatory landscape of ShcD by identifying its upstream and alternative promoters that reveal tissue-specific adaptation as well as dynamic usage across embryonic and postnatal development. Contribution of ShcD to signaling pathways implicated in glioma progression, as reported here, can be used to delineate neurogenesis as well as to understand instances of disease occurrence and progression, including cancer and neurodegenerative disorders. The data presented in this thesis can, therefore, serve as a foundation to identify ShcD as a potential diagnostic marker and/or a therapeutic target. 2021-01-01

Cell signal transduction requires an organized cascade of enzymatic conversions and molecular recognition events to convert external messages into physiological responses. Central participants in such pathways are the transmembrane Receptor Tyrosine Kinases (RTKs), including the well-characterized Epidermal Growth Factor (EGF) Receptors, which situate at the cell surface to intercept incoming signals. Upon stimulation, the intracellular tail of the RTK recruits proteins such as those of the Shc adaptor family, which help to nucleate the developing transduction complex. Additionally, Shc proteins have been implicated in post-stimulus receptor endocytosis owing to a short sequence, known as the AP2 adaptin binding motif, which is conserved across the majority of Shc proteins. ShcD is the fourth and most-recently discovered Shc homolog, sharing substantial amino acid identity with its better-known counterpart, ShcA, as well as possessing some distinguishing characteristics that have not been investigated. We now report that ShcD challenges the canonical signalling paradigm by promoting ligand-independent EGFR activation, and sequestering the phosphorylated receptor in the perinuclear area, thereby reducing cellular EGF uptake and suppressing the downstream activation of Erk. EGFR hyperphosphorylation is a cell autonomous effect that requires the intrinsic EGFR kinase and the ShcD PhosphoTyrosine Binding (PTB) domain, and it primarily affects residues Y1068, Y1148 and Y1173. Meanwhile, a central residue in receptor fate determination, Y1045, appears to remain unmodified in the presence of ShcD. To assess the molecular determinants of subcellular localization, we transplanted the ShcA AP2 binding motif into the analogous region of ShcD, and discovered that the resulting chimeric protein retains the capacity to facilitate ligand-independent EGFR phosphorylation, but restores ligand uptake in the cell, suggesting that ShcD possess several molecular permutations that subvert the canonical EGFR cascade. We have additionally found evidence of transcriptional ShcD upregulation in brain tumours, which are also frequently associated with dysregulated EGFR signalling. In our cellular model of a grade IV glioma, EGFR-ShcD synergy can be recapitulated. In light of our findings, we challenge the designation of ShcD as a traditional phosphotyrosine signalling adaptor, and suggest instead that it is actively involved in sculpting the transduction framework. Natural Sciences and Engineering Research Council of Canada (Discovery Grant RG327372), the Brain Tumour Foundation of Canada, Canadian Institutes of Health Research Vanier Canada Graduate Scholarship, the University of Guelph Brock Doctoral Scholarship.