Date of Award
Doctor of Philosophy (PhD)
Biochemistry & Molecular Biophysics
Mark A. Lemmon
Signaling by receptor tyrosine kinases (RTKs) typically involves binding of extracellular ligands and subsequent activation of intracellular kinase domain, which in turn mediates autophosphorylation and downstream signal transduction. However, several RTKs, such as ErbB3 (HER3) and Ryk/Derailed, lack key conserved (and catalytically important) residues in their intracellular kinase domain, and are thought to be inactive `pseudo'-kinases. Much less is known regarding how these receptor tyrosine `pseudo'-kinases mediate transmembrane signaling. The work described in this dissertation attempts to understand the non-canonical signaling by ErbB3 and Ryk/Derailed using a combination of biophysical, biochemical and cell biology methods. ErbB3 is one of four members of the human epidermal growth factor receptor (EGFR) family. ErbB3 signals primarily by heterodimerizing with ErbB2 (HER2) and mediates resistance of tumor cells to EGFR/ErbB2-targeted therapeutics. We have investigated the role of ErbB3 `pseudo'-kinase domain in its signaling. In vitro enzymology study shows that, despite the sequence alterations, ErbB3 kinase domain binds ATP with high affinity and possesses weak kinase activity to trans-autophosphorylate its intracellular region. We also describe a crystal structure of ErbB3 kinase, which resembles the inactive EGFR and ErbB4 kinase domains. Whereas mutations that destabilize this configuration activate EGFR and ErbB4 (and promote EGFR-dependent lung cancers), a similar mutation conversely compromises ErbB3's ability to activate downstream Akt signaling in our cellular studies. These findings suggest that ErbB3 kinase may be crucial for signaling and could represent an important therapeutic target. Ryk and its Drosophila homolog Derailed (Drl) contain an extracellular Wnt Inhibitory Factor (WIF) domain and participate in non-canonical Wnt signaling. We attempt to understand, for the first time, the molecular mechanism of Ryk/Drl-Wnt interaction using Drl as a model system. We show that Drl binds directly to Drosophila Wnt5 (DWnt5) with submicromolar affinity using surface plasmon resonance (SPR). X-ray crystallography study has identified a conserved binding site of Drl for DWnt5. Mutating this site abolishes DWnt5 binding by Drl. This study serves as a starting point for our understanding of Wnt signaling by Ryk/Drl at a molecular level.
Shi, Fumin, "Signaling by Non-Canonical Receptor Tyrosine Kinases ERBB3 and RYK" (2012). Publicly Accessible Penn Dissertations. 699.