Online

Schmoker, Anna

Biology

2020

Ph. D.

Cells communicate to drive all biological processes during organismal development, homeostasis, and disease. Communication, or signaling, is carried out through an orchestration of complex sequential molecular interactions. A signal is typically initiated by an extracellular cue binding to a receptor on the cell membrane, which induces an intracellular response, resulting ultimately in cellular phenotypes such as growth, proliferation, migration, apoptosis or survival. Adaptor proteins are critical to signal transduction, as they facilitate the formation of protein complexes that transduce signals. CT10 regulator of kinase (CRK) and CRK-like (CRKL) form a family of adaptors that facilitate complex formation during developmental signaling, bridging phosphorylated tyrosine residues on membrane localized proteins with downstream signaling molecules. We previously identified Discoidin, CUB, and LCCL domain-containing 2 (DCBLD2), a protein with similar structure to the neuronal guidance receptors neuropilins, as an interactor of CRK adaptors. DCBLD1 and DCBLD2 constitute a family of type-I transmembrane orphan receptors that play roles in development and cancer. How DCBLD proteins modulate cellular processes including proliferation, migration, and growth on a molecular scale are unknown. This dissertation begins by further characterizing the interactions between CRK adaptors and DCBLD family members. We identify the cytoplasmic tyrosine kinases FYN and ABL as regulators of the DCBLD/CRKL interaction. FYN- and ABL-driven DCBLD1 and 2 tyrosine, serine, and threonine phosphorylation sites and the resulting changes in protein interactors are quantified with multiple quantitative mass spectrometry approaches. Next, we identify a mechanism by which DCBLD2 serves as a scaffold for signaling pathways initiated by the receptor tyrosine kinases PDGFR[Beta] and insulin receptor (INSR), and alters downstream signaling events and cell proliferation. We find that activation of both receptors induces DCBLD2 tyrosine phosphorylation at multiple sites including CRKL binding sites, and demonstrate direct phosphorylation of DCBLD2 by PDGFR[Beta] and ABL. Finally, we describe an original way to predict how bridging molecules find targets to facilitate signaling complex formation. This "in silico proteomics" approach uses publicly available webtools to predict and prioritize CRK adaptor interactions. Our approach identified known CRK-interactors, including DCBLD2, as well as many novel interactors, several of which we have biochemically validated. We further extended our approach to incorporate any modified motif-driven binding interaction to empower the signaling community in their studies of a wide array of signaling systems. Together, this dissertation expands our understanding of cellular signaling mechanisms by delineating DCBLD receptor signaling and by predicting and characterizing many novel signaling interactions dependent on post-translationally modified motifs, particularly those involving the critical and versatile CRK family adaptors.