The Caenorhabditis elegans Eph RTK, VAB-1, has known roles in neuronal and epidermal morphogenesis as well as oocyte maturation through interaction with its ephrin ligands. In humans, Eph receptors are involved in nervous and vascular system development and have been implicated in cancer formation and progression. DAF-18, a C. elegans ortholog of the human tumour suppressor gene, PTEN, has been identified as an interacting partner with the Eph RTK, VAB-1. Mutations in human PTEN have been associated with numerous cancers and in the worm, DAF-18 is a well studied member of the DAF-2/Insulin receptor-like signaling pathway which has roles in dauer formation, thermotolerance and adult longevity. Our lab has previously shown that VAB-1/EphR binds DAF-18. To further investigate the significance of this interaction as well as offer additional function to the proteins involved, I have shown that VAB-1/EphR is a negative regulator of DAF-18/PTEN at the protein level. Western blotting reveals that endogenous expression of DAF-18/PTEN is low in wild-type animals and expression is increased in a vab-1/ephR mutant. Additionally, VAB-1/EphR and DAF-18/PTEN are expressed in head neurons, oocytes and the germline precursor cells, Z2/Z3. vab-1/ephR mutants show increases longevity and sensitivity to dauer conditions which is consistent with increased DAF-18/PTEN activity. Lastly, daf-18(ok480) is able to suppress the oocyte maturation phenotype and increased MAPK expression displayed by vab-1(dx31) animals, providing genetic evidence of an interaction. By identifying the tissues where these proteins are co-expressed and substantiating the interaction with multiple analyses, novel roles may be proposed for each: VAB-1/EphR in DAF-2/Insulin signaling and DAF-18/PTEN in oocyte maturation downstream of VAB-1/EphR signaling. This work provides further understanding of how an organism coordinates complex developmental processes and reiterates the notion that cellular signaling is a complex network of interacting players. As many signaling pathways are evolutionarily conserved, my research in C. elegans may provide a mechanism on how Eph RTKs and PTEN are regulated in more complex organisms, including humans. / Thesis (Master, Biology) -- Queen's University, 2009-02-27 17:09:10.582

The generation of a functional nervous system requires that neuronal cells and axons navigate precisely to their appropriate targets. The Eph Receptor Tyrosine Kinases (RTKs) and their ephrin ligands have emerged as one of the important guidance cues for neuronal and axon navigation. However, the molecular mechanisms of how Eph RTKs regulate these processes are still incomplete. The purpose of this work was to contribute to the understanding of how Eph receptors regulate axon guidance by identifying and characterizing components of the Caenorhabditis elegans Eph RTK (VAB-1) signaling pathway. To achieve this objective I utilized a hyper active form of the VAB-1 Eph RTK (MYR-VAB-1) that caused penetrant axon guidance defects in the PLM mechanosensory neurons, and screened for suppressors of the MYR-VAB-1 phenotype. Through a candidate gene approach, I identified the adaptor NCK-1 as a downstream effector of VAB-1. Molecular and genetic analysis revealed that the nck-1 gene encodes for two isoforms (NCK-1A and NCK-1B) that share similar expression patterns in parts of the nervous system, but also have independent expression patterns in other tissues. Genetic rescue experiments showed that both NCK-1 isoforms can function in axon guidance, but each isoform also has specific functions. In vitro binding assays showed that NCK-1 binds to VAB-1 in a kinase dependent manner. In addition to NCK-1, WSP-1/N-WASP was also identified as an effector of VAB-1 signaling. Phenotypic analysis showed that nck-1 and wsp-1 mutants had PLM axon over extension defects similar to vab-1 animals. Furthermore, VAB-1, NCK-1 and WSP-1 formed a complex in vitro. Intriguingly, protein binding assays showed that NCK-1 can also bind to the actin regulator UNC-34/Ena, but genetic experiments suggest that unc-34 is an inhibitor of nck-1 function. Through various genetic and biochemical experiments, I provide evidence that VAB-1 can disrupt the NCK-1/UNC-34 complex, and negatively regulate UNC-34. Taken together, my work provides a model of how VAB-1 RTK signaling can inhibit axon extension. I propose that activated VAB-1 can prevent axon extension by inhibiting growth cone filopodia formation. This is accomplished by inhibiting UNC-34/Ena activity, and simultaneously activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex. / Thesis (Ph.D, Biology) -- Queen's University, 2011-07-28 16:20:31.957