Identification of Genes Involved in the C. elegans VAB-1 Eph Receptor Tyrosine Kinase Signaling Pathway

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.

FES KINASE SIGNALING PROMOTES MAST CELL RECRUITMENT TO TUMOURS

FES protein-tyrosine kinase (PTK) activation downstream of the KIT receptor in mast cells (MC) promotes cell polarization and migration towards the KIT ligand Stem cell factor (SCF). A variety of tumours secrete SCF to promote MC recruitment and release of mediators that enhance tumour vascularization and growth. This study investigates whether FES promotes MC migration via regulation of microtubules (MTs), and if FES is required for MC recruitment to the tumour microenvironment. MT binding assays showed that FES has at least two MT binding sites, which likely contribute to the partial co-localization of FES with MTs in polarized bone marrow-derived mast cells (BMMCs). Live cell imaging revealed a significant defect in chemotaxis of FES-deficient BMMCs towards SCF embedded within an agarose drop, which correlated with less MT organization compared to control cells. To extend these results to a tumour model, mouse mammary carcinoma AC2M2 cells were engrafted under the skin and into the mammary fat pads of immune compromised control (nu/nu) or FES-deficient (nu/nu:fes-/-) mice. A drastic reduction in tumour-associated MCs was observed in FES-deficient mice compared to control in both mammary and skin tissue sections. This correlated with a trend towards reduced tumour volumes in FES-deficient mice. These results implicate FES signaling downstream of KIT, in promoting MT reorganization during cell polarization and for chemotaxis of MCs towards tumour-derived SCF. Thus, FES is a potential therapeutic target to limit recruitment of stromal mast cells or macrophages to solid tumours that enhance tumour progression.

An immune modulatory role for the fes proto-oncogene

The Fes protein tyrosine kinase is abundantly expressed in phagocytic immune cells, including tumor associated macrophages. Fes knockout mice (fes-/-) display enhanced sensitivity to LPS, and this was shown to be associated with increased NF-κB signaling and TNFα production from fes-/- macrophages. Interestingly, tumor onset in the mouse mammary tumor virus (MMTV-Neu) transgenic mouse model of breast cancer is significantly delayed in fes-/- mice, and this was associated with increased frequency of CD11b+ myeloid and CD3+ T cells in the premalignant mammary glands. Recent studies have also implicated Fes in cross-talk between MHC-I and the NF-κB and IRF-3 pathways in macrophages. Signal 3, the production of inflammatory cytokines and Type I interferons downstream of NF-κB and IRF-3 pathways in antigen presenting cells, is considered an important component of T-cell activation, after engagement of T cell receptor by MHC presented antigen (Signal 1) and co-receptors by their ligands (Signal 2). Using a lymphocytic choriomeningitis virus (LCMV) model of immune activation, I show that LPS stimulated fes-/- macrophages promote more robust activation of LCMV antigenspecific CD8+ T cells than wild type macrophages (fes+/+). Furthermore, LPS stimulated fes-/- macrophages showed increased phosphorylation of NF-B and IRF-3. I also showed that Fes colocalizes with MHC-I in dynamic vesicular structures within macrophages. These observations are consistent with a model where Fes regulates Signal 3 in antigen presenting cells through roles in cross-talk between MHC-I and the NF-kB and IRF-3 signaling pathways. This suggests that Fes plays an immune checkpoint role at the level of Signal 3, and that Fes inhibition could promote tumor immunity through increased Signal 3 driven T cell activation.