Stimulation of the receptor tyrosine kinase KIT by Stem Cell Factor (SCF) triggers activation of RAS and its downstream effectors. Proper KIT activation is essential for the maturation, proliferation and survival of mast cells. In addition, SCF activation of KIT is critical for recruiting mast cells to sites of infection or injury, where they release a mix of pro-inflammatory substances. RIN3, a RAS effector and RAB5-directed guanine nucleotide exchange factor (GEF), is highly expressed and enriched in human mast cells. SCF treatment of mast cells increased the amount of GTP-bound RAB5, and the degree of RAB5 activation correlated with the expression level of RIN3. SCF treatment also caused the dissociation of a pre-formed complex of RIN3 with BIN2, a membrane bending protein. Silencing of RIN3 increased the rate of SCF-induced KIT internalization, while persistent RIN3 over-expression led to KIT downregulation. These observations strongly support a role for RIN3 in coordinating the early steps in KIT endocytosis. Importantly, RIN3 also functions as an inhibitor of mast cell migration toward SCF. Finally, we demonstrate that elevated RIN3 levels sensitize mastocytosis cells to treatment with a KIT tyrosine kinase inhibitor, suggesting the value of a two-pronged inhibitor approach for this difficult to treat malignancy. These findings directly connect KIT activation with a mast cell-specific RAS effector that regulates the cellular response to SCF and provide new insight for the development of more effective mastocytosis treatments.

Activation of cell surface receptors by ligands leads to the recruitment of a number of signaling molecules that mediate cellular responses. RAS-GTPases are signaling proteins that bring about major changes in cell growth, differentiation, gene expression and cytoskeletal organization upon receptor stimulation. The RAS effector RIN1 is involved in receptor trafficking and signaling. RIN1 is recruited to the receptor following ligand stimulation, but the function of its individual effector domains in this process remained uncharacterized. RIN1 acts as a guanine nucleotide exchange factor (GEF) for the small GTPase RAB5, which is involved in the formation of early endosomes. RIN1 can also directly bind and activate ABL tyrosine kinases, which have been implicated in cellular actin remodeling and receptor trafficking. RIN1 also forms a complex with STAM, an ESCRT complex protein, which favors maturation of receptor-containing endosomes. As all of RIN1's known effectors are involved in modulating receptor trafficking, we resorted to examine the role of individual domains in receptor internalization and fate. Chapters two and three debrief the mechanisms by which RIN1 effectors function in an integrated manner to regulate receptor trafficking and corresponding cellular responses.

Using domain-specific mutants of RIN1, we show that the RAB5 and ABL signaling pathways mediate opposing functions, maintaining a balance in the route and rate of receptor internalization. RAB5 promotes actin remodeling, EGF induced macropinocytosis and migration, facilitating receptor degradation. ABL kinases, on the other hand, prevent receptor degradation and migration towards EGF. Our study also shows that regulation of RIN1 localization by the 14-3-3 proteins is a major factor in determining signaling intensity. We characterized a novel binding partner of RIN1, namely, BIN1, a BAR domain protein involved in membrane bending.

Intracellular pathogens invade host cells by exploiting host cell surface receptors and signaling pathways. Listeria monocytogenes in a food-borne pathogen that binds host MET to invade epithelial cells and modulates RAB5 GTPases at several stages of the infection. The effect of host RAB5 regulators on pathogenesis remained uncharacterized. We analyzed the role of RIN1, a major RAB5-GEF in epithelial cells, in the process of bacterial invasion and spread (chapter four). This study leads the way to determining the mechanism of pathogenesis of several pathogens that depend on host RAB5-GTPases for effective infection progress. Finally, chapter five discusses the significance of our studies from a therapeutic viewpoint in the context of cancers and infectious diseases.

The small G protein, RAS, transduces signals from receptor tyrosine kinases at the plasma membrane to the interior of the cell, mediating cell proliferation, differentiation, apoptosis and senescence. Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) tightly regulate RAS activation. Dysregulation of this process through activating mutations in RAS is responsible for 16% of all human tumors. Prior research has focused on regulation of RAS activity by GEFs and GAPs, but recently scientists have begun to uncover a role for posttranslational modifications in RAS regulation. In Chapter Two we describe the identification of a novel ABL tyrosine phosphorylation site on RAS (RAS-Y137) that allosterically regulates RAS activation and effector binding. Furthermore, phosphorylation at this site is significantly enhanced by overexpression of the RAS effector RIN1, which binds to and activates its effector ABL. This suggests that RAS-stimulated RIN1 can drive ABL-mediated RAS modification and regulation in a novel feedback circuit.

In response to activation by RAS binding, RIN1 signals through two downstream effectors - the small GTPase RAB5 and the non-receptor tyrosine kinase ABL - and mediates endocytosis and cytoskeleton remodeling. Consistent with this role, RIN1 localizes to the cytoplasm and can be recruited to the plasma membrane by activated RAS. However, previous studies have sporadically reported nuclear localization of RIN1. Chapter Three describes the novel cell-cycle dependent nuclear localization of RIN1. RIN1 nuclear localization peaks in G2 phase, and is regulated by three nuclear localization sequences and three serine residues. Multidimensional protein identification technology (MudPIT) analysis found that during G2 phase nuclear RIN1 binds to chaperones, nucleic acid binding proteins and ribonucleoproteins. These data suggest a novel pathway by which this RAS effector influences signal transduction from the plasma membrane to the nucleus.

RIN1 binding to its effector ABL relieves ABL autoinhibition and stimulates its kinase activity. Importantly, RIN1 also binds to the leukemogenic fusion protein, BCR-ABL1. Although BCR-ABL1 is considered to be constitutively active, previous work in the lab has demonstrated that RIN1 enhances BCR-ABL1 kinase activity and accelerates BCR-ABL1- induced leukemias in mice. We extend these studies in Chapter Four, examining the requirement for RIN1 in BCR-ABL1 leukemias. We demonstrate that RIN1 is required for BCR-ABL1 bone marrow transformation ex vivo and that RIN1 silencing sensitizes drug-resistant cells to the tyrosine kinase inhibitor imatinib. However, we found that RIN1 is not required for BCR-ABL1- induced leukemias in mice, suggesting that while BCR-ABL1 remains responsive to RIN1, this interaction is not required for leukemogensis.

That RIN1 silencing increases sensitivity to the ABL kinase inhibitor imatinib, even in drug-resistant cells, suggested that small molecule inhibitors of the RIN1::BCR-ABL1 interaction might be an effective therapy in combination with existing kinase active site-directed inhibitors. Chapter Five describes the design and implementation of a TR-FRET-based high throughput screen to identify inhibitors of this protein-protein interaction, as well as the identification of two lead scaffolds.