UCLA

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.