In response to extracellular stimuli, a diverse network of signaling pathways involved in cell growth, survival, and differentiation are activated and this process is prominently regulated by the Ras family of small guanosine triphosphatases (GTPases). Chapter 2 shows that RIT1, a Ras-related GTPase that regulates cell survival and stress response, can directly associate with core components of the spindle assembly checkpoint (SAC). The SAC functions as a sensor of unattached kinetochores that delays mitotic progression into anaphase until proper chromosome segregation is guaranteed. Disruptions to this safety mechanism lead to genomic instability and aneuploidy, which serve as the genetic cause of embryonic demise, congenital birth defects, intellectual disability, and cancer. However, despite the understanding of the fundamental mechanisms that control the SAC, it remains unknown how signaling pathways directly interact with and regulate the mitotic checkpoint activity. Through various biochemical and cell biological approaches, I demonstrate that RIT1 is essential for timely progression through mitosis and proper chromosome segregation. RIT1 dissociates from the plasma membrane (PM) during mitosis and interacts directly with SAC proteins MAD2 and p31comet in a process that is regulated by cyclin-dependent kinase 1 (CDK1) activity. Furthermore, RIT1 oncoproteins silence the SAC and accelerate transit through mitosis by sequestering MAD2 from the mitotic checkpoint complex (MCC). Moreover, SAC suppression by pathogenic RIT1 promotes chromosome segregation errors and aneuploidy. These results highlight a unique function of RIT1 compared to other Ras GTPases and elucidate a direct link between a signaling pathway and the SAC through a novel regulatory mechanism. RIT1 gain-of-function mutations are found in lung cancer, leukemia, and in the germline of Noonan syndrome individuals; these mutations promote RAF/MEK/ERK pathway activation, yet the mechanism by which RIT1 activates RAF remains poorly understood. Chapter 3 outlines a set of biophysical and biochemical approaches employed to characterize the association of RIT1 with plasma membrane lipids and its interaction with RAF kinases. We identify critical residues present in the RIT1 hypervariable region that facilitate interaction with negatively charged membrane lipids and show that these are necessary for association with RAF kinases. Furthermore, despite direct interaction with RAF kinases, RIT1 is unable to activate RAF/MEK/ERK signaling in the absence of classical Ras proteins. Lastly, consistent with RIT1-mediated RAF activation as a driver of disease, we show that MEK1/2 inhibition alleviates cardiac hypertrophy in a mouse model of RIT1-mutant Noonan syndrome.

With an estimated 3.9 billion people at risk of dengue fever, dengue is a major disease threat. Other flaviviruses such as Yellow fever virus (YFV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), West Nile virus (WNV) amongst others, also have a significant burden of disease. For this reason, a model was built that optimizes viral infection in dendritic cells (DCs). DCs are the link between the innate and adaptive immunity and flaviviruses such as DENV, ZIKV YFV, JEV are capable of infecting DCs. Therefore, identifying mechanisms that mediate infection is important to understanding disease progression. We cell sorted populations of infected and bystander cells and collected RNA of DCs of all these populations. RNA-sequencing (RNA-seq) results indicated that despite similarities in the genome of ZIKV and DENV, they upregulated different pathways. ZIKV upregulated lipid metabolism and DENV upregulated inflammatory pathways when compared against mock. Infection models were similarly created with YFV and JEV vaccine strains. Our infection model, when coupled to a cell sort method provides a powerful method for identifying pro- or anti-viral mechanism from pure infected and uninfected populations.