Uncovering the Role Select Residues Play in Mediating RasC Activation and Interaction With mTORC2

Abstract

Cellular migration toward a chemical gradient is chemotaxis and is a crucial biological function of embryonic development, immune response, and wound healing. On a cellular level, chemotaxis begins when a cell surface receptor binds a chemical messenger and transmits the signal across the plasma membrane. This signal transduction initiates the chemotaxis signaling network that culminates with cell polarization toward the chemical gradient and cellular protrusions in that direction. In Dictyostelium, two key players in this signaling network are thought to directly interact, RasC and mechanistic target of rapamycin complex 2 (mTORC2), and the work of this dissertation is to understand which residues of RasC play a role in this interaction. To this end, we designed mutations to RasC, expressed the mutant RasC proteins in Dictyostelium cells lacking rasC (rasC-), and analyzed the downstream effects. We found that mutation of A31 to aspartic acid prevents RasC activation by its guanosine exchange factor (GEF). We also found that disruption of membrane anchoring residues in the hypervariable region (HVR) prevents RasC from localizing to the membrane and participating in downstream signaling. Some residue changes in both the effector domain and allosteric domain of RasC disrupt chemotactic signaling and prevent Dictyostelium from developing and activating mTORC2 in the same way as RasCWT. We also began working on purification methods of RasC using both bacterial and Dictyostelium expression, and labeling RasC with a tetra-cysteine tag suitable for fluorescent labeling. This work on understanding the in vivo activity of RasC, purifying RasC from multiple expression vectors, and developing fluorescently tagged RasC has helped us understand the RasC-GEF interaction and has given insight into the RasC-mTORC2 signaling pathway. We found that RasC does not express as a soluble product in bacteria, and that purification from Dictyostelium is difficult to scale. The work on fluorescently tagging RasC is ongoing.