Type of Thesis
Joseph J. Falke
Jeffrey C. Cameron
Robin D. Dowell
H-Ras is a G-protein responsible for the activation of multiple signaling pathways that control cell growth. Our primary interest is examining its role in the regulation of the phosphoinositide-3-kinase (PI3K) signaling cascade, as H-Ras has been previously shown to recruit PI3K to the plasma membrane where this lipid kinase phosphorylates PIP2 to PIP3, a signal for cellular growth. The H-Ras-PI3K-PIP3 signaling pathway is known to be highly oncogenic, with constitutive activation of H-Ras leading to increased PIP3 production and cell growth. We hypothesize that disease-linked mutations on the binding interface between Ras and PI3K increase the affinity for complex formation, thereby recruiting more PI3K to the membrane and increasing PIP3 production. The affinity of five H-Ras mutants for the PI3Ky Ras binding domain (PI3Ky-RBD) were measured using Micro-Scale Thermophoresis (MST). The findings reveal that the H-Ras Y64H and E63K mutations trigger significant increases in the affinity of H-Ras binding to PI3Ky-RBD. Unexpectedly, the common cancer-linked mutation H-Ras G12S may also increase the affinity of the H-Ras-PI3Ky-RBD complex, potentially adding a second mechanism to the current model of G12S-triggered oncogenesis. Two control mutations (D38E and Y40C) are observed to significantly weaken H-Ras binding to PI3Ky-RBD as predicted. Collectively our results support the existence of a novel disease mechanism by which disease-linked Ras mutations stimulate PI3K and PIP3 signaling by increasing the affinity of Ras binding to PI3K, in turn recruiting additional PI3K to the membrane and triggering increased PIP3 production and the associated unregulated cell growth.
Martyr, Justin, "Effects of H-Ras Disease Mutations on Binding to the Ras Binding Domain of PI3Ky Measured by Micro-Scale Thermophoresis" (2019). Undergraduate Honors Theses. 1870.