The small GTPase Ras proteins play an essential role in transducing extracellular signals that regulate cell growth, survival, and differentiation. Growth factors and cytokines activate Ras by recruiting proteins such as Son of Sevenless (SOS), which acts as guanine nucleotide exchange factors and converts inactive GDP-Ras to active GTP-Ras. Ras then transmits signals to downstream signaling pathways such as the mitogen-activated protein (MAP) kinase pathway. The MAPK pathway is composed of three protein kinases- Raf, MEK and MAP kinase. These kinases transduce Ras signals through a phosphorylation cascade. The MAPKs include c-Jun-NH2-terminal kinase (JNK), extracellular-regulated kinase (ERK) and p38. These MAPK regulate expression of diverse transcription factors that regulate cell growth, survival, and differentiation. From a yeast two-hybrid assay, we cloned a novel JNK-interacting protein that we call Ras Signaling Modifier (RSM). We find that growth factors and TNF-alpha induce phosphorylation of RSM leading to the association of RSM with Sos and Raf. This has two important consequences, the facilitation of Sos-mediated activation of Ras followed by Ras activation of Raf leading to an enhanced magnitude and duration of ERK activation. Oxidative stress activates the JNK pathway causing apoptosis while the ERK pathway protects cells from apoptosis and we observed that RSM protects fibroblasts from TNF-alpha-induced apoptosis. In the lung, oxidative stress has been shown to induce apoptosis of wound fibroblasts. Therefore, RSM may protect against oxidative stress injury in lung fibroblasts, which express abundant RSM, by activating the ERK pathway and inhibiting JNK activation. In addition, the ERK pathway mediates growth factor-induced proliferation and migration of fibroblasts, processes that are critical for wound healing so that RSM may augment the proliferative and migratory response to growth factors in lung fibroblasts. We view RSM as a dynamic molecular scaffolding protein in the Ras pathway. Our proposed studies will examine how RSM regulates Ras signal transduction and affects the response of lung fibroblasts to oxidative stress. We put forth three Aims: (1) Analysis of RSM in the JNK pathway; (2) Characterize the mechanism of RSM activation of the Raf/ERK pathway; and (3) Examine RSM function in lung fibroblasts. These studies will elucidate the biological function of a novel and unique regulator of Ras signal transduction.
