DECIPHERING HOW EGFR-GRB2-SOS1 COMPLEX REGULATES KRAS4B ACTIVATION AND LEADS TO HIPPO SIGNALING THROUGH RASSF5

Abstract

Ras is a small GTPase, which regulates cell proliferation and apoptosis. Its bifunctional switch is controlled by the nucleotide state: GTP-bound – switch on; GDP-bound – switch off. Among the three Ras isoforms, HRas, NRas, and KRas (with two splice variants of KRas4A and KRas4B), KRas4B is highly oncogenic, the most frequently mutated in lung, colorectal, and pancreatic cancers. However, Ras was thought to be “undruggable” due to the lack of effective pharmacological inhibitors over the past three decades. Most of the current focus has been directed at inhibiting the activation of Ras signaling. Ras proteins transduce signals between cell surface receptors and multiple intracellular signaling cascades. In response to epidermal growth factor receptor (EGFR) activation, growth factor receptor bound protein 2 (Grb2) establishes the connection between EGFR and Ras-specific nucleotide exchange factor (RasGEF), son of sevenless 1 (SOS1). SOS1 activates Ras by exchanging GDP to GTP. In addition to Ras major effectors and pathways, e.g. MAPK and PI3Kα/Akt, which are cell growth related, GTP-bound Ras associating with RASSF5 activates the Hippo pathway, which acts to suppress cell proliferation. In this serial study, we use NMR measurement and molecular dynamics (MD) simulation to investigate the interactions of Grb2–SOS1, SOS1–KRas4B, KRas4B–RASSF5, and the EGFR effects on the binding of Grb2–SOS1. Our findings successfully uncovered (1) a novel Grb2 binding site PKLPPKTYKREH on SOS1 and the most probable binding mode of Grb2–SOS1, (2) strong SOS1 peptide binders induce a closed conformation of Grb2 nSH3 domain but unchanged conformation of Grb2 cSH3 domain, (3) full length Grb2 performs high affinities for one-site SOS1 peptides, and the EGFR segment may facilitate the binding of Grb2 to the particular two-site SOS1 peptide, (4) KRas4B binding to SOS1 allosteric site induces the conformational changes of catalytic site and accelerate the KRas4B activation cycle, (5) the hypothesized mechanism that RASSF5 is a tumor suppressor in vivo but opposite in vitro, and (6) the dynamic mechanism of RASSF5 auto-inhibition. Our effort in elucidating the mechanism of Ras and Ras effectors results in 8 publications and offers a new venues for future therapeutic strategies.