The RAS-Effector Interface: Isoform-Specific Differences in the Effector Binding Regions

Abstract

RAS effectors specifically interact with the GTP-bound form of RAS in response to extracellular signals and link them to downstream signaling pathways. The molecular nature of effector interaction by RAS is well-studied but yet still incompletely understood in a comprehensive and systematic way. Here, structure-function relationships in the interaction between different RAS proteins and various effectors were investigated in detail by combining our in vitro data with in silico data. Equilibrium dissociation constants were determined for the binding of HRAS, KRAS, NRAS, RRAS1 and RRAS2 to both the RAS binding (RB) domain of CRAF and PI3Kα, and the RAS association (RA) domain of RASSF5, RALGDS and PLCε, respectively, using fluorescence polarization. An interaction matrix, constructed on the basis of available crystal structures, allowed identification of hotspots as critical determinants for RAS-effector interaction. New insights provided by this study are the dissection of the identified hotspots in five distinct regions (R1 to R5) in spite of high sequence variability not only between, but also within, RB/RA domain-containing effectors proteins. Finally, we propose that intermolecular β-sheet interaction in R1 is a central recognition region while R3 may determine specific contacts of RAS versus RRAS isoforms with effectors.

Pharmacological Induction of RAS-GTP Confers RAF Inhibitor Sensitivity in KRAS Mutant Tumors

“…The fact that elevated RAS-GTP levels observed upon treatment with MEK or PI3K inhibitors did not appear to activate PI3K signaling suggests an apparent preference toward MAPK pathway activation over that of the PI3K pathway. Interestingly, two reports have shown that RAS-GTP interacts with the RBD of the RAF kinases with orders of magnitude greater affinity versus that of the PI3Ks (Burd et al, 2014;Nakhaeizadeh et al, 2016). Hence, we hypothesize that elevated RAS-GTP levels result in greater association of RAS-GTP with RAF versus PI3K irrespective of whether that activation was mediated through MEK or PI3K inhibition.…”

Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids

“…Furthermore, the CRAF Ras binding domain (RBD) crystallized in complex with either HRAS or RAP1A, indicating that both interactions are sufficiently strong to yield co-crystal structures (5,6). This multi-specificity of Raf kinases relates to the conservation of the effector binding site and RBD at the atomic molecular level (7). Thus, reagents that interfere with the Ras/Raf interaction are likely to efficiently inhibit the activation of growth signals over a wide range of conditions.…”

From basic researches to new achievements in therapeutic strategies of KRAS-driven cancers

“…Other than these therapeutic strategies, progress has been made in generating alternative agents to inhibit KRAS onc -RAF interaction which is needed to stimulate RAS-dependent oncogenic signaling 40,76 . Thus, a better understanding of the detailed interactions of KRAS onc with RAS binding domains and RAS association domains of its downstream effectors provides alternative opportunities for the inhibition of intermolecular interactions 77,78 . Table 2 provides a summary of studies examining the direct inhibition of KRAS mutant from 1997 to 2017.…”

“…Because of the unsuccessful EGFR targeted therapy for KRAS onc -dependent cancers and the difficulty associated with targeting KRAS onc directly, a great deal of effort has been applied to target downstream effector pathways. The specific interaction of RAS family proteins with downstream effectors regulates various cellular functions 3,77,96,97 . Constitutive activation of downstream effector pathways by oncogenic KRAS results in the uncontrolled growth, proliferation, and survival of cancer cells 98 .…”