The Ras–Membrane Interface: Isoform-Specific Differences in the Catalytic Domain

Parker, Jillian A.; Mattos, Carla

doi:10.1158/1541-7786.mcr-14-0535

Cited by 49 publications

(52 citation statements)

References 77 publications

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“…Consistent with this finding, previous MD simulation studies of K-RAS4B on neutral membrane bilayers did not recapitulate the H-RASspecific orientations (3). Importantly, the RAS isoforms H-RAS, N-RAS, and even the splice variant K-RAS4A have C-terminal HVR sequences and lipidation motifs distinct from those of K-RAS4B (1), as well as isoform-specific substitutions in the C-terminal lobe of the GTPase domain (14), which likely lead to different orientational equilibria of the GTPase domain. Interaction with RAS-Binding Domains Requires K-RAS4B Reorientation to Expose Effector-Binding Site.…”

Section: K-ras4b Activation State Determines Population Of Two Majormentioning

confidence: 63%

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site

Mazhab-Jafari

Marshall

Smith

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

202

292

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K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery. KRAS | nuclear magnetic resonance | lipid bilayer nanodisc | oncogenic mutation | Noonan syndrome T he K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) protein product of the KRAS gene undergoes posttranslational farnesylation and C-terminal processing, which, in conjunction with a poly-basic hypervariable region (HVR), targets K-RAS4B to anionic lipid rafts on the intracellular side of the plasma membrane (Fig. 1A) (1). This localization is essential for K-RAS4B function and enhances signaling fidelity (2). Although the significance of membrane tethering of K-RAS4B is well appreciated, a high-resolution map of how K-RAS4B interacts with the membrane is lacking. Because membrane-anchored RAS presents a major challenge to crystallization, current structural insights into the behavior of membrane-anchored RAS have come from a variety of lower-resolution techniques including in vivo FRET-based studies (3), fluorescence and infrared spectroscopic studies (4-6), and in silico models (3). These pioneering studies suggested that the K-RAS4B GTPase domain adopts certain p...

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Section: K-ras4b Activation State Determines Population Of Two Majormentioning

confidence: 63%

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site

Mazhab-Jafari

Marshall

Smith

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

202

292

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“…HRAS, KRAS and NRAS exhibit remarkable differences beyond their common interaction interfaces for regulators and effectors [74,75,76], especially at their C-terminal hypervariable region (S3 Fig), which has different features, including protein-protein interaction [77,78]. An interesting issue, which is increasingly appreciated, is a RAS-membrane interaction that appears to generate RAS isoform specificity with respect to effector interactions [79,80,81]. This is likely achieved by RAS-specific scaffold proteins, including CaM, GAL1, GAL3, IQGAPs, NPM1, NCL, SHOC2/SUR8 [78,82], which may modulate isoform specificity at specific site of the cell.…”

Section: Discussionmentioning

confidence: 99%

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

Nakhaeizadeh¹,

Amin²,

Nakhaei‐Rad³

et al. 2016

PLoS ONE

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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.

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“…Whether these sequence differences contribute to the functional differences of RAS isoforms is still largely understudied. However, the allosteric lobe has been suggested to play a role in SII conformation and membrane orientation (Parker and Mattos, 2015), and RAS membrane orientation has been shown to regulate effector utilization (Abankwa et al, 2008(Abankwa et al, , 2010.…”

Section: Ras Isoform Differences and Post-translational Modificationsmentioning

confidence: 99%

RAS isoforms and mutations in cancer at a glance

Hobbs

Der

Rossman

2016

Journal of Cell Science

779

709

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RAS proteins (KRAS4A, KRAS4B, NRAS and HRAS) function as GDP-GTP-regulated binary on-off switches, which regulate cytoplasmic signaling networks that control diverse normal cellular processes. Gain-of-function missense mutations in RAS genes are found in ∼25% of human cancers, prompting interest in identifying anti-RAS therapeutic strategies for cancer treatment. However, despite more than three decades of intense effort, no anti-RAS therapies have reached clinical application. Contributing to this failure has been an underestimation of the complexities of RAS. First, there is now appreciation that the four human RAS proteins are not functionally identical. Second, with >130 different missense mutations found in cancer, there is an emerging view that there are mutation-specific consequences on RAS structure, biochemistry and biology, and mutation-selective therapeutic strategies are needed. In this Cell Science at a Glance article and accompanying poster, we provide a snapshot of the differences between RAS isoforms and mutations, as well as the current status of anti-RAS drug-discovery efforts.

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The Ras–Membrane Interface: Isoform-Specific Differences in the Catalytic Domain

Cited by 49 publications

References 77 publications

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site

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

RAS isoforms and mutations in cancer at a glance

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