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Title: Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

Journal Article · · Proceedings of the National Academy of Sciences of the United States of America
 [1];  [2];  [3];  [4];  [2];  [2];  [5];  [6];  [2];  [7]
  1. Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Oregon Health and Science Univ., Portland, OR (United States)
  2. Univ. of California, San Francisco, CA (United States)
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, San Francisco, CA (United States)
  4. Oregon Health and Science Univ., Portland, OR (United States)
  5. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  6. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Oregon Health and Science Univ., Portland, OR (United States); Univ. of California, San Francisco, CA (United States)
  7. Stanford Univ., Stanford, CA (United States)
+ Show Author Affiliations

Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.

Research Organization:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Organization:
USDOE Office of Science (SC)
Grant/Contract Number:
AC02-05CH11231
OSTI ID:
1221216
Journal Information:
Proceedings of the National Academy of Sciences of the United States of America, Vol. 112, Issue 26; ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)Copyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 188 works
Citation information provided by
Web of Science

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Cited By (40)

The renewed battle against RAS-mutant cancers journal February 2016
Inhibition of RAS function through targeting an allosteric regulatory site journal November 2016
Direct small-molecule inhibitors of KRAS: from structural insights to mechanism-based design journal July 2016
The dimer-dependent catalytic activity of RAF family kinases is revealed through characterizing their oncogenic mutants journal June 2018
Targeting the α4–α5 dimerization interface of K-RAS inhibits tumor formation in vivo journal December 2018
KRAS-specific inhibition using a DARPin binding to a site in the allosteric lobe journal June 2019
Phosphorylated Rho–GDP directly activates mTORC2 kinase towards AKT through dimerization with Ras–GTP to regulate cell migration journal July 2019
A model for RAS mutation patterns in cancers: finding the sweet spot journal November 2018
CRISPR-Cas9 Mediated Labelling Allows for Single Molecule Imaging and Resolution journal August 2017
Investigation of GTP-dependent dimerization of G12X K-Ras variants using ultraviolet photodissociation mass spectrometry journal January 2019
Membrane-associated Ras dimers are isoform-specific: K-Ras dimers differ from H-Ras dimers journal June 2016
Seeing is believing: Ras dimers observed in live cells journal July 2015
Conformational resolution of nucleotide cycling and effector interactions for multiple small GTPases determined in parallel journal May 2019
Phosphatidylserine and GTPase activation control Cdc42 nanoclustering to counter dissipative diffusion journal June 2018
High-throughput single-particle tracking reveals nested membrane nanodomain organization that dictates Ras diffusion and trafficking journal March 2019
Conformational resolution of nucleotide cycling and effector interactions for multiple small GTPases in parallel journal March 2019
Deciphering lipid codes: K‐Ras as a paradigm journal December 2017
Direct targeting of oncogenic RAS mutants with a tumor-specific cytosol-penetrating antibody inhibits RAS mutant–driven tumor growth journal January 2020
RAF1/BRAF dimerization integrates the signal from RAS to ERK and ROKα journal March 2017
Phosphorylation of the C-Raf N Region Promotes Raf Dimerization journal July 2017
MicroRNAs in Pancreatic Cancer: biomarkers, prognostic, and therapeutic modulators journal November 2019
Assessment of mutation probabilities of KRAS G12 missense mutants and their long-timescale dynamics by atomistic molecular simulations and Markov state modeling journal September 2018
KRAS mutant allele-specific expression knockdown in pancreatic cancer model with systemically delivered bi-shRNA KRAS lipoplex journal May 2018
Avidity‐driven polarity establishment via multivalent lipid– GTP ase module interactions journal December 2018
Principles of K-Ras effector organization and the role of oncogenic K-Ras in cancer initiation through G1 cell cycle deregulation journal October 2015
KRAS: A Promising Therapeutic Target for Cancer Treatment journal November 2019
Insights from the Cold Transcriptome and Metabolome of Dendrobium officinale: Global Reprogramming of Metabolic and Gene Regulation Networks during Cold Acclimation journal November 2016
Mitogen-Activated Protein Kinase Inhibitors and T-Cell-Dependent Immunotherapy in Cancer journal January 2020
Specific cancer-associated mutations in the switch III region of Ras increase tumorigenicity by nanocluster augmentation journal August 2015
High-throughput, single-particle tracking reveals nested membrane domains that dictate KRasG12D diffusion and trafficking journal November 2019
Presence or Absence of Ras Dimerization Shows Distinct Kinetic Signature in Ras-Raf Interaction journal April 2020
MicroRNAs in Pancreatic Cancer: biomarkers, prognostic, and therapeutic modulators text January 2019
PDEδ Binding to Ras Isoforms Provides a Route to Proper Membrane Localization journal June 2017
Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers journal January 2017
Ras functional proximity proteomics establishes mTORC2 as new direct ras effector journal August 2019
Ras Dimer Formation as a New Signaling Mechanism and Potential Cancer Therapeutic Target journal February 2016
Early and Late Induction of KRAS and HRAS Proto-Oncogenes by Reactive Oxygen Species in Primary Astrocytes journal June 2017
RAS Nanoclusters: Dynamic Signaling Platforms Amenable to Therapeutic Intervention journal March 2021
Molecular Dynamics Simulations and Dynamic Network Analysis Reveal the Allosteric Unbinding of Monobody to H-Ras Triggered by R135K Mutation journal October 2017
Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution journal January 2016

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Ras dimer
MAPK signaling
cancer
single molecule imaging
superresolution microscopy