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. 2015 Jun 30;112(26):7996-8001.
doi: 10.1073/pnas.1509123112. Epub 2015 Jun 16.

Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway (VSports注册入口)

Xiaolin Nan  1 , Tanja M Tamgüney  2 , Eric A Collisson  3 , Li-Jung Lin  4 , Cameron Pitt  2 , Jacqueline Galeas  2 , Sophia Lewis  5 , Joe W Gray  6 , Frank McCormick  7 , Steven Chu  8
Affiliations

Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway

Xiaolin Nan (VSports最新版本) et al. Proc Natl Acad Sci U S A. .

V体育2025版 - Abstract

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 VSports手机版. 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 KRas(G12D), a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRas(G12D) 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. .

Keywords: MAPK signaling; Ras dimer; cancer; single molecule imaging; superresolution microscopy. V体育安卓版.

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"V体育2025版" Conflict of interest statement

The authors declare no conflict of interest.

Figures (VSports注册入口)

Fig. 1.
Fig. 1.
KRasG12D forms dimers at expression levels that activate Raf-MAPK. We used a lentiviral vector construct with a CMV-TO (TetOn) promoter (A) to express PAmCherry1-KRas under tetracycline or doxycycline (Dox) regulation. (B) Western blotting demonstrated dose-dependent induction of PAmCherry1-KRasG12D expression, leading to varying degrees of activation of Raf/MAPK (as indicated by ppErk) in a single clone of BHK21 cells. Confocal fluorescence microscopy showed a clear membrane localization of the induced PAmCherry1-KRasG12D proteins (C). (D–F), PALM imaging and cluster analysis of PAmCherry1-KRasG12D molecules in cells from the same clone as in B induced at 1 and 2 ng/mL Dox. Each dot in the PALM images represents one PAmCherry1-tagged KRasG12D molecule. White arrows indicate dimers (see also the inset in the bottom PALM image). (Scale bars, 5 µm in C and 200 nm in D.)
Fig. S1.
Fig. S1.
Biological activity of PAmCherry1-KRas expression constructs. NIH 3T3 cells stably expressing PAmCherry1-KRas WT were not transformed (B) compared with the original NIH 3T3 cells (A). By contrast, stable expression of PAmCherry1-KRas G12D mutant caused marked transformation of the cells as indicated by the formation of numerous colonies (C). Stable cell lines were established by lentiviral infection as described in Materials and Methods. Cells were grown past confluence to allow colony formation.
Fig. S2.
Fig. S2.
Western blotting of BHK21 cells stably expressing PAmCherry1-KRas wild type under Dox induction. Cells were incubated with various concentrations of Dox (0, 0.5, 1, 2, 5, and 0 ng/mL, lanes 1–6, respectively) and serum starved overnight. Cells for lane 6 was treated with 10 ng/mL EGF for 15 min, and those for the other lanes were untreated; cells were then harvested for Western blotting.
Fig. S3.
Fig. S3.
Formation of KRas clusters at high expression levels. PALM images of BHK21 cells (originated from the same clone as used in Fig.1) stably expressing PAmCherry1-KRas G12D under 5 ng/mL Dox (Top), and those (parent BHK21 cells) transiently overexpressing PAmCherry1-KRas G12D (Middle) or PAmCherry1-KRas wild type (Bottom) were taken under the same conditions as described in SI Materials and Methods.
Fig. S4.
Fig. S4.
Dox-induced expression of PAmCherry1-KRas G12D and Raf-MAPK activation in 293TRex cells. Stable cell 293TRex cell line expressing PAmCherry1-KRas G12D was established using the same method as described for BHK21 cells. Single cell clones were isolated and assayed with both Western blotting (A) and PALM microscopy (B). Ripley’s K test (C) and SAD analysis (D) both suggested formation of Ras dimers and a small population of trimers at 2 ng/mL Dox, when Raf-MAPK activation was observed in the Western blot (A).
Fig. 2.
Fig. 2.
Artificial dimerization of monomeric KRasG12D leads to Raf-MAPK activation. (A) Schematics of the artificial dimerization system, where a dimerization domain (DD) is genetically fused to the N terminus of PAmCherry1-KRasG12D. A small molecule, AP20187, forces PAmCherry1-KRasG12D to dimerize by binding to two DD domains at once. (B) Western blot showing Dox-induced expression of DD-PAmCherry1-KRasG12D and effects of AP20187. Induction at 1 ng/mL Dox yielded detectable levels of DD-PAmCherry1-KRasG12D but no Raf/MAPK activation (lane 3) until AP20187 was added (lane 4). AP20187 also increased ppErk in cells treated with 2 ng/mL Dox but to a lesser extent (lanes 5–6). (C) PALM imaging and cluster size analysis of DD-PAmCherry1-KRasG12D expressed in cells induced at 1 ng/mL Dox, confirming a monomer to dimer conversion before and after AP20187 treatment. (D) Western blotting demonstrated Raf/MAPK activation by AP20187 in BHK21 cells expressing low levels of DD-PAmCherry1-KRasG12D under a weak, PGK promoter. (Scale bars, 200 nm.)
Fig. S5.
Fig. S5.
Artificial dimerization of KRas G12D in TRex-293 cells activates Raf-MAPK. We established a stable TRex-293 cell line expressing DD-PAmCherry1-KRas G12D (i.e., a “dimerizable” KRas mutant) and isolated single cell clones for imaging and Western blotting.
Fig. S6.
Fig. S6.
Artificial dimerization of KRas G12D rescues TRex-293 cells from serum starvation (A) and MAPK inhibition (B). TRex-293 cells expressing DD-PAmCherry1-KRas G12D under 1 ng/mL Dox were grown to confluency and serum starved (A) or treated with a MEK inhibitor Trametinib (GSK1120212; 50 nM) (B) in the absence (Left) or presence (Center) of 100 nM AP20187. To quantify the number of viable cells, we trypsinzed the cells and used mild (1,000 × g) centrifugation to pellet the cells. The pelleted cells were then resuspended in PBS, stained with trypan blue and counted (unstained cells) using a hemocytometer (Right).
Fig. 3.
Fig. 3.
Ras dimerization and signaling depend on an intact CAAX motif. PALM images of PAmCherry1-KRas wild type (A) and PAmCherry1-CAAX (CAAX = last 21 amino acids of KRas) (B) taken on cells expressing the proteins at membrane densities around 70 ± 19 molecules per µm2. White arrows indicate dimers and occasional higher order clusters. (C) Cluster size analysis of both PAmCherry1-KRas (green) and PAmCherry1-CAAX (blue) indicated similar clustering properties between the two proteins as well as to PAmCherry1-KRasG12D (Fig. 1E). (D) Western blot comparing the response of DD-PAmCherry1-KRasG12D (lanes 1–2) and the DD-PAmCherry1-KrasG12D/C185S double mutant (lanes 3–4) to artificial dimerization by AP20187. Both proteins were expressed at low levels under a PGK promoter. (Scale bars, 200 nm.)
Fig. S7.
Fig. S7.
Western blotting of TRex-293 cells expressing DD-PAmCherry1-KRas G12D/Y64A double mutant. We generated TRex-293 cells stably expressing DD-PAmCherry1-KRas G12D/Y64A double mutant under tetracycline regulation by lentiviral infection, similarly to those expressing the G12D single mutant. A pool of infected cells were exposed to 0, 2, and 4 ng/mL Dox for 48 h and serum starved for 4 h before treatment with mock or AP20187 (100 nM; 15 min). DD-PAmCherry1-KRas G12D activated MAPK in the absence of AP20187 at high concentrations (e.g., 4 ng/mL) of Dox and in response to AP20187 at low Dox concentrations (e.g., 2 ng/mL).
Fig. S8.
Fig. S8.
Comparing biological activities of DD-KRas G12D and DD-PAmCherry1-KRas G12D in TRex-293 cells. TRex-293 cells stably expressing DD-KRas G12D or DD-PAmCherry1-KRas G12D under tetracycline regulation were generated in parallel by lentiviral infection and single clones were isolated, similarly to that described in Materials and Methods. Cells from single clones were exposed to 0, 2, and 4 ng/mL Dox for 48 h and serum starved for 4 h before treatment with mock or AP20187 (100 nM; 15 min). Both DD-KRas G12D and DD-PAmCherry1-KRas G12D activated MAPK in the absence of AP20187 at high concentrations (e.g., 4 ng/mL) of Dox. Activation of MAPK was minimal at low Dox concentrations (e.g., 0–2 ng/mL) but was significantly enhanced by incubating cells with AP20187 in both cell lines.
Fig. 4.
Fig. 4.
Dimer model for Ras-mediated activation of Raf/MAPK. Ras attaches to the cell membrane via the C-terminal HVR including the CAAX motif. In the GDP-bound state, Ras is unable to bind Raf, leaving Raf auto-inhibited in the cytosol even when Ras-GDP forms dimers (Left). GTP-loaded Ras can bind to Raf (Upper Right) or form a Ras-GTP dimer (Lower Center); either event alone does not activate Raf. When two Ras-Raf complexes further dimerize or when a Ras-GTP dimer recruits two Raf molecules, the event results in a Raf-Raf dimer that in turn activates the Raf kinase and subsequently MEK/Erk (MAPK) (Lower Right). It is presently unclear how the Ras-Ras dimer formation and the Ras-Raf binding processes are ordered and coupled in Ras-mediated Raf activation. GAP, GTPase activating proteins; GEF, guanine-nucleotide exchange factor; KD, Kinase domain; RBD, Ras binding domain.
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Comment in

  • Seeing is believing: Ras dimers observed in live cells.
    Philips MR, Der CJ. Philips MR, et al. Proc Natl Acad Sci U S A. 2015 Aug 11;112(32):9793-4. doi: 10.1073/pnas.1511805112. Epub 2015 Jul 30. Proc Natl Acad Sci U S A. 2015. PMID: 26229079 Free PMC article. No abstract available.

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