Specific cancer-associated mutations in the switch III region of Ras increase tumorigenicity by nanocluster augmentation
- Åbo Akademi University, Finland
- University of Helsinki, Finland
- University of Texas Health Science Center at Houston, United States
- University of Turku, Finland
Figures
Figure 1 with 1 supplement
Intramolecular switch III-conformer coupling suggests altered nanoclustering in switch III mutants.
(A) Schematic representation of conformer-nanocluster coupling and its effect on H-ras signaling. For GTP-Ras a conformational equilibrium is assumed, which can be shifted by mutations as indicated …
Figure 1—figure supplement 1
Nucleotide-dependent H-ras conformers on the membrane.
Computational modeling-derived structures of the predominant H-ras-GTP- (left) and H-ras-GDP- (right) conformers on the membrane. Conformer stabilizing residues R128,R135 on helix α4 and R169,K170 …
Figure 2 with 2 supplements
Computational modeling-derived switch III mutations D47A,E49A in H-ras increase nanoclustering and RBD-recruitment.
(A) Electron microscopic nanoclustering analysis of mGFP-H-rasG12V and mGFP-H-rasG12V-D47A,E49A in BHK cells. Normalized univariate K-functions, where maximal L(r)-r values above the 99% CI for …
Figure 2—figure supplement 1
The computational modeling-derived switch III H-ras mutant exhibits stronger Gal-1 complexation and remains sensitive to GAP-mediated hydrolysis.
(A) Schematic representation of the Gal-1-complexation FRET assay in which complexation of mRFP-Gal-1 with mGFP-tagged mutants of Ras was measured in intact BHK cells using FRET (left). …
Figure 2—figure supplement 2
SOS-mediated nucleotide exchange kinetics, as well as mantGTPγS dissociation constants of wt H-ras and mutants.
SOS-induced Eu3+-GTP association (A) and dissociation (B) kinetics with wt H-ras (black), H-ras-D47A,E49A (red), H-ras-G48R (blue), and H-ras-G48R,D92N (orange) monitored using the quenching …
Figure 3 with 1 supplement
Orientation-switch III mutations in oncogenic Ras isoforms that are reported in cancer genome databases.
Cancer-associated point mutations (missense, nonsense, and silent) in the orientation-switch III regions as reported in COSMIC, cBioPortal, and ICGC databases. Schematic representations of the …
Figure 3—figure supplement 1
Phylogenetic analysis of the Ras switch III region.
(A) ClustalW sequence alignment of 18 Ras proteins from different species as annotated on the left. Consensus sequence is shaded in dark and switch III region elements β2-β3-loop (residues 40–56) …
Figure 4 with 1 supplement
Increased nanoclustering and effector recruitment by a tumor-derived switch III mutation in H-ras.
(A) Representative confocal images of mCFP-H-rasG12V co-localization with the switch III mutants mCherry-H-rasG12V-G48R (top) or mCherry-H-rasG12V-G48R,D92N (bottom) in BHK cells. The colocalization …
Figure 4—figure supplement 1
Cancer-associated H-ras switch III mutations have no relevant effect on its biochemical properties.
(A) RBD-recruitment FRET data of indicated H-ras mutants transiently expressed in BHK cells. (B) Gal-1-complexation FRET analysis of H-rasG12V-G48R and its parent construct. (A, B) Numbers in bars …
Figure 5 with 1 supplement
Tumor-derived N-ras switch III mutations increase cell proliferation and transforming activity by increased nanoclustering.
(A) Representative confocal images of mCFP-N-rasG12V co-localization with the switch III mutants mCherry-N-rasG12V-T50I, mCherry-N-rasG12V-E49K, or mCherry-N-rasG12V-C51Y in BHK cells. The Manders' …
Figure 5—figure supplement 1
Cancer-associated mutations in switch III of N-ras do not alter its biochemical properties and Gal-1 complexation.
(A) Gal-1-complexation FRET data of indicated N-ras mutants and parent construct transiently expressed in BHK cells. (B) RBD-recruitment FRET analysis of indicated N-ras mutant and parent constructs …
Figure 6 with 1 supplement
K-ras with colorectal cancer-associated mutation R164Q displays increased nanoclustering to drive oncogenic transformation.
(A) Colocalization of mCFP-K-rasG12V and switch III mutants mCherry-K-rasG12V-V152G and mCherry-K-rasG12V-R164Q in BHK cells. The Manders' coefficient (R) that quantifies colocalization is marked on …
Figure 6—figure supplement 1
K-ras with mutation R164Q has unaltered Gal-1-complexation and biochemical properties.
(A) Gal-1-complexation FRET analysis of K-ras mutants and its parent construct transiently expressed in BHK cells. (B) RBD-recruitment FRET analysis of K-ras mutants and its parent construct …
Figure 7
Summary table of Ras switch III mutant properties studied in this work.
The table summarizes major experimental results obtained in different assays that were used to characterize switch III mutants of H-ras (highlighted in green), N-ras (violet), and K-ras4B (blue). …
Additional files
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Supplementary file 1
Thermodynamic and kinetic parameters of H-ras mutant in vitro experiments. Table contains background corrected GEF-dependent Eu3+-GTP association (kon), and dissociation (koff) kinetics of wt H-ras and mutants. The Kon, Koff, and Kd derived from Koff/Kon values are calculated from corrected association and dissociation data as described in ‘Materials and methods’. Dissociation constants (Kd) between mantGTPγS and wt H-ras and its mutants (in the presence of EDTA and in the absence of Mg2+). The absence of Mg2+ increases the Kd, which is otherwise in the pM range (John et al., 1988, 1990). Dissociation constants (Kd) of C-Raf-RBD and mantGTPγS-bound wt H-ras and its mutants were measured by fluorescence anisotropy as described in ‘Materials and methods’. Raw data are shown in Figure 2—figure supplement 2.
- https://doi.org/10.7554/eLife.08905.017
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Supplementary file 2
Distribution of isoform specific coding mutations in the switch III region by cancer tissue type. Table details the frequency of amino acid changes due to coding mutations observed in switch III region of each isoform in different tissue types of cancer. Point mutations leading to different amino acid residue changes at the same coding position have been added to indicate the number of changes at that position. Summary for total mutations observed in all isoforms and total mutations per isoform have also been provided. Gray highlighted cells are the tissue types and switch III regions having the highest number of coding mutations at that position. Red and bold highlighted numbers indicate coding mutations observed in patient samples with the corresponding cancer tissue type.
- https://doi.org/10.7554/eLife.08905.018
