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Specific cancer-associated mutations in the switch III region of Ras increase tumorigenicity by nanocluster augmentation.

Šolman M, Ligabue A, Blaževitš O, Jaiswal A, Zhou Y, Liang H, Lectez B, Kopra K, Guzmán C, Härmä H, Hancock JF, Aittokallio T, Abankwa D - Elife (2015)

Bottom Line: Here, we show that several cancer-associated mutations in the switch III region moderately increase Ras activity in all isoforms.Nanoclustering dictates downstream effector recruitment, MAPK-activity, and tumorigenic cell proliferation.Our results describe an unprecedented mechanism of signaling protein activation in cancer.

View Article: PubMed Central - PubMed

Affiliation: Turku Centre for Biotechnology, Åbo Akademi University, Turku, Finland.

ABSTRACT
Hotspot mutations of Ras drive cell transformation and tumorigenesis. Less frequent mutations in Ras are poorly characterized for their oncogenic potential. Yet insight into their mechanism of action may point to novel opportunities to target Ras. Here, we show that several cancer-associated mutations in the switch III region moderately increase Ras activity in all isoforms. Mutants are biochemically inconspicuous, while their clustering into nanoscale signaling complexes on the plasma membrane, termed nanocluster, is augmented. Nanoclustering dictates downstream effector recruitment, MAPK-activity, and tumorigenic cell proliferation. Our results describe an unprecedented mechanism of signaling protein activation in cancer.

No MeSH data available.


Related in: MedlinePlus

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 resonance energy transfer (QRET) technique. Dots represent data points obtained from individual reactions, with a total of 360 individual data points for each H-ras. (C) Fluorescence anisotropy binding data for the derivation of dissociation constants (Kd) between mantGTPγS and wt H-ras (black), H-ras-D47A,E49A (red), H-ras-G48R (blue), and H-ras-G48R,D92N (orange). Details are described in the ‘Materials and methods’.DOI:http://dx.doi.org/10.7554/eLife.08905.007
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fig2s2: 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 resonance energy transfer (QRET) technique. Dots represent data points obtained from individual reactions, with a total of 360 individual data points for each H-ras. (C) Fluorescence anisotropy binding data for the derivation of dissociation constants (Kd) between mantGTPγS and wt H-ras (black), H-ras-D47A,E49A (red), H-ras-G48R (blue), and H-ras-G48R,D92N (orange). Details are described in the ‘Materials and methods’.DOI:http://dx.doi.org/10.7554/eLife.08905.007

Mentions: (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 complete spatial randomness indicate clustering at that value of r (number of membrane sheets analyzed per condition, n = 17). (B) Schematic representation of nanoclustering-FRET analysis, where mGFP-tagged and mCherry-tagged Ras constructs were co-expressed in cells. (C) The nanoclustering-FRET response of H-rasG12V-D47A,E49A and its parent construct in dependence of the dose of the nanocluster scaffold Gal-1 in BHK cells. (D) Representative nanoclustering-FRET fluorescence lifetime images of BHK cells expressing FRET-pairs (or donor only, left column) of constructs indicated on the left, under Gal-1 conditions as annotated on the top. Color look up table to the right shows fluorescence lifetimes. (E) RBD-recruitment FRET data of H-rasG12V-D47A,E49A and its parent construct at three different Gal-1 doses analyzed using FRET-imaging of transiently transfected BHK cells. (C, E) Numbers in bars give numbers of analyzed cells from three independent experiments. Error bars represent the standard error of the mean (±SEM). Statistical analysis vs parent RasG12V was performed as described in ‘Materials and methods’ (*p < 0.05; **p < 0.01; ***p < 0.001). (F) Western blot analysis of C-Raf, MEK, and ERK phosphorylation and C-Raf in BHK cells transiently expressing mGFP-H-rasG12V-D47A,E49K, its parent or an empty vector control. Equal expression of Ras constructs can be seen in the mGFP row, equal loading in the Actin row. (G) In vitro RBD binding to mant-GTPγS loaded wild-type (wt) H-ras or H-ras-D47A,E49A measured with a fluorescence anisotropy assay. Details on the fitting function are in the ‘Materials and methods’. Data are averages ±SEM of three repeats. (H) RBD-pulldown experiments in BHK cells transiently expressing indicated H-ras mutants or wt H-ras. Top panel shows level of active Ras after EGF-stimulation (100 ng/ml). Bottom panel shows GAP sensitivity of wt and mutant H-ras proteins. See also Figure 2—figure supplement 1 and Figure 2—figure supplement 2.


Specific cancer-associated mutations in the switch III region of Ras increase tumorigenicity by nanocluster augmentation.

Šolman M, Ligabue A, Blaževitš O, Jaiswal A, Zhou Y, Liang H, Lectez B, Kopra K, Guzmán C, Härmä H, Hancock JF, Aittokallio T, Abankwa D - Elife (2015)

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 resonance energy transfer (QRET) technique. Dots represent data points obtained from individual reactions, with a total of 360 individual data points for each H-ras. (C) Fluorescence anisotropy binding data for the derivation of dissociation constants (Kd) between mantGTPγS and wt H-ras (black), H-ras-D47A,E49A (red), H-ras-G48R (blue), and H-ras-G48R,D92N (orange). Details are described in the ‘Materials and methods’.DOI:http://dx.doi.org/10.7554/eLife.08905.007
© Copyright Policy
Related In: Results  -  Collection

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fig2s2: 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 resonance energy transfer (QRET) technique. Dots represent data points obtained from individual reactions, with a total of 360 individual data points for each H-ras. (C) Fluorescence anisotropy binding data for the derivation of dissociation constants (Kd) between mantGTPγS and wt H-ras (black), H-ras-D47A,E49A (red), H-ras-G48R (blue), and H-ras-G48R,D92N (orange). Details are described in the ‘Materials and methods’.DOI:http://dx.doi.org/10.7554/eLife.08905.007
Mentions: (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 complete spatial randomness indicate clustering at that value of r (number of membrane sheets analyzed per condition, n = 17). (B) Schematic representation of nanoclustering-FRET analysis, where mGFP-tagged and mCherry-tagged Ras constructs were co-expressed in cells. (C) The nanoclustering-FRET response of H-rasG12V-D47A,E49A and its parent construct in dependence of the dose of the nanocluster scaffold Gal-1 in BHK cells. (D) Representative nanoclustering-FRET fluorescence lifetime images of BHK cells expressing FRET-pairs (or donor only, left column) of constructs indicated on the left, under Gal-1 conditions as annotated on the top. Color look up table to the right shows fluorescence lifetimes. (E) RBD-recruitment FRET data of H-rasG12V-D47A,E49A and its parent construct at three different Gal-1 doses analyzed using FRET-imaging of transiently transfected BHK cells. (C, E) Numbers in bars give numbers of analyzed cells from three independent experiments. Error bars represent the standard error of the mean (±SEM). Statistical analysis vs parent RasG12V was performed as described in ‘Materials and methods’ (*p < 0.05; **p < 0.01; ***p < 0.001). (F) Western blot analysis of C-Raf, MEK, and ERK phosphorylation and C-Raf in BHK cells transiently expressing mGFP-H-rasG12V-D47A,E49K, its parent or an empty vector control. Equal expression of Ras constructs can be seen in the mGFP row, equal loading in the Actin row. (G) In vitro RBD binding to mant-GTPγS loaded wild-type (wt) H-ras or H-ras-D47A,E49A measured with a fluorescence anisotropy assay. Details on the fitting function are in the ‘Materials and methods’. Data are averages ±SEM of three repeats. (H) RBD-pulldown experiments in BHK cells transiently expressing indicated H-ras mutants or wt H-ras. Top panel shows level of active Ras after EGF-stimulation (100 ng/ml). Bottom panel shows GAP sensitivity of wt and mutant H-ras proteins. See also Figure 2—figure supplement 1 and Figure 2—figure supplement 2.

Bottom Line: Here, we show that several cancer-associated mutations in the switch III region moderately increase Ras activity in all isoforms.Nanoclustering dictates downstream effector recruitment, MAPK-activity, and tumorigenic cell proliferation.Our results describe an unprecedented mechanism of signaling protein activation in cancer.

View Article: PubMed Central - PubMed

Affiliation: Turku Centre for Biotechnology, Åbo Akademi University, Turku, Finland.

ABSTRACT
Hotspot mutations of Ras drive cell transformation and tumorigenesis. Less frequent mutations in Ras are poorly characterized for their oncogenic potential. Yet insight into their mechanism of action may point to novel opportunities to target Ras. Here, we show that several cancer-associated mutations in the switch III region moderately increase Ras activity in all isoforms. Mutants are biochemically inconspicuous, while their clustering into nanoscale signaling complexes on the plasma membrane, termed nanocluster, is augmented. Nanoclustering dictates downstream effector recruitment, MAPK-activity, and tumorigenic cell proliferation. Our results describe an unprecedented mechanism of signaling protein activation in cancer.

No MeSH data available.


Related in: MedlinePlus