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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

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 give number of analyzed cells from three independent experiments. Error bars represent the standard error of the mean (±SEM). Statistical analysis was performed as described in ‘Materials and methods’ (NS, non-significant; ***p < 0.001). (C) RBD-pulldown experiment quantification of the active, GTP-bound forms of H-ras mutants. +EGF denotes stimulation with 100 ng/ml EGF. −EGF serum starved cells. +GAP incubation with GAP domain of NF1, to assay for GAP sensitivity. The graphs represent the averages of active H-ras-G48R and H-ras-G48R,D92N normalized to wt H-ras + EGF-stimulation from three independent experiments. Blue vertical line annotates the activity of wt H-ras when stimulated with EGF. Error bars represent the standard error of the mean (±SEM). Statistical analysis was performed as described in ‘Materials and methods’ (NS, non-significant).DOI:http://dx.doi.org/10.7554/eLife.08905.011
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fig4s1: 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 give number of analyzed cells from three independent experiments. Error bars represent the standard error of the mean (±SEM). Statistical analysis was performed as described in ‘Materials and methods’ (NS, non-significant; ***p < 0.001). (C) RBD-pulldown experiment quantification of the active, GTP-bound forms of H-ras mutants. +EGF denotes stimulation with 100 ng/ml EGF. −EGF serum starved cells. +GAP incubation with GAP domain of NF1, to assay for GAP sensitivity. The graphs represent the averages of active H-ras-G48R and H-ras-G48R,D92N normalized to wt H-ras + EGF-stimulation from three independent experiments. Blue vertical line annotates the activity of wt H-ras when stimulated with EGF. Error bars represent the standard error of the mean (±SEM). Statistical analysis was performed as described in ‘Materials and methods’ (NS, non-significant).DOI:http://dx.doi.org/10.7554/eLife.08905.011

Mentions: Confocal colocalization analysis confirmed that the localization of both mutants in BHK cells was unaltered as compared to parent H-rasG12V (Figure 4A). Assuming that the G48R,D92N mutations were gain of function, we combined these cancer-associated mutations with the inactivating mutation R128A,R135A on helix α4. This new mutant showed normalized H-ras activity (Figure 4—figure supplement 1A), comparable to the modeling-derived orientation-switch III mutants (Figure 1D). This suggested that the cancer-associated mutation is also coupled to the reorientation mechanism of membrane bound H-ras and may thus also impact on its nanoclustering. Indeed, electron microscopic analysis of plasma membrane sheets derived from BHK cells expressing the single or double-mutant revealed a strong and significant increase in nanoclustering of both mutants as compared to the parent H-rasG12V (Figure 4B). This was confirmed by nanoclustering-FRET analysis in cells, where consistent with the increased Gal-1 complexation of the G48R-mutant (Figure 4—figure supplement 1B), the Gal-1-dose-dependent nanoclustering response was significantly increased for both the G48R and G48R,D92N mutants at all Gal-1 levels (Figure 4C). Moreover, the Gal-1-dose-dependent RBD recruitment response of both mutants was increased in cells (Figure 4D), while RBD-binding in vitro remained indistinguishable from the parent Ras (Figure 4E, Supplementary file 1). The correlation of the RBD- and nanoclustering-response pattern again suggested that augmented nanoclustering increased H-ras activity by enhancing effector recruitment. Together with the absence of any RBD-binding defect, this led us to the conclusion that introduction of mutations G48R or G48R,D92N enhances effector recruitment due to increased nanoclustering.10.7554/eLife.08905.010Figure 4.Increased nanoclustering and effector recruitment by a tumor-derived switch III mutation in H-ras.


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)

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 give number of analyzed cells from three independent experiments. Error bars represent the standard error of the mean (±SEM). Statistical analysis was performed as described in ‘Materials and methods’ (NS, non-significant; ***p < 0.001). (C) RBD-pulldown experiment quantification of the active, GTP-bound forms of H-ras mutants. +EGF denotes stimulation with 100 ng/ml EGF. −EGF serum starved cells. +GAP incubation with GAP domain of NF1, to assay for GAP sensitivity. The graphs represent the averages of active H-ras-G48R and H-ras-G48R,D92N normalized to wt H-ras + EGF-stimulation from three independent experiments. Blue vertical line annotates the activity of wt H-ras when stimulated with EGF. Error bars represent the standard error of the mean (±SEM). Statistical analysis was performed as described in ‘Materials and methods’ (NS, non-significant).DOI:http://dx.doi.org/10.7554/eLife.08905.011
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fig4s1: 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 give number of analyzed cells from three independent experiments. Error bars represent the standard error of the mean (±SEM). Statistical analysis was performed as described in ‘Materials and methods’ (NS, non-significant; ***p < 0.001). (C) RBD-pulldown experiment quantification of the active, GTP-bound forms of H-ras mutants. +EGF denotes stimulation with 100 ng/ml EGF. −EGF serum starved cells. +GAP incubation with GAP domain of NF1, to assay for GAP sensitivity. The graphs represent the averages of active H-ras-G48R and H-ras-G48R,D92N normalized to wt H-ras + EGF-stimulation from three independent experiments. Blue vertical line annotates the activity of wt H-ras when stimulated with EGF. Error bars represent the standard error of the mean (±SEM). Statistical analysis was performed as described in ‘Materials and methods’ (NS, non-significant).DOI:http://dx.doi.org/10.7554/eLife.08905.011
Mentions: Confocal colocalization analysis confirmed that the localization of both mutants in BHK cells was unaltered as compared to parent H-rasG12V (Figure 4A). Assuming that the G48R,D92N mutations were gain of function, we combined these cancer-associated mutations with the inactivating mutation R128A,R135A on helix α4. This new mutant showed normalized H-ras activity (Figure 4—figure supplement 1A), comparable to the modeling-derived orientation-switch III mutants (Figure 1D). This suggested that the cancer-associated mutation is also coupled to the reorientation mechanism of membrane bound H-ras and may thus also impact on its nanoclustering. Indeed, electron microscopic analysis of plasma membrane sheets derived from BHK cells expressing the single or double-mutant revealed a strong and significant increase in nanoclustering of both mutants as compared to the parent H-rasG12V (Figure 4B). This was confirmed by nanoclustering-FRET analysis in cells, where consistent with the increased Gal-1 complexation of the G48R-mutant (Figure 4—figure supplement 1B), the Gal-1-dose-dependent nanoclustering response was significantly increased for both the G48R and G48R,D92N mutants at all Gal-1 levels (Figure 4C). Moreover, the Gal-1-dose-dependent RBD recruitment response of both mutants was increased in cells (Figure 4D), while RBD-binding in vitro remained indistinguishable from the parent Ras (Figure 4E, Supplementary file 1). The correlation of the RBD- and nanoclustering-response pattern again suggested that augmented nanoclustering increased H-ras activity by enhancing effector recruitment. Together with the absence of any RBD-binding defect, this led us to the conclusion that introduction of mutations G48R or G48R,D92N enhances effector recruitment due to increased nanoclustering.10.7554/eLife.08905.010Figure 4.Increased nanoclustering and effector recruitment by a tumor-derived switch III mutation in H-ras.

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