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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 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 transiently expressed in BHK cells with (+) or without (−) 5 μM compactin treatment for 48 hr. Compactin treatment relocalized N-ras mutant/RBD complexes to the cytoplasm, allowing us to assess binding in solution. (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). (C) RBD-pulldown experiment quantification of the active, GTP-bound forms of wt and mutant N-ras. +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 N-ras mutants normalized to wt N-ras + EGF-stimulation from two independent experiments. Blue vertical line annotates the activity of wt N-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). (D) Western blot analysis of phosphorylated MEK, ERK, and AKT and total MEK, ERK, and AKT in BHK cells transiently expressing EGFP only, mCit-N-ras-wt, mCit-N-ras-T50I, mCit-N-ras-E49K, or mCit-N-ras-C51Y. Cells were serum starved and collected without EGF stimulation (−EGF) or after stimulation with 100 ng/ml EGF for 10 min (+EGF). Equal expression of Ras constructs can be seen in the GFP row, equal loading in the β-actin row. (E) Validation of comparable protein expression levels of HA-tagged N-ras mutants stably expressed in NIH/3T3 cells. Anti-HA-tag Western blot.DOI:http://dx.doi.org/10.7554/eLife.08905.013
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fig5s1: 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 transiently expressed in BHK cells with (+) or without (−) 5 μM compactin treatment for 48 hr. Compactin treatment relocalized N-ras mutant/RBD complexes to the cytoplasm, allowing us to assess binding in solution. (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). (C) RBD-pulldown experiment quantification of the active, GTP-bound forms of wt and mutant N-ras. +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 N-ras mutants normalized to wt N-ras + EGF-stimulation from two independent experiments. Blue vertical line annotates the activity of wt N-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). (D) Western blot analysis of phosphorylated MEK, ERK, and AKT and total MEK, ERK, and AKT in BHK cells transiently expressing EGFP only, mCit-N-ras-wt, mCit-N-ras-T50I, mCit-N-ras-E49K, or mCit-N-ras-C51Y. Cells were serum starved and collected without EGF stimulation (−EGF) or after stimulation with 100 ng/ml EGF for 10 min (+EGF). Equal expression of Ras constructs can be seen in the GFP row, equal loading in the β-actin row. (E) Validation of comparable protein expression levels of HA-tagged N-ras mutants stably expressed in NIH/3T3 cells. Anti-HA-tag Western blot.DOI:http://dx.doi.org/10.7554/eLife.08905.013

Mentions: As observed for H-ras switch III mutations, none of the three N-ras mutations altered the subcellular distribution of N-ras (Figure 5A). We next assessed nanoclustering and effector recruitment of these mutants in BHK cells. Gal-1 is not known to impact on N-ras nanoclustering and binding to it remained unaltered by the mutations in N-ras (Figure 5—figure supplement 1A). We therefore investigated the N-ras mutants only at endogenous Gal-1 concentrations. While mutation T50I had no significant effect, both mutation E49K and C51Y significantly increased nanoclustering (Figure 5B,C) and ensuing Ras effector recruitment as compared to parent N-rasG12V (Figure 5D).10.7554/eLife.08905.012Figure 5.Tumor-derived N-ras switch III mutations increase cell proliferation and transforming activity by increased nanoclustering.


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 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 transiently expressed in BHK cells with (+) or without (−) 5 μM compactin treatment for 48 hr. Compactin treatment relocalized N-ras mutant/RBD complexes to the cytoplasm, allowing us to assess binding in solution. (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). (C) RBD-pulldown experiment quantification of the active, GTP-bound forms of wt and mutant N-ras. +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 N-ras mutants normalized to wt N-ras + EGF-stimulation from two independent experiments. Blue vertical line annotates the activity of wt N-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). (D) Western blot analysis of phosphorylated MEK, ERK, and AKT and total MEK, ERK, and AKT in BHK cells transiently expressing EGFP only, mCit-N-ras-wt, mCit-N-ras-T50I, mCit-N-ras-E49K, or mCit-N-ras-C51Y. Cells were serum starved and collected without EGF stimulation (−EGF) or after stimulation with 100 ng/ml EGF for 10 min (+EGF). Equal expression of Ras constructs can be seen in the GFP row, equal loading in the β-actin row. (E) Validation of comparable protein expression levels of HA-tagged N-ras mutants stably expressed in NIH/3T3 cells. Anti-HA-tag Western blot.DOI:http://dx.doi.org/10.7554/eLife.08905.013
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fig5s1: 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 transiently expressed in BHK cells with (+) or without (−) 5 μM compactin treatment for 48 hr. Compactin treatment relocalized N-ras mutant/RBD complexes to the cytoplasm, allowing us to assess binding in solution. (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). (C) RBD-pulldown experiment quantification of the active, GTP-bound forms of wt and mutant N-ras. +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 N-ras mutants normalized to wt N-ras + EGF-stimulation from two independent experiments. Blue vertical line annotates the activity of wt N-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). (D) Western blot analysis of phosphorylated MEK, ERK, and AKT and total MEK, ERK, and AKT in BHK cells transiently expressing EGFP only, mCit-N-ras-wt, mCit-N-ras-T50I, mCit-N-ras-E49K, or mCit-N-ras-C51Y. Cells were serum starved and collected without EGF stimulation (−EGF) or after stimulation with 100 ng/ml EGF for 10 min (+EGF). Equal expression of Ras constructs can be seen in the GFP row, equal loading in the β-actin row. (E) Validation of comparable protein expression levels of HA-tagged N-ras mutants stably expressed in NIH/3T3 cells. Anti-HA-tag Western blot.DOI:http://dx.doi.org/10.7554/eLife.08905.013
Mentions: As observed for H-ras switch III mutations, none of the three N-ras mutations altered the subcellular distribution of N-ras (Figure 5A). We next assessed nanoclustering and effector recruitment of these mutants in BHK cells. Gal-1 is not known to impact on N-ras nanoclustering and binding to it remained unaltered by the mutations in N-ras (Figure 5—figure supplement 1A). We therefore investigated the N-ras mutants only at endogenous Gal-1 concentrations. While mutation T50I had no significant effect, both mutation E49K and C51Y significantly increased nanoclustering (Figure 5B,C) and ensuing Ras effector recruitment as compared to parent N-rasG12V (Figure 5D).10.7554/eLife.08905.012Figure 5.Tumor-derived N-ras switch III mutations increase cell proliferation and transforming activity by increased nanoclustering.

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