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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

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). Note that in addition extensive biochemical characterization data of the H-ras mutants can be found in Supplementary file 1. Black circle dot indicates that no significant change was observed. Black arrows represent significant increase (up) or decrease (down) of quantified parameters as compared to the parent RasG12V control. The percentage of these changes is given in addition. The following columns (italics) report on: Conformation—predicted H-ras mutant conformation, according to Figure 1A; GAP—sensitivity of mutants to GAP-stimulated GTP hydrolysis; RBD + soluble Ras—binding of the C-Raf-RBD to Ras mutants in solution, which was either measured by fluorescence anisotropy with purified proteins or in BHK cells treated with compactin using FRET (annotated with *); Localization—change in colocalization of mutants relative to their parent constructs in BHK cells determined by confocal microscopy; Nanoclustering-EM—changes in Ras nanoclustering obtained by electron microscopy or -FRET in BHK cells; RBD + membrane Ras—binding of the C-Raf-RBD to Ras mutants in BHK cells measured by FRET; PC12—results of PC12-cell neurite outgrowth assay; last three columns report on Ras-mutant transformed NIH/3T3 cell proliferation, focus formation (transformation) and anchorage-independent growth (soft agar).DOI:http://dx.doi.org/10.7554/eLife.08905.016
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fig7: 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). Note that in addition extensive biochemical characterization data of the H-ras mutants can be found in Supplementary file 1. Black circle dot indicates that no significant change was observed. Black arrows represent significant increase (up) or decrease (down) of quantified parameters as compared to the parent RasG12V control. The percentage of these changes is given in addition. The following columns (italics) report on: Conformation—predicted H-ras mutant conformation, according to Figure 1A; GAP—sensitivity of mutants to GAP-stimulated GTP hydrolysis; RBD + soluble Ras—binding of the C-Raf-RBD to Ras mutants in solution, which was either measured by fluorescence anisotropy with purified proteins or in BHK cells treated with compactin using FRET (annotated with *); Localization—change in colocalization of mutants relative to their parent constructs in BHK cells determined by confocal microscopy; Nanoclustering-EM—changes in Ras nanoclustering obtained by electron microscopy or -FRET in BHK cells; RBD + membrane Ras—binding of the C-Raf-RBD to Ras mutants in BHK cells measured by FRET; PC12—results of PC12-cell neurite outgrowth assay; last three columns report on Ras-mutant transformed NIH/3T3 cell proliferation, focus formation (transformation) and anchorage-independent growth (soft agar).DOI:http://dx.doi.org/10.7554/eLife.08905.016

Mentions: Here, we have described an unprecedented mechanism for the activation of a signaling protein in cancer. We showed that rare cancer-associated mutations in the switch III regions of H-, N-, and K-ras can increase effector engagement, while not changing the affinity to the effector. Instead the increase of the nanoscale concentration of Ras in nanocluster hyperactivates Ras to promote tumorigenic growth (Figure 7).10.7554/eLife.08905.016Figure 7.Summary table of Ras switch III mutant properties studied in this work.


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)

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). Note that in addition extensive biochemical characterization data of the H-ras mutants can be found in Supplementary file 1. Black circle dot indicates that no significant change was observed. Black arrows represent significant increase (up) or decrease (down) of quantified parameters as compared to the parent RasG12V control. The percentage of these changes is given in addition. The following columns (italics) report on: Conformation—predicted H-ras mutant conformation, according to Figure 1A; GAP—sensitivity of mutants to GAP-stimulated GTP hydrolysis; RBD + soluble Ras—binding of the C-Raf-RBD to Ras mutants in solution, which was either measured by fluorescence anisotropy with purified proteins or in BHK cells treated with compactin using FRET (annotated with *); Localization—change in colocalization of mutants relative to their parent constructs in BHK cells determined by confocal microscopy; Nanoclustering-EM—changes in Ras nanoclustering obtained by electron microscopy or -FRET in BHK cells; RBD + membrane Ras—binding of the C-Raf-RBD to Ras mutants in BHK cells measured by FRET; PC12—results of PC12-cell neurite outgrowth assay; last three columns report on Ras-mutant transformed NIH/3T3 cell proliferation, focus formation (transformation) and anchorage-independent growth (soft agar).DOI:http://dx.doi.org/10.7554/eLife.08905.016
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4563131&req=5

fig7: 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). Note that in addition extensive biochemical characterization data of the H-ras mutants can be found in Supplementary file 1. Black circle dot indicates that no significant change was observed. Black arrows represent significant increase (up) or decrease (down) of quantified parameters as compared to the parent RasG12V control. The percentage of these changes is given in addition. The following columns (italics) report on: Conformation—predicted H-ras mutant conformation, according to Figure 1A; GAP—sensitivity of mutants to GAP-stimulated GTP hydrolysis; RBD + soluble Ras—binding of the C-Raf-RBD to Ras mutants in solution, which was either measured by fluorescence anisotropy with purified proteins or in BHK cells treated with compactin using FRET (annotated with *); Localization—change in colocalization of mutants relative to their parent constructs in BHK cells determined by confocal microscopy; Nanoclustering-EM—changes in Ras nanoclustering obtained by electron microscopy or -FRET in BHK cells; RBD + membrane Ras—binding of the C-Raf-RBD to Ras mutants in BHK cells measured by FRET; PC12—results of PC12-cell neurite outgrowth assay; last three columns report on Ras-mutant transformed NIH/3T3 cell proliferation, focus formation (transformation) and anchorage-independent growth (soft agar).DOI:http://dx.doi.org/10.7554/eLife.08905.016
Mentions: Here, we have described an unprecedented mechanism for the activation of a signaling protein in cancer. We showed that rare cancer-associated mutations in the switch III regions of H-, N-, and K-ras can increase effector engagement, while not changing the affinity to the effector. Instead the increase of the nanoscale concentration of Ras in nanocluster hyperactivates Ras to promote tumorigenic growth (Figure 7).10.7554/eLife.08905.016Figure 7.Summary table of Ras switch III mutant properties studied in this work.

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