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Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering.

Blaževitš O, Mideksa YG, Šolman M, Ligabue A, Ariotti N, Nakhaeizadeh H, Fansa EK, Papageorgiou AC, Wittinghofer A, Ahmadian MR, Abankwa D - Sci Rep (2016)

Bottom Line: We show that it indirectly forms a complex with GTP-H-ras via a high-affinity interaction with the Ras binding domain (RBD) of Ras effectors.Consistently, interference with H-rasG12V-effector interactions basically abolishes H-ras nanoclustering.Based on our results the Gal-1/effector interface represents a potential drug target site in diseases with aberrant Ras signalling.

View Article: PubMed Central - PubMed

Affiliation: Turku Centre for Biotechnology, Åbo Akademi University, Tykistökatu 6B, 20520 Turku, Finland.

ABSTRACT
Galectin-1 (Gal-1) dimers crosslink carbohydrates on cell surface receptors. Carbohydrate-derived inhibitors have been developed for cancer treatment. Intracellularly, Gal-1 was suggested to interact with the farnesylated C-terminus of Ras thus specifically stabilizing GTP-H-ras nanoscale signalling hubs in the membrane, termed nanoclusters. The latter activity may present an alternative mechanism for how overexpressed Gal-1 stimulates tumourigenesis. Here we revise the current model for the interaction of Gal-1 with H-ras. We show that it indirectly forms a complex with GTP-H-ras via a high-affinity interaction with the Ras binding domain (RBD) of Ras effectors. A computationally generated model of the Gal-1/C-Raf-RBD complex is validated by mutational analysis. Both cellular FRET as well as proximity ligation assay experiments confirm interaction of Gal-1 with Raf proteins in mammalian cells. Consistently, interference with H-rasG12V-effector interactions basically abolishes H-ras nanoclustering. In addition, an intact dimer interface of Gal-1 is required for it to positively regulate H-rasG12V nanoclustering, but negatively K-rasG12V nanoclustering. Our findings suggest stacked dimers of H-ras, Raf and Gal-1 as building blocks of GTP-H-ras-nanocluster at high Gal-1 levels. Based on our results the Gal-1/effector interface represents a potential drug target site in diseases with aberrant Ras signalling.

No MeSH data available.


Related in: MedlinePlus

H-rasG12V nanoclustering largely depends on effector interactions, while Gal-1 interacts with Raf-effectors.(A) Electron microscopic nanoclustering analysis of mGFP-H-rasG12V and mGFP-H-rasG12V-D38A with or without antisense-mediated knockdown of Gal-1 in BHK21 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 analysed per condition, n ≥ 10). (B) Complexation between indicated mGFP-tagged H-ras mutants and mRFP-tagged C-Raf-RBD or Gal-1 was determined using FLIM-FRET in HEK293-EBNA cells transiently expressing above constructs (two independent biological repeats). (C) Complexation between indicated EGFP-tagged full-length Raf proteins and mRFP-tagged Gal-1 measured by FLIM-FRET in HEK293-EBNA cells (three independent biological repeats). Examples of FLIM-FRET images of cells, coexpressing indicated FRET-pairs or EGFP-tagged C-Raf-RBD as donor-only control. Image colour look-up table on the right shows fluorescence lifetimes. (B,C) Plotted values correspond to the mean ± SEM. Numbers inside and above the bars indicate total number of cells imaged. The Methods section describes the indicated statistical comparisons (***p < 0.001). Samples with coexpressed fluorescent proteins mGFP and mRFP (B), or EGFP and mRFP (C) served as FRET controls. Note that non-control sample FRET-values were all significantly different from the (FRET-)control sample. (D) Analysis of the interaction between endogenous Raf isoforms and Gal-1 in BHK21 cells using in situ proximity ligation assay (PLA). Representative confocal microscopy images of indicated proteins are shown. The sample with siRNA-mediated Gal-1 depletion served as a negative control. Cell nuclei were stained with DAPI. Red foci indicate positive signals for protein interactions and their quantification is shown in the graph. Scale bar is 21 μm.
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f2: H-rasG12V nanoclustering largely depends on effector interactions, while Gal-1 interacts with Raf-effectors.(A) Electron microscopic nanoclustering analysis of mGFP-H-rasG12V and mGFP-H-rasG12V-D38A with or without antisense-mediated knockdown of Gal-1 in BHK21 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 analysed per condition, n ≥ 10). (B) Complexation between indicated mGFP-tagged H-ras mutants and mRFP-tagged C-Raf-RBD or Gal-1 was determined using FLIM-FRET in HEK293-EBNA cells transiently expressing above constructs (two independent biological repeats). (C) Complexation between indicated EGFP-tagged full-length Raf proteins and mRFP-tagged Gal-1 measured by FLIM-FRET in HEK293-EBNA cells (three independent biological repeats). Examples of FLIM-FRET images of cells, coexpressing indicated FRET-pairs or EGFP-tagged C-Raf-RBD as donor-only control. Image colour look-up table on the right shows fluorescence lifetimes. (B,C) Plotted values correspond to the mean ± SEM. Numbers inside and above the bars indicate total number of cells imaged. The Methods section describes the indicated statistical comparisons (***p < 0.001). Samples with coexpressed fluorescent proteins mGFP and mRFP (B), or EGFP and mRFP (C) served as FRET controls. Note that non-control sample FRET-values were all significantly different from the (FRET-)control sample. (D) Analysis of the interaction between endogenous Raf isoforms and Gal-1 in BHK21 cells using in situ proximity ligation assay (PLA). Representative confocal microscopy images of indicated proteins are shown. The sample with siRNA-mediated Gal-1 depletion served as a negative control. Cell nuclei were stained with DAPI. Red foci indicate positive signals for protein interactions and their quantification is shown in the graph. Scale bar is 21 μm.

Mentions: When studying nanoclustering of H-rasG12V mutants, we serendipitously found that the effector-site mutation D38A48 reduced nanoclustering to a similar extent as knockdown of Gal-1, and a complete loss of nanoclustering was observed when knockdown and mutation were combined (Fig. 2A). We therefore tested, whether residue D38A, which is at the centre of the Ras/RBD interface48, affects the FRET between mRFP-Gal-1 and mGFP-H-rasG12V in HEK293-EBNA cells. Indeed, the Ras effector-site mutation did not only abrogate FRET between H-rasG12V and the C-Raf-RBD, but also of H-rasG12V and Gal-1 (Fig. 2B). Note that FRET-levels were significantly lower than those of the non-farnesylatable CAAX-mutant (Supplementary Fig. 1B), supporting that farnesylation was not required for an interaction with either the RBD or Gal-1 in cells, while an intact effector-site on H-ras was required. These data therefore indicated that GTP-H-ras/Gal-1 complex formation depends on binding of an effector to H-ras and may therefore proceed indirectly.


Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering.

Blaževitš O, Mideksa YG, Šolman M, Ligabue A, Ariotti N, Nakhaeizadeh H, Fansa EK, Papageorgiou AC, Wittinghofer A, Ahmadian MR, Abankwa D - Sci Rep (2016)

H-rasG12V nanoclustering largely depends on effector interactions, while Gal-1 interacts with Raf-effectors.(A) Electron microscopic nanoclustering analysis of mGFP-H-rasG12V and mGFP-H-rasG12V-D38A with or without antisense-mediated knockdown of Gal-1 in BHK21 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 analysed per condition, n ≥ 10). (B) Complexation between indicated mGFP-tagged H-ras mutants and mRFP-tagged C-Raf-RBD or Gal-1 was determined using FLIM-FRET in HEK293-EBNA cells transiently expressing above constructs (two independent biological repeats). (C) Complexation between indicated EGFP-tagged full-length Raf proteins and mRFP-tagged Gal-1 measured by FLIM-FRET in HEK293-EBNA cells (three independent biological repeats). Examples of FLIM-FRET images of cells, coexpressing indicated FRET-pairs or EGFP-tagged C-Raf-RBD as donor-only control. Image colour look-up table on the right shows fluorescence lifetimes. (B,C) Plotted values correspond to the mean ± SEM. Numbers inside and above the bars indicate total number of cells imaged. The Methods section describes the indicated statistical comparisons (***p < 0.001). Samples with coexpressed fluorescent proteins mGFP and mRFP (B), or EGFP and mRFP (C) served as FRET controls. Note that non-control sample FRET-values were all significantly different from the (FRET-)control sample. (D) Analysis of the interaction between endogenous Raf isoforms and Gal-1 in BHK21 cells using in situ proximity ligation assay (PLA). Representative confocal microscopy images of indicated proteins are shown. The sample with siRNA-mediated Gal-1 depletion served as a negative control. Cell nuclei were stained with DAPI. Red foci indicate positive signals for protein interactions and their quantification is shown in the graph. Scale bar is 21 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834570&req=5

f2: H-rasG12V nanoclustering largely depends on effector interactions, while Gal-1 interacts with Raf-effectors.(A) Electron microscopic nanoclustering analysis of mGFP-H-rasG12V and mGFP-H-rasG12V-D38A with or without antisense-mediated knockdown of Gal-1 in BHK21 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 analysed per condition, n ≥ 10). (B) Complexation between indicated mGFP-tagged H-ras mutants and mRFP-tagged C-Raf-RBD or Gal-1 was determined using FLIM-FRET in HEK293-EBNA cells transiently expressing above constructs (two independent biological repeats). (C) Complexation between indicated EGFP-tagged full-length Raf proteins and mRFP-tagged Gal-1 measured by FLIM-FRET in HEK293-EBNA cells (three independent biological repeats). Examples of FLIM-FRET images of cells, coexpressing indicated FRET-pairs or EGFP-tagged C-Raf-RBD as donor-only control. Image colour look-up table on the right shows fluorescence lifetimes. (B,C) Plotted values correspond to the mean ± SEM. Numbers inside and above the bars indicate total number of cells imaged. The Methods section describes the indicated statistical comparisons (***p < 0.001). Samples with coexpressed fluorescent proteins mGFP and mRFP (B), or EGFP and mRFP (C) served as FRET controls. Note that non-control sample FRET-values were all significantly different from the (FRET-)control sample. (D) Analysis of the interaction between endogenous Raf isoforms and Gal-1 in BHK21 cells using in situ proximity ligation assay (PLA). Representative confocal microscopy images of indicated proteins are shown. The sample with siRNA-mediated Gal-1 depletion served as a negative control. Cell nuclei were stained with DAPI. Red foci indicate positive signals for protein interactions and their quantification is shown in the graph. Scale bar is 21 μm.
Mentions: When studying nanoclustering of H-rasG12V mutants, we serendipitously found that the effector-site mutation D38A48 reduced nanoclustering to a similar extent as knockdown of Gal-1, and a complete loss of nanoclustering was observed when knockdown and mutation were combined (Fig. 2A). We therefore tested, whether residue D38A, which is at the centre of the Ras/RBD interface48, affects the FRET between mRFP-Gal-1 and mGFP-H-rasG12V in HEK293-EBNA cells. Indeed, the Ras effector-site mutation did not only abrogate FRET between H-rasG12V and the C-Raf-RBD, but also of H-rasG12V and Gal-1 (Fig. 2B). Note that FRET-levels were significantly lower than those of the non-farnesylatable CAAX-mutant (Supplementary Fig. 1B), supporting that farnesylation was not required for an interaction with either the RBD or Gal-1 in cells, while an intact effector-site on H-ras was required. These data therefore indicated that GTP-H-ras/Gal-1 complex formation depends on binding of an effector to H-ras and may therefore proceed indirectly.

Bottom Line: We show that it indirectly forms a complex with GTP-H-ras via a high-affinity interaction with the Ras binding domain (RBD) of Ras effectors.Consistently, interference with H-rasG12V-effector interactions basically abolishes H-ras nanoclustering.Based on our results the Gal-1/effector interface represents a potential drug target site in diseases with aberrant Ras signalling.

View Article: PubMed Central - PubMed

Affiliation: Turku Centre for Biotechnology, Åbo Akademi University, Tykistökatu 6B, 20520 Turku, Finland.

ABSTRACT
Galectin-1 (Gal-1) dimers crosslink carbohydrates on cell surface receptors. Carbohydrate-derived inhibitors have been developed for cancer treatment. Intracellularly, Gal-1 was suggested to interact with the farnesylated C-terminus of Ras thus specifically stabilizing GTP-H-ras nanoscale signalling hubs in the membrane, termed nanoclusters. The latter activity may present an alternative mechanism for how overexpressed Gal-1 stimulates tumourigenesis. Here we revise the current model for the interaction of Gal-1 with H-ras. We show that it indirectly forms a complex with GTP-H-ras via a high-affinity interaction with the Ras binding domain (RBD) of Ras effectors. A computationally generated model of the Gal-1/C-Raf-RBD complex is validated by mutational analysis. Both cellular FRET as well as proximity ligation assay experiments confirm interaction of Gal-1 with Raf proteins in mammalian cells. Consistently, interference with H-rasG12V-effector interactions basically abolishes H-ras nanoclustering. In addition, an intact dimer interface of Gal-1 is required for it to positively regulate H-rasG12V nanoclustering, but negatively K-rasG12V nanoclustering. Our findings suggest stacked dimers of H-ras, Raf and Gal-1 as building blocks of GTP-H-ras-nanocluster at high Gal-1 levels. Based on our results the Gal-1/effector interface represents a potential drug target site in diseases with aberrant Ras signalling.

No MeSH data available.


Related in: MedlinePlus