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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

A dimerization deficient Galectin-1 mutant loses its effect on Ras nanoclustering and signalling.(A) Nanoclustering-FRET response of H-rasG12V in dependence of Gal-1 or its dimerization deficient mutant N-Gal-1 in HEK293-EBNA cells expressing mGFP-/mCherry-H-rasG12V. (B) RBD-recruitment FRET response of H-rasG12V in dependence of Gal-1 or its dimerization deficient mutant N-Gal-1 (mGFP-H-rasG12V and mRFP-C-Raf-RBD expressed in HEK293-EBNA) to assess effector translocation from the cytoplasm to active H-ras in plasma membrane nanoclusters. (C) Left, Western blot analysis of HEK293-EBNA lysates expressing mGFP-tagged H-ras and mRFP-tagged Gal-1 constructs as indicated. Serum-starved cells were stimulated with 100 ng/ml EGF for the indicated times. Total ERK and phospho-ERK (pERK) levels were then determined by immunoblotting. β-actin is the loading control. Right, Quantification of three independent repeats of Western blot data as shown on left. The pERK-signal was normalized to the total ERK-signal. (D) The nanoclustering-FRET response of H-rasG12V, N-rasG12V and K-rasG12V with increasing concentration of Gal-1. BHK21 cells were transiently co-transfected with mGFP-/and mCherry-tagged Ras constructs alone (1.0) or with antisense-Gal-1 (0.5) or non-labelled Gal-1 (3.4). The cellular total Gal-1 concentration relative to endogenous Gal-1 in control BHK21 cells ([Gal-1]rel.) is displayed to the left of the data. (E) The nanoclustering-FRET response of K-rasG12V. HEK293-EBNA cells transiently expressed mGFP-/mCherry-K-rasG12V and if indicated non-labelled Gal-1 or N-Gal-1. Note that in (D) the FRET-levels for K-rasG12V are lower than in (E), due to the higher Gal-1 level in BHK21 as compared to HEK293-EBNA cells (Supplementary Fig. 4E). (A,B,D,E) Plotted values correspond to the mean ± SEM of three independent biological experiments. Numbers inside the bars indicate total number of cells imaged. The Methods section describes the indicated statistical comparisons (ns, non significant; *p < 0.05; ***p < 0.001); comparisons in (D) were done against the 1.0 parent-control. Samples with coexpressed fluorescent proteins mGFP and mCherry (A,E) or mGFP and mRFP (B) served as a FRET control. Note that non-control sample FRET-values were all significantly different from the (FRET-)control sample.
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f5: A dimerization deficient Galectin-1 mutant loses its effect on Ras nanoclustering and signalling.(A) Nanoclustering-FRET response of H-rasG12V in dependence of Gal-1 or its dimerization deficient mutant N-Gal-1 in HEK293-EBNA cells expressing mGFP-/mCherry-H-rasG12V. (B) RBD-recruitment FRET response of H-rasG12V in dependence of Gal-1 or its dimerization deficient mutant N-Gal-1 (mGFP-H-rasG12V and mRFP-C-Raf-RBD expressed in HEK293-EBNA) to assess effector translocation from the cytoplasm to active H-ras in plasma membrane nanoclusters. (C) Left, Western blot analysis of HEK293-EBNA lysates expressing mGFP-tagged H-ras and mRFP-tagged Gal-1 constructs as indicated. Serum-starved cells were stimulated with 100 ng/ml EGF for the indicated times. Total ERK and phospho-ERK (pERK) levels were then determined by immunoblotting. β-actin is the loading control. Right, Quantification of three independent repeats of Western blot data as shown on left. The pERK-signal was normalized to the total ERK-signal. (D) The nanoclustering-FRET response of H-rasG12V, N-rasG12V and K-rasG12V with increasing concentration of Gal-1. BHK21 cells were transiently co-transfected with mGFP-/and mCherry-tagged Ras constructs alone (1.0) or with antisense-Gal-1 (0.5) or non-labelled Gal-1 (3.4). The cellular total Gal-1 concentration relative to endogenous Gal-1 in control BHK21 cells ([Gal-1]rel.) is displayed to the left of the data. (E) The nanoclustering-FRET response of K-rasG12V. HEK293-EBNA cells transiently expressed mGFP-/mCherry-K-rasG12V and if indicated non-labelled Gal-1 or N-Gal-1. Note that in (D) the FRET-levels for K-rasG12V are lower than in (E), due to the higher Gal-1 level in BHK21 as compared to HEK293-EBNA cells (Supplementary Fig. 4E). (A,B,D,E) Plotted values correspond to the mean ± SEM of three independent biological experiments. Numbers inside the bars indicate total number of cells imaged. The Methods section describes the indicated statistical comparisons (ns, non significant; *p < 0.05; ***p < 0.001); comparisons in (D) were done against the 1.0 parent-control. Samples with coexpressed fluorescent proteins mGFP and mCherry (A,E) or mGFP and mRFP (B) served as a FRET control. Note that non-control sample FRET-values were all significantly different from the (FRET-)control sample.

Mentions: While wt Gal-1 significantly increased H-rasG12V nanoclustering (Fig. 5A) and RBD-effector recruitment to H-rasG12V (Fig. 5B) as observed before10, N-Gal-1 did not support either increase (Fig. 5A,B). Of note, FRET between the C-Raf-RBD and N-Gal-1 was significantly increased in cells (Supplementary Fig. 4C). This observation rules out that a loss in affinity for the C-Raf-RBD is responsible for the loss in RBD recruitment. Consistent with this loss-of-function in supporting H-rasG12V nanoclustering and RBD recruitment, N-Gal-1 did not potentiate EGF-induced ppERK-signalling when H-ras was overexpressed (Fig. 5C). Hence, our data show that an intact Gal-1 dimer interface is required for H-ras nanocluster augmentation.


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)

A dimerization deficient Galectin-1 mutant loses its effect on Ras nanoclustering and signalling.(A) Nanoclustering-FRET response of H-rasG12V in dependence of Gal-1 or its dimerization deficient mutant N-Gal-1 in HEK293-EBNA cells expressing mGFP-/mCherry-H-rasG12V. (B) RBD-recruitment FRET response of H-rasG12V in dependence of Gal-1 or its dimerization deficient mutant N-Gal-1 (mGFP-H-rasG12V and mRFP-C-Raf-RBD expressed in HEK293-EBNA) to assess effector translocation from the cytoplasm to active H-ras in plasma membrane nanoclusters. (C) Left, Western blot analysis of HEK293-EBNA lysates expressing mGFP-tagged H-ras and mRFP-tagged Gal-1 constructs as indicated. Serum-starved cells were stimulated with 100 ng/ml EGF for the indicated times. Total ERK and phospho-ERK (pERK) levels were then determined by immunoblotting. β-actin is the loading control. Right, Quantification of three independent repeats of Western blot data as shown on left. The pERK-signal was normalized to the total ERK-signal. (D) The nanoclustering-FRET response of H-rasG12V, N-rasG12V and K-rasG12V with increasing concentration of Gal-1. BHK21 cells were transiently co-transfected with mGFP-/and mCherry-tagged Ras constructs alone (1.0) or with antisense-Gal-1 (0.5) or non-labelled Gal-1 (3.4). The cellular total Gal-1 concentration relative to endogenous Gal-1 in control BHK21 cells ([Gal-1]rel.) is displayed to the left of the data. (E) The nanoclustering-FRET response of K-rasG12V. HEK293-EBNA cells transiently expressed mGFP-/mCherry-K-rasG12V and if indicated non-labelled Gal-1 or N-Gal-1. Note that in (D) the FRET-levels for K-rasG12V are lower than in (E), due to the higher Gal-1 level in BHK21 as compared to HEK293-EBNA cells (Supplementary Fig. 4E). (A,B,D,E) Plotted values correspond to the mean ± SEM of three independent biological experiments. Numbers inside the bars indicate total number of cells imaged. The Methods section describes the indicated statistical comparisons (ns, non significant; *p < 0.05; ***p < 0.001); comparisons in (D) were done against the 1.0 parent-control. Samples with coexpressed fluorescent proteins mGFP and mCherry (A,E) or mGFP and mRFP (B) served as a FRET control. Note that non-control sample FRET-values were all significantly different from the (FRET-)control sample.
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f5: A dimerization deficient Galectin-1 mutant loses its effect on Ras nanoclustering and signalling.(A) Nanoclustering-FRET response of H-rasG12V in dependence of Gal-1 or its dimerization deficient mutant N-Gal-1 in HEK293-EBNA cells expressing mGFP-/mCherry-H-rasG12V. (B) RBD-recruitment FRET response of H-rasG12V in dependence of Gal-1 or its dimerization deficient mutant N-Gal-1 (mGFP-H-rasG12V and mRFP-C-Raf-RBD expressed in HEK293-EBNA) to assess effector translocation from the cytoplasm to active H-ras in plasma membrane nanoclusters. (C) Left, Western blot analysis of HEK293-EBNA lysates expressing mGFP-tagged H-ras and mRFP-tagged Gal-1 constructs as indicated. Serum-starved cells were stimulated with 100 ng/ml EGF for the indicated times. Total ERK and phospho-ERK (pERK) levels were then determined by immunoblotting. β-actin is the loading control. Right, Quantification of three independent repeats of Western blot data as shown on left. The pERK-signal was normalized to the total ERK-signal. (D) The nanoclustering-FRET response of H-rasG12V, N-rasG12V and K-rasG12V with increasing concentration of Gal-1. BHK21 cells were transiently co-transfected with mGFP-/and mCherry-tagged Ras constructs alone (1.0) or with antisense-Gal-1 (0.5) or non-labelled Gal-1 (3.4). The cellular total Gal-1 concentration relative to endogenous Gal-1 in control BHK21 cells ([Gal-1]rel.) is displayed to the left of the data. (E) The nanoclustering-FRET response of K-rasG12V. HEK293-EBNA cells transiently expressed mGFP-/mCherry-K-rasG12V and if indicated non-labelled Gal-1 or N-Gal-1. Note that in (D) the FRET-levels for K-rasG12V are lower than in (E), due to the higher Gal-1 level in BHK21 as compared to HEK293-EBNA cells (Supplementary Fig. 4E). (A,B,D,E) Plotted values correspond to the mean ± SEM of three independent biological experiments. Numbers inside the bars indicate total number of cells imaged. The Methods section describes the indicated statistical comparisons (ns, non significant; *p < 0.05; ***p < 0.001); comparisons in (D) were done against the 1.0 parent-control. Samples with coexpressed fluorescent proteins mGFP and mCherry (A,E) or mGFP and mRFP (B) served as a FRET control. Note that non-control sample FRET-values were all significantly different from the (FRET-)control sample.
Mentions: While wt Gal-1 significantly increased H-rasG12V nanoclustering (Fig. 5A) and RBD-effector recruitment to H-rasG12V (Fig. 5B) as observed before10, N-Gal-1 did not support either increase (Fig. 5A,B). Of note, FRET between the C-Raf-RBD and N-Gal-1 was significantly increased in cells (Supplementary Fig. 4C). This observation rules out that a loss in affinity for the C-Raf-RBD is responsible for the loss in RBD recruitment. Consistent with this loss-of-function in supporting H-rasG12V nanoclustering and RBD recruitment, N-Gal-1 did not potentiate EGF-induced ppERK-signalling when H-ras was overexpressed (Fig. 5C). Hence, our data show that an intact Gal-1 dimer interface is required for H-ras nanocluster augmentation.

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