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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

Galectin-1 neither binds to a farnesylated Ras-peptide nor to H-ras in solution experiments.(A,B) Fluorescence polarization binding assay of 0.25 μM fluorescein labelled and farnesylated Rheb peptide titrated with increasing concentrations of (A) the farnesyl-binding protein PDEδ or (B) purified Gal-1. (C) Sensitized acceptor FRET binding experiment of 250 nM H-ras and 250 nM Gal-1 or 250 nM C-Raf–RBD (RBD) fluorescently labelled using the ACP-tag technology. The legend to the left shows interaction partners schematically. H-ras was either GTPγS (GTP) or GDP loaded, as indicated. Fluorescent labelling substrates, coenzyme A (CoA)-linked ATTO-488 as a FRET-donor and DY-547 as FRET-acceptor, are represented by green and red stars, respectively. Control sample was a 1:1 mix of fluorescent labelling substrates each at 100 nM. Error bars indicate measurement error. (D,E) Interaction between H-ras and Gal-1 or the C-Raf-RBD (RBD) as indicated by the legend in (C) was determined by FLIM-FRET. Purified proteins as in (C) were incubated with fluorescent protein tagged proteins derived from BHK21 cell lysates (indicated with dotted cell outline). (D) Proteins from cell lysates were mRFP-tagged (red circle). (E) H-rasG12V labelled with mGFP (green circle) from lysates was used. Control is either mRFP-tagged C-Raf-RBD (upper column) or Gal-1 (lower column) incubated with 1 μM of CoA-488 in (D) and mGFP-H-rasG12V incubated with 1 μM of CoA-547 label in (E). (C–E) Binding of GTP-H-ras and the C-Raf-RBD served as a positive control. (D,E) Plotted values correspond to the mean ± SEM from three independent biological repeats. Numbers inside or above the bars indicate total number of imaged regions. The Methods section describes indicated statistical comparisons (ns, non-significant; ***p < 0.001).
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f1: Galectin-1 neither binds to a farnesylated Ras-peptide nor to H-ras in solution experiments.(A,B) Fluorescence polarization binding assay of 0.25 μM fluorescein labelled and farnesylated Rheb peptide titrated with increasing concentrations of (A) the farnesyl-binding protein PDEδ or (B) purified Gal-1. (C) Sensitized acceptor FRET binding experiment of 250 nM H-ras and 250 nM Gal-1 or 250 nM C-Raf–RBD (RBD) fluorescently labelled using the ACP-tag technology. The legend to the left shows interaction partners schematically. H-ras was either GTPγS (GTP) or GDP loaded, as indicated. Fluorescent labelling substrates, coenzyme A (CoA)-linked ATTO-488 as a FRET-donor and DY-547 as FRET-acceptor, are represented by green and red stars, respectively. Control sample was a 1:1 mix of fluorescent labelling substrates each at 100 nM. Error bars indicate measurement error. (D,E) Interaction between H-ras and Gal-1 or the C-Raf-RBD (RBD) as indicated by the legend in (C) was determined by FLIM-FRET. Purified proteins as in (C) were incubated with fluorescent protein tagged proteins derived from BHK21 cell lysates (indicated with dotted cell outline). (D) Proteins from cell lysates were mRFP-tagged (red circle). (E) H-rasG12V labelled with mGFP (green circle) from lysates was used. Control is either mRFP-tagged C-Raf-RBD (upper column) or Gal-1 (lower column) incubated with 1 μM of CoA-488 in (D) and mGFP-H-rasG12V incubated with 1 μM of CoA-547 label in (E). (C–E) Binding of GTP-H-ras and the C-Raf-RBD served as a positive control. (D,E) Plotted values correspond to the mean ± SEM from three independent biological repeats. Numbers inside or above the bars indicate total number of imaged regions. The Methods section describes indicated statistical comparisons (ns, non-significant; ***p < 0.001).

Mentions: In an effort to explain this contradictory observation, we scrutinized the structural basis of the specific Gal-1/H-ras interaction. We first studied their complex formation in solution outside of the membrane. In order to test whether Gal-1 could indeed bind farnesylated Ras-proteins, we employed a fluorescence polarization binding assay that was recently used to demonstrate the ability of the Ras trafficking chaperone PDEδ to solubilize farnesylated proteins42. Incubation of a fluorescently labelled peptide derived from the Ras-family protein Rheb with increasing concentrations of PDEδ increased the polarization signal, in agreement with binding of the rotationally highly mobile peptide to the relatively immobile PDEδ protein (Fig. 1A). By contrast, no change in polarization was observed, if the peptide was incubated with purified Gal-1 at concentrations up to 50 μM (Fig. 1B), confirming very recently published results43.


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)

Galectin-1 neither binds to a farnesylated Ras-peptide nor to H-ras in solution experiments.(A,B) Fluorescence polarization binding assay of 0.25 μM fluorescein labelled and farnesylated Rheb peptide titrated with increasing concentrations of (A) the farnesyl-binding protein PDEδ or (B) purified Gal-1. (C) Sensitized acceptor FRET binding experiment of 250 nM H-ras and 250 nM Gal-1 or 250 nM C-Raf–RBD (RBD) fluorescently labelled using the ACP-tag technology. The legend to the left shows interaction partners schematically. H-ras was either GTPγS (GTP) or GDP loaded, as indicated. Fluorescent labelling substrates, coenzyme A (CoA)-linked ATTO-488 as a FRET-donor and DY-547 as FRET-acceptor, are represented by green and red stars, respectively. Control sample was a 1:1 mix of fluorescent labelling substrates each at 100 nM. Error bars indicate measurement error. (D,E) Interaction between H-ras and Gal-1 or the C-Raf-RBD (RBD) as indicated by the legend in (C) was determined by FLIM-FRET. Purified proteins as in (C) were incubated with fluorescent protein tagged proteins derived from BHK21 cell lysates (indicated with dotted cell outline). (D) Proteins from cell lysates were mRFP-tagged (red circle). (E) H-rasG12V labelled with mGFP (green circle) from lysates was used. Control is either mRFP-tagged C-Raf-RBD (upper column) or Gal-1 (lower column) incubated with 1 μM of CoA-488 in (D) and mGFP-H-rasG12V incubated with 1 μM of CoA-547 label in (E). (C–E) Binding of GTP-H-ras and the C-Raf-RBD served as a positive control. (D,E) Plotted values correspond to the mean ± SEM from three independent biological repeats. Numbers inside or above the bars indicate total number of imaged regions. The Methods section describes indicated statistical comparisons (ns, non-significant; ***p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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f1: Galectin-1 neither binds to a farnesylated Ras-peptide nor to H-ras in solution experiments.(A,B) Fluorescence polarization binding assay of 0.25 μM fluorescein labelled and farnesylated Rheb peptide titrated with increasing concentrations of (A) the farnesyl-binding protein PDEδ or (B) purified Gal-1. (C) Sensitized acceptor FRET binding experiment of 250 nM H-ras and 250 nM Gal-1 or 250 nM C-Raf–RBD (RBD) fluorescently labelled using the ACP-tag technology. The legend to the left shows interaction partners schematically. H-ras was either GTPγS (GTP) or GDP loaded, as indicated. Fluorescent labelling substrates, coenzyme A (CoA)-linked ATTO-488 as a FRET-donor and DY-547 as FRET-acceptor, are represented by green and red stars, respectively. Control sample was a 1:1 mix of fluorescent labelling substrates each at 100 nM. Error bars indicate measurement error. (D,E) Interaction between H-ras and Gal-1 or the C-Raf-RBD (RBD) as indicated by the legend in (C) was determined by FLIM-FRET. Purified proteins as in (C) were incubated with fluorescent protein tagged proteins derived from BHK21 cell lysates (indicated with dotted cell outline). (D) Proteins from cell lysates were mRFP-tagged (red circle). (E) H-rasG12V labelled with mGFP (green circle) from lysates was used. Control is either mRFP-tagged C-Raf-RBD (upper column) or Gal-1 (lower column) incubated with 1 μM of CoA-488 in (D) and mGFP-H-rasG12V incubated with 1 μM of CoA-547 label in (E). (C–E) Binding of GTP-H-ras and the C-Raf-RBD served as a positive control. (D,E) Plotted values correspond to the mean ± SEM from three independent biological repeats. Numbers inside or above the bars indicate total number of imaged regions. The Methods section describes indicated statistical comparisons (ns, non-significant; ***p < 0.001).
Mentions: In an effort to explain this contradictory observation, we scrutinized the structural basis of the specific Gal-1/H-ras interaction. We first studied their complex formation in solution outside of the membrane. In order to test whether Gal-1 could indeed bind farnesylated Ras-proteins, we employed a fluorescence polarization binding assay that was recently used to demonstrate the ability of the Ras trafficking chaperone PDEδ to solubilize farnesylated proteins42. Incubation of a fluorescently labelled peptide derived from the Ras-family protein Rheb with increasing concentrations of PDEδ increased the polarization signal, in agreement with binding of the rotationally highly mobile peptide to the relatively immobile PDEδ protein (Fig. 1A). By contrast, no change in polarization was observed, if the peptide was incubated with purified Gal-1 at concentrations up to 50 μM (Fig. 1B), confirming very recently published results43.

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