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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

Comparison of current and our new model for the mechanism of action of Gal-1 as a nanocluster scaffold.(A) Current model. Direct interaction of active H-ras and Gal-1 stabilizes nanocluster. H-ras (depicted as yellow oval) activation supposedly makes the C-terminal farnesyl chain of H-ras more accessible for the prenyl-binding pocket of Gal-1 (depicted as a blue hexagon). This mechanistic step has not been described for other, similar trafficking chaperones, such as GDIs or PDEδ. Instead the spontaneous, activation state independent dissociation from the membrane is the basis for complexation by such chaperones in the cytoplasm7475. (B) In the new model proposed here, Raf effectors (depicted as violet rectangles) are recruited to active H-ras in nanoclusters on the plasma membrane. At higher concentrations Gal-1 can dimerize. Gal-1 binds directly to the Ras binding domain (RBD) of effectors, such as Raf. Thus, dimeric Gal-1 could stabilize effector (Raf)-dimers, which then act as the actual ‘scaffold’ for H-ras nanocluster. Note that effector and Gal-1 can form complexes already in the cytoplasm. Stacked dimers of H-ras + effector (Raf) + Gal-1 (box to right) would be nucleating the growth of H-ras nanocluster, a process that may be supported by the membrane environment.
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f6: Comparison of current and our new model for the mechanism of action of Gal-1 as a nanocluster scaffold.(A) Current model. Direct interaction of active H-ras and Gal-1 stabilizes nanocluster. H-ras (depicted as yellow oval) activation supposedly makes the C-terminal farnesyl chain of H-ras more accessible for the prenyl-binding pocket of Gal-1 (depicted as a blue hexagon). This mechanistic step has not been described for other, similar trafficking chaperones, such as GDIs or PDEδ. Instead the spontaneous, activation state independent dissociation from the membrane is the basis for complexation by such chaperones in the cytoplasm7475. (B) In the new model proposed here, Raf effectors (depicted as violet rectangles) are recruited to active H-ras in nanoclusters on the plasma membrane. At higher concentrations Gal-1 can dimerize. Gal-1 binds directly to the Ras binding domain (RBD) of effectors, such as Raf. Thus, dimeric Gal-1 could stabilize effector (Raf)-dimers, which then act as the actual ‘scaffold’ for H-ras nanocluster. Note that effector and Gal-1 can form complexes already in the cytoplasm. Stacked dimers of H-ras + effector (Raf) + Gal-1 (box to right) would be nucleating the growth of H-ras nanocluster, a process that may be supported by the membrane environment.

Mentions: Ras nanoclustering is indispensable for Ras signalling8 and we have recently described it as target of cancer associated Ras-mutations57, underscoring its significance for the signalling architecture of Ras. Only a handful of nanocluster regulators, so called Ras nanocluster scaffolds are known, and Gal-1 has so far been the one scaffold that was functionally and mechanistically best understood. We here presented data, which question the existing model of Gal-1 binding directly to the farnesyl-lipid on the C-terminus of Ras proteins (Fig. 6A). We did neither observe binding of Gal-1 to a farnesylated Ras-peptide, nor directly to the G-domain of Ras (Fig. 1). Instead, we found that Gal-1 indirectly couples to Ras via a direct association with the RBD-domain of effectors (Figs 2, 3, 4) and that an intact Gal-1 dimer interface is required for Gal-1 to modulate Ras nanoclustering (Fig. 5).


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)

Comparison of current and our new model for the mechanism of action of Gal-1 as a nanocluster scaffold.(A) Current model. Direct interaction of active H-ras and Gal-1 stabilizes nanocluster. H-ras (depicted as yellow oval) activation supposedly makes the C-terminal farnesyl chain of H-ras more accessible for the prenyl-binding pocket of Gal-1 (depicted as a blue hexagon). This mechanistic step has not been described for other, similar trafficking chaperones, such as GDIs or PDEδ. Instead the spontaneous, activation state independent dissociation from the membrane is the basis for complexation by such chaperones in the cytoplasm7475. (B) In the new model proposed here, Raf effectors (depicted as violet rectangles) are recruited to active H-ras in nanoclusters on the plasma membrane. At higher concentrations Gal-1 can dimerize. Gal-1 binds directly to the Ras binding domain (RBD) of effectors, such as Raf. Thus, dimeric Gal-1 could stabilize effector (Raf)-dimers, which then act as the actual ‘scaffold’ for H-ras nanocluster. Note that effector and Gal-1 can form complexes already in the cytoplasm. Stacked dimers of H-ras + effector (Raf) + Gal-1 (box to right) would be nucleating the growth of H-ras nanocluster, a process that may be supported by the membrane environment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Comparison of current and our new model for the mechanism of action of Gal-1 as a nanocluster scaffold.(A) Current model. Direct interaction of active H-ras and Gal-1 stabilizes nanocluster. H-ras (depicted as yellow oval) activation supposedly makes the C-terminal farnesyl chain of H-ras more accessible for the prenyl-binding pocket of Gal-1 (depicted as a blue hexagon). This mechanistic step has not been described for other, similar trafficking chaperones, such as GDIs or PDEδ. Instead the spontaneous, activation state independent dissociation from the membrane is the basis for complexation by such chaperones in the cytoplasm7475. (B) In the new model proposed here, Raf effectors (depicted as violet rectangles) are recruited to active H-ras in nanoclusters on the plasma membrane. At higher concentrations Gal-1 can dimerize. Gal-1 binds directly to the Ras binding domain (RBD) of effectors, such as Raf. Thus, dimeric Gal-1 could stabilize effector (Raf)-dimers, which then act as the actual ‘scaffold’ for H-ras nanocluster. Note that effector and Gal-1 can form complexes already in the cytoplasm. Stacked dimers of H-ras + effector (Raf) + Gal-1 (box to right) would be nucleating the growth of H-ras nanocluster, a process that may be supported by the membrane environment.
Mentions: Ras nanoclustering is indispensable for Ras signalling8 and we have recently described it as target of cancer associated Ras-mutations57, underscoring its significance for the signalling architecture of Ras. Only a handful of nanocluster regulators, so called Ras nanocluster scaffolds are known, and Gal-1 has so far been the one scaffold that was functionally and mechanistically best understood. We here presented data, which question the existing model of Gal-1 binding directly to the farnesyl-lipid on the C-terminus of Ras proteins (Fig. 6A). We did neither observe binding of Gal-1 to a farnesylated Ras-peptide, nor directly to the G-domain of Ras (Fig. 1). Instead, we found that Gal-1 indirectly couples to Ras via a direct association with the RBD-domain of effectors (Figs 2, 3, 4) and that an intact Gal-1 dimer interface is required for Gal-1 to modulate Ras nanoclustering (Fig. 5).

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