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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 directly binds to the Ras binding domain of effectors.(A) Interaction of Gal-1 with fragments of C-Raf (as can be derived from Supplementary Fig. 2A) or with the RBD of PI3Kα studied by FLIM-FRET in BHK21 cells, transiently expressing mCit-tagged Gal-1 and mRFP-tagged RBD-constructs (three independent biological repeats). Fluorescence lifetimes of FRET-samples were all significantly different from the donor-control. 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 mRFP and mCit served as FRET controls. Note that non-control sample FRET-values were all significantly different from the (FRET-)control sample. (B) GST pull-down experiments were performed by mixing bacterially purified Gal-1 with GST, GST-C-Raf-RBD or GST-PI3Kα-RBD immobilized on glutathione sepharose beads. GST was used as a negative control. Proteins retained on the beads were resolved by SDS-PAGE and Western blotted using a monoclonal antibody (M01) against Gal-1 for detection. (C) Corrected sensitized acceptor emission FRET data of 100 nM ATTO-488-labelled Gal-1 titrated with increasing concentrations of DY-547-labelled C-Raf-RBD (scheme on the left). Both proteins were purified from bacteria and labelled with the ACP-tag method. The dissociation constant (Kd) was determined from the shown curve fit on the dataset of EmFRET as described in the Methods section. Plotted values correspond to the mean ± SEM.
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f3: Galectin-1 directly binds to the Ras binding domain of effectors.(A) Interaction of Gal-1 with fragments of C-Raf (as can be derived from Supplementary Fig. 2A) or with the RBD of PI3Kα studied by FLIM-FRET in BHK21 cells, transiently expressing mCit-tagged Gal-1 and mRFP-tagged RBD-constructs (three independent biological repeats). Fluorescence lifetimes of FRET-samples were all significantly different from the donor-control. 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 mRFP and mCit served as FRET controls. Note that non-control sample FRET-values were all significantly different from the (FRET-)control sample. (B) GST pull-down experiments were performed by mixing bacterially purified Gal-1 with GST, GST-C-Raf-RBD or GST-PI3Kα-RBD immobilized on glutathione sepharose beads. GST was used as a negative control. Proteins retained on the beads were resolved by SDS-PAGE and Western blotted using a monoclonal antibody (M01) against Gal-1 for detection. (C) Corrected sensitized acceptor emission FRET data of 100 nM ATTO-488-labelled Gal-1 titrated with increasing concentrations of DY-547-labelled C-Raf-RBD (scheme on the left). Both proteins were purified from bacteria and labelled with the ACP-tag method. The dissociation constant (Kd) was determined from the shown curve fit on the dataset of EmFRET as described in the Methods section. Plotted values correspond to the mean ± SEM.

Mentions: Next, we wanted to identify the minimal domain of Raf that mediates the interaction with Gal-1. The D38A mutation reduces the affinity of GTP-H-ras to the C-Raf-RBD by approximately 100-fold to 1300 nM, thus basically abrogating their interaction48. Based on our observations that this mutation also blocks H-rasG12V complexation with Gal-1 (Fig. 2B), we reasoned that RBD containing Raf-fragments would bind to Gal-1. Indeed, both the C-Raf-RBD and the same extended by the cysteine rich domain (CRD) showed identically high FRET with Gal-1 when expressed in BHK21 cells (Fig. 3A). Moreover, the structurally related RBD from PI3Kα showed FRET with Gal-1, albeit significantly less than the C-Raf-RBD (Fig. 3A, Supplementary Fig. 3B), suggesting that Gal-1 directly interacts with the Ras binding domain of effectors. In order to confirm these FRET results, we performed co-immunoprecipitation experiments, which showed that GST-labelled RBD-fragments were able to pull-down bacterially purified Gal-1 (Fig. 3B) or vice versa (Supplementary Fig. 2D). Finally, we provided evidence for direct binding of Gal-1 to the RBD. We purified Gal-1 and the C-Raf-RBD, both with N-terminal A1-tags in order to label them fluorescently for FRET-based binding experiments (Fig. 3C). Analysis of our FRET-binding data established a dissociation constant for Gal-1/C-Raf-RBD of Kd = 106 ± 40 nM.


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 directly binds to the Ras binding domain of effectors.(A) Interaction of Gal-1 with fragments of C-Raf (as can be derived from Supplementary Fig. 2A) or with the RBD of PI3Kα studied by FLIM-FRET in BHK21 cells, transiently expressing mCit-tagged Gal-1 and mRFP-tagged RBD-constructs (three independent biological repeats). Fluorescence lifetimes of FRET-samples were all significantly different from the donor-control. 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 mRFP and mCit served as FRET controls. Note that non-control sample FRET-values were all significantly different from the (FRET-)control sample. (B) GST pull-down experiments were performed by mixing bacterially purified Gal-1 with GST, GST-C-Raf-RBD or GST-PI3Kα-RBD immobilized on glutathione sepharose beads. GST was used as a negative control. Proteins retained on the beads were resolved by SDS-PAGE and Western blotted using a monoclonal antibody (M01) against Gal-1 for detection. (C) Corrected sensitized acceptor emission FRET data of 100 nM ATTO-488-labelled Gal-1 titrated with increasing concentrations of DY-547-labelled C-Raf-RBD (scheme on the left). Both proteins were purified from bacteria and labelled with the ACP-tag method. The dissociation constant (Kd) was determined from the shown curve fit on the dataset of EmFRET as described in the Methods section. Plotted values correspond to the mean ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Galectin-1 directly binds to the Ras binding domain of effectors.(A) Interaction of Gal-1 with fragments of C-Raf (as can be derived from Supplementary Fig. 2A) or with the RBD of PI3Kα studied by FLIM-FRET in BHK21 cells, transiently expressing mCit-tagged Gal-1 and mRFP-tagged RBD-constructs (three independent biological repeats). Fluorescence lifetimes of FRET-samples were all significantly different from the donor-control. 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 mRFP and mCit served as FRET controls. Note that non-control sample FRET-values were all significantly different from the (FRET-)control sample. (B) GST pull-down experiments were performed by mixing bacterially purified Gal-1 with GST, GST-C-Raf-RBD or GST-PI3Kα-RBD immobilized on glutathione sepharose beads. GST was used as a negative control. Proteins retained on the beads were resolved by SDS-PAGE and Western blotted using a monoclonal antibody (M01) against Gal-1 for detection. (C) Corrected sensitized acceptor emission FRET data of 100 nM ATTO-488-labelled Gal-1 titrated with increasing concentrations of DY-547-labelled C-Raf-RBD (scheme on the left). Both proteins were purified from bacteria and labelled with the ACP-tag method. The dissociation constant (Kd) was determined from the shown curve fit on the dataset of EmFRET as described in the Methods section. Plotted values correspond to the mean ± SEM.
Mentions: Next, we wanted to identify the minimal domain of Raf that mediates the interaction with Gal-1. The D38A mutation reduces the affinity of GTP-H-ras to the C-Raf-RBD by approximately 100-fold to 1300 nM, thus basically abrogating their interaction48. Based on our observations that this mutation also blocks H-rasG12V complexation with Gal-1 (Fig. 2B), we reasoned that RBD containing Raf-fragments would bind to Gal-1. Indeed, both the C-Raf-RBD and the same extended by the cysteine rich domain (CRD) showed identically high FRET with Gal-1 when expressed in BHK21 cells (Fig. 3A). Moreover, the structurally related RBD from PI3Kα showed FRET with Gal-1, albeit significantly less than the C-Raf-RBD (Fig. 3A, Supplementary Fig. 3B), suggesting that Gal-1 directly interacts with the Ras binding domain of effectors. In order to confirm these FRET results, we performed co-immunoprecipitation experiments, which showed that GST-labelled RBD-fragments were able to pull-down bacterially purified Gal-1 (Fig. 3B) or vice versa (Supplementary Fig. 2D). Finally, we provided evidence for direct binding of Gal-1 to the RBD. We purified Gal-1 and the C-Raf-RBD, both with N-terminal A1-tags in order to label them fluorescently for FRET-based binding experiments (Fig. 3C). Analysis of our FRET-binding data established a dissociation constant for Gal-1/C-Raf-RBD of Kd = 106 ± 40 nM.

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