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

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.

(B) Interaction between non-farnesylatable mGFP-H-rasG12V-C186S and mRFPtagged Gal-1, as well as mGFP-H-rasG12V and putative farnesyl-pocket mutant Gal-1-K29T studied using FLIM-FRET in HEK293-EBNA cells transiently expressing indicated constructs. As compared to the positive control H-rasG12V and C-Raf-RBD, the binding between H-rasG12V-C186S and C-Raf-RBD was decreased due to the loss of H-ras plasma membrane anchorage, where H-rasG12V exhibits a higher recruitment efficiency 1 . However, significant FRET between H-rasG12V-C186S and Gal-1 was still retained. In addition, the mutation K29T in Gal-1 could not disrupt the complexation between H-rasG12V and Gal-1, as the FRET compared to the (FRET-) control sample was statistically significant (not indicated). Samples coexpressing mGFP and mRFP served as a FRET control.
(C) Schematic representation of ACP-tag protein expression and fluorescence labelling for in vitro experiments. Recombinantly expressed and purified proteins were used for studies in solution. The protein expression plasmid pQE-A1 was constructed on the basis of commercial pQE-30 Xa vector (Qiagen). The pQE-A1-plasmid has BglII and KpnI restriction sites for sub-cloning of different genes of interest. The final fusion protein consists of four functional elements: 1) the Nterminus contains 6-His affinity tag for purification on a nickel chelate column; 2) the PreScission protease recognition site for a site-specific protease cleavage (indicated by the scissors); 3) the A1-tag is an alternative acyl carrier protein (ACP)-tag (NEB) with a short amino acid sequence (12 aa) used for selective fluorescent labelling; 4) the final element is the sequence of the protein of interest. ACP labelling reaction: ACP synthase (4´-phosphopantetheinyl transferase) catalyzes the covalent transfer of substituents from derivatized coenzyme A (CoA) to A1-tagged fusion proteins in solution. The A1-tag is a small tag (8 kDa) based on the acyl carrier protein (ACP) that allows the specific, covalent attachment of virtually any molecule to a protein of interest. Substrates for labelling are derivatives of coenzyme A (CoA). In the labelling reaction, ACP Synthase covalently attaches the substituted phosphopantetheine group of CoA to a serine residue on the A1-tag. Statistical significance between treated samples and marked control (dark grey) was determined as described in Methods (ns, nonsignificant; ***, p < 0.001).
(C) Multiple sequence alignment of human A-, B-and C-Raf-and PI3Kα-RBDs.
Top panel: Shown here are the Ras-binding region, which harbour major binding residues with Ras, and candidate C-Raf-RBD/ Gal-1 interface residues at distance less than 5Å (red arrowheads) from Gal-1. Key residues that abolished RBD/ Gal-1 complex formation in experiments after they were mutated to alanine are marked with asterisks. Nuclear import (NLS) and export (NES) sequence stretches with high activity score (cNLS mapper) are marked in light yellow and blue shaded boxes, respectively. Bottom panel: Optimal pairwise alignment of C-Raf-and PI3Kα-RBDs identifies a conserved residue, which computational modelling and experimental data suggest as being central to complex formation between Gal-1 and the C-Raf-RBD.
(D) C-Raf-RBD directly binds GST-Gal-1. Pull-down experiments were performed by mixing bacterially purified C-Raf-RBD and GST-Gal-1 immobilized on glutathione sepharose beads. GST was used as a control. Proteins retained on the beads (output) were resolved by SDS-PAGE Laemmli buffer and processed for SDS-PAGE gel, which was stained using coomassie brilliant blue. Standard proteins were used as molecular weight markers. (C) Sequence alignment of Rattus norvegicus Gal-1 and human Gal-3. Critical residues recognizing the carbohydrate ligand are highly conserved among mammalian galectins (green shaded boxes). Displayed above the sequence by red arrowheads are 'hot spot' interfacial residues identified in this study. Corresponding predicted key residues that facilitate Gal-1/ RBD complex formation were mutated to alanine (asterisks). A bipartite NLS stretch is centred at the C-terminal end of Gal-1 (light yellow shaded box).
Mutant residues used in this study for both proteins in the complex are marked here by asterisks. Numbering of residues is according to sequences deposited in UniProt (P09382 -Gal-1_Homo sapiens, P04049 -C-Raf_Homo sapiens). The loop that undergoes major conformational and stereochemical changes (loop4) between apoand liganded Gal-1 is coloured orange.