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

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.

SPRED1 Interferes with K-ras but Not H-ras Membrane Anchorage and Signaling

“…5A). This observation is consistent with the negative effect of H-rasG12V on K-rasG12V nanoscale clustering (62), which may be further augmented by increased nanoscale clustering of H-ras by Gal-1 (39). A similar negative effect on K-ras FRET was observed for SPRED1 alone (Fig.…”

“…Gal-1 can increase this negative effect on K-ras nanoclustering and signaling, probably via its clustering effect on GTP-H-ras ( and C) (39). We also recently described dimeric Gal-1 as a candidate Raf dimer stabilizer, which binds to the Ras binding domain (RBD) of Raf proteins (39). Our data suggest that by stabilizing B-/C-Raf dimers, Gal-1 facilitates plasma membrane localization of SPRED1, where the latter may then act on the membrane organization of K-ras, thus increasing the already Gal-1-associated inhibition of K-ras ( Fig.…”

“…We have recently described the small, dimeric protein galectin-1 (Gal-1) as a candidate Raf dimer stabilizer (39). We therefore explored whether expression of Gal-1 in HEK cells is sufficient to induce SPRED1 translocation.…”

“…Galectins also have a role in tumor progression and metastasis, and their expression is often associated with a poor prognosis (38). We have recently shown that dimeric Gal-1 binds to the Ras binding domain (RBD) of effectors, and both the intact Gal-1 dimer interface and that to the RBD are required for Gal-1 to positively regulate the nanoscale clustering of GTP-H-ras and negatively regulate that of GTP-Kras (39). These results therefore imply the striking possibility that Gal-1 is an endogenous Raf dimer stabilizer.…”

Common mechanisms of catalysis in small and heterotrimeric GTPases and their respective GAPs

“…However, it remains unclear whether this is primarily due to the general effects of galectin on the membrane or to a specific galectin-Ras interaction. Notably, a recent report describes the direct interaction of galectin-1 dimers with Raf effectors, indicating an indirect interaction with Ras (Blaževitš et al, 2016). Further, the GEF SOS binds two Ras molecules (Margarit et al, 2003;Sondermann et al, 2004;Gureasko et al, 2008), and the effector BRAF dimerizes and also potentially binds to two Ras molecules (Poulikakos et al, 2010).…”