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

Sci Rep. 2016 Apr 18:6:24165. doi: 10.1038/srep24165.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Computer Simulation
  • Cricetinae
  • Dimerization
  • Galectin 1 / chemistry
  • Galectin 1 / genetics
  • Galectin 1 / metabolism*
  • Humans
  • Mutation
  • Protein Binding
  • Signal Transduction
  • raf Kinases / genetics
  • raf Kinases / metabolism*
  • ras Proteins / chemistry
  • ras Proteins / genetics
  • ras Proteins / metabolism*

Substances

  • Galectin 1
  • raf Kinases
  • ras Proteins