K-Ras Nanoclustering Is Subverted by Overexpression of the Scaffold Protein Galectin-3 (original) (raw)
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Galectin-1 Is a Novel Structural Component and a Major Regulator of H-Ras Nanoclusters
Molecular Biology of the Cell, 2008
The organization of Ras proteins into nanoclusters on the inner plasma membrane is essential for Ras signal transduction, but the mechanisms that drive nanoclustering are unknown. Here we show that epidermal growth factor receptor activation stimulates the formation of H-Ras.GTP-Galectin-1 (Gal-1) complexes on the plasma membrane that are then assembled into transient nanoclusters.
Cancer Research, 2004
Ras biological activity necessitates membrane anchorage that depends on the Ras farnesyl moiety and is strengthened by Ras/galectin-1 interactions. We identified a hydrophobic pocket in galectin-1, analogous to the Cdc42 geranylgeranyl-binding cavity in RhoGDI, possessing homologous isoprenoid-binding residues, including the critical L11, whose RhoGDI L77 homologue changes dramatically on Cdc42 binding. By substituting L11A, we obtained a dominant interfering galectin-1 that possessed normal carbohydrate-binding capacity but inhibited H-Ras GTP-loading and extracellular signal-regulated kinase activation, dislodged H-Ras(G12V) from the cell membrane, and attenuated H-Ras(G12V) fibroblast transformation and PC12-cell neurite outgrowth. Thus, independently of carbohydrate binding, galectin-1 cooperates with Ras, whereas galectin-1(L11A) inhibits it. cell membranes is vital for its biological activities (6 -8). Exchange of GDP for GTP promotes activation by Ras of its effectors, and its activity is terminated by GTPase-activating proteins that facilitate GTP hydrolysis (9 -11). The strength, duration, and selectivity of the Ras signal is also determined by the spatio-temporal organization of Ras in the plasma membrane and other internal membranes .
Galectin-1 binds oncogenic H-Ras to mediate Ras membrane anchorage and cell transformation
Oncogene, 2001
Ras genes, frequently mutated in human tumors, promote malignant transformation. Ras transformation requires membrane anchorage, which is promoted by Ras farnesylcysteine carboxymethylester and by a second signal. Previously we showed that the farnesylcysteine mimetic, farnesylthiosalicylic acid (FTS) disrupts Ras membrane anchorage. To understand how this disruption contributes to inhibition of cell transformation we searched for new Ras-interacting proteins and identified galectin-1, a lectin implicated in human tumors, as a selective binding partner of oncogenic H-Ras(12V). The observed size of H-Ras(12V)-galectin-1 complex, which is equal to the sum of the molecular weights of Ras and galectin-1 indicates a direct binding interaction between the two proteins. FTS disrupted H-Ras(12V)-galectin-1 interactions. Overexpression of galectin-1 increased membrane-associated Ras, Ras-GTP, and active ERK resulting in cell transformation, which was blocked by dominant negative Ras. Galecti...
Multivalent scaffolds induce galectin-3 aggregation into nanoparticles
Beilstein Journal of Organic Chemistry, 2014
Galectin-3 meditates cell surface glycoprotein clustering, cross linking, and lattice formation. In cancer biology, galectin-3 has been reported to play a role in aggregation processes that lead to tumor embolization and survival. Here, we show that lactose-functionalized dendrimers interact with galectin-3 in a multivalent fashion to form aggregates. The glycodendrimer-galectin aggregates were characterized by dynamic light scattering and fluorescence microscopy methodologies and were found to be discrete particles that increased in size as the dendrimer generation was increased. These results show that nucleated aggregation of galectin-3 can be regulated by the nucleating polymer and provide insights that improve the general understanding of the binding and function of sugar-binding proteins.
Extracellular and intracellular small-molecule galectin-3 inhibitors
Scientific Reports, 2019
Galectin-3 is a carbohydrate binding protein which has important roles in cancer and immunity. Potent galectin-3 inhibitors have been synthesized, for experimental purposes and potential clinical use. As galectin-3 is implicated in both intra-and extracellular activities, permeability of galectin-3 inhibitors is an important parameter determining biological effects. We compared the cellular uptake of galectin-3 inhibitors and their potency in the intracellular or extracellular space. The inhibitors differed in their polar surface area (PSA), but had similar affinities for galectin-3. Using a well-established permeability assay, we confirmed that the uptake was significantly higher for the inhibitor with the lowest PSA, as expected. To analyze intracellular activity of the inhibitors, we developed a novel assay based on galectin-3 accumulation around damaged intracellular vesicles. The results show striking differences between the inhibitors intracellular potency, correlating with their PSAs. To test extracellular activity of the inhibitors, we analyzed their potency to block binding of galectin-3 to cell surfaces. All inhibitors were equally able to block galectin-3 binding to cells and this was proportional to their affinity for galectin-3. These inhibitors may serve as useful tools in exploring biological roles of galectin-3 and may further our understanding of intracellular versus extracellular roles of galectin-3. The galectin family of carbohydrate binding proteins have gained increasing interest as therapeutic targets in several diseases, such as chronic inflammation and cancer 1-4. Galectins are soluble proteins synthesized on free ribosomes in the cytosol. Even though they lack the classical characteristics of secreted proteins, they are rapidly translocated to the extracellular space through a yet unknown pathway 5. Once in the extracellular environment, the galectins are exposed to a large variety of glycan structures, where they recognize and bind specific β-galactosides. As some galectins are able to form multivalent structures or are multivalent in nature, they are able to cross-link glycoconjugates and form lattices. Formation of galectin/glycoconjugate lattices on the plasma membrane has been observed to influence the expression time, localization, and activity of several cell surface receptors, thus influencing numerous biological functions such as cell signaling, cell migration, and cell adherence 5,6. Furthermore, galectins can quickly (within minutes) be recycled back to the inside of cells trough the endocytic pathway, regulating sorting of both soluble and membrane bound glycoconjugates 5,7. Apart from the extracellular activities of the galectin family, mediated through glycan binding, galectins also play important roles in the intracellular compartments. Several studies have reported that galectins may influence cell signaling by interacting with signaling proteins in the cytosol, e.g. RAS proteins and β-catenin, and RNA splicing through binding of components of the spliceosome complex in the nucleus 8-12. As complex glycans are not found in the intracellular compartment, these activities are most likely mediated trough protein-protein interactions. Interestingly, however, several of these reported intracellular activates are inhibited by molecules interacting with the galectin carbohydrate binding site, such as lactose 8,10,13. Additionally, galectins play an important role at the interface between the cytosolic and intravesicular compartments by monitoring the integrity of vesicular membranes. It is now well established that several galectins (e.g. galectin-3 and-8 in particular) accumulate around disrupted vesicles by binding to exposed glycan structures and induce clearance of the damaged organelles by selective autophagy 14-20. The diverse roles of the galectins on a cellular level have consequences for several physiological
RAS Nanoclusters: Dynamic Signaling Platforms Amenable to Therapeutic Intervention
Biomolecules, 2021
RAS proteins are mutated in approximately 20% of all cancers and are generally associated with poor clinical outcomes. RAS proteins are localized to the plasma membrane and function as molecular switches, turned on by partners that receive extracellular mitogenic signals. In the on-state, they activate intracellular signal transduction cascades. Membrane-bound RAS molecules segregate into multimers, known as nanoclusters. These nanoclusters, held together through weak protein–protein and protein–lipid associations, are highly dynamic and respond to cellular input signals and fluctuations in the local lipid environment. Disruption of RAS nanoclusters results in downregulation of RAS-mediated mitogenic signaling. In this review, we discuss the propensity of RAS proteins to display clustering behavior and the interfaces that are associated with these assemblies. Strategies to therapeutically disrupt nanocluster formation or the stabilization of signaling incompetent RAS complexes are d...
Intracellular Galectins: Platforms for Assembly of Macromolecular Complexes
ACS Symposium Series, 2012
Although members of the galectin family were initially isolated on the basis of their saccharide-binding activity and cell surface localization, at least 11 of the 15 galectins identified in mammalian systems have been reported to be in the nucleus, as well as in the cytoplasm of cells. More interestingly, the precise intracellular localization and activity of the various galectins also cover a wide range. In most instances, distinct galectins give rise to the same or similar cellular responses. At the inner surface of plasma membranes, for example, galectin-1 (Gal1) and galectin-3 (Gal3) exhibit similar activities by binding to the Ras oncogene product to direct effector usage and signaling pathway. Gal1 and Gal3 also interact with Gemin4 and TFII-I as a part of ribonucleoprotein complexes and the two galectins have been documented to be required, but redundant, factors in pre-mRNA splicing. On the other hand, however, it appears that, although Gal3 and galectin-7 (Gal7) both bind to the apoptosis repressor protein Bcl-2, the ultimate cellular response is opposite: Gal3 is anti-apoptotic while Gal7 is pro-apoptotic. In all of these interactions, the ligands bind to the galectins through protein-protein interactions rather than via protein-carbohydrate recognition. The emergent general theme
Galectin-3 interacts with membrane lipids and penetrates the lipid bilayer
Biochemical and biophysical research …, 2005
The precise mechanism by which galectin-3 and other cytosolic proteins that lack signal peptides are secreted is yet to be elucidated. In the present analyses, we determined that galectin-3, a β-galactoside binding protein, can interact directly with membrane lipids in solid phase binding assays. More interestingly, we determined by spectrophotometric methods that it can spontaneously penetrate the lipid bilayer of liposomes in either direction. These findings suggest that galectin-3 on its own has the capacity to traverse the lipid bilayer. Whereas the situation is rather simplified in liposomes, the interaction of galectin-3 with the plasma membrane may involve cholesterol-rich membrane domains where galectin-3 can be concentrated and form multimers or interact covalently with other proteins.
H-Ras Nanocluster Stability Regulates the Magnitude of MAPK Signal Output
PLoS ONE, 2010
H-Ras is a binary switch that is activated by multiple co-factors and triggers several key cellular pathways one of which is MAPK. The specificity and magnitude of downstream activation is achieved by the spatio-temporal organization of the active H-Ras in the plasma membrane. Upon activation, the GTP bound H-Ras binds to Galectin-1 (Gal-1) and becomes transiently immobilized in short-lived nanoclusters on the plasma membrane from which the signal is propagated to Raf. In the current study we show that stabilizing the H-Ras-Gal-1 interaction, using bimolecular fluorescence complementation (BiFC), leads to prolonged immobilization of H-Ras.GTP in the plasma membrane which was measured by fluorescence recovery after photobleaching (FRAP), and increased signal out-put to the MAPK module. EM measurements of Raf recruitment to the H-Ras.GTP nanoclusters demonstrated that the enhanced signaling observed in the BiFC stabilized H-Ras.GTP nanocluster was attributed to increased H-Ras immobilization rather than to an increase in Raf recruitment. Taken together these data demonstrate that the magnitude of the signal output from a GTP-bound H-Ras nanocluster is proportional to its stability. Citation: Rotblat B, Belanis L, Liang H, Haklai R, Elad-Zefadia G, et al. (2010) H-Ras Nanocluster Stability Regulates the Magnitude of MAPK Signal Output. PLoS ONE 5(8): e11991.