Galectin-1 is a novel structural component and a major regulator of H-Ras nanoclusters (original) (raw)
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Single-molecule imaging analysis of Ras activation in living cells
Proceedings of the National Academy of Sciences, 2004
A single-molecule fluorescence resonance energy transfer (FRET) method has been developed to observe the activation of the small G protein Ras at the level of individual molecules. KB cells expressing H-or K-Ras fused with YFP (donor) were microinjected with the fluorescent GTP analogue BodipyTR-GTP (acceptor), and the epidermal growth factor-induced binding of BodipyTR-GTP to YFP-(H or K)-Ras was monitored by single-molecule FRET. On activation, Ras diffusion was greatly suppressed͞immobilized, suggesting the formation of large, activated Ras-signaling complexes. These complexes may work as platforms for transducing the Ras signal to effector molecules, further suggesting that Ras signal transduction requires more than simple collisions with effector molecules. GAP334-GFP recruited to the membrane was also stationary, suggesting its binding to the signaling complex. The single-molecules FRET method developed here provides a powerful technique to study the signal-transduction mechanisms of various G proteins.
K-Ras Nanoclustering Is Subverted by Overexpression of the Scaffold Protein Galectin-3
Cancer Research, 2008
The spatial organization of K-Ras proteins into nanoclusters on the plasma membrane is essential for high fidelity signal transduction. The mechanism underlying K-Ras nanoclustering is unknown. We show here that K-Ras.GTP recruits Galectin-3 (Gal-3) from the cytosol to the plasma membrane where it becomes an integral nanocluster component. Importantly we demonstrate that the cytosolic level of Gal-3 determines the magnitude of K-Ras.GTP nanoclustering and signal output. The βsheet layers of the Gal-3 carbohydrate recognition domain (CRD) contain a hydrophobic pocket that may accommodate the farnesyl group of K-Ras. V125A substitution within this hydrophobic pocket yields a dominant negative Gal-3(V125A) mutant that inhibits K-Ras activity. Gal-3(V125A) interaction with K-Ras.GTP, reduces K-Ras.GTP nanocluster formation, which abrogates signal output from the Raf/MEK/ERK pathway. Gal-3(V125A) negatively regulates cell growth and reduces cellular transformation. Thus regulation of K-Ras nanocluster formation and signal output by Gal-3 critically depends on the integrity of the Gal-3 hydrophobic pocket. These results show that Gal-3 over-expression in breast cancer cells, which increases K-Ras signal output represents oncogenic subversion of plasma membrane nanostructure.
Thrombosis and Haemostasis, 2007
Theadventoffluorescent proteins has revolutionizedsignaling research,shifting focus from biochemical assays to analysis of live cells,organized tissuesand even entireorganisms.Modern applications of fluorescent proteins go beyond their usea ss pecific markersofcells or tissues,allowing the researchertovisualize intracellular translocations as well as biochemical reactions. In
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.
Improved Binding of Raf to Ras·GDP Is Correlated with Biological Activity
Journal of Biological Chemistry, 2009
The GTP-binding protein Ras plays a central role in the regulation of various cellular processes, acting as a molecular switch that triggers signaling cascades. Only Ras bound to GTP is able to interact strongly with effector proteins like Raf kinase, phosphatidylinositol 3-kinase, and RalGDS, whereas in the GDP-bound state, the stability of the complex is strongly decreased, and signaling is interrupted. To determine whether this process is only controlled by the stability of the complex, we used computer-aided protein design to improve the interaction between Ras and effector. We challenged the Ras⅐Raf complex in this study because Raf among all effectors shows the highest Ras affinity and the fastest association kinetics. The proposed mutations were characterized as to their changes in dynamics and binding strength. We demonstrate that Ras-Raf interaction can only be improved at the cost of a loss in specificity of Ras⅐GTP versus Ras⅐GDP. As shown by NMR spectroscopy, the Raf mutation A85K leads to a shift of Ras switch I in the GTP-bound as well as in the GDP-bound state, thereby increasing the complex stability. In a luciferase-based reporter gene assay, Raf A85K is associated with higher signaling activity, which appears to be a mere matter of Ras-Raf affinity. Signal transduction across the cell is mediated by a network of interacting proteins leading to a controlled level of cellular response. Within the superfamily of small GTP-binding proteins, Ras appears to be a "master regulator" involved in cell proliferation, cell cycle progression, cell division, and apoptosis (1, 2). Attached to the inner leaflet of the cell membrane, Ras comes in two states; the inactive GDP-bound and the active GTP-bound state. The activation of Ras occurs by GDP/GTP nucleotide exchange, whereas hydrolysis of GTP leads to inactivation and interruption of signaling (3, 4). The activation of a distinct pathway occurs by the interaction of Ras with the responsible effector. Therefore, Ras has a large number of effector proteins, such as Raf kinase (5, 6), RalGDS (7), phosphatidylinositol 3-kinase (8-10), and Nore1A (11, 12), representing different signal directions. These effectors have in common the so-called Ras binding domain (RBD), 2 enabling them to interact with Ras. Only the GTP-bound form of Ras binds strongly to effectors and leads to their activation. Zooming more into the molecular detail of the Ras/effector interactions reveals two regions of Ras to be responsive to the nucleotide state and thereby convey specific recognition by the effectors. The flexible region switch I (residues 30-38) is mainly responsible for the interaction with the RBD of the effectors (13-15), whereas only a few effectors like Nore1A contact also the second flexible region of Ras, switch II (residues 60-67) (16). The RBDs from most effectors comprise 80-100 amino acids, and despite poor sequence homology, they all show the same topology (i.e. the ubiquitin fold) (17). In addition, the structures of various effector RBDs in complex with Ras show the same mode of binding, namely an intermolecular, antiparallel -sheet established by 1 and 2 of the RBD and 2 and 3 of Ras (13, 18, 19). Despite the structural similarities, the range of affinities of Ras/effector spans 2 orders of magnitude. Except Nore1A with a small dissociation rate constant, k off (16), the values for this constant are similar for the other effectors (ϳ10 s Ϫ1), with the differences in affinity being governed by variations in k on (17, 20-22). Another common feature observed in all Ras⅐RBD complex structures is the high charge complementarity between the two proteins. The contact area of Ras is mainly negatively charged, whereas the binding interface of the RBDs is mainly positive (13, 15, 17-19, 23, 24). The general influence of the charge complementarity on protein-protein interactions was investigated in great detail by Schreiber and Fersht (29), which resulted in the concept of electrostatic steering; the process of protein complex formation is favored by complementary
Cytometry Part A, 2013
We have revealed a reorientation of ectodomain I of the epidermal growth factor receptor (EGFR; ErbB1; Her1) in living CHO cells expressing the receptor, upon binding of the native ligand EGF. The state of the unliganded, nonactivated EGFR was compared to that exhibited after ligand addition in the presence of a kinase inhibitor that prevents endocytosis but does not interfere with binding or the ensuing conformational rearrangements. To perform these experiments, we constructed a transgene EGFR with an acyl carrier protein sequence between the signal peptide and the EGFR mature protein sequence. This protein, which behaves similarly to wild-type EGFR with respect to EGF binding, activation, and internalization, can be labeled at a specific serine in the acyl carrier tag with a fluorophore incorporated into a 4 0-phosphopantetheine (P-pant) conjugate transferred enzymatically from the corresponding CoA derivative. By measuring F€ orster resonance energy transfer between a molecule of Atto390 covalently attached to EGFR in this manner and a novel lipid probe NR12S distributed exclusively in the outer leaflet of the plasma membrane, we determined the apparent relative separation of ectodomain I from the membrane under nonactivating and activating conditions. The data indicate that the unliganded domain I of the EGFR receptor is situated much closer to the membrane before EGF addition, supporting the model of a selfinhibited configuration of the inactive receptor in quiescent cells.