title: H-Ras nanocluster stability regulates the magnitude of MAPK signal output.
abstract: 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.
authors.affiliation: Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
authors.forename: Barak
authors.initials: B
authors.lastname: Rotblat
authors.forename: Liron
authors.initials: L
authors.lastname: Belanis
authors.forename: Hong
authors.initials: H
authors.lastname: Liang
authors.forename: Roni
authors.initials: R
authors.lastname: Haklai
authors.forename: Galit
authors.initials: G
authors.lastname: Elad-Zefadia
authors.forename: John F
authors.initials: JF
authors.lastname: Hancock
authors.forename: Yoel
authors.initials: Y
authors.lastname: Kloog
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Plowman
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D006224:Cricetinae; D036681:Fluorescence Recovery After Photobleaching; D037483:Galectin 1 / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D020935:MAP Kinase Signaling System; D049329:Nanostructures / Q000737:chemistry*; D055550:Protein Stability; D013997:Time Factors; D048490:raf Kinases / Q000737:chemistry / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D037483:Galectin 1; D055767:Immobilized Proteins; D006160:Guanosine Triphosphate; D048490:raf Kinases; D018631:ras Proteins