H-Ras nanocluster stability regulates the magnitude of MAPK signal output.
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.
Rotblat
Barak
B
Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
Belanis
Liron
L
Liang
Hong
H
Haklai
Roni
R
Elad-Zefadia
Galit
G
Hancock
John F
JF
Kloog
Yoel
Y
Plowman
Sarah J
SJ
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*
D037483:Galectin 1; D055767:Immobilized Proteins; D006160:Guanosine Triphosphate; D048490:raf Kinases; D018631:ras Proteins