Structural determinants of Ras-Raf interaction analyzed in live cells.
The minimum structure of the Raf-1 serine/threonine kinase that recognizes active Ras was used to create a green fluorescent fusion protein (GFP) for monitoring Ras activation in live cells. In spite of its ability to bind activated Ras in vitro, the Ras binding domain (RBD) of Raf-1 (Raf-1[51-131]GFP) failed to detect Ras in Ras-transformed NIH 3T3 fibroblasts and required the addition of the cysteine-rich domain (CRD) (Raf-1[51-220]GFP) to show clear localization to plasma membrane ruffles. In normal NIH 3T3 cells, (Raf-1[51-220]GFP) showed minimal membrane localization that was enhanced after stimulation with platelet-derived growth factor or phorbol-12-myristate-13-acetate. Mutations within either the RBD (R89L) or CRD (C168S) disrupted the membrane localization of (Raf-1[51-220]GFP), suggesting that both domains contribute to the recruitment of the fusion protein to Ras at the plasma membrane. The abilities of the various constructs to localize to the plasma membrane closely correlated with their inhibitory effects on mitogen-activated protein kinase kinase1 and mitogen-activated protein kinase activation. Membrane localization of full-length Raf-1-GFP was less prominent than that of (Raf-1[51-220]GFP) in spite of its strong binding to RasV12 and potent activation of mitogen-activated protein kinase. These finding indicate that both RBD and CRD are necessary to recruit Raf-1 to active Ras at the plasma membrane, and that these domains are not fully exposed in the Raf-1 molecule. Visualization of activated Ras in live cells will help to better understand the dynamics of Ras activation under various physiological and pathological conditions.
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
Tzvetanka
T
Bondeva
András
A
Balla
Péter
P
Várnai
Tamas
T
Balla
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D022081:Cell Surface Extensions / Q000378:metabolism; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D048369:MAP Kinase Kinase 1; D051379:Mice; D018613:Microscopy, Confocal; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D009154:Mutation; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; C095477:platelet-derived growth factor AB; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D019950:Mitogen-Activated Protein Kinase 1; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins