Ras membrane orientation and nanodomain localization generate isoform diversity.
The structural elements encoding functional diversity among Ras GTPases are poorly defined. The orientation of the G domain of H-ras with respect to the plane of the plasma membrane is recognized by the Ras binding domain of C-Raf, coupling orientation to MAPK activation. We now show that two other proteins, phosphoinositide-3-kinase-alpha and the structurally unrelated galectin-1, also recognize G-domain orientation. These results rationalize the role of galectin-1 in generating active GTP-H-ras signaling nanoclusters. However, molecular dynamics simulations of K-ras membrane insertion and fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) imaging of the effector interactions of N-Ras, K-Ras, and M-ras suggest that there are two hyperactive, signaling-competent orientations of the Ras G domain. Mutational and functional analyses establish a clear relationship between effector binding and the amphilicities of helix alpha4 and the C-terminal hypervariable region, thus confirming that these structural elements critically tune the orientation of the Ras G domain. Finally, we show that G-domain orientation and nanoclustering synergize to generate Ras isoform specificity with respect to effector interactions.
Abankwa
Daniel
D
The University of Queensland, Institute for Molecular Bioscience, Brisbane 4072, Australia. d.abankwa@uq.edu.au
Gorfe
Alemayehu A
AA
Inder
Kerry
K
Hancock
John F
JF
D000595:Amino Acid Sequence; D031541:Fluorescence Resonance Energy Transfer; D037483:Galectin 1 / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D036103:Nanotechnology; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D018631:ras Proteins / Q000737:chemistry / Q000378:metabolism*
D037483:Galectin 1; D020033:Protein Isoforms; D006160:Guanosine Triphosphate; D019869:Phosphatidylinositol 3-Kinases; D018631:ras Proteins