Figure 2. Computational modeling-derived switch III mutations D47A,E49A in H-ras increase nanoclustering and RBD-recruitment.

(A) Electron microscopic nanoclustering analysis of mGFP-H-rasG12V and mGFP-H-rasG12V-D47A,E49A in BHK cells. Normalized univariate K-functions, where maximal L(r)-r values above the 99% CI for complete spatial randomness indicate clustering at that value of r (number of membrane sheets analyzed per condition, n = 17). (B) Schematic representation of nanoclustering-FRET analysis, where mGFP-tagged and mCherry-tagged Ras constructs were co-expressed in cells. (C) The nanoclustering-FRET response of H-rasG12V-D47A,E49A and its parent construct in dependence of the dose of the nanocluster scaffold Gal-1 in BHK cells. (D) Representative nanoclustering-FRET fluorescence lifetime images of BHK cells expressing FRET-pairs (or donor only, left column) of constructs indicated on the left, under Gal-1 conditions as annotated on the top. Color look up table to the right shows fluorescence lifetimes. (E) RBD-recruitment FRET data of H-rasG12V-D47A,E49A and its parent construct at three different Gal-1 doses analyzed using FRET-imaging of transiently transfected BHK cells. (C, E) Numbers in bars give numbers of analyzed cells from three independent experiments. Error bars represent the standard error of the mean (±SEM). Statistical analysis vs parent RasG12V was performed as described in ‘Materials and methods’ (*p < 0.05; **p < 0.01; ***p < 0.001). (F) Western blot analysis of C-Raf, MEK, and ERK phosphorylation and C-Raf in BHK cells transiently expressing mGFP-H-rasG12V-D47A,E49K, its parent or an empty vector control. Equal expression of Ras constructs can be seen in the mGFP row, equal loading in the Actin row. (G) In vitro RBD binding to mant-GTPγS loaded wild-type (wt) H-ras or H-ras-D47A,E49A measured with a fluorescence anisotropy assay. Details on the fitting function are in the ‘Materials and methods’. Data are averages ±SEM of three repeats. (H) RBD-pulldown experiments in BHK cells transiently expressing indicated H-ras mutants or wt H-ras. Top panel shows level of active Ras after EGF-stimulation (100 ng/ml). Bottom panel shows GAP sensitivity of wt and mutant H-ras proteins. See also Figure 2—figure supplement 1 and Figure 2—figure supplement 2.

DOI: http://dx.doi.org/10.7554/eLife.08905.005

Figure 2—figure supplement 1. The computational modeling-derived switch III H-ras mutant exhibits stronger Gal-1 complexation and remains sensitive to GAP-mediated hydrolysis.

(A) Schematic representation of the Gal-1-complexation FRET assay in which complexation of mRFP-Gal-1 with mGFP-tagged mutants of Ras was measured in intact BHK cells using FRET (left). Gal-1-complexation FRET analysis of H-rasG12V-D47A,E49A and its parent construct. Numbers in bars give number of analyzed cells from three independent experiments. Error bars represent the standard error of the mean (±SEM). Statistical analysis of differences vs H-rasG12V was performed as described in ‘Materials and methods’ (*p < 0.05) (right). (B) Relative amounts of phosphorylated C-Raf, MEK and ERK in BHK cells transiently expressing vector control, mGFP-H-rasG12V or mGFP-H-rasG12V-D47A,E49A determined by western blotting from three independent repeats. Error bars represent the standard error of the mean (±SEM). Quantification of band intensities and statistical analysis was performed as described in ‘Materials and methods’ (*p < 0.05, **p < 0.01, ***p < 0.001). (C) RBD-pulldown experiment quantification of the active, GTP-bound fraction of H-ras-D47A,E49A in BHK cells. +EGF denotes stimulation with 100 ng/ml EGF. −EGF serum starved cells. +GAP incubation with GAP domain of NF1, to assay for GAP-sensitivity. The graphs represent the averages of active H-ras-D47A,E49A normalized to wt H-ras + EGF-stimulation from three independent experiments. Blue vertical line annotates the activity of wt H-ras when stimulated with EGF. Error bars represent the standard error of the mean (±SEM). Statistical analysis was performed as described in ‘Materials and methods’ (NS, non-significant; **p < 0.01, *p < 0.05).

DOI: http://dx.doi.org/10.7554/eLife.08905.006

Figure 2—figure supplement 2. SOS-mediated nucleotide exchange kinetics, as well as mantGTPγS dissociation constants of wt H-ras and mutants.

SOS-induced Eu³⁺-GTP association (A) and dissociation (B) kinetics with wt H-ras (black), H-ras-D47A,E49A (red), H-ras-G48R (blue), and H-ras-G48R,D92N (orange) monitored using the quenching resonance energy transfer (QRET) technique. Dots represent data points obtained from individual reactions, with a total of 360 individual data points for each H-ras. (C) Fluorescence anisotropy binding data for the derivation of dissociation constants (Kd) between mantGTPγS and wt H-ras (black), H-ras-D47A,E49A (red), H-ras-G48R (blue), and H-ras-G48R,D92N (orange). Details are described in the ‘Materials and methods’.

DOI: http://dx.doi.org/10.7554/eLife.08905.007