Membrane-dependent signal integration by the Ras activator Son of sevenless


Jodi Gureasko, William J. Galush, Sean Boykevisch, Holger Sondermann, Dafna Bar-Sagi, Jay T. Groves and John Kuriyan


Nat Struct Mol Biol. 15(5):452-61. (local copy)

Abstract / Figures from the paper / Supplementary Information


Abstract:

The kinetics of Ras activation by Son of sevenless (SOS) changes profoundly when Ras is tethered to membranes, instead of being in solution. SOS has two binding sites for Ras, one of which is an allosteric site that is distal to the active site. The activity of the SOS catalytic unit (SOScat) is up to 500-fold higher when Ras is on membranes compared to rates in solution, because the allosteric Ras site anchors SOScat to the membrane. This effect is blocked by the N-terminal segment of SOS, which occludes the allosteric site. We show that SOS responds to the membrane density of Ras molecules, to their state of GTP loading and to the membrane concentration of phosphatidylinositol-4,5-bisphosphate (PIP2), and that the integration of these signals potentiates the release of autoinhibition.

Illustrations from the paper.

Click on the small image to get a bigger one.


Figure 1. SOS Structure. (a) Domain organization of SOS. (b) Model for SOS localization at the membrane. The two basic residues in the PH domain that are crucial for PIP2 binding and mutated in this work (K456 and R459) are indicated by gray spheres.



Figure 2. The rate of SOScat-catalyzed nucleotide exchange is increased dramatically when Ras is tethered to membranes. The rate of fluorescently labeled mant-dGDP release from Ras in solution (black) and Ras tethered to lipid vesicles (green) in the absence and presence of SOScat is compared (mant-dGDP exchanged for GDP). The bulk volume concentration of Ras and SOS (when present) is 1 μM in all reactions. The Ras surface density is 5,300 molecules per μm2.



Figure 3. The membrane-dependent increase in the rate of SOScat-catalyzed Ras exchange is a function of the surface density of Ras. (a) The rate for SOScat-catalyzed nucleotide exchange for Ras coupled to lipid vesicles is shown as a function of Ras surface density (Ras per μm2). Although the surface density of Ras is varied, the bulk volume concentrations of SOS and Ras are the same for each measurement (1 μM). Mant-dGDP is displaced by unlabeled GDP. Experimental rates are shown as points. The solid line represents the rates predicted from the kinetic scheme (Supplementary Discussion). (b) The rate for SOScat-catalyzed nucleotide exchange for Ras coupled to supported lipid bilayers is shown as a function of Ras surface density. In these experiments there is a large excess of SOS molecules versus membrane-bound Ras ([SOS] = 10 nM). BODIPY-GDP is used as the fluorescent nucleotide. Experimental data are indicated by points, and the solid line represents the rates predicted by the kinetic model, using the same set of parameters as in a. Two data points, between densities of 3,000–4,000 Ras per μm2, have apparently aberrant values, but are included for completeness.




Figure 4. Membrane localization of SOScat by allosteric Ras binding in cells. (a) Left, colocalization of SOScat and Ras(A59G D38E), a Ras variant that binds only to the allosteric site of SOS, at the plasma membrane. Immunofluorescence studies reveal that when SOScat (red, above left) and Ras(A59G D38E) (green, below left) are co-transfected into COS1 cells, both proteins colocalize at the plasma membrane (yellow, inset). Right, the redistribution of SOS to the membrane is not seen when a mutant form of SOScat, SOScat(L687E R688A), is coexpressed with Ras(A59G D38E) (green, below right). SOScat(L687E R688A) is impaired in binding to Ras at the allosteric site. (b) SOScat(L687E R688A) is defective in ERK–MAP kinase activation, but targeting SOScat(L687E R688A) to the membrane using the Ras-derived membrane anchoring sequence, CAAX, fully restores SOS signaling activity. Error bars in the bar graph represent the s.d. from three independent experiments. The levels of ERK activation, as represented by ERK phosphorylation (P-ERK; green (appears yellow due to overlap)), were quantified by densitometry and normalized to levels of total ERK (ERK; red). P-ERK and ERK comigrate, so the appearance of P-ERK gives an apparently yellow color. a.u., arbitrary units; WCL, whole cell lysate.




Figure 5. The substrate Ras molecule and the activating Ras molecule both need to be tethered to the membrane for maximal SOS activity. A mutant form of Ras, RasY64A, which binds to the allosteric site of SOS, but not to the catalytic site, is used in these experiments. (a) All components are in solution. (b) Ras–mant-dGDP molecules are tethered to lipid membranes, and RasY64A-GppNp (allosteric activator) and SOScat are both in solution. (c) RasY64A-GppNp is tethered to lipid membranes, and Ras–mant-dGDP (substrate Ras) and SOScat are both in solution.




Figure 6. Activity of SOS constructs containing N-terminal regulatory domains. (a) The rates for SOScat(W729E)-, SOSDPC- and SOSHDPC-catalyzed nucleotide release from Ras-coupled vesicles are shown as a function of Ras surface density. Although the Ras density is varied, the bulk volume concentrations of Ras and SOS are the same for each measurement (1 μM). Error bars indicate the means with standard error from at least two independent experiments. (b) The activities of SOSDPC (gray), SOSHDPC (purple) and SOScat (green) toward Ras coupled to vesicles when mant-dGDP is exchanged for either unlabeled GDP or unlabeled GTP are compared for an equal surface density of Ras (1,300 Ras per μm2; bulk concentration of Ras and SOS is 1 μM). Note that the replacement of GDP for GTP on membrane-bound Ras results in a strong enhancement in the activity of all SOS constructs.




Figure 7. PIP2-dependent activation of SOS. (a) The activities of SOSDPC, SOSDPC(PH mutant), SOSHDPC, SOSHDPC(R552G) and SOScat toward Ras in solution when mant-dGDP is exchanged for unlabeled GDP and GTP are compared (bulk concentration of Ras and SOS is 1 μM). SOSDPC(PH mutant) is a mutant form of SOSDPC that contains mutations (K456E and R459E) that abolish binding to PIP2. SOSHDPC(R552G) contains the mutation associated with Noonan syndrome. (b) Nucleotide exchange by the indicated SOS constructs is shown in the absence and presence of PIP2 in Ras-coupled lipid vesicles. (Ras surface densities of 4,550 molecules per μm2 and 5,000 molecules per μm2, respectively; [SOS] = 10 nM, that is, 100-fold lower than in a; [Ras] (by volume) = 1 μM). In these reactions, mant-dGDP is displaced by unlabeled GTP in solution. Note that inclusion of 3% PIP2 into Ras-coupled vesicles results in a substantial increase in the activity of the Noonan syndrome mutant (SOSHDPC(R552G)) and SOSDPC toward membrane-bound Ras. (c) Inclusion of 2% PIP2 into Ras-coupled supported bilayers results in a marked increase in the activity of SOSDPC ([SOS] = 10 nM; Ras–BODIPY-GDP exchanged for GDP). Data for SOScat in the presence of 2% PIP2 is shown for comparison.




Figure 8. The Noonan syndrome mutant, SOSHDPC(R552G), is responsive to the membrane density of PIP2. The nucleotide exchange rates of 10 nM SOSHDPC(R552G) toward Ras tethered to lipid vesicles containing 1% and 3% PIP2 are compared when mant-dGDP is displaced by unlabeled GTP (6,000 Ras per μm2 and 4,810 Ras per μm2, respectively; [Ras] (by volume) = 1 μM).




Figure 9. The integration of several membrane-localization signals in the activation of Ras and SOS. (a) At low Ras-GDP (orange) surface densities and low surface concentrations of PIP2 (purple circles), the N-terminal regulatory segment maintains SOS in an inactive state by inhibiting the localization of SOS to the membrane by allosteric Ras. Ras binding to the allosteric site causes a conformational change at the active site, promoting substrate engagement. (b) The activity of autoinhibited SOS is stimulated by increasing Ras density at the membrane, the replacement of Ras-bound GDP by GTP (green) and by increasing PIP2 concentrations in the membrane. (c) Further anchorage of SOS to the membrane, such as by the coupling to activated receptors, in combination with high levels of Ras density, the generation of Ras-GTP and high levels of PIP2, results in effective release of autoinhibition.




Supplementary Information

Supplementary Information