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Allosteric Gating of Son of Sevenless Activity by the Histone Domain

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Allosteric Gating of Son of Sevenless Activity by the Histone Domain Allosteric gating of Son of sevenless activity by the histone domain Kamlesh K Yadava and Dafna Bar-Sagia,b,1 aCell and Molecular Biology Program and bDepartment of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, NY 10016 Communicated by John Kuriyan, University of California, Berkeley, CA, December 13, 2009 (received for review October 7, 2009) Regulated activation of Ras by receptor tyrosine kinases (RTK) Results and Discussion constitutes a key transduction step in signaling processes that con- The Histone Domain Displays Lipid-Dependent Membrane Binding trol an array of fundamental cellular functions including prolifer- Activity. An initial unbiased screen for potential interactions be- ation, differentiation, and survival. The principle mechanism by tween Sos-H and phospholipids indicated preferential binding to which Ras is activated down stream of RTKs involves the stimula- phosphatidic acid (PA) (Fig. S1A). To validate this observation, tion of guanine nucleotide exchange by the ubiquitous guanine we used a lipid vesicle-sedimentation assay in which the partition- nucleotide exchange factor Son of sevenless (Sos). In resting con- ing of purified Sos-H between the vesicle-containing pellet ditions, Sos activity is constrained by intramolecular interactions (bound) and the supernatant (free) is assessed post ultracentri- that maintain the protein in an autoinhibited conformation. Struc- fugation. As illustrated in Fig. 1B, Sos-H bound to PA-containing tural, biochemical, and genetic studies have implicated the histone vesicles in a concentration-dependent manner, with detectable domain (Sos-H), which comprises the most N-terminal region binding observed at a concentration of 0.01 mM PA. In contrast, of Sos, in the regulation of Sos autoinhibition. However, the mo- no binding was observed to vesicles composed of phosphatidyl- lecular underpinnings of this regulatory function are not well choline only or to vesicles containing phosphatidylserine, another understood. In the present study we demonstrate that Sos-H pos- acidic phospholipid (Fig. S1B). Consistent with the observation of sesses in vitro and in vivo membrane binding activity that is Gureasko et al. (9), Sos-H also bound to PIP2-containing vesicles mediated, in part, by the interactions between a cluster of basic (Fig. S1B). Because the binding of Sos-H to PA was significantly residues and phosphatidic acid. This interaction is required for higher relative to PIP2 binding, we have focused our analysis on BIOCHEMISTRY Sos-dependent activation of Ras following EGF stimulation. The in- the Sos-H/PA interactions. The modulation of Sos function by in- ducible association of Sos-H with membranes contributes to the teractions with membrane components other than PA, i.e. PIP2, is catalytic activity of Sos by forcing the domain to adopt a conforma- the subject of the accompanying study by Gureasko et al (9). tion that destabilizes the autoinhibitory state. Thus, Sos-H plays a Whether or not these interactions play a redundant or comple- critical role in governing the catalytic output of Sos through the mentary role with that of PA remains to be determined. coupling of membrane recruitment to the release of autoinhibition. The solvent exposed surface of Sos-H contains three promi- ∣ ∣ ∣ nent basic patches surrounding a negatively charged E108 residue Ras guanine nucleotide exchange factor Noonan syndrome (Fig. 1C, Left). Significantly, all of these patches and E108 are phosphatidic acid conserved across Sos sequences (Fig. S1D). To assess the indivi- dual contribution of these patches to PA-binding, lysine and as proteins are essential transducers of signals that originate arginine residues within each patch were mutated. As illustrated Rat the cell surface following the ligand-dependent stimulation in Fig. 1C Right , each set of mutations compromised PA-binding. of receptor tyrosine kinases (RTK). The activation of Ras by RTK However, the Sos-H mutant in which AEA residues were proceeds through the inducible conversion of Ras-GDP to substituted for 97RKR99 (Sos-HRKR∕AEA) displayed the most pro- Ras-GTP by the multidomain guanine nucleotide exchange factor nounced reduction in binding activity indicating that this basic Son of sevenless (Sos) (Fig. 1A). Structural and biochemical stu- cluster serves as a major PA-binding determinant. To substantiate dies have assigned specific functions to the different domains of this conclusion, the binding affinities of PA for Sos-H and Sos in regulating guanine nucleotide exchange on Ras. The cat- Sos-HRKR∕AEA were measured. Sos-H bound to PA-containing alytic region consisting of the Rem and Cdc25 domains harbors vesicles with a Kd of 0.2 μM, nearly 600 times stronger than PC- two Ras-binding sites, one for Ras-GDP and the other for Ras- containing vesicles (Kd ¼ 110 μM) (Fig. 1D). By comparison, the GTP (1). The Ras-GDP binding site (also referred to as the cat- ∕ binding of Sos-HRKR AEA to PA-containing vesicles was 200-fold alytic site) mediates nucleotide dissociation, and the Ras-GTP ∕ weaker (Kd ¼ 40 μM). Of note, the binding of Sos-HRKR AEA to binding site (also referred to as the allosteric site) promotes PIP2 was not altered relative to Sos-H suggesting that PA and the release of Sos from an inactive autoinhibited state imposed PIP2 interact at distinct sites (Fig. S1C). by the intramolecular interaction between the DH domain and To test whether PA-binding could play a role in the membrane the Rem segment of the catalytic domain (Fig. 1A). The allosteric association of Sos-H in vivo, GFP-fusion constructs of Sos-H and site has also been implicated in the membrane recruitment of Sos Sos-HRKR∕AEA were transiently transfected into COS-1 cells and along with the proline-rich C-terminal region and the PH domain their subcellular distribution analyzed by fluorescence micro- (2–4). The N-terminus of Sos (residues 1–198) contains a seg- ment with sequence and structural similarity to histones and ac- scopy. In unstimulated cells, Sos-H displayed a basal level of cordingly has been termed the histone domain (Sos-H) (5). The membrane localization plausibly due to charge-mediated binding only known function ascribed to date to this domain is in the to membrane phospholipids (Fig. 1E). The extent of this con- maintenance of the autoinhibited conformation of Sos through stitutive membrane association was dependent on the levels of the in cis binding to a helical linker that connects the DH-PH module of Sos to the catalytic domain (3, 6–8) (Fig. 1A). Analysis Author contributions: K.K.Y. and D.B.-S. designed research; K.K.Y. performed research and of the surface electrostatic potential of Sos-H revealed positively analyzed data; and K.K.Y. and D.B.-S.wrote the paper. charged regions and it has been proposed that these regions may The authors declare no conflict of interest. mediate the association with negatively charged membrane com- 1To whom correspondence should be addressed. E-mail: [email protected]. ponents (7). In the present study we have sought to determine the This article contains supporting information online at www.pnas.org/cgi/content/full/ existence and significance of Sos-H membrane interactions. 0914315107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0914315107 PNAS Early Edition ∣ 1of5 Downloaded by guest on September 23, 2021 Fig. 1. The histone domain of Sos (Sos-H) binds to membrane lipids. (A) The domain arrangement of Sos. H, Histone; DH, Dbl homology; PH, Pleckstrin homology; L, helical Linker; REM, Ras exchange motif; CDC25, yeast CDC25 homology; and PxxP, proline-rich region. Below, a cartoon depiction of the relative orientation of the various domains of Sos in the autoinhibited state. The red bars indicate intramolecular interactions. The blue dotted line circle indicates the binding site for allosteric Ras, which is occluded by the DH domain. (B) His-tagged Sos-H (0.5 μM) was mixed with the indicated concentrations of lipid vesicles comprised PC∶PA (90∶10). Vesicles were pelleted and the amount of Sos-H in the pellet (Bound) or in the supernatant (Free) was detected by immunoblotting (IB). (C) Surface electrostatic potential map (blue, positive; red, negative) showing the 3 basic patches in Sos-H and the residues mutated (see Materials and Methods). Right, Sos-H constructs containing the indicated mutations were mixed with 1 mM PC∶PA vesicles (90∶10) and binding was determined as in B.(D) Increasing amounts of Sos-H were incubated with 1 mM PC∶PA (closed circles) or PC lipid vesicles (open triangles) and Sos-HRKR∕AEA with PC∶PA vesicles (closed triangles). Kd was determined as previously described (2). Data presented in panels B–D are representative of three independent experiments. (E) COS-1 cells were transfected with GFP-tagged Sos-H and membrane localization scored in either starved cells or upon incubation with PA (100 μM) for 20 min. The image represents a single slice of 0.25 μm from a serial Z-section of the cell. The boxed areas are enlarged in the left and right adjacent images with arrowheads showing the relative increase in fluorescence intensity at the membrane. Results (bar graph) are mean Æs:d: of three independent experiments with at least 100 expressing cells counted for each condition (see Materials and Methods). Scale bar: 10 μm. ectopically expressed constructs making it difficult to assess its RNA interference was used to knock-down PLD2. Suppression of physiological relevance. The addition of membrane-permeable PLD2 expression impaired the ability of Sos-H to translocate to PA to serum-deprived cells stimulated the translocation of Sos-H, the plasma membrane in response to EGF stimulation (Fig. S2). but not Sos-HRKR∕AEA, from the cytoplasm to the membrane as Together, these data indicate that the affinity of Sos-H to the evident by a rim of fluorescence at the cell periphery (Fig.
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