Phospholipase D2-generated phosphatidic acid couples EGFR stimulation to Ras activation by Sos

The activation of Ras by the guanine nucleotide-exchange factor Son of sevenless (Sos) constitutes the rate-limiting step in the transduction process that links receptor tyrosine kinases to Ras-triggered intracellular signalling pathways. A prerequisite for the function of Sos in this context is its ligand-dependent membrane recruitment, and the prevailing model implicates both the Sos carboxy-terminal proline-rich motifs and amino-terminal pleckstrin homology (PH) domain in this process. Here, we describe a previously unrecognized pathway for the PH domain-dependent membrane recruitment of Sos that is initiated by the growth factor-induced generation of phosphatidic acid via the signalling enzyme phospholipase D2 (PLD2). Phosphatidic acid interacts with a defined site in the Sos PH domain with high affinity and specificity. This interaction is essential for epidermal growth factor (EGF)-induced Sos membrane recruitment and Ras activation. Our findings establish a crucial role for PLD2 in the coupling of extracellular signals to Sos-mediated Ras activation, and provide new insights into the spatial coordination of this activation event.     

Identification and characterization of a new family of guanine nucleotide exchange factors for the ras-related GTPase Ral

Guanine nucleotide exchange factors (GEFs) are responsible for coupling cell surface receptors to Ras protein activation. Here we describe the characterization of a novel family of differentially expressed GEFs, identified by database sequence homology searching. These molecules share the core catalytic domain of other Ras family GEFs but lack the catalytic non-conserved (conserved non-catalytic/Ras exchange motif/structurally conserved region 0) domain that is believed to contribute to Sos1 integrity. In vitro binding and in vivo nucleotide exchange assays indicate that these GEFs specifically catalyze the GTP loading of the Ral GTPase when overexpressed in 293T cells. A central proline-rich motif associated with the Src homology (SH)2/SH3-containing adapter proteins Grb2 and Nck in vivo, whereas a pleckstrin homology (PH) domain was located at the GEF C terminus. We refer to these GEFs as RalGPS 1A, 1B, and 2 (Ral GEFs with PH domain and SH3 binding motif). The PH domain was required for in vivo GEF activity and could be functionally replaced by the Ki-Ras C terminus, suggesting a role in membrane targeting. In the absence of the PH domain RalGPS 1B cooperated with Grb2 to promote Ral activation, indicating that SH3 domain interaction also contributes to RalGPS regulation. In contrast to the Ral guanine nucleotide dissociation stimulator family of Ral GEFs, the RalGPS proteins do not possess a Ras-GTP-binding domain, suggesting that they are activated in a Ras-independent manner.     

Structural basis of phosphoinositide binding to kindlin-2 protein pleckstrin homology domain in regulating integrin activation

Kindlins are a subclass of FERM-containing proteins that have recently emerged as key regulators of integrin receptor activation and signaling. As compared with the conventional FERM domain, the kindlin FERM domain contains an inserted pleckstrin homology (PH) domain that recognizes membrane phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). Using NMR spectroscopy, we show that PIP3 site-specifically binds to kindlin-2 PH with substantial chemical shift changes that are much larger than PIP2. This suggests an enhanced association of kindlin-2 with membrane as mediated by PIP3 upon its conversion from PIP2 by phosphoinositide-3 kinase, a known regulator of integrin activation. We determined the NMR structure of the kindlin-2 PH domain bound to the head group of PIP3, inositol 1,3,4,5-tetraphosphate (IP4). The structure reveals a canonical PH domain fold, yet with a distinct IP4 binding pocket that appears highly conserved for the kindlin family members. Functional experiments demonstrate that although wild type kindlin-2 is capable of cooperating with integrin activator talin to induce synergistic integrin α(IIb)β(3) activation, this ability is significantly impaired for a phosphoinositide binding-defective kindlin-2 mutant. These results define a specific PIP3 recognition mode for the kindlin PH domain. Moreover, they shed light upon a mechanism as to how the PH domain mediates membrane engagement of kindlin-2 to promote its binding to integrin and cooperation with talin for regulation of integrin activation.     

Multiscale Simulations Suggest a Mechanism for the Association of the Dok7 PH Domain with PIP-Containing Membranes

Dok7 is a peripheral membrane protein that is associated with the MuSK receptor tyrosine kinase. Formation of the Dok7/MuSK/membrane complex is required for the activation of MuSK. This is a key step in the complex exchange of signals between neuron and muscle, which lead to neuromuscular junction formation, dysfunction of which is associated with congenital myasthenic syndromes. The Dok7 structure consists of a Pleckstrin Homology (PH) domain and a Phosphotyrosine Binding (PTB) domain. The mechanism of the Dok7 association with the membrane remains largely unknown. Using multi-scale molecular dynamics simulations we have explored the formation of the Dok7 PH/membrane complex. Our simulations indicate that the PH domain of Dok7 associates with membranes containing phosphatidylinositol phosphates (PIPs) via interactions of the β1/β2, β3/β4, and β5/β6 loops, which together form a positively charged surface on the PH domain and interact with the negatively charged headgroups of PIP molecules. The initial encounter of the Dok7 PH domain is followed by formation of additional interactions with the lipid bilayer, and especially with PIP molecules, which stabilizes the Dok7 PH/membrane complex. We have quantified the binding of the PH domain to the model bilayers by calculating a density landscape for protein/membrane interactions. Detailed analysis of the PH/PIP interactions reveal both a canonical and an atypical site to be occupied by the anionic lipid. PH domain binding leads to local clustering of PIP molecules in the bilayer. Association of the Dok7 PH domain with PIP lipids is therefore seen as a key step in localization of Dok7 to the membrane and formation of a complex with MuSK.     

Structure-based mutagenesis reveals distinct functions for Ras switch 1 and switch 2 in Sos-catalyzed guanine nucleotide exchange

Ras GTPases function as binary switches in signaling pathways controlling cell growth and differentiation. The guanine nucleotide exchange factor Sos mediates the activation of Ras in response to extracellular signals. We have previously solved the crystal structure of nucleotide-free Ras in complex with the catalytic domain of Sos (Boriack-Sjodin, P. A., Margarit, S. M., Bar-Sagi, D., and Kuriyan, J. (1998) Nature 394, 337-343). The structure demonstrates that Sos induces conformational changes in two loop regions of Ras known as switch 1 and switch 2. In this study, we have employed site-directed mutagenesis to investigate the functional significance of the conformational changes for the catalytic function of Sos. Switch 2 of Ras is held in a very tight embrace by Sos, with almost every external side chain coordinated by Sos. Mutagenesis of contact residues at the switch 2-Sos interface shows that only a small set of side chains affect binding, with the most important contact being mediated by tyrosine 64, which is buried in a hydrophobic pocket of Sos in the Ras.Sos complex. Substitutions of Ras and Sos side chains that are inserted into the Mg(2+)- and nucleotide phosphate-binding site of switch 2 (Ras Ala(59) and Sos Leu(938) and Glu(942)) have no effect on the catalytic function of Sos. These results indicate that the interaction of Sos with switch 2 is necessary for tight binding, but is not the critical driving force for GDP displacement. The structural distortion of switch 1 induced by Sos is mediated by a small number of specific contacts between highly conserved residues on both Ras and Sos. Mutations of a subset of these residues (Ras Tyr(32) and Tyr(40)) result in an increase in the intrinsic rate of nucleotide dissociation from Ras and impair the binding of Ras to Sos. Based on this analysis, we propose that the interactions of Sos with the switch 1 and switch 2 regions of Ras have distinct functional consequences: the interaction with switch 2 mediates the anchoring of Ras to Sos, whereas the interaction with switch 1 leads to disruption of the nucleotide-binding site and GDP dissociation.     

Molecular mechanism of an oncogenic mutation that alters membrane targeting: Glu17Lys modifies the PIP lipid specificity of the AKT1 PH domain

The protein kinase AKT1 regulates multiple signaling pathways essential for cell function. Its N-terminal PH domain (AKT1 PH) binds the rare signaling phospholipid phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)], resulting in plasma membrane targeting and phosphoactivation of AKT1 by a membrane-bound kinase. Recently, it was discovered that the Glu17Lys mutation in the AKT1 PH domain is associated with multiple human cancers. This mutation constitutively targets the AKT1 PH domain to the plasma membrane by an unknown mechanism, thereby promoting constitutive AKT1 activation and oncogenesis. To elucidate the molecular mechanism underlying constitutive plasma membrane targeting, this work compares the membrane docking reactions of the isolated wild-type and E17K AKT1 PH domains. In vitro studies reveal that the E17K mutation dramatically increases the affinity for the constitutive plasma membrane lipid PI(4,5)P(2). The resulting PI(4,5)P(2) equilibrium affinity is indistinguishable from that of the standard PI(4,5)P(2) sensor, PLCdelta1 PH domain. Kinetic studies indicate that the effects of E17K on PIP lipid binding arise largely from electrostatic modulation of the dissociation rate. Membrane targeting analysis in live cells confirms that the constitutive targeting of E17K AKT1 PH to plasma membrane, like PLCdelta1 PH, stems from PI(4,5)P(2) binding. Overall, the evidence indicates that the molecular mechanism underlying E17K oncogenesis is a broadened target lipid selectivity that allows high-affinity binding to PI(4,5)P(2). Moreover, the findings strongly implicate the native Glu17 side chain as a key element of PIP lipid specificity in the wild-type AKT1 PH domain. Other PH domains may employ an analogous anionic residue to control PIP specificity.     

Identification of the ras GTPase-activating protein GAP1(m) as a phosphatidylinositol-3,4,5-trisphosphate-binding protein in vivo

GAP1(m) is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) [1]. In vitro, it has been shown to bind inositol 1, 3,4,5-tetrakisphosphate (IP4), the water-soluble inositol head group of the lipid second messenger phosphatidylinositol 3,4, 5-trisphosphate (PIP3) [2] [3]. This has led to the suggestion that GAP1(m) might function as a PIP3 receptor in vivo [4]. Here, using rat pheochromocytoma PC12 cells transiently transfected with a plasmid expressing a chimera of green fluorescent protein fused to GAP1(m) (GFP-GAP1(m)), we show that epidermal growth factor (EGF) induces a rapid (less than 60 seconds) recruitment of GFP-GAP1(m) from the cytosol to the plasma membrane. This recruitment required a functional GAP1(m) pleckstrin homology (PH) domain, because a specific point mutation (R629C) in the PH domain that inhibits IP4 binding in vitro [5] totally blocked EGF-induced GAP1(m) translocation. Furthermore, the membrane translocation was dependent on PI 3-kinase, and the time course of translocation paralleled the rate by which EGF stimulates the generation of plasma membrane PIP3 [6]. Significantly, the PIP3-induced recruitment of GAP1(m) did not appear to result in any detectable enhancement in its basal Ras GAP activity. From these results, we conclude that GAP1(m) binds PIP3 in vivo, and it is recruited to the plasma membrane, but does not appear to be activated, following agonist stimulation of PI 3-kinase.     

Tandem histone folds in the structure of the N-terminal segment of the ras activator Son of Sevenless

The Ras activator Son of Sevenless (Sos) contains a Cdc25 homology domain, responsible for nucleotide exchange, as well as Dbl/Pleckstrin homology (DH/PH) domains. We have determined the crystal structure of the N-terminal segment of human Sos1 (residues 1-191) and show that it contains two tandem histone folds. While the N-terminal domain is monomeric in solution, its structure is surprisingly similar to that of histone dimers, with both subunits of the histone "dimer" being part of the same peptide chain. One histone fold corresponds to the region of Sos that is clearly similar in sequence to histones (residues 91-191), whereas the other is formed by residues in Sos (1-90) that are unrelated in sequence to histones. Residues that form a contiguous patch on the surface of the histone domain of Sos are conserved from C. elegans to humans, suggesting a potential role for this domain in protein-protein interactions.     

The Ca2+-activated cation channel TRPM4 is regulated by phosphatidylinositol 4,5-biphosphate

Transient receptor potential (TRP) channel, melastatin subfamily (TRPM)4 is a Ca2+-activated monovalent cation channel that depolarizes the plasma membrane and thereby modulates Ca2+ influx through Ca2+-permeable pathways. A typical feature of TRPM4 is its rapid desensitization to intracellular Ca2+ ([Ca2+]i). Here we show that phosphatidylinositol 4,5-biphosphate (PIP2) counteracts desensitization to [Ca2+]i in inside-out patches and rundown of TRPM4 currents in whole-cell patch-clamp experiments. PIP2 shifted the voltage dependence of TRPM4 activation towards negative potentials and increased the channel's Ca2+ sensitivity 100-fold. Conversely, activation of the phospholipase C (PLC)-coupled M1 muscarinic receptor or pharmacological depletion of cellular PIP2 potently inhibited currents through TRPM4. Neutralization of basic residues in a C-terminal pleckstrin homology (PH) domain accelerated TRPM4 current desensitization and strongly attenuated the effect of PIP2, whereas mutations to the C-terminal TRP box and TRP domain had no effect on the PIP2 sensitivity. Our data demonstrate that PIP2 is a strong positive modulator of TRPM4, and implicate the C-terminal PH domain in PIP2 action. PLC-mediated PIP2 breakdown may constitute a physiologically important brake on TRPM4 activity.     

Crystal structure of kindlin-2 PH domain reveals a conformational transition for its membrane anchoring and regulation of integrin activation

Kindlin-2 belongs to a subfamily of FERM domain containing proteins, which plays key roles in activating integrin transmembrane receptors and mediating cell adhesion. Compared to conventional FERM domains, kindlin-2 FERM contains an inserted pleckstrin homology (PH) domain that specifically binds to phosphatidylinositol (3,4,5) trisphosphate (PIP3) and regulates the kindlin-2 function. We have determined the crystal structure of kindlin-2 PH domain at 1.9 ? resolution, which reveals a conserved PH domain fold with a highly charged and open binding pocket for PIP3 head group. Structural comparison with a previously reported solution structure of kindlin-2 PH domain bound to PIP3 head group reveals that upon PIP3 insertion, there is a significant conformational change of both the highly positively charged loop at the entry of the PIP3 binding pocket and the entire β barrel of the PH domain. We propose that such “induced-fit” type change is crucial for the tight binding of PIP3 to anchor kindlin-2 onto the membrane surface, thereby promoting its binding to integrins. Our results provide important structural insight into kindlin-2-mediated membrane anchoring and integrin activation.     

Regulation of Sos Activity by Intramolecular Interactions

The guanine nucleotide exchange factor Sos mediates the coupling of receptor tyrosine kinases to Ras activation. To investigate the mechanisms that control Sos activity, we have analyzed the contribution of various domains to its catalytic activity. Using human Sos1 (hSos1) truncation mutants, we show that Sos proteins lacking either the amino or the carboxyl terminus domain, or both, display a guanine nucleotide exchange activity that is significantly higher compared with that of the full-length protein. These results demonstrate that both the amino and the carboxyl terminus domains of Sos are involved in the negative regulation of its catalytic activity. Furthermore, in vitro Ras binding experiments suggest that the amino and carboxyl terminus domains exert negative allosteric control on the interaction of the Sos catalytic domain with Ras. The guanine nucleotide exchange activity of hSos1 was not augmented by growth factor stimulation, indicating that Sos activity is constitutively maintained in a downregulated state. Deletion of both the amino and the carboxyl terminus domains was sufficient to activate the transforming potential of Sos. These findings suggest a novel negative regulatory role for the amino terminus domain of Sos and indicate a cooperation between the amino and the carboxyl terminus domains in the regulation of Sos activity.     

Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity

The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-gamma1 with translational elongation factor (EF)-1alpha, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with various domains of PLC-gamma1 followed by peptide sequence analysis, we identified EF-1alpha as a binding partner of a split PH domain of PLC-gamma1. Analysis by site-directed mutagenesis of the PH domain revealed that the beta2-sheet of a split PH domain is critical for the interaction with EF-1alpha. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)). So the PH domain of PLC-gamma1 binds to both EF-1alpha and PIP(2). The binding affinity of EF-1alpha to the GST.PH domain fusion protein increased in the presence of PIP(2), although PIP(2) does not bind to EF-1alpha directly. This suggests that EF-1alpha may control the binding affinity between the PH domain and PIP(2). PLC-gamma1 is substantially activated in the presence of EF-1alpha with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-gamma1 (Y509A/F510A) that lost its binding affinity to EF-1alpha shows basal level activity. Taken together, our data show that EF-1alpha plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-gamma1 activity via a split PH domain.     

Regulation of presynaptic phosphatidylinositol 4,5-biphosphate by neuronal activity

Phosphatidylinositol 4,5-biphosphate (PIP2) has been implicated in a variety of cellular processes, including synaptic vesicle recycling. However, little is known about the spatial distribution of this phospholipid in neurons and its dynamics. In this study, we have focused on these questions by transiently expressing the phospholipase C (PLC)-delta1 pleckstrin homology (PH) domain fused to green fluorescent protein (GFP) in cultured hippocampal neurons. This PH domain binds specifically and with high affinity to PIP2. Live confocal imaging revealed that in resting cells, PH-GFP is localized predominantly on the plasma membrane. Interestingly, no association of PH-GFP with synaptic vesicles in quiescent neurons was observed, indicating the absence of detectable PIP2 on mature synaptic vesicles. Electrical stimulation of hippocampal neurons resulted in a decrease of the PH-GFP signal at the plasma membrane, most probably due to a PLC-mediated hydrolysis of PIP2. This was accompanied in the majority of presynaptic terminals by a marked increase in the cytoplasmic PH-GFP signal, localized most probably on freshly endocytosed membranes. Further investigation revealed that the increase in PH-GFP signal was dependent on the activation of N-methyl-D-aspartate receptors and the consequent production of nitric oxide (NO). Thus, PIP2 in the presynaptic terminal appears to be regulated by postsynaptic activity via a retrograde action of NO.     

Phosphoinositide binding by the pleckstrin homology domains of Ipl and Tih1

The Ipl protein consists of a single pleckstrin homology (PH) domain with short N- and C-terminal extensions. This protein is highly conserved among vertebrates, and it acts to limit placental growth in mice. However, its biochemical function is unknown. The closest paralogue of Ipl is Tih1, another small PH domain protein. By sequence comparisons, Ipl and Tih1 define an outlying branch of the PH domain superfamily. Here we describe phosphatidylinositol phosphate (PIP) binding by these proteins. Ipl and Tih1 bind to immobilized PIPs with moderate affinity, but this binding is weaker and more promiscuous than that of prototypical PH domains from the general receptor for phosphoinositides (GRP1), phospholipase C delta1, and dual adaptor for phosphoinositides and phosphotyrosine 1. In COS7 cells exposed to epidermal growth factor, green fluorescent protein (GFP)-Ipl and GFP-Tih1 accumulate at membrane ruffles without clearing from the cytoplasm, whereas control GFP-GRP1 translocates rapidly to the plasma membrane and clears from the cytoplasm. Ras*-Ipl and Ras*-Tih1 fusion proteins both rescue cdc25ts Saccharomyces cerevisiae, but Ras*-Ipl rescues more efficiently in the presence of phosphatidylinositol 3-kinase (PI3K), whereas PI3K-independent rescue is more efficient with Ras*-Tih1. Site-directed mutagenesis defines amino acids in the beta1-loop1-beta2 regions of Ipl and Tih1 as essential for growth rescue in this assay. Thus, Ipl and Tih1 are bona fide PH domain proteins, with broad specificity and moderate affinity for PIPs.     

Membrane docking geometry of GRP1 PH domain bound to a target lipid bilayer: an EPR site-directed spin-labeling and relaxation study

The second messenger lipid PIP(3) (phosphatidylinositol-3,4,5-trisphosphate) is generated by the lipid kinase PI3K (phosphoinositide-3-kinase) in the inner leaflet of the plasma membrane, where it regulates a broad array of cell processes by recruiting multiple signaling proteins containing PIP(3)-specific pleckstrin homology (PH) domains to the membrane surface. Despite the broad importance of PIP(3)-specific PH domains, the membrane docking geometry of a PH domain bound to its target PIP(3) lipid on a bilayer surface has not yet been experimentally determined. The present study employs EPR site-directed spin labeling and relaxation methods to elucidate the membrane docking geometry of GRP1 PH domain bound to bilayer-embedded PIP(3). The model target bilayer contains the neutral background lipid PC and both essential targeting lipids: (i) PIP(3) target lipid that provides specificity and affinity, and (ii) PS facilitator lipid that enhances the PIP(3) on-rate via an electrostatic search mechanism. The EPR approach measures membrane depth parameters for 18 function-retaining spin labels coupled to the PH domain, and for calibration spin labels coupled to phospholipids. The resulting depth parameters, together with the known high resolution structure of the co-complex between GRP1 PH domain and the PIP(3) headgroup, provide sufficient constraints to define an optimized, self-consistent membrane docking geometry. In this optimized geometry the PH domain engulfs the PIP(3) headgroup with minimal bilayer penetration, yielding the shallowest membrane position yet described for a lipid binding domain. This binding interaction displaces the PIP(3) headgroup from its lowest energy position and orientation in the bilayer, but the headgroup remains within its energetically accessible depth and angular ranges. Finally, the optimized docking geometry explains previous biophysical findings including mutations observed to disrupt membrane binding, and the rapid lateral diffusion observed for PIP(3)-bound GRP1 PH domain on supported lipid bilayers.     

Role of phosphatidylinositol 4,5-bisphosphate in the activation of cytosolic phospholipase A2-alpha

Cytosolic phospholipase A(2)-alpha (cPLA(2)) plays an important role in the release of arachidonic acid and in cell injury. Activation of cPLA(2) is dependent on a rise in cytosolic Ca(2+) concentration, membrane association via the Ca(2+)-dependent lipid binding (CaLB) domain, and phosphorylation. This study addresses the activation of cPLA(2) via potential association with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), including the role of a "pleckstrin homology (PH)-like" region of cPLA(2) (amino acids 263-354). In cells incubated with complement, phorbol myristate acetate+the Ca(2+) ionophore, A23187, or epidermal growth factor+A23187, expression of the PH domain of phospholipase C-delta1 (which sequesters membrane PIP(2)) attenuated cPLA(2) activity. Stimulated cPLA(2) activity was also attenuated by the expression of cPLA(2) 135-366, or cPLA(2) 2-366, and expression of a PIP(2)-specific 5'-phosphatase. However, in a yeast-based assay that tests the ability of proteins to bind to membrane lipids, including PIP(2), with high affinity, only cPLA(2) 1-200 (CaLB domain) was able to interact with membrane lipids, whereas cPLA(2)s 135-366, 2-366, 201-648, and 1-648 were unable to do so. Therefore, cPLA(2) activity can be modulated by sequestration or depletion of cellular PIP(2), although the interaction of cPLA(2) with membrane PIP(2) appears to be indirect, or of weak affinity.     

Acanthamoeba myosin IC colocalizes with phosphatidylinositol 4,5-bisphosphate at the plasma membrane due to the high concentration of negative charge

The tail of Acanthamoeba myosin IC (AMIC) has a basic region (BR), which contains a putative pleckstrin homology (PH) domain, followed by two Gly/Pro/Ala (GPA)-rich regions separated by a Src homology 3 (SH3) domain. Cryoelectron microscopy had shown that the tail is folded back on itself at the junction of BR and GPA1, and nuclear magnetic resonance spectroscopy indicated that the SH3 domain may interact with the putative PH domain. The BR binds to acidic phospholipids, and the GPA region binds to F-actin. We now show that the folded tail does not affect the affinity of AMIC for acidic phospholipids. AMIC binds phosphatidylinositol 4,5-bisphosphate (PIP2) with high affinity (approximately 1 microm), but binding is not stereospecific. When normalized to net negative charge, AMIC binds with equal affinity to phosphatidylserine (PS) and PIP2. This and other data show that the putative PH domain of AMIC is not a typical PIP2-specific PH domain. We have identified a 13-residue sequence of basic-hydrophobic-basic amino acids within the putative PH domain that may be a major determinant of binding of AMIC to acidic phospholipids. Despite the lack of stereospecificity, AMIC binds 10 times more strongly to vesicles containing 5% PIP2 plus 25% PS than to vesicles containing only 25% PS, suggesting that AMIC may be targeted to PIP2-enriched regions of the plasma membrane. In agreement with this, AMIC colocalizes with PIP2 at dynamic, protrusive regions of the plasma membrane. We discuss the possibility that AMIC binding to PIP2 may initiate the formation of a multiprotein complex at the plasma membrane.     

Son of sevenless directly links the Robo receptor to rac activation to control axon repulsion at the midline

Son of sevenless (Sos) is a dual specificity guanine nucleotide exchange factor (GEF) that regulates both Ras and Rho family GTPases and thus is uniquely poised to integrate signals that affect both gene expression and cytoskeletal reorganization. Here, using genetics, biochemistry, and cell biology, we demonstrate that Sos is recruited to the plasma membrane, where it forms a ternary complex with the Roundabout receptor and the SH3-SH2 adaptor protein Dreadlocks (Dock) to regulate Rac-dependent cytoskeletal rearrangement in response to the Slit ligand. Intriguingly, the Ras and Rac-GEF activities of Sos can be uncoupled during Robo-mediated axon repulsion; Sos axon guidance function depends on its Rac-GEF activity, but not its Ras-GEF activity. These results provide in vivo evidence that the Ras and RhoGEF domains of Sos are separable signaling modules and support a model in which Robo recruits Sos to the membrane via Dock to activate Rac during midline repulsion.     

Unexpected complexity in the mechanisms that target assembly of the spectrin cytoskeleton

The spectrin cytoskeleton assembles within discrete regions of the plasma membrane in a wide range of animal cell types. Although recent studies carried out in vertebrate systems indicate that spectrin assembly occurs indirectly through the adapter protein ankyrin, recent studies in Drosophila have established that spectrin can also assemble through a direct ankyrin-independent mechanism. Here we tested specific regions of the spectrin molecule for a role in polarized assembly and function. First, we tested mutant beta-spectrins lacking ankyrin binding activity and/or the COOH-terminal pleckstrin homology (PH) domain for their assembly competence in midgut, salivary gland, and larval brain. Remarkably, three different assembly mechanisms operate in these three cell types: 1) neither site was required for assembly in salivary gland; 2) only the PH domain was required in midgut copper cells; and 3) either one of the two sites was sufficient for spectrin assembly in larval brain. Further characterization of the PH domain revealed that it binds strongly to lipid mixtures containing phosphatidylinositol 4,5-bisphosphate (PIP(2)) but not phosphatidylinositol 3,4,5-trisphosphate. A K8Q mutation in the lipid binding region of the PH domain eliminated the PIP(2) interaction in vitro, yet the mutant protein retained full biological function in vivo. Reporter gene studies revealed that PIP(2) and the spectrin PH domain codistribute with one another in cells but not with authentic wild type alphabeta-spectrin. Thus, it appears that the PH domain imparts membrane targeting activity through a second mechanism that takes precedence over its PIP(2) binding activity.     

Assembly of the Sos1-Grb2-Gab1 ternary signaling complex is under allosteric control

Allostery has evolved as a form of local communication between interacting protein partners allowing them to quickly sense changes in their immediate vicinity in response to external cues. Herein, using isothermal titration calorimetry (ITC) in conjunction with circular dichroism (CD) and macromolecular modeling (MM), we show that the binding of Grb2 adaptor—a key signaling molecule involved in the activation of Ras GTPase—to its downstream partners Sos1 guanine nucleotide exchange factor and Gab1 docker is under tight allosteric regulation. Specifically, our findings reveal that the binding of one molecule of Sos1 to the nSH3 domain allosterically induces a conformational change within Grb2 such that the loading of a second molecule of Sos1 onto the cSH3 domain is blocked and, in so doing, allows Gab1 access to the cSH3 domain in an exclusively non-competitive manner to generate the Sos1-Grb2-Gab1 ternary signaling complex.