Bivalent binding drives the formation of the Grb2-Gab1 signaling complex in a noncooperative manner

Although the growth factor receptor binder 2 (Grb2)-Grb2-associated binder (Gab)1 macromolecular complex mediates a multitude of cellular signaling cascades, the molecular basis of its assembly has hitherto remained largely elusive. Herein, using an array of biophysical techniques, we show that, whereas Grb2 exists in a monomer-dimer equilibrium, the proline-rich (PR) domain of Gab1 is a monomer in solution. Of particular interest is the observation that although the PR domain appears to be structurally disordered, it nonetheless adopts a more or less compact conformation reminiscent of natively folded globular proteins. Importantly, the structurally flexible conformation of the PR domain appears to facilitate the binding of Gab1 to Grb2 with a 1:2 stoichiometry. More specifically, the formation of the Grb2-Gab1 signaling complex is driven via a bivalent interaction through the binding of the C-terminal homology 3 (cSH3) domain within each monomer of Grb2 homodimer to two distinct RXXK motifs, herein designated G1 and G2, located within the PR domain of Gab1. Strikingly, in spite of the key role of bivalency in driving this macromolecular assembly, the cSH3 domains bind to the G1 and G2 motifs in an independent manner with zero cooperativity. Taken together, our findings shed new light on the physicochemical forces driving the assembly of a key macromolecular signaling complex that is relevant to cellular health and disease.     

Allostery mediates ligand binding to Grb2 adaptor in a mutually exclusive manner

Allostery plays a key role in dictating the stoichiometry and thermodynamics of multi‐protein complexes driving a plethora of cellular processes central to health and disease. Herein, using various biophysical tools, we demonstrate that although Sos1 nucleotide exchange factor and Gab1 docking protein recognize two non‐overlapping sites within the Grb2 adaptor, allostery promotes the formation of two distinct pools of Grb2–Sos1 and Grb2–Gab1 binary signaling complexes in concert in lieu of a composite Sos1–Grb2–Gab1 ternary complex. Of particular interest is the observation that the binding of Sos1 to the nSH3 domain within Grb2 sterically blocks the binding of Gab1 to the cSH3 domain and vice versa in a mutually exclusive manner. Importantly, the formation of both the Grb2–Sos1 and Grb2–Gab1 binary complexes is governed by a stoichiometry of 2:1, whereby the respective SH3 domains within Grb2 homodimer bind to Sos1 and Gab1 via multivalent interactions. Collectively, our study sheds new light on the role of allostery in mediating cellular signaling machinery. Copyright © 2013 John Wiley & Sons, Ltd.     

Identification of the mitogen-activated protein kinase phosphorylation sites on human Sos1 that regulate interaction with Grb2.

The Son of sevenless proteins (Sos) are guanine nucleotide exchange factors involved in the activation of Ras by cytoplasmic and receptor tyrosine kinases. Growth factor stimulation rapidly induces the phosphorylation of Sos on multiple serine and threonine sites. Previous studies have demonstrated that growth factor-induced Sos phosphorylation occurs at the C-terminal region of the protein and is mediated, in part, by mitogen-activated protein (MAP) kinase. In this report, we describe the identification of five MAP kinase sites (S-1137, S-1167, S-1178, S-1193, and S-1197) on hSos1. We demonstrate that four of these sites, S-1132, S-1167, S-1178, and S-1193, become phosphorylated following growth factor stimulation. The MAP kinase phosphorylation sites are clustered within a region encompassing three proline-rich SH3-binding sites in the C-terminal domain of hSos1. Replacing the MAP kinase phosphorylation sites with alanine residues results in an increase in the binding affinity of Grb2 to hSos1. Interestingly, hSos2 contains only one MAP kinase phosphorylation site and, as demonstrated previously, has an increased affinity toward Grb2 compared with hSos1. These results suggest a role for MAP kinase in the regulation of Grb2-Sos interactions. Since the binding of Grb2 is important for Sos function, the phosphorylation-dependent modulation of Grb2-Sos association may provide a means of controlling Ras activation.     

Binding of the Grb2 SH2 domain to phosphotyrosine motifs does not change the affinity of its SH3 domains for Sos proline-rich motifs.

Phosphotyrosine peptide binding to Grb2 induces tryptophan fluorescence changes in the Src homology 2 (SH2) domain. Affinities are in the nanomolar range, the Shc peptide having the highest affinity, followed by peptides mimicking Grb2 binding sites on EGF and HGF receptors, the putative sites on insulin and IGF-1 receptors having much lower affinities. Proline-rich peptide binding to the SH3 domains induces fluorescence changes mainly in the C-terminal SH3. Affinities are in the micromolar range, the highest affinity peptides mimicking the first proline-rich motif of the Sos C-terminus. Additional residues before this PVPPPVPP motif provide a minor contribution to the binding, but the two residues after this motif are important and may contribute to specificity. The affinity of each SH3 for each proline-rich motif is too low to account for the high stability of the Grb2-Sos complex, suggesting that Grb2 recognizes other structural features in the Sos C-terminus. Binding of a phosphotyrosine peptide to the SH2 has no effect on the SH3s. Thus the binding of Grb2 to a receptor or to an associated protein phosphorylated on tyrosines is unlikely to activate the exchange factor activity of Sos through a conformational change transmitted from the SH2 to the SH3 domains.     

Unusual binding of Grb2 protein to a bivalent polyproline-ligand immobilized on a SPR sensor: Intermolecular bivalent binding

The Grb2 adapter protein is involved in the activation of the Ras signaling pathway. It recruits the Sos protein by binding of its two SH3 domains to Sos polyproline sequences. We observed that the binding of Grb2 to a bivalent ligand, containing two Sos-derived polyproline-sequences immobilized on a SPR sensor, shows unusual kinetic behavior. SPR-kinetic analysis and supporting data from other techniques show major contributions of an intermolecular bivalent binding mode. Each of the two Grb2 SH3 domains binds to one polyproline-sequence of two different ligand molecules, facilitating binding of a second Grb2 molecule to the two remaining free polyproline binding sites. A molecular model based on the X-ray structure of the Grb2 dimer shows that Grb2 is flexible enough to allow this binding mode. The results fit with a role of Grb2 in protein aggregation, achieving specificity by multivalent interactions, despite the relatively low affinity of single SH3 interactions.     

Structural basis of the differential binding of the SH3 domains of Grb2 adaptor to the guanine nucleotide exchange factor Sos1

Grb2-Sos1 interaction, mediated by the canonical binding of N-terminal SH3 (nSH3) and C-terminal SH3 (cSH3) domains of Grb2 to a proline-rich sequence in Sos1, provides a key regulatory switch that relays signaling from activated receptor tyrosine kinases to downstream effector molecules such as Ras. Here, using isothermal titration calorimetry in combination with site-directed mutagenesis, we show that the nSH3 domain binds to a Sos1-derived peptide containing the proline-rich consensus motif PPVPPR with an affinity that is nearly threefold greater than that observed for the binding of cSH3 domain. We further demonstrate that such differential binding of nSH3 domain relative to the cSH3 domain is largely due to the requirement of a specific acidic residue in the RT loop of the β-barrel fold to engage in the formation of a salt bridge with the arginine residue in the consensus motif PPVPPR. While this role is fulfilled by an optimally positioned D15 in the nSH3 domain, the chemically distinct and structurally non-equivalent E171 substitutes in the case of the cSH3 domain. Additionally, our data suggest that salt tightly modulates the binding of both SH3 domains to Sos1 in a thermodynamically distinct manner. Our data further reveal that, while binding of both SH3 domains to Sos1 is under enthalpic control, the nSH3 binding suffers from entropic penalty in contrast to entropic gain accompanying the binding of cSH3, implying that the two domains employ differential thermodynamic mechanisms for Sos1 recognition. Our new findings are rationalized in the context of 3D structural models of SH3 domains in complex with the Sos1 peptide. Taken together, our study provides structural basis of the differential binding of SH3 domains of Grb2 to Sos1 and a detailed thermodynamic profile of this key protein-protein interaction pertinent to cellular signaling and cancer.     

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.     

Quantifying intramolecular binding in multivalent interactions: a structure-based synergistic study on Grb2-Sos1 complex

Numerous signaling proteins use multivalent binding to increase the specificity and affinity of their interactions within the cell. Enhancement arises because the effective binding constant for multivalent binding is larger than the binding constants for each individual interaction. We seek to gain both qualitative and quantitative understanding of the multivalent interactions of an adaptor protein, growth factor receptor bound protein-2 (Grb2), containing two SH3 domains interacting with the nucleotide exchange factor son-of-sevenless 1 (Sos1) containing multiple polyproline motifs separated by flexible unstructured regions. Grb2 mediates the recruitment of Sos1 from the cytosol to the plasma membrane where it activates Ras by inducing the exchange of GDP for GTP. First, using a combination of evolutionary information and binding energy calculations, we predict an additional polyproline motif in Sos1 that binds to the SH3 domains of Grb2. This gives rise to a total of five polyproline motifs in Sos1 that are capable of binding to the two SH3 domains of Grb2. Then, using a hybrid method combining molecular dynamics simulations and polymer models, we estimate the enhancement in local concentration of a polyproline motif on Sos1 near an unbound SH3 domain of Grb2 when its other SH3 domain is bound to a different polyproline motif on Sos1. We show that the local concentration of the Sos1 motifs that a Grb2 SH3 domain experiences is approximately 1000 times greater than the cellular concentration of Sos1. Finally, we calculate the intramolecular equilibrium constants for the crosslinking of Grb2 on Sos1 and use thermodynamic modeling to calculate the stoichiometry. With these equilibrium constants, we are able to predict the distribution of complexes that form at physiological concentrations. We believe this is the first systematic analysis that combines sequence, structure, and thermodynamic analyses to determine the stoichiometry of the complexes that are dominant in the cellular environment.     

High affinity IgG receptor activation of Src family kinases is required for modulation of the Shc-Grb2-Sos complex and the downstream activation of the nicotinamide adenine dinucleotide phosphate (reduced) oxidase

We used the U937 cell line to examine the modulation of adaptor protein interactions (Shc, Grb2, and Cbl) after high affinity IgG receptor (FcgammaRI) cross-linking, leading to the formation of the Grb2-Sos complex, the activation of Ras, and the regulation of the respiratory burst. Cross-linking of FcgammaRI induced the conversion of GDP-Ras to GTP-Ras reaching a maximum 5 min after stimulation. Concomitant with Ras activation, Sos underwent an electrophoretic mobility shift and the Sos-Grb2 association was increased (6-fold). The Grb2-Sos complex was present only in the membrane fraction and was augmented after FcgammaRI stimulation. Tyrosine-phosphorylated Shc, mainly the p52 isoform, was observed to transiently onload to the membrane Grb2-Sos complex on FcgammaRI stimulation. Cross-linking of FcgammaRI induces the tyrosine phosphorylation of Cbl, which forms a complex with Grb2 and Shc via the Cbl C terminus. Kinetic experiments confirm that Cbl-Grb2 is relatively stable, whereas Grb2-Sos, Grb2-Shc, and Cbl-Shc interactions are highly inducible. The Src family tyrosine kinase inhibitor, PP1, was shown to completely inhibit Shc tyrosine phosphorylation, the Shc-Grb2 interaction, and the FcgammaR-induced respiratory burst. Our results provide the first evidence that the upstream activation of Src kinases is required for the modulation of the Shc-Grb2 interaction and the myeloid NADPH oxidase response.     

Binding of laminin alpha1-chain LG4-5 domain to alpha-dystroglycan causes tyrosine phosphorylation of syntrophin to initiate Rac1 signaling

Previously, a signaling pathway was described [Oak, Zhou, and Jarrett (2003) J. Biol. Chem. 278, 39287-39295] that links matrix laminin binding on the outside of the sarcolemma to Grb2 binding to syntrophin on the inside surface of the sarcolemma and by way of Grb2-Sos1-Rac1-PAK1-JNK ultimately results in the phosphorylation of c-jun on Ser(65). How this signaling is initiated was investigated. Grb2-binding to syntrophin is increased by the addition of either laminin-1 or the isolated laminin alpha1 globular domain modules LG4-5, a protein referred to as E3. This identifies the LG4-5 sequences as the region of laminin responsible for signaling. Since laminin alpha1 LG4 is known to bind alpha-dystroglycan, this directly implicates alpha-dystroglycan as the laminin-signaling receptor. E3 or laminin-1 increase Grb2-binding and Rac1 activation. In the presence of E3 or laminin-1, syntrophin is phosphorylated on a tyrosine residue, and this increases and alters Grb2 binding. The alpha-dystroglycan antibody, IIH6, which blocks binding of laminins to alpha-dystroglycan, blocks both the laminin-induced Sos1/2 recruitment and syntrophin phosphorylation, showing that it is alpha-dystroglycan binding the LG4-5 region of laminin that is responsible. The C-terminal SH3 domain of Grb2 (C-SH3) binds only to nonphosphorylated syntrophin, and phosphorylation causes the Grb2 SH2 domain to bind and prevents SH3 binding. Syntrophin, tyrosine phosphate, beta-dystroglycan, and Rac1 all co-localize to the sarcolemma of rat muscle sections. A model for how this phosphorylation may initiate downstream events in laminin signaling is presented.     

A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase.

Autophosphorylated growth factor receptors provide binding sites for the src homology 2 domains of intracellular signaling molecules. In response to epidermal growth factor (EGF), the activated EGF receptor binds to a complex containing the signaling protein GRB2 and the Ras guanine nucleotide-releasing factor Sos, leading to activation of the Ras signaling pathway. We have investigated whether the platelet-derived growth factor (PDGF) receptor binds GRB2-Sos. In contrast with the EGF receptor, the GRB2 does not bind to the PDGF receptor directly. Instead, PDGF stimulation induces the formation of a complex containing GRB2; 70-, 80-, and 110-kDa tyrosine-phosphorylated proteins; and the PDGF receptor. Moreover, GRB2 binds directly to the 70-kDa protein but not to the PDGF receptor. Using a panel of PDGF beta-receptor mutants with altered tyrosine phosphorylation sites, we identified Tyr-1009 in the PDGF receptor as required for GRB2 binding. Binding is inhibited by a phosphopeptide containing a YXNX motif. The protein tyrosine phosphatase Syp/PTP1D/SHPTP2/PTP2C is approximately 70 kDa, binds to the PDGF receptor via Tyr-1009, and contains several YXNX sequences. We found that the 70-kDa protein that binds to the PDGF receptor and to GRB2 comigrates with Syp and is recognized by anti-Syp antibodies. Furthermore, both GRB2 and Sos coimmunoprecipitate with Syp from lysates of PDGF-stimulated cells, and GRB2 binds directly to tyrosine-phosphorylated Syp in vitro. These results indicate that GRB2 interacts with different growth factor receptors by different mechanisms and the cytoplasmic phosphotyrosine phosphatase Syp acts as an adapter between the PDGF receptor and the GRB2-Sos complex.     

Tyrosine phosphorylation of caldesmon is required for binding to the Shc.Grb2 complex

S3-v-erbB is a retroviral oncogene that encodes a ligand-independent, transforming mutant of the epidermal growth factor receptor. This oncogene has been shown to be sarcomagenic in vivo and to transform fibroblasts in vitro. Our previous studies (McManus, M. J., Lingle, W. L., Salisbury, J. L., and Maihle, N. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11351-11356) showed that expression of S3-v-erbB in primary fibroblasts results in the tyrosine phosphorylation of caldesmon (CaD), an actin- and calmodulin-binding protein. This phosphorylation is transformation-associated, and the phosphorylated form of CaD is associated with a signaling complex consisting of Shc, Grb2, and Sos in transformed fibroblasts. To identify the tyrosine phosphorylation site(s) in the CaD molecule and to further elucidate the functional role of CaD tyrosine phosphorylation in S3-v-ErbB oncogenic signaling, we have generated a series of mutant CaDs in which one or more tyrosine residues have been replaced with phenylalanine. Using a CaD null cell line, DF1 cells (an immortalized chicken embryo fibroblast cell line), and transient transfection assays, we demonstrated that Tyr-27 and Tyr-393 are the major sites of tyrosine phosphorylation on CaD. Interestingly, Tyr-27 is located within the myosin binding domain of CaD, and Tyr-393 is adjacent to one of the major actin binding and actomyosin ATPase inhibitory domains. Our studies also show that the tyrosine phosphorylation of CaD enhances its binding to the Shc.Grb2 complex. Specifically, replacement of Tyr-27, but not of Tyr-165 or Tyr-393, significantly reduces the ability of CaD to interact with the Shc. Grb2 complex. Together, these studies demonstrate that the major sites of tyrosine phosphorylation on CaD are located in the myosin and actin binding domains of CaD and that Tyr-27 is the major tyrosine phosphorylation site through which CaD interacts with the Shc.Grb2 complex.     

Molecular cloning and characterization of an adaptor protein Shc isoform from Xenopus laevis oocytes

In order to gain further insight into IGF-1 receptor signaling in Xenopus laevis oocytes and embryos, we have undertaken the characterization of the adapter protein Shc and studied its implication in oocyte maturation induced after IGF-1 receptor activation, especially since expression of this molecule has been indirectly evidenced in Xenopus oocytes, eggs and embryos. We report herein the cloning from Xenopus postvitellogenic oocytes of a complementary DNA encoding a protein of 470 amino acids which shows the higher identity with the mammalian adaptor protein p52(ShcA). Western blot analysis using homologous antibodies evidenced a 60-kDa protein, p60(Xl)(Shc), that is predominantly expressed in oocytes and in early embryos. We also demonstrate that, like p60(Xl)(Shc), Grb2 and the guanine nucleotide exchange factor Sos are expressed in oocytes throughout vitellogenesis and in early embryos and that overexpression of a dominant-negative form of Grb2 specifically inhibits insulin-induced resumption of meiosis. We finally show that Grb2 binds to p60(Shc) in oocytes specifically upon insulin treatment. Altogether, these results suggest that Shc and Grb2-Sos are implicated in ras-dependent Xenopus oocyte maturation induced by insulin/IGF-1; they also indicate that inability of insulin/IGF-1 to activate the Ras-MAPK cascade in vitellogenic oocytes does not result from an insufficient expression level of Shc, Grb2 and Sos.     

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.     

Dopamine activates ERKs in alveolar epithelial cells via Ras-PKC-dependent and Grb2/Sos-independent mechanisms

Recently it has been described that dopamine (DA), via dopaminergic type 2 receptors (D(2)R), activates the mitogen-activated protein kinase extracellular signal-regulated kinase (MAPK/ERK) proteins in alveolar epithelial cells (AEC), which results in the upregulation of Na(+)-K(+)-ATPase. In the present report, we used AEC to investigate the signaling pathway that links DA with ERK activation. Incubation of AEC with DA resulted in rapid and transient stimulation of ERK activity, which was mediated by Ras proteins and the serine/threonine kinase Raf-1. Pretreatment of AEC with Src homology 3 binding peptide, which blocks the interaction between Grb2 and Sos, did not prevent DA activation of ERK. Diacylglycerol (DAG)-dependent protein kinase C (PKC) isoenzymes, involved in the DA-mediated activation of ERK proteins as pretreatment with either bisindolylmaleimide or Ro-31-8220, prevented the phosphorylation of Elk-1, and quinpirole, a D(2)R activator, stimulates the translocation of PKCepsilon. Together, the data suggest that DA activated MAPK/ERK via Ras, Raf-1 kinase, and DAG-dependent PKC isoenzymes, but, importantly and contrary to the classical model, this pathway did not involve the Grb2-Sos complex formation.     

Lymphocyte antigen receptor signal integration and regulation by the SHC adaptor

The Shc adaptor protein transduces signals from transmembrane receptors to the Ras pathway of cell activation by providing binding sites for the recruitment to the submembrane compartment of the Grb2/Sos G-nucleotide exchange complex. The need for Shc in this process is however unclear since Grb2 can be recruited directly to phosphotyrosine containing membrane receptors through its src-homology-2 domain. Evidence from studies in lymphocytes indicates that Shc is multifunctional and is involved in the integration of independent signals to the Ras pathway. Furthermore, Shc may be a key control point at which signaling can be modulated both by interfering signals and by feedback mechanisms. Here we review recent literature to support these functions for Shc.     

LAT and NTAL mediate immunoglobulin E-induced sustained extracellular signal-regulated kinase activation critical for mast cell survival

Immunoglobulin E (IgE) induces mast cell survival in the absence of antigen (Ag) through the high-affinity IgE receptor, Fcepsilon receptor I (FcepsilonRI). Although we have shown that protein tyrosine kinase Syk and sustained extracellular signal-regulated kinase (Erk) activation are required for IgE-induced mast cell survival, how Syk couples with sustained Erk activation is still unclear. Here, we report that the transmembrane adaptors LAT and NTAL are phosphorylated slowly upon IgE stimulation and that sustained but not transient Erk activation induced by IgE was inhibited in LAT(-/-) NTAL(-/-) bone marrow-derived mast cells (BMMCs). IgE-induced survival requires Ras activation, and both were impaired in LAT(-/-) NTAL(-/-) BMMCs. Sos was preferentially required for FcepsilonRI signals by IgE rather than IgE plus Ag. Survival impaired in LAT(-/-) NTAL(-/-) BMMCs was restored to levels comparable to those of the wild type by membrane-targeted Sos, which bypasses the Grb2-mediated membrane recruitment of Sos. The IgE-induced survival of BMMCs lacking Gads, an adaptor critical for the formation of the LAT-SLP-76-phospholipase Cgamma (PLCgamma) complex, was observed to be normal. IgE stimulation induced the membrane retention of Grb2-green fluorescent protein fusion proteins in wild-type but not LAT(-/-) NTAL(-/-) BMMCs. These results suggest that LAT and NTAL contribute to the maintenance of Erk activation and survival through the membrane retention of the Ras-activating complex Grb2-Sos and, further, that the LAT-Gads-SLP-76-PLCgamma and LAT/NTAL-Grb2-Sos pathways are differentially required for degranulation and survival, respectively.     

Gene dosage-dependent functions for phosphotyrosine-Grb2 signaling during mammalian tissue morphogenesis

BACKGROUND: The mammalian Grb2 adaptor protein binds pTyr-X-Asn motifs through its SH2 domain, and engages downstream targets such as Sos1 and Gab1 through its SH3 domains. Grb2 thereby couples receptor tyrosine kinases to the Ras-MAP kinase pathway, and potentially to phosphatidylinositol (PI) 3'-kinase. By creating a null (Delta) allele of mouse Grb2, we have shown that Grb2 is required for endoderm differentiation at embryonic day 4.0. RESULTS: Grb2 likely has multiple embryonic and postnatal functions. To address this issue, a hypomorphic mutation, first characterized in the Caenorhabditis elegans Grb2 ortholog Sem-5, was engineered into the mouse Grb2 gene. This mutation (E89K) reduces phosphotyrosine binding by the SH2 domain. Embryos that are compound heterozygous for the null and hypomorphic alleles exhibit defects in placental morphogenesis and in the survival of a subset of migrating neural crest cells required for branchial arch formation. Furthermore, animals homozygous for the hypomorphic mutation die perinatally because of clefting of the palate, a branchial arch-derived structure. Analysis of E89K/Delta Grb2 mutant fibroblasts revealed a marked defect in ERK/MAP kinase activation and Gab1 tyrosine phosphorylation following growth factor stimulation. CONCLUSIONS: We have created an allelic series within mouse Grb2, which has revealed distinct functions for phosphotyrosine-Grb2 signaling in tissue morphogenesis and cell viability necessary for mammalian development. The placental defects in E89K/Delta mutant embryos are reminiscent of those seen in receptor tyrosine kinase-, Sos1-, and Gab1-deficient embryos, consistent with the finding that endogenous Grb2 is required for efficient RTK signaling to the Ras-MAP kinase and Gab1 pathways.     

Tyrosine phosphorylation of Grb2 by Bcr/Abl and epidermal growth factor receptor: a novel regulatory mechanism for tyrosine kinase signaling.

Growth factor receptor-binding protein-2 (Grb2) plays a key role in signal transduction initiated by Bcr/Abl oncoproteins and growth factors, functioning as an adaptor protein through its Src homology 2 and 3 (SH2 and SH3) domains. We found that Grb2 was tyrosine-phosphorylated in cells expressing BCR/ABL and in A431 cells stimulated with epidermal growth factor (EGF). Phosphorylation of Grb2 by Bcr/Abl or EGF receptor reduced its SH3-dependent binding to Sos in vivo, but not its SH2-dependent binding to Bcr/Abl. Tyr209 within the C-terminal SH3 domain of Grb2 was identified as one of the tyrosine phosphorylation sites, and phosphorylation of Tyr209 abolished the binding of the SH3 domain to a proline-rich Sos peptide in vitro. In vivo expression of a Grb2 mutant where Tyr209 was changed to phenylalanine enhanced BCR/ABL-induced ERK activation and fibroblast transformation, and potentiated and prolonged Grb2-mediated activation of Ras, mitogen-activated protein kinase and c-Jun N-terminal kinase in response to EGF stimulation. These results suggest that tyrosine phosphorylation of Grb2 is a novel mechanism of down-regulation of tyrosine kinase signaling.