Binding Of Src Homology 2 Domain Research Articles

Optimization of a Novel DEL Hit That Binds in the Cbl-b SH2 Domain and Blocks Substrate Binding.

We were attracted to the therapeutic potential of inhibiting Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING E3 ligase that plays a critical role in regulating the activation of T cells. However, given that only protein-protein interactions were involved, it was unclear whether inhibition by a small molecule would be a viable approach. After screening an ∼6 billion member DNA-encoded library (DEL) using activated Cbl-b, we identified compound 1 as a hit for which the cis-isomer (2) was confirmed by biochemical and surface plasmon resonance (SPR) assays. Our hit optimization effort was greatly accelerated when we obtained a cocrystal structure of 2 with Cbl-b, which demonstrated induced binding at the substrate binding site, namely, the Src homology-2 (SH2) domain. This was quite noteworthy given that there are few reports of small molecule inhibitors that bind to SH2 domains and block protein-protein interactions. Structure- and property-guided optimization led to compound 27, which demonstrated measurable cell activity, albeit only at high concentrations.

Lipid Binding of SH2 Domains.

The Src homology 2 (SH2) domain is a modular protein interaction domain that specifically recognizes the phosphotyrosine (pY) motif of a target molecule. We recently reported that a large majority of human SH2 domains tightly bind membrane lipids, and many show high lipid specificity. Most of them can bind a lipid and the pY motif coincidently because their lipid-binding sites are topologically distinct from pY-binding pockets. Lipid binding of SH2 domain-containing kinases and phosphatases is functionally important because it exerts exquisite spatiotemporal control on protein-protein interaction and cell signaling activities mediated by these proteins. Here, we describe two assays, surface plasmon resonance analysis and fluorescence quenching analysis, which allow quantitative determination of the affinity and specificity of SH2-lipid interaction and high-throughput screening for SH2 domain-lipid-binding inhibitors.

SH2 Domain Binding: Diverse FLVRs of Partnership.

The Src homology 2 (SH2) domain has a special role as one of the cornerstone examples of a “modular” domain. The interactions of this domain are very well-conserved, and have long been described as a bidentate, or “two-pronged plug” interaction between the domain and a phosphotyrosine (pTyr) peptide. Recent work has, however, highlighted unusual features of the SH2 domain that illustrate a greater diversity than was previously appreciated. In this review we discuss some of the novel and unusual characteristics across the SH2 family, including unusual peptide binding pockets, multiple pTyr recognition sites, recognition sites for unphosphorylated peptides, and recently identified variability in the conserved FLVR motif.

MK‐STYX alters localization dynamics of autophagosomes and phenotypic dynamics of lysosomes

MK‐STYX[MAPK (mitogen‐activated protein kinase)phosphoserine/threonine/tyrosine‐binding protein] is a pseudophosphatase, a member of the dual‐specificity family subfamily of MAPK phosphatases (MKPs)that lack the essential nucleophilic cysteine in its signature motif required for catalytic activity. MK‐STYX maintains its three dimensional fold and ability to bind proteins; it is involved in cellular pathways such as those for stress response, apoptosis, and neuronal differentiation. Previously, were ported that MK‐STYX interacts with G3BP‐1 [Ras‐GAP (GTPAse‐activating protein) SH3 (Src homology 3) domain binding protein‐1], and inhibits stress granule formation. Stress granules, cytoplasmic storage sites for mRNA, form as a protective mechanism against stress caused by UV irradiation, hypoxia, and heat shock. Stress induces stress granules, and involves many cellular mechanisms such as post‐translational modifications, protein‐protein interactions, and microtubule networks. Furthermore, stress granules are targeted and cleared by autophagy, an initiated response to cellular stress. They are responsible for the degradation of cellular components. Therefore, autophagy is essential for cellular degradation. Since autophagy and MK‐STYX each negatively affect stress granule assembly, we sought to determine whether MK‐STYX has a role in autophagy. Pursuing the role of MK‐STYX in regulating autophagy will enhance our understanding of MK‐STYX's mechanism in the stress response pathway. Our studies show that MK‐STYX causes cytosolic TFEB (Transcription factor E‐Box; the autophagy “master switch”) to form perinuclear aggregates independent of nutrient status. Whereas, MK‐STYXactive (active mutant in which catalytic activity has been “restored”), only increased TFEB cytosolic aggregates in serum starved cells, suggesting that the catalytic signature motif of MK‐STYX may play a role in stress responses. TFEB is also found to localize to the lysosomal surface, acting as negative regulator of lysosomal and autophagosomal biogenesis. MK‐STYX alters lysosomal and autophagosomal dynamics; oversized lysosomes are observed in the presence of MK‐STYX. Furthermore, autophagosomes localize to the distal ends of HEK/293 cellular extensions in the presence of MK‐STYX. Autophagy plays a major role in major cellular processes ranging from initial developmental stages to the onset of progressive human pathologies such as neurodegenerative diseases and cancer. These studies suggest that MK‐STYX may have an important role in autophagy.Support or Funding InformationThis work was supported by the National Science Foundation Grant MCB1113167 to S.D.H.; Coco Award to S.D.H.; Howard Hughes Medical Research Institute grant through the Undergraduate Science Education Program to the College of William and Mary (HHMI Summer Fellowships to P.C. and A.M.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Src homology 2 domains enhance tyrosine phosphorylation in vivo by protecting binding sites in their target proteins from dephosphorylation

Phosphotyrosine (pTyr)-dependent signaling is critical for many cellular processes. It is highly dynamic, as signal output depends not only on phosphorylation and dephosphorylation rates but also on the rates of binding and dissociation of effectors containing phosphotyrosine-dependent binding modules such as Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains. Previous in vitro studies suggested that binding of SH2 and PTB domains can enhance protein phosphorylation by protecting the sites bound by these domains from phosphatase-mediated dephosphorylation. To test whether this occurs in vivo, we used the binding of growth factor receptor bound 2 (GRB2) to phosphorylated epidermal growth factor receptor (EGFR) as a model system. We analyzed the effects of SH2 domain overexpression on protein tyrosine phosphorylation by quantitative Western and far-Western blotting, mass spectrometry, and computational modeling. We found that SH2 overexpression results in a significant, dose-dependent increase in EGFR tyrosine phosphorylation, particularly of sites corresponding to the binding specificity of the overexpressed SH2 domain. Computational models using experimentally determined EGFR phosphorylation and dephosphorylation rates, and pTyr-EGFR and GRB2 concentrations, recapitulated the experimental findings. Surprisingly, both modeling and biochemical analyses suggested that SH2 domain overexpression does not result in a major decrease in the number of unbound phosphorylated SH2 domain-binding sites. Our results suggest that signaling via SH2 domain binding is buffered over a relatively wide range of effector concentrations and that SH2 domain proteins with overlapping binding specificities are unlikely to compete with one another for phosphosites in vivo.

Combining biophysical methods to analyze the disulfide bond in SH2 domain of C-terminal Src kinase.

The Src Homology 2 (SH2) domain is a structurally conserved protein domain that typically binds to a phosphorylated tyrosine in a peptide motif from the target protein. The SH2 domain of C-terminal Src kinase (Csk) contains a single disulfide bond, which is unusual for most SH2 domains. Although the global motion of SH2 domain regulates Csk function, little is known about the relationship between the disulfide bond and binding of the ligand. In this study, we combined X-ray crystallography, solution NMR, and other biophysical methods to reveal the interaction network in Csk. Denaturation studies have shown that disulfide bond contributes significantly to the stability of SH2 domain, and crystal structures of the oxidized and C122S mutant showed minor conformational changes. We further investigated the binding of SH2 domain to a phosphorylated peptide from Csk-binding protein upon reduction and oxidation using both NMR and fluorescence approaches. This work employed NMR, X-ray cryptography, and other biophysical methods to study a disulfide bond in Csk SH2 domain. In addition, this work provides in-depth understanding of the structural dynamics of Csk SH2 domain.

Selectivity and Mechanism of Action of a Growth Factor Receptor-Bound Protein 2 Src Homology 2 Domain Binding Antagonist

We have shown previously that a potent synthetic antagonist of growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain binding (1) blocks growth factor stimulated motility, invasion, and angiogenesis in cultured cell models, as well as tumor metastasis in animals. To characterize the selectivity of 1 for the SH2 domain of Grb2 over other proteins containing similar structural binding motifs, we synthesized a biotinylated derivative (3) that retained high affinity Grb2 SH2 domain binding and potent biological activity. To investigate the selectivity of 1 and 3 for Grb2, the biotinylated antagonist 3 was used to immobilize target proteins from cell extracts for subsequent identification by mass spectrometry. Non-specific binding was identified in parallel using a biotinylated analogue that lacked a single critical binding determinant. The mechanism of action of the antagonist was further characterized by immunoprecipitation, immunoblotting, and light microscopy. This approach to defining protein binding antagonist selectivity and molecular basis of action should be widely applicable in drug development.

Avian and 1918 Spanish Influenza A Virus NS1 Proteins Bind to Crk/CrkL Src Homology 3 Domains to Activate Host Cell Signaling

NS1 (nonstructural protein 1) is an important virulence factor of the influenza A virus. We observed that NS1 proteins of the 1918 pandemic virus (A/Brevig Mission/1/18) and many avian influenza A viruses contain a consensus Src homology 3 (SH3) domain-binding motif. Screening of a comprehensive human SH3 phage library revealed the N-terminal SH3 of Crk and CrkL as the preferred binding partners. Studies with recombinant proteins confirmed avid binding of NS1 proteins of the 1918 virus and a representative avian H7N3 strain to Crk/CrkL SH3 but not to other SH3 domains tested, including p85alpha and p85beta. Endogenous CrkL readily co-precipitated NS1 from cells infected with the H7N3 virus. In transfected cells association with CrkL was observed for NS1 of the 1918 and H7N3 viruses but not A/Udorn/72 or A/WSN/33 NS1 lacking this sequence motif. SH3 binding was dispensable for suppression of interferon-induced gene expression by NS1 but was associated with enhanced phosphatidylinositol 3-kinase signaling, as evidenced by increased Akt phosphorylation. Thus, the Spanish Flu virus resembles avian influenza A viruses in its ability to recruit Crk/CrkL to modulate host cell signaling.

Motif Decomposition of the Phosphotyrosine Proteome Reveals a New N-terminal Binding Motif for SHIP2

Advances in mass spectrometry-based proteomics have yielded a substantial mapping of the tyrosine phosphoproteome and thus provided an important step toward a systematic analysis of intracellular signaling networks in higher eukaryotes. In this study we decomposed an uncharacterized proteomics data set of 481 unique phosphotyrosine (Tyr(P)) peptides by sequence similarity to known ligands of the Src homology 2 (SH2) and the phosphotyrosine binding (PTB) domains. From 20 clusters we extracted 16 known and four new interaction motifs. Using quantitative mass spectrometry we pulled down Tyr(P)-specific binding partners for peptides corresponding to the extracted motifs. We confirmed numerous previously known interaction motifs and found 15 new interactions mediated by phosphosites not previously known to bind SH2 or PTB. Remarkably, a novel hydrophobic N-terminal motif ((L/V/I)(L/V/I)pY) was identified and validated as a binding motif for the SH2 domain-containing inositol phosphatase SHIP2. Our decomposition of the in vivo Tyr(P) proteome furthermore suggests that two-thirds of the Tyr(P) sites mediate interaction, whereas the remaining third govern processes such as enzyme activation and nucleic acid binding.

Reciprocal Regulation of SH3 and SH2 Domain Binding via Tyrosine Phosphorylation of a Common Site in CD3ε

Recruitment of cellular signaling proteins by the CD3 polypeptides of the TCR complex mediates T cell activation. We have screened a human Src homology 3 (SH3) domain phage display library for proteins that can bind to the proline-rich region of CD3epsilon. This screening identified Eps8L1 (epidermal growth factor receptor pathway substrate 8-like 1) together with the N-terminal SH3 domain of Nck1 and Nck2 as its preferred SH3 partners. Studies with recombinant proteins confirmed strong binding of CD3epsilon to Eps8L1 and Nck SH3 domains. CD3epsilon bound well also to Eps8 and Eps8L3, and modestly to Eps8L2, but not detectably to other SH3 domains tested. Interestingly, binding of Nck and Eps8L1 SH3 domains was mapped to a PxxDY motif that shared its tyrosine residue (Y166) with the ITAM of CD3epsilon. Phosphorylation of this residue abolished binding of Eps/Nck SH3 domains in peptide spot filter assays, as well as in cells cotransfected with a dominantly active Lck kinase. TCR ligation-induced binding and phosphorylation-dependent loss of binding were also demonstrated between Eps8L1 and endogenous CD3epsilon in Jurkat T cells. Thus, phosphorylation of Y166 serves as a molecular switch during T cell activation that determines the capacity of CD3epsilon to interact with either SH3 or SH2 domain-containing proteins.

Vav1 Acidic Region Tyrosine 174 Is Required for the Formation of T Cell Receptor-induced Microclusters and Is Essential in T Cell Development and Activation

Vav proteins are multidomain signaling molecules critical for mediating signals downstream of several surface receptors, including the antigen receptors of T and B lymphocytes. The catalytic guanine nucleotide exchange factor (GEF) activity of the Vav Dbl homology (DH) domain is thought to be controlled by an intramolecular autoinhibitory mechanism involving an N-terminal extension and phosphorylation of tyrosine residues in the acidic region (AC). Here, we report that the sequences surrounding the Vav1 AC: Tyr(142), Tyr(160), and Tyr(174) are evolutionarily conserved, conform to consensus SH2 domain binding motifs, and bind several proteins implicated in TCR signaling, including Lck, PI3K p85alpha, and PLCgamma1, through direct interactions with their SH2 domains. In addition, the AC tyrosines regulate tyrosine phosphorylation of Vav1. We also show that Tyr(174) is required for the maintenance of TCR-signaling microclusters and for normal T cell development and activation. In this regard, our data demonstrate that while Vav1 Tyr(174) is essential for maintaining the inhibitory constraint of the DH domain in both developing and mature T cells, constitutively activated Vav GEF disrupts TCR-signaling microclusters and leads to defective T cell development and proliferation.

Src Homology 2 Domain-based High Throughput Assays for Profiling Downstream Molecules in Receptor Tyrosine Kinase Pathways

Src homology 2 (SH2) domains are evolutionary conserved small protein modules that bind specifically to tyrosine-phosphorylated peptides. More than 100 SH2 domains have been identified in proteins encoded by the human genome. The binding specificity of these domains plays a critical role in signaling within the cell, mediating the relocalization and interaction of proteins in response to changes in tyrosine phosphorylation states. Here we developed an SH2 domain profiling method based on a multiplexed fluorescent microsphere assay in which various SH2 domains are used to probe the global state of tyrosine phosphorylation within a cell and to screen synthetic peptides that specifically bind to each SH2 domain. The multiplexed, fluorescent microsphere-based assay is a recently developed technology that can potentially detect a wide variety of interactions between biological molecules. We constructed 25-plex SH2 domain-GST fusion protein-conjugated fluorescent microsphere sets to investigate phosphorylation-mediated cell signaling through the specific binding of SH2 domains to activated target proteins. The response of HeLa, COS-1, A431, and 293 cells and four breast cancer cell lines to epidermal growth factor and insulin were quantitatively profiled using this novel microsphere-based, multiplexed, high throughput assay system.

Regulation of Bin1 SH3 domain binding by phosphoinositides.

Bin1/M-amphiphysin-II is an amphiphysin-II isoform highly expressed in transverse tubules of adult striated muscle and is implicated in their biogenesis. Bin1 contains a basic unique amino-acid sequence, Exon10, which interacts with certain phosphoinositides such as phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), to localize to membranes. Here we found that Exon10 also binds to the src homology 3 (SH3) domain of Bin1 itself, and hence blocks the binding of the SH3 domain to its canonical PxxP ligands, including dynamin. This blockage was released by addition of PI(4,5)P(2) in vitro or in cells overexpressing phosphatidylinositol 4-phosphate 5-kinase. The Exon10-binding interface of the Bin1 SH3 domain largely overlapped with its PxxP-binding interface. We also show that the PLCdelta pleckstrin homology domain, another PI(4,5)P(2)-binding module, cannot substitute for Exon10 in Bin1 function in transverse tubule formation, and suggest the importance of the dual biochemical properties of Exon10 in myogenesis. Our results exemplify a novel mechanism of SH3 domain regulation, and suggest that the SH3-mediated protein-protein interactions of Bin1 are regulated by Exon10 so that it may only occur when Bin1 localizes to certain submembrane areas.

Binding of cytoplasmic proteins to the CD19 intracellular domain is high affinity, competitive, and multimeric.

CD19 is required for the development of B1 and marginal zone B cells, for Ab responses, and for B cell memory. CD19 immunoprecipitates contain a complex of cytoplasmic proteins, including Lyn, Vav, phospholipase Cgamma2 (PLCgamma2), Grb2, and the p85 subunit of phosphatidylinositol 3-kinase. Which of these bind directly to CD19 and the strengths of the interactions are unknown. These issues are important in understanding the signaling functions of CD19, which are crucial for normal B cell physiology. Using purified, recombinant proteins, we now show that each of these signaling proteins contains at least one Src homology 2 (SH2) domain that interacts directly with the phosphorylated CD19 cytoplasmic domain. The affinities of binding of the SH2 domains of Vav, p85, and Grb2 to CD19 are each in the nanomolar range by surface plasmon resonance (Biacore) analysis. Binding of Lyn and PLCgamma2 do not fit 1:1 modeling. However, analyses of binding data (Lyn) and competition experiments (PLCgamma2) suggest that these bind with comparable affinity. Competition experiments demonstrate that SH2 domains whose binding is dependent on the same CD19 tyrosine(s) compete for binding, but these SH2 domains do not impede binding of different SH2 domains to other CD19 tyrosines. We conclude that binding to the CD19 cytoplasmic domain is multimeric, high affinity, and competitive. The high affinity of the interactions also suggests that tyrosines that were nonessential in vivo are nevertheless functional. A preliminary structural model suggests that CD19 forms a signaling complex containing multiple cytoplasmic proteins in close proximity to each other and to the plasma membrane.

Analysis of SH2 ligands and identification of sites of interaction.

AbstractThe molecular analysis of cell signaling pathways requires an understanding of what each component in the pathway is, the manner in which they interact, and how these interactions serve to propagate the signal. Cytokine signaling begins with the binding of an extracellular cytokine to a specific cell-surface receptor. This usually results in the activation of intracellular protein kinases, which phosphorylate protein substrates important for the propagation of a cell signal. One mechanism by which protein phosphorylation regulates the transmission of a cell signal is through the creation of docking sites recognized by other signaling proteins. Such sites promote the association between protein molecules leading to the formation of active signaling complexes. Src homology 2 (SH2) domains are protein modules of approx 100 amino acids that recognize and bind to specific phosphotyrosine-containing polypeptide sequences (1). SH2 domains are recruited to tyrosine-phosphorylated proteins in their normal physiological setting; however, short synthetic phosphopeptides derived from these cellular docking sites contain all of the necessary binding determinants to interact with SH2 domains. This fact has allowed the identification of SH2 binding sites on tyrosine-phosphorylated proteins based on the analysis of SH2 domains interacting with phosphopeptides. Structural and functional characterization of these interactions has led to the observation that a central phosphotyrosine (pY) residue is the defining feature of ligands recognized by all SH2 domains; however, specific binding of a particular SH2 domain to a tyrosine-phosphorylated protein is dependent on the primary sequence in the immediate vicinity of the pY residue, particularly the flanking sequence immediately C-terminal to the pY residue (2).KeywordsDocking SiteRelative Binding AffinitySoluble PeptideBiosensor ChipIntracellular Protein KinaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Synthesis of a phosphotyrosyl analogue having χ1, χ2 and φ angles constrained to values observed for an SH2 domain-bound phosphotyrosyl residue

Src homology 2 (SH2) domains provide connectivity in protein-tyrosine kinase (PTK)-dependent signaling through their high affinity association with phosphotyrosyl (pTyr)-containing peptide sequences. Because recognition of pTyr residues is central to SH2 domain-binding affinity, design of pTyr-mimicking residues has been one component of SH2 domain signaling antagonist development. Reported herein is the synthesis of (±)-( rel-1 R,2 R,5 S)-3-acetyl-1,2,3,4,5,6-hexahydro-8- O-phosphoryl-1,5-methano-3-benzazocine-2-carboxylic acid methyl ester ( 3c ) as a monomeric pTyr-mimicking analogue that constrains three torsion angles ( χ 1=168°; χ 2=−85°; φ 1=−113°) to values approximating those observed for a pTyr residue bound to the Grb2 SH2 domain ( χ 1=182°; χ 2=−89°; φ 1=−132°). Compound 3c differs from our previously reported analogue, (±)-( rel-1 R,2 R,5 S)-3-acetyl-1,2,3,4,5,6-hexahydro-1-methyl-1,5-methano-3-benzazocin-8-ol, in lacking a methyl substituent at the bridgehead 1-position. Molecular modeling studies had indicated that this methyl group could potentially hinder SH2 domain binding. Synthesis of the desmethyl derivative was achieved by formation of the methanobenzazocine ring system using an intramolecular electrophilic cyclization that proceeds through an activated acyliminium intermediate. Importantly, the correct relative (2 R) stereochemistry at the ‘α-carboxyl’-bearing carbon is obtained through base-catalyzed equilibration of a (2 S/2 R) diastereomeric mixture that results from intramolecular ring closure. Comparison of Grb2 SH2 domain-binding affinity of 3c (IC 50=1167 μM) with conformationally flexible phosphorylated (±)- N-acetyl-tyrosine methyl ester ( 15 ; IC 50=1469 μM) revealed no apparent enhancement in affinity. This apparent ineffectiveness of ‘local conformational constraint’ on SH2 domain-binding affinity of the monomeric pTyr mimetic is consistent with previous reports obtained by conformationally constraining pTyr-mimicking residues that were contained within peptide platforms. Although not providing high binding affinity in its current form, the novel 1,5-methano-3-benzazocine ring system may afford a novel platform for further elaboration and development of small molecule SH2 domain signaling antagonists.

Inhibitory effects of rosmarinic acid on Lck SH2 domain binding to a synthetic phosphopeptide.

In the course of screening inhibitors from the methanol (MeOH) extracts of 168 medicinal plants against lymphocyte cell-specific kinase (Lck) Src -homology 2 (SH2) binding to a synthetic phosphotyrosine-containing peptide (phosphopeptide), we isolated rosmarinic acid from the MeOH extract of Prunella vulgaris, which showed specific inhibitory activity. The IC 50 value for Lck SH2 binding to phosphopeptide (SGSGEEPQpYEEIPI) of hamster polyomavirus middle-sized tumor (hmT pY324) was 7 microM. However, even at concentrations of 0.1 to 1000 microM, no significant inhibitions were observed against other SH2 domains binding such as the growth factor receptor binding protein 2 (Grb2) SH2 domain to phosphopeptide of Shc and phospholipase Cgamma1 (PLCgamma1) SH2 domain to translational elongation factor 1alpha (EF1alpha) C-terminal. Rosmarinic acid inhibited interleukin-2 (IL-2) gene expression by 50 % at a concentration of 8 microM in Jurkat cells stimulated with anti-CD3 and anti-CD4 antibodies. FK506 and cyclosporin A (CsA) employed as positive controls showed less than 30 % inhibition at the same concentration. In addition, rosmarinic acid inhibited the intracellular [Ca 2+] i increase in Jurkat cells after T cell activation in a dose-dependent manner at concentrations of 1.4 to 140 microM of rosmarinic acid, which is one of the earliest responses of antigen-specific T cell receptor (TCR) and of the upstream pathway of IL-2 expression. Taken together, these results suggest that rosmarinic acid has the potential to specifically inhibit Lck SH2 domain binding to its cognate ligand, including ZAP-70, Cbl, HS-1, and PLCgamma1, and Lck-dependent Ca 2+ signaling pathway of its downstream effector and finally to modulate IL-2 gene expression after T cell activation.

Binding of SAP SH2 domain to FynT SH3 domain reveals a novel mechanism of receptor signalling in immune regulation.

SAP (or SH2D1A), an adaptor-like molecule expressed in immune cells, is composed almost exclusively of a Src homology 2 (SH2) domain. In humans, SAP is mutated and either absent or non-functional in X-linked lymphoproliferative (XLP) syndrome, a disease characterized by an inappropriate response to Epstein-Barr virus (EBV) infection. Through its SH2 domain, SAP associates with tyrosines in the cytoplasmic domain of the SLAM family of immune cell receptors, and is absolutely required for the function of these receptors. This property results from the ability of SAP to promote the selective recruitment and activation of FynT, a cytoplasmic Src-related protein tyrosine kinase (PTK). Here, we demonstrate that SAP operates in this pathway by binding to the SH3 domain of FynT, through a second region in the SAP SH2 domain distinct from the phosphotyrosine-binding motif. We demonstrate that this interaction is essential for SAP-mediated signalling in T cells, and for the capacity of SAP to modulate immune cell function. These observations characterize a biologically important signalling mechanism in which an adaptor molecule composed only of an SH2 domain links a receptor devoid of intrinsic catalytic activity to the kinase required for its function.

Specific binding of the C-terminal Src homology 2 domain of the p85alpha subunit of phosphoinositide 3-kinase to phosphatidylinositol 3,4,5-trisphosphate. Localization and engineering of the phosphoinositide-binding motif.

Phosphoinositide second messengers, generated from the action of phosphoinositide 3-kinase (PI3K), mediate an array of signaling pathways through the membrane recruitment and activation of downstream effector proteins. Although pleckstrin domains of many target proteins have been shown to bind phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and/or phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) with high affinity, published data concerning the phosphoinositide binding specificity of Src homology 2 (SH2) domains remain conflicting. Using three independent assays, we demonstrated that the C-terminal (CT-)SH2 domain, but not the N-terminal SH2 domain, on the PI3K p85alpha subunit displayed discriminative affinity for PIP(3). However, the binding affinity diminished precipitously when the acyl chain of PIP(3) was shortened. In addition, evidence suggests that the charge density on the phosphoinositol ring represents a key factor in determining the phosphoinositide binding specificity of the CT-SH2 domain. In light of the largely shared structural features between PIP(3) and PI(4,5)P(2), we hypothesized that the PIP(3)-binding site on the CT-SH2 domain encompassed a sequence that recognized PI(4,5)P(2). Based on a consensus PI(4,5)P(2)-binding sequence (KXXXXXKXKK; K denotes Arg, Lys, and His), we proposed the sequence (18)RNKAENLLRGKR(29) as the PIP(3)-binding site. This binding motif was verified by using a synthetic peptide and site-directed mutagenesis. More importantly, neutral substitution of flanking Arg(18) and Arg(29) resulted in a switch of ligand specificity of the CT-SH2 domain to PI(4,5)P(2) and PI(3,4)P(2), respectively. Together with computer modeling, these mutagenesis data suggest a pseudosymmetrical relationship in the recognition of the phosphoinositol head group at the binding motif.

The ORF3 Protein of Hepatitis E Virus Binds to Src Homology 3 Domains and Activates MAPK

The hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The biology and pathogenesis of HEV remain poorly understood. We have used in vitro binding assays to show that the HEV ORF3 protein (pORF3) binds to a number of cellular signal transduction pathway proteins. This includes the protein tyrosine kinases Src, Hck, and Fyn, the p85alpha regulatory subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma, and the adaptor protein Grb2. A yeast two-hybrid assay was used to further confirm the pORF3-Grb2 interaction. The binding involves a proline-rich region in pORF3 and the src homology 3 (SH3) domains in the cellular proteins. Competition assays and computer-assisted modeling was used to evaluate the binding surfaces and interaction energies of the pORF3.SH3 complex. In pORF3-expressing cells, pp60(src) was found to associate with an 80-kDa protein, but no activation of the Src kinase was observed in these cells. However, there was increased activity and nuclear localization of ERK in the pORF3-expressing cells. These studies suggest that pORF3 is a viral regulatory protein involved in the modulation of cell signaling. The ORF3 protein of HEV appears to be the first example of a SH3 domain-binding protein encoded by a virus that causes an acute and primarily self-limited infection.