Tandem SH2 Domains Research Articles

CLEC-2 activates Syk through dimerization

AbstractThe C-type lectin receptor CLEC-2 activates platelets through Src and Syk tyrosine kinases, leading to tyrosine phosphorylation of downstream adapter proteins and effector enzymes, including phospholipase-C γ2. Signaling is initiated through phosphorylation of a single conserved tyrosine located in a YxxL sequence in the CLEC-2 cytosolic tail. The signaling pathway used by CLEC-2 shares many similarities with that used by receptors that have 1 or more copies of an immunoreceptor tyrosine-based activation motif, defined by the sequence Yxx(L/I)x6-12Yxx(L/I), in their cytosolic tails or associated receptor chains. Phosphorylation of the conserved immunoreceptor tyrosine-based activation motif tyrosines promotes Syk binding and activation through binding of the Syk tandem SH2 domains. In this report, we present evidence using peptide pull-down studies, surface plasmon resonance, quantitative Western blotting, tryptophan fluorescence measurements, and competition experiments that Syk activation by CLEC-2 is mediated by the cross-linking through the tandem SH2 domains with a stoichiometry of 2:1. In support of this model, cross-linking and electron microscopy demonstrate that CLEC-2 is present as a dimer in resting platelets and converted to larger complexes on activation. This is a unique mode of activation of Syk by a single YxxL-containing receptor.

The Syk Kinase SmTK4 of Schistosoma mansoni Is Involved in the Regulation of Spermatogenesis and Oogenesis

The signal transduction protein SmTK4 from Schistosoma mansoni belongs to the family of Syk kinases. In vertebrates, Syk kinases are known to play specialized roles in signaling pathways in cells of the hematopoietic system. Although Syk kinases were identified in some invertebrates, their role in this group of animals has not yet been elucidated. Since SmTK4 is the first Syk kinase from a parasitic helminth, shown to be predominantly expressed in the testes and ovary of adult worms, we investigated its function. To unravel signaling cascades in which SmTK4 is involved, yeast two-/three-hybrid library screenings were performed with either the tandem SH2-domain, or with the linker region including the tyrosine kinase domain of SmTK4. Besides the Src kinase SmTK3 we identified a new Src kinase (SmTK6) acting upstream of SmTK4 and a MAPK-activating protein, as well as mapmodulin acting downstream. Their identities and colocalization studies pointed to a role of SmTK4 in a signaling cascade regulating the proliferation and/or differentiation of cells in the gonads of schistosomes. To confirm this decisive role we performed biochemical and molecular approaches to knock down SmTK4 combined with a novel protocol for confocal laser scanning microscopy for morphological analyses. Using the Syk kinase-specific inhibitor Piceatannol or by RNAi treatment of adult schistosomes in vitro, corresponding phenotypes were detected in the testes and ovary. In the Xenopus oocyte system it was finally confirmed that Piceatannol suppressed the activity of the catalytic kinase domain of SmTK4. Our findings demonstrate a pivotal role of SmTK4 in gametogenesis, a new function for Syk kinases in eukaryotes.

Switching between low and high affinity for the Syk tandem SH2 domain by irradiation of azobenzene containing ITAM peptidomimetics

Spleen tyrosine kinase (Syk) plays an essential role in IgE receptor signaling (FcepsilonRI), which leads to mast cell degranulation. Divalent binding of the tandem SH2 domain (tSH2) of Syk to the intracellular ITAM motif of FcepsilonRI activates the kinase domain of Syk, and thereby initiates cell degranulation. The inter SH2 domain distance in Syk tSH2 might be important for Syk kinase activation. In this study, photoswitchable ITAM peptidomimetics containing an azobenzene moiety were synthesized. Irradiation of these constructs changes the distance between the two SH2 binding epitopes and therefore, they may be used as photoswitches. The affinity of the cis- and trans-isomer for tSH2 was assayed with SPR. The ITAM peptidomimetic with the smallest linker displayed the largest difference in affinity between the two isomers (at least 100-fold), and the affinity of the cis-isomer was comparable to monovalent binding. The ITAM mimics with larger photoswitchable linkers displayed modest differences. These results indicate that Syk tSH2 is able to adapt the inter SH2 domain distance to ligands larger than native ITAM, but not to smaller ones.

Characterization of Phospholipase Cγ Enzymes with Gain-of-Function Mutations

Phospholipase C gamma isozymes (PLC gamma 1 and PLC gamma 2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLC gamma 2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLC gamma 1 and PLC gamma 2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC gamma 2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC gamma 2 and corresponding PLC gamma 1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.

Isoform-Selective Interaction of the Adaptor Protein Tks5/FISH with Sos1 and Dynamins

The adaptor protein Tks5/FISH (tyrosine kinase substrate 5/five SH3 domains, hereafter termed Tks5) is a crucial component of a protein network that controls the invasiveness of cancer cells and progression of Alzheimer's disease. Tks5 consists of an amino-terminal PX domain that is followed by five SH3 domains (SH3A–E), and two different splice variants are expressed. We identified son of sevenless-1 (Sos1) as a novel binding partner of Tks5 and found colocalization of Tks5 with Sos1 in human epithelial lung carcinoma (A549) cells and in podosomes of Src-transformed NIH 3T3 cells. We observe synergistic binding of SH3A and SH3B to Sos1 when peptide arrays are used, indicating that the tandem SH3A and SH3B domains of Tks5 can potentially bind in a superSH3 binding mode, as was described for the homologous protein p47phox. These results are further corroborated by pull-down assays and isothermal titration calorimetry showing that both intact SH3 domains are required for efficient binding to the entire proline-rich domain of Sos1. The presence of a basic insertion between the SH3A and SH3B domains in the long splice variant of Tks5 decreases the affinity to Sos1 isoforms about 10-fold as determined by analytical ultracentrifugation. Furthermore, it leads to an alteration in the recognition of binding motifs for the interaction with Sos1: While the insertion abrogates the interaction with the majority of peptides derived from the proline-rich domains of Sos1 and dynamin that are recognized by the short splice isoform, it enables binding to a different set of peptides including a sequence comprising the splice insertion in the long isoform of Sos1 (Sos1_2). In the absence of the basic insertion, Tks5 was found to bind a range of Sos1 and dynamin peptides including conventional proline-rich motifs and atypical recognition sequences. Hereby, the tandem SH3 domains in Tks5 employ two distinct types of binding modes: One class of peptides is recognized by single SH3 domains, whereas a second class of peptides requires the presence of both domains to bind synergistically. We conclude that the tandem SH3A and SH3B domains of Tks5 constitute a versatile module for the implementation of isoform-specific protein–protein interactions.

A region N-terminal to the tandem SH3 domain of p47phox plays a crucial role in the activation of the phagocyte NADPH oxidase

The superoxide-producing NADPH oxidase in phagocytes is crucial for host defence; its catalytic core is the membrane-integrated protein gp91phox [also known as Nox2 (NADPH oxidase 2)], which forms a stable heterodimer with p22phox. Activation of the oxidase requires membrane translocation of the three cytosolic proteins p47phox, p67phox and the small GTPase Rac. At the membrane, these proteins assemble with the gp91phox-p22phox heterodimer and induce a conformational change of gp91phox, leading to superoxide production. p47phox translocates to membranes using its two tandemly arranged SH3 domains, which directly interact with p22phox, whereas p67phox is recruited in a p47phox-dependent manner. In the present study, we show that a short region N-terminal to the bis-SH3 domain is required for activation of the phagocyte NADPH oxidase. Alanine substitution for Ile152 in this region, a residue that is completely conserved during evolution, results in a loss of the ability to activate the oxidase; and the replacement of Thr153 also prevents oxidase activation, but to a lesser extent. In addition, the corresponding isoleucine residue (Ile155) of the p47phox homologue Noxo1 (Nox organizer 1) participates in the activation of non-phagocytic oxidases, such as Nox1 and Nox3. The I152A substitution in p47phox, however, does not affect its interaction with p22phox or with p67phox. Consistent with this, a mutant p47phox (I152A), as well as the wild-type protein, is targeted upon cell stimulation to membranes, and membrane recruitment of p67phox and Rac normally occurs in p47phox (I152A)-expressing cells. Thus the Ile152-containing region of p47phox plays a crucial role in oxidase activation, probably by functioning at a process after oxidase assembly.

Investigation of the Regulatory Mechanism of the ZAP-70 Immunological Signaling Enzyme

ZAP-70 is a critical Syk-family protein tyrosine kinase (PTK) that functions in the initial step of T-cell receptor (TCR) signaling. Its importance is highlighted by mutations in ZAP-70 that cause severe combined immunodeficiency (SCID). Moreover, elevated levels of ZAP-70 are associated with T-cell proliferative diseases and chronic lymphocytic leukemia (CLL). Under normal conditions, T-cell activation by foreign antigen results in co-localization with ZAP-70, due to binding of its associated tandem SH2 domains (tSH2) to phosphorylated ITAM (Immunoreceptor Tyrosine Activation Motif) sequences present on the TCR ζ subunits. This leads to up-regulation of kinase activity and downstream initiation of an immune response. Experimental structures reveal that upon ITAM binding, the tSH2 must undergo a large conformational change. Of interest, therefore, is how ITAM-binding controls the tSH2 conformational equilibrium, and the subsequent proposed disassembly of the kinase domain. To this end, we have carried out 1.5 μs of molecular dynamics (MD) simulations of various ZAP-70 systems, composed of 50,000 to 70,000 atoms. These include the isolated tSH2, and variants of the ITAM-bound state. Collectively, our results suggest that one phosphotyrosine site regulates the tSH2 conformational equilibrium, whilst the other controls localization of the tSH2 to membrane-proximal ITAM targets. Using these results, along with those from targeted MD simulations, we have identified a set of order parameters that describe the tSH2 conformational transition. We have subsequently performed multidimensional potential of mean force (PMF) calculations, via umbrella-sampling, to describe the transition; a single such PMF for the tSH2 “switch” constitutes over 3 μs of simulation time. Thus, we have achieved an atomic level characterization of the free-energy landscape of the tSH2 in the presence and absence of the catalytic domain, with important consequences for the mechanism of signaling and autoinhibition in Syk-family kinases.

ITAM-derived phosphopeptide-containing dendrimers as multivalent ligands for Syk tandem SH2 domain

Spleen tyrosine kinase (Syk) is activated when its tandem SH2 domain (tSH2) binds to a diphosphorylated ITAM motif of e.g. the FcepsilonRI receptor. In this divalent interaction each SH2 domain binds to a phosphotyrosine-containing tetrapeptide motif in ITAM. One of those tetrapeptide sequences was synthesized and conjugated to dendrimers via 'click' chemistry to create a series of functional phosphopeptide-containing dendrimers ranging from a monovalent to an octavalent dendrimer. The affinity of the functionalized dendrimers for Syk tSH2 has been assayed in SPR competition experiments. Both the tetra- and octavalent dendrimer had an affinity in the high nanomolar range, which is approximately 100-fold enhanced compared to the monovalent tetrapeptide, indicating a multivalency effect.

A photoswitchable ITAM peptidomimetic: Synthesis and real time surface plasmon resonance (SPR) analysis of the effects of cis– trans isomerization on binding

The Syk protein plays an important role in immune receptor signaling. The Syk tandem SH2 domain (tSH2)–ITAM interaction is important for recruiting Syk to the receptor complex and for Syk kinase activation. A peptidomimetic ligand for tSH2 was synthesized in which a photoswitchable azobenzene moiety was incorporated. Such a photoswitchable moiety may regulate the distance between the two phosphotyrosine containing ITAM sequences, which bind to tSH2. Different affinities of the cis and trans isomer of the ligand were found by surface plasmon resonance (SPR). By in situ irradiation during SPR measurements the effect of the cis– trans isomerization on binding could be monitored in real time.

Interdomain A is crucial for ITAM-dependent and -independent regulation of Syk

Non-receptor type protein tyrosine kinase (PTK) Syk is essential for the signaling via the B cell antigen receptor (BCR). Upon BCR crosslinking, Syk is recruited via its tandem SH2 domains to tyrosine-phosphorylated Ig-α/Ig-β constituting components of BCR, and is then activated. The interdomain A lying between the two SH2 domains is highly conserved among different species of Syk and between Syk and ZAP-70. The mutant Syk carrying a deletion in the interdomain A (Δ140–159) became phosphorylated regardless of BCR ligation and did not induce Ca 2+ mobilization upon crosslinking of BCR. Furthermore, in vitro binding assay revealed that deletion of a part of the interdomain A abolished its binding activity to phosphorylated Ig-α/Ig-β. These results indicate that the interdomain A of Syk is required for activation of Syk by binding to the phosphorylated Ig-α/Ig-β upon BCR ligation and inhibition of spontaneous activation at the resting state.

SLP-65 Signal Transduction Requires Src Homology 2 domain-mediated Membrane Anchoring and a Kinase-independent Adaptor Function of Syk

The family of SLPs (Src homology 2 domain-containing leukocyte adaptor proteins) are cytoplasmic signal effectors of lymphocyte antigen receptors. A main function of SLP is to orchestrate the assembly of Ca(2+)-mobilizing enzymes at the inner leaflet of the plasma membrane. For this purpose, SLP-76 in T cells utilizes the transmembrane adaptor LAT, but the mechanism of SLP-65 membrane anchoring in B cells remains an enigma. We now employed two genetic reconstitution systems to unravel structural requirements of SLP-65 for the initiation of Ca(2+) mobilization and subsequent activation of gene transcription. First, mutational analysis of SLP-65 in DT40 B cells revealed that its C-terminal Src homology 2 domain controls efficient tyrosine phosphorylation by the kinase Syk, plasma membrane recruitment, as well as downstream signaling to NFAT activation. Second, we dissected these processes by expressing SLP-65 in SLP-76-deficient T cells and found that a kinase-independent adaptor function of Syk is required to link phosphorylated SLP-65 to Ca(2+) mobilization. These approaches unmask a mechanistic complexity of SLP-65 activation and coupling to signaling cascades in that Syk is upstream as well as downstream of SLP-65. Moreover, membrane anchoring of the SLP-65-assembled Ca(2+) initiation complex, which appears to be fundamentally different from that of closely related SLP-76, does not necessarily involve a B cell-specific component.

The C-type Lectin Receptors CLEC-2 and Dectin-1, but Not DC-SIGN, Signal via a Novel YXXL-dependent Signaling Cascade

The two lectin receptors, CLEC-2 and Dectin-1, have been shown to signal through a Syk-dependent pathway, despite the presence of only a single YXXL in their cytosolic tails. In this study, we show that stimulation of CLEC-2 in platelets and in two mutant cell lines is dependent on the YXXL motif and on proteins that participate in signaling by immunoreceptor tyrosine-based activation motif receptors, including Src, Syk, and Tec family kinases, and on phospholipase Cgamma. Strikingly, mutation of either Src homology (SH) 2 domain of Syk blocks signaling by CLEC-2 despite the fact that it has only a single YXXL motif. Furthermore, signaling by CLEC-2 is only partially dependent on the BLNK/SLP-76 family of adapter proteins in contrast to that of immunoreceptor tyrosine-based activation motif receptors. The C-type lectin receptor, Dectin-1, which contains a YXXL motif preceded by the same four amino acids as for CLEC-2 (DEDG), signals like CLEC-2 and also requires the two SH2 domains of Syk and is only partially dependent on the BLNK/SLP-76 family of adapters. In marked contrast, the C-type lectin receptor, DC-SIGN, which has a distinct series of amino acids preceding a single YXXL, signals independent of this motif. A mutational analysis of the DEDG sequence of CLEC-2 revealed that the glycine residue directly upstream of the YXXL tyrosine is important for CLEC-2 signaling. These results demonstrate that CLEC-2 and Dectin-1 signal through a single YXXL motif that requires the tandem SH2 domains of Syk but is only partially dependent on the SLP-76/BLNK family of adapters.

Molecular dynamics study on the ligand recognition by tandem SH3 domains of p47 phox, regulating NADPH oxidase activity

The phagocyte NADPH oxidase complex plays a crucial role in host defense against microbial infection through the production of superoxides. Chronic granulomatous disease (CGD) is an inherited immune deficiency caused by the absence of certain components of the NADPH oxidase. Key to the activation of the NADPH oxidase is the cytoplasmic subunit p47 phox, which includes the tandem SH3 domains (N-SH3 and C-SH3). In active phagocytes, p47 phox forms a stable complex with the cytoplasmic region of membrane subunit p22 phox that forms a left-handed polyproline type-II (PPII) helix conformation. In this report, we have analyzed the conformational changes of p47 phox–p22 phox complexes of wild-type and three mutants, which have been detected in CGD patients, using molecular dynamics simulations. We have found that in the wild-type, two basal planes of PPII prism in cytoplasmic region of p22 phox interacted with N-SH3 and C-SH3. In contrast, in the modeled mutants, the residue at the ape of PPII helix, which interacts simultaneously with both of the tandem SH3 domains in the wild-type, moved toward C-SH3. Furthermore, interaction energies of the cytoplasmic region of p22 phox with C-SH3 tend to decrease in these mutants. All these findings led us to conclude that interactions between N-SH3 of p47 phox and PPII helix, which is formed by cytoplasmic region of p22 phox, may play a significant role in the activation of the NADPH oxidase.

Small-Angle X-ray Scattering Reveals an Extended Organization for the Autoinhibitory Resting State of the p47phox Modular Protein

In response to microbial infection, neutrophiles promote the assembly of the NADPH oxidase complex in order to produce superoxide anions. This reaction is activated by the association of cytosolic factors, p47(phox), p67(phox), p40(phox), and a small G protein Rac with the membranous heterodimeric flavocytochrome b(558), composed of gp91(phox) and p22(phox). In the activation process, p47(phox) plays a central role as the target of phosphorylations and as a scaffolding protein conducting the translocation and assembly of cytosolic factors onto the membranous components. The PX and tandem SH3s of p47(phox) have been highlighted as being key determinants for the interaction with membrane lipids and the p22(phox) component, respectively. In the resting state, the two corresponding interfaces are thought to be masked allowing its cytoplasmic localization. However, the resting state modular organization of p47(phox) and its autoinhibition mode are still not fully understood despite available structural information on separate modules. More precisely, it raises the question of the mutual arrangement of the PX domain and the tandem SH3 domains in the resting state. To address this question, we have engaged a study of the entire p47(phox) molecule in solution using small-angle X-ray scattering. Despite internal autoinhibitory interactions, p47(phox) adopts an extended conformation. First insights about the domain arrangement in whole p47(phox) can be derived. Our data allow to discard the usual representation of a globular and compact autoinhibited resting state.

Intramolecular Regulatory Switch in ZAP-70: Analogy with Receptor Tyrosine Kinases

ZAP-70, a Syk family cytoplasmic protein tyrosine kinase (PTK), is required to couple the activated T-cell antigen receptor (TCR) to downstream signaling pathways. It contains two tandem SH2 domains that bind to phosphorylated TCR subunits and a C-terminal catalytic domain. The region connecting the SH2 domains with the kinase domain, termed interdomain B, has previously been shown to have striking regulatory effects on ZAP-70 function, presumed to be due to the recruitment of key substrates. Paradoxically, deletion of interdomain B preserves ZAP-70 function. Recent structural studies of several receptor tyrosine kinases (RTKs) revealed that their juxtamembrane regions negatively regulate their catalytic activities. In EphB2 and several other RTKs, this autoinhibition depends upon interaction between the kinase domain and tyrosine residues within the juxtamembrane region. Autoinhibition is released when these tyrosines become phosphorylated following receptor stimulation. Sequence homology suggested analogous regulation for ZAP-70. Based on mutagenesis analysis of ZAP-70 interdomain B, we find that this region downregulates ZAP-70 catalytic activity in a similar manner as the juxtamembrane region of EphB2. Similar regulation was also noted for the related Syk kinase. These findings suggest that a general autoinhibitory mechanism employed by RTKs is also used by some cytoplasmic tyrosine kinases.

Peptide microarrays for the detection of molecular interactions in cellular signal transduction

The formation of protein complexes is a hallmark of cellular signal transduction. Here, we show that peptide microarrays provide a robust and quantitative means to detect signalling-dependent changes of molecular interactions. Recruitment of a protein into a complex upon stimulation of a cell leads to the masking of an otherwise exposed binding site. In cell lysates this masking can be detected by reduced binding to a microarray carrying a peptide that corresponds to the binding motif of the respective interaction domain. The method is exemplified for the lymphocyte-specific tyrosine kinase 70 kDa zeta-associated protein binding to a bis-phosphotyrosine-motif of the activated T-cell receptor via its tandem SH2 domain. Compared to established techniques, the method provides a significant shortcut to the detection of molecular interactions.

Transient transfection of human peripheral blood basophils

The human basophil has resisted previous attempts at transient transfection. Basophils were transfected by nucleoporation and to test whether there was sufficient expression to modify cell function, the cells were transfected with a syk kinase tandem SH2 construct linked to GFP. This approach was taken because in RBL cells and murine mast cells syk kinase is known to play a very early role in signal transduction and previous studies in RBL cells demonstrated that expression of the tandem SH2 domains of syk would inhibit signaling, presumably by competition with endogenous syk for binding to ITAMs. Results from basophil transfections with SH2 syk were compared to an empty construct. Basophils were stimulated with anti-IgE antibody and analyzed for single cell changes in cytosolic calcium levels. Basophils expressing the empty GFP construct showed a cytosolic calcium response similar to non-expressing cells. In contrast, basophils expressing the GFP-tandem SH2 syk construct, on average, showed an anti-IgE-induced calcium response that was completely ablated. The transfection frequency was 8% (median), with an average viable recovery of 12% ( n=18). While the procedure is not benign and is not always successful, these studies indicate that with gating techniques, the human basophil, a non-dividing primary leukocyte, can be transiently transfected to express high enough levels of an inhibitory protein to alter an IgE-mediated response.

Inhibition of FcγRIIA Mediated Platelet Phagocytosis with Syk-Specific siRNA Affects PECAM-1 Mediated Signal Transduction and Cell-Cell Detachment in Human Macrophages.

We have previously shown that Syk-specific siRNA inhibits FcγRIIA mediated platelet phagocytosis in the human macrophage cell line, THP-1. Surprisingly, we observed that THP-1 cell-cell interactions were affected by Syk-specific siRNA suggesting that Syk may be important in the signaling pathway for physiological cell spreading. As reported by Brown et al. ( Nature 418:200, 2002), homophilic ligation of PECAM-1 on macrophages triggers cell-cell detachment, prompting us to speculate that Syk may be involved in PECAM-1 mediated signaling. PECAM-1 is a member of the immunoglobulin superfamily that contains an immunoreceptor tyrosine inhibitory motif (ITIM) required for inhibitory signaling and cell spreading via binding protein tyrosine phosphatase. The structure of Syk reveals considerable flexibility in the relative orientation of its tandem SH2 domains providing a molecular basis for the potential involvement of Syk in a variety of signal transduction pathways. Thus, the five tyrosine residues present in the cytoplasmic domain of human PECAM-1 could serve as docking sites for recruitment of other intracellular signaling molecules such as the protein tyrosine kinase, Syk. The present studies test the hypothesis that Syk may mediate an activation influence on intercellular interaction upon PECAM-1 ligation.METHODS: Association of Syk with PECAM-1 was studied in THP-1 cells by immunoprecipitation and immunoblotting with anti-PECAM-1 monoclonal antibody with or without Syk-specific siRNA. The effect of Syk-specific siRNA on THP-1 cells was evaluated by morphology, cell growth kinetics, Trypan Blue exclusion, annexin V staining, and DNA fragmentation analysis. A series of synthetic peptides including sequences within the ITIM region of PECAM-1 were evaluated for their ability to bind Syk directly in THP-1 lysates and to phosphorylate Syk intracellularly.RESULTS: First, Syk protein was co-immunoprecipitated from THP-1 cell lysates by anti-PECAM-1 monoclonal antibody, and Syk-specific siRNA knocked down PECAM-1 associated Syk in THP-1 cells. Second, knock down of Syk by siRNA resulted in decreased cell spreading and cell proliferation. Trypan Blue exclusion, annexin V staining, and DNA fragmentation were identical in THP-1 cells transfected with control dsRNA and Syk-specific siRNA. Third, the synthetic peptide of residues 658-691 within the ITIM region of PECAM-1 that contains two phosphotyrosines (N-S-D-V-Q-pY-T-E-V-Q-V-S-S-A-E-S-H-K-D-L-G-K-K- D-T-E-T-V-pY-S-E-V-R-K) bound directly to Syk. Fourth, ligation of PECAM-1 on THP-1 cells by anti-PECAM-1 monoclonal antibody resulted in Syk phosphorylation. Fifth, the dual phosphotyrosine 658-691 PECAM-1 peptide triggered direct phosphorylation of Syk in permeabilized THP-1 cells.CONCLUSIONS: Our novel observations suggest that signal transduction after PECAM-1 ligation may trigger an activation pathway through the tyrosine kinase, Syk, and facilitate intercellular adhesion. The decreased proliferation observed in THP-1 cells transfected with Syk-specific siRNA is not a result of cell death or apoptosis. Based on our findings, we propose that PECAM-1 may exhibit both ITIM (inhibitory) and ITAM (activation) properties and that the balance of phosphatase and Syk activity triggered by PECAM-1 homophilic binding may affect the fate of intercellular interaction. These processes are expected to influence the net degree of phagocytosis of opsonized platelets by the reticuloendothelial system in patients with immune thrombocytopenia.

Solution Structure of the Tandem Src Homology 3 Domains of p47phox in an Autoinhibited Form

The phagocyte NADPH oxidase is a multisubunit enzyme responsible for the generation of superoxide anions (O(2).) that kill invading microorganisms. p47(phox) is a cytosolic subunit of the phagocyte NADPH oxidase, which plays a crucial role in the assembly of the activated NADPH oxidase complex. The molecular shapes of the p47(phox) tandem SH3 domains either with or without a polybasic/autoinhibitory region (PBR/AIR) at the C terminus were studied using small angle x-ray scattering. The tandem SH3 domains with PBR/AIR formed a compact globular structure, whereas the tandem SH3 domains lacking the PBR/AIR formed an elongated structure. Alignment anisotropy analysis by NMR based on the residual dipolar couplings revealed that the tandem SH3 domains with PBR/AIR were in good agreement with a globular module corresponding to the split half of the intertwisted dimer in crystalline state. The structure of the globular module was elucidated to represent a solution structure of the tandem SH3 domain in the autoinhibited form, where the PBR/AIR bundled the tandem SH3 domains and the linker forming a closed structure. Once PBR/AIR is released by phosphorylation, rearrangements of the SH3 domains may occur, forming an open structure that binds to the cytoplasmic proline-rich region of membrane-bound p22(phox).

T Cell Receptor Signaling: Beyond Complex Complexes

The adaptive phase of the immune response begins with engagement on CD4 + helper T cells of the T cell antigen receptor (TCR) 1 by its ligand, a small foreign peptide bound to a cell surface protein of the class II major histocompatibility complex (peptide-MHC) expressed on an antigen-presenting cell. This engagement initiates a series of biochemical events that can differentially signal the naive T cell to: 1) enter into a pathway leading to generation of effector T cells with the onset of rapid proliferation and production of effector cytokines; 2) enter into a state of antigenic non-responsiveness known as anergy; or 3) die by apoptosis. The type of response elicited depends on multiple factors including the affinity of the interaction, the duration of the interaction, and the presence or absence of various costimulatory signaling inputs such as those provided by the CD4 coreceptor and the CD28 costimulatory receptor. In this review we provide an overview of the signaling events that are associated with the first of these outcomes: T cell activation. To present an overview of sufficiently broad scope, the depth of discussion of each aspect ofTCR signaling is by necessity limited, and the reader is referred to the reviews cited throughout this text for consideration of these events in more detail.