Lipid rafts orchestrate signaling by the platelet receptor glycoprotein VI

The platelet collagen receptor glycoprotein VI (GPVI) couples to the immune receptor adaptor Fc receptor gamma-chain (FcRgamma) and signals using many of the same intracellular signaling molecules as immune receptors. Studies of immune receptor signaling have revealed a critical role for specialized areas of the cell membrane known as lipid rafts, which are enriched in essential signaling molecules. However, the role of lipid rafts in signaling in nonimmune cells such as platelets remains poorly defined. This study shows that GPVI-FcRgamma does not constitutively associate with rafts, but is recruited to lipid rafts following receptor stimulation in both GPVI-expressing RBL-2H3 cells and human platelets. FcRgamma is required for GPVI association with lipid rafts, as mutant GPVI receptors that do not couple to FcRgamma were unable to associate with lipid rafts after receptor clustering. Following GPVI stimulation in platelets, virtually all phosphorylated FcRgamma was found in lipid rafts, but inhibition of FcRgamma phosphorylation did not block receptor association with lipid rafts. This work demonstrates that lipid rafts orchestrate GPVI receptor signaling in platelets in a manner analogous to immune cell receptors and supports a model of GPVI signaling in which FcRgamma phosphorylation is controlled by ligand-dependent association with lipid rafts.     

CD14 controls the LPS-induced endocytosis of Toll-like receptor 4

The transport of Toll-like Receptors (TLRs) to various organelles has emerged as an essential means by which innate immunity is regulated. While most of our knowledge is restricted to regulators that promote the transport of newly synthesized receptors, the regulators that control TLR transport after microbial detection remain unknown. Here, we report that the plasma membrane localized Pattern Recognition Receptor (PRR) CD14 is required for the microbe-induced endocytosis of TLR4. In dendritic cells, this CD14-dependent endocytosis pathway is upregulated upon exposure to inflammatory mediators. We identify the tyrosine kinase Syk and its downstream effector PLCγ2 as important regulators of TLR4 endocytosis and signaling. These data establish that upon microbial detection, an upstream PRR (CD14) controls the trafficking and signaling functions of a downstream PRR (TLR4). This innate immune trafficking cascade illustrates how pathogen detection systems operate to induce both membrane transport and signal transduction.     

IL-7 receptor mediates tyrosine phosphorylation but does not activate the phosphatidylinositol-phospholipase C-gamma 1 pathway.

IL-7 is a glycoprotein involved in the regulation of lymphocyte precursor growth. In addition, it has a comitogenic effect on mature T cells but not on mature B cells. The exact mechanism whereby IL-7R mediates these cell growth properties remains unknown. Because many growth factor receptor systems on various cell types transduce signals by activating a tyrosine kinase, we have studied here the effect of IL-7R ligation on protein tyrosine phosphorylation. We found that human rIL-7 consistently induced tyrosine phosphorylation of five major proteins, of 175, 155, 135, 110, and 85 kDa, and five minor proteins. The effect of human rIL-7 on tyrosine phosphorylation of these substrates was concentration and time dependent. One of the known substrates that is phosphorylated on tyrosine residues after binding of growth factors to their receptors is the phosphoinositide-specific phospholipase C. Several phospholipase C isozymes have been recently recognized; one isozyme, phospholipase C-gamma 1, was demonstrated to be phosphorylated rapidly after ligand binding to the platelet-derived growth factor receptor and the T cell Ag receptor. We show here that, in contrast to Ag receptor ligation, activation of IL-7R does not induce tyrosine phosphorylation on phospholipase C-gamma 1. Consistent with these results, human rIL-7 failed to increase phosphatidylinositol turnover and did not induce a rise in cytosolic free Ca2+ in the thymocytes, mature T cells, or pre-pre-B cells. The results indicate that the IL-7R mediates the activation of the tyrosine phosphorylation pathway but does not induce the phosphatidylinositol-phospholipase C pathway.     

Innate recognition of microbial-derived signals in immunity and inflammation

Microbes generate a vast array of different types of conserved structural components called pathogen-associated molecular patterns(PAMPs),which canbe recognized by cells of the innate immune system.This recognition of "nonself" signatures occurs through host pattern recognition receptors(PRRs),suggesting that microbial-derived signals are good targets for innate immunity to discriminate between self- and nonself.Such PAMP-PRR interactions trigger multiple but distinct downstream signaling cascades,subsequently leading to production of proinflammatory cytokines and interferons that tailor immune responses to particular microbes.Aberrant PRR signals have been associated with various inflammatory diseases and fine regulation of PRR signaling is essential for avoiding excessive inflammatory immune responses and maintaining immune homeostasis.In this review we summarize the ligands and signal transduction pathways of PRRs and highlight recent progress of the mechanisms involved in microbe-specific innate immune recognition during immune responses and inflammation,which may provide new targets for therapeutic intervention to the inflammatory disorders.     

CD2/LFA-3 ligation induces phospholipase-C gamma 1 tyrosine phosphorylation and regulates CD3 signaling.

Activation of T cells through the TCR/CD3 receptor complex with either specific Ag or antibody results in tyrosine phosphorylation of intracellular protein substrates and phosphatidylinositol-phospholipase C (PLC) signaling, leading to the generation of PI breakdown products and the mobilization of intracellular calcium. Stimulation of the T cell surface receptor CD2 similarly propagates early signals through phosphatidylinositol-PLC activation. Previous reports have shown that CD3 activation leads to tyrosine phosphorylation of the PLC isozyme PLC gamma 1. In this report, we investigated the potential similarity between CD3-induced signaling through PLC gamma 1 and that induced by CD2. We show that stimulation of CD2 receptors on T cells caused tyrosine phosphorylation of PLC gamma 1. Cross-linking of CD2 with CD3 receptors augmented the phosphorylation of PLC gamma 1 on tyrosine, whereas ligation of the CD45 tyrosine phosphatase with CD2 receptors prevented PLC gamma 1 tyrosine phosphorylation. T cells stimulated by ligation of CD2 with its counter-receptor in the form of a soluble LFA-3/Ig fusion protein cross-linked on the cell surface, resulted in a low, but detectable level of PLC gamma 1 phosphorylation with prolonged kinetics, whereas that induced by cross-linking with anti-CD2 was stronger but transient. Co-ligation of LFA-3/Ig with suboptimal concentrations of anti-CD3 resulted in profound augmentation of PLC gamma 1 tyrosine phosphorylation, mobilization of intracellular calcium and T cell proliferation. To explore the relationship between CD3- and CD2-stimulated signaling, T cells were desensitized through 1 h incubation with anti-CD3. CD3 receptor modulation potently down-regulated CD2-induced PLC gamma 1 tyrosine phosphorylation and calcium mobilization. In contrast, PMA or ionomycin treatment did not alter CD2-stimulated tyrosine phosphorylation of PLC gamma 1, suggesting that tyrosine kinase inhibition by CD3 receptor modulation was not caused by signaling events downstream of PLC gamma 1. Taken together, these results support the hypothesis that CD2 provides a potent co-stimulatory signal for CD3-induced T cell activation that is associated with tyrosine kinase(s) and PLC gamma 1.     

Toll-like receptors are key participants in innate immune responses

During an infection, one of the principal challenges for the host is to detect the pathogen and activate a rapid defensive response. The Toll-like family of receptors (TLRs), among other pattern recognition receptors (PRR), performs this detection process in vertebrate and invertebrate organisms. These type I transmembrane receptors identify microbial conserved structures or pathogen-associated molecular patterns (PAMPs). Recognition of microbial components by TLRs initiates signaling transduction pathways that induce gene expression. These gene products regulate innate immune responses and further develop an antigen-specific acquired immunity. TLR signaling pathways are regulated by intracellular adaptor molecules, such as MyD88, TIRAP/Mal, between others that provide specificity of individual TLR- mediated signaling pathways. TLR-mediated activation of innate immunity is involved not only in host defense against pathogens but also in immune disorders. The involvement of TLR-mediated pathways in auto-immune and inflammatory diseases is described in this review article.     

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

Scavenger Receptor SREC-I Mediated Entry of TLR4 into Lipid Microdomains and Triggered Inflammatory Cytokine Release in RAW 264.7 Cells upon LPS Activation

Scavenger receptor associated with endothelial cells I (SREC-I) was shown to be expressed in immune cells and to play a role in the endocytosis of peptides and antigen presentation. As our previous studies indicated that SREC-I required intact Toll-like receptor 4 (TLR4) expression for its functions in tumor immunity, we examined potential interactions between these two receptors. We have shown here that SREC-I became associated with TLR4 on binding bacterial lipopolysaccharides (LPS) in RAW 264.7 and HEK 293 cells overexpressing these two receptors. The receptors then became internalized together in intracellular endosomes. SREC-I promoted TLR4-induced signal transduction through the NF-kB and MAP kinase pathways, leading to enhanced inflammatory cytokine release. Activation of inflammatory signaling through SREC-I/TLR4 complexes appeared to involve recruitment of the receptors into detergent-insoluble, cholesterol-rich lipid microdomains that contained the small GTPase Cdc42 and the non-receptor tyrosine kinase c-src. Under conditions of SREC-I activation by LPS, TLR4 activity required Cdc42 as well as cholesterol and actin polymerization for signaling through NF-kB and MAP kinase pathways in RAW 264.7 cells. SREC-I appeared to respond differently to another ligand, the molecular chaperone Hsp90 that, while triggering SREC-I-TLR4 binding caused only faint activation of the NF-kB pathway. Our experiments therefore indicated that SREC-I could bind LPS and might be involved in innate inflammatory immune responses to extracellular danger signals in RAW 264.7 cells or bone marrow-derived macrophages.     

The role of SLAM family receptors in immune cell signaling.

The signaling lymphocyte-activating molecule (SLAM) family immunoreceptors are expressed in a wide array of immune cells, including both T and B lymphocytes. By virtue of their ability to transduce tyrosine phosphorylation signals through the so-called ITSM (immunoreceptor tyrosine-based switch motif) sequences, they play an important part in regulating both innate and adaptive immune responses. The critical role of the SLAM immunoreceptors in mediating normal immune reactions was highlighted in recent findings that SAP, a SLAM-associated protein, modulates the activities of various immune cells through interactions with different members of the SLAM family expressed in these cells. Importantly, mutations or deletions of the sap gene in humans result in the X-linked lymphoproliferative syndrome. In this review, we summarize current knowledge and survey the latest developments in signal transduction events triggered by the activation of SLAM family receptors in different cell types.     

Modeling of T cell contact-dependent B cell activation. IL-4 and antigen receptor ligation primes quiescent B cells to mobilize calcium in response to Ia cross-linking.

The generation of antibody secretory cells from resting B lymphocytes after immunization with most protein Ag requires B cell signaling by Ag, direct Th cell contact and lymphokines. Previous studies suggest that cell contact-mediated signals may be transduced by Ia after Ia binding by alpha beta TCR and/or CD4. Seemingly inconsistent with this concept are findings that cross-linking of Ia molecules on quiescent B cells leads to cAMP generation that is antagonistic for B cell mitogenesis. Here we show that ligand binding to IL-4 and Ag receptors on quiescent B cells induce transition of these cells into a competent state in which Ia molecules transduce signals via a distinct mechanism. This mechanism involves the tyrosine kinase-dependent activation of phospholipase C leading to Ca2+ mobilization from intracellular stores and the extracellular space. This competence, which is seen within 4 h of priming, is not simply a function of increased Ia expression by the B cell because the response can be induced by cross-linking of less than 5% of cell surface Ia molecules on primed cells. Finally, cross-linking of Ia molecules leads to more than fivefold greater increase in [Ca2+]i than is induced by membrane Ig ligation. These findings are consistent with alpha beta TCR/CD4 delivery via Ia of proliferative signals mediated by tyrosine kinase activation, phosphoinositide hydrolysis and Ca2+ mobilization.     

TIR, CARD and PYRIN: three domains for an antimicrobial triad

Innate immunity to microorganisms in mammals has gained a substantial interest during the last decade. The discovery of the Toll-like receptor (TLR) family has allowed the identification of a class of membrane-spanning receptors dedicated to microbial sensing. TLRs transduce downstream signaling via their intracellular Toll-interleukin-1 receptor (TIR) domain. More recently, the role of intracellular microbial sensors has been uncovered. These molecules include the Nod-like receptors Nod1, Nod2, Ipaf and Nalps, together with the helicase domain-containing antiviral proteins RIG-I and Mda-5. The intracellular microbial sensors lack the TIR domain, but instead transduce downstream signals via two domains also implicated in homophilic protein-protein interactions, the caspase activation and recruitment domain (CARD) and PYRIN domains. In light with these recent findings, we propose that TIR, CARD and PYRIN domains represent the three arms of innate immune detection of microorganisms in mammals.     

An oncogenic epidermal growth factor receptor signals via a p21-activated kinase-caldesmon-myosin phosphotyrosine complex

Many ligand-independent receptor tyrosine kinases are tumorigenic. The biochemical signals that mediate ligand-independent transformation of cells by these transmembrane receptors are poorly defined. In this report, we demonstrate that a constitutively activated mutant epidermal growth factor receptor (v-ErbB) induces the formation of a transformation-specific signaling module that complexes with myosin II. The components of this signaling complex include the signal adapter proteins Shc, Grb2, and Nck, and tyrosine-phosphorylated forms of p21-activated kinase (Pak), caldesmon, and myosin light chain kinase. Transformation-specific, tyrosine phosphorylation of Pak enhances the catalytic activity of this serine/threonine kinase. Furthermore, the tyrosine phosphorylation of Pak is Rho-, but not Ras-, Rac-, or Cdc42-dependent. These results demonstrate that a ligand-independent epidermal growth factor receptor mutant can transduce oncogenic signals that are distinct from ligand-dependent, mitogenic signals. In addition, these data provide evidence for the coupling of oncogenic receptor tyrosine kinases with the actomyosin molecular motor. This myosin-associated signaling module may mediate some of the biochemical changes of myosin found in v-ErbB- transformed fibroblasts, thereby contributing to the regulation of the mechanical forces governing cellular adhesion, cytoskeletal tension, and, hence, anchorage-independent cell growth.     

The CD3-gamma delta epsilon transducing module mediates CD38-induced protein-tyrosine kinase and mitogen-activated protein kinase activation in Jurkat T cells.

We have examined the ability of the CD3-gamma delta epsilon and CD3-zeta signaling modules of the T cell receptor (TCR) to couple CD38 to intracellular signaling pathways. The results demonstrated that in TCR+ T cells that express the whole set of CD3 subunits CD38 ligation led to complete tyrosine phosphorylation of both CD3-zeta and CD3-epsilon polypeptide chains. In contrast, in TCR+ cells with a defective CD3-zeta association CD38 engagement caused tyrosine phosphorylation of CD3-epsilon but not of CD3-zeta. Despite these differences, in both cell types CD38 ligation resulted in protein-tyrosine kinase and mitogen-activated protein kinase activation. However, in cells expressing chimerical CD25-zeta or CD25-epsilon receptors or in a TCR-beta- Jurkat T cell line, CD38 ligation did not result in tyrosine phosphorylation of the chimeric receptors, or CD3 subunits, or protein-tyrosine kinase or mitogen-activated protein kinase activation. In summary, these results support a model in which CD38 transduces activating signals inside the cell by means of CD3-epsilon and CD3-zeta tyrosine phosphorylation. Moreover, these data identify the CD3-gamma delta epsilon signaling module as a necessary and sufficient component of the TCR/CD3 complex involved in T cell activation through CD38.     

Regulation of cytosolic phospholipase A2 activation and cyclooxygenase 2 expression in macrophages by the beta-glucan receptor

Phagocytosis of non-opsonized microorganisms by macrophages initiates innate immune responses for host defense against infection. Cytosolic phospholipase A(2) is activated during phagocytosis, releasing arachidonic acid for production of eicosanoids, which initiate acute inflammation. Our objective was to identify pattern recognition receptors that stimulate arachidonic acid release and cyclooxygenase 2 (COX2) expression in macrophages by pathogenic yeast and yeast cell walls. Zymosan- and Candida albicans-stimulated arachidonic acid release from resident mouse peritoneal macrophages was blocked by soluble glucan phosphate. In RAW264.7 cells arachidonic acid release, COX2 expression, and prostaglandin production were enhanced by overexpressing the beta-glucan receptor, dectin-1, but not dectin-1 lacking the cytoplasmic tail. Pure particulate (1, 3)-beta-D-glucan stimulated arachidonic acid release and COX2 expression, which were augmented in a Toll-like receptor 2 (TLR2)-dependent manner by macrophage-activating lipopeptide-2. However, arachidonic acid release and leukotriene C(4) production stimulated by zymosan and C. albicans were TLR2-independent, whereas COX2 expression and prostaglandin production were partially blunted in TLR2(-/-) macrophages. Inhibition of Syk tyrosine kinase blocked arachidonic acid release and COX2 expression in response to zymosan, C. albicans, and particulate (1, 3)-beta-D-glucan. The results suggest that cytosolic phospholipase A(2) activation triggered by the beta-glucan component of yeast is dependent on the immunoreceptor tyrosine-based activation motif-like domain of dectin-1 and activation of Syk kinase, whereas both TLR2 and Syk kinase regulate COX2 expression.     

Induction of phosphorylation and intracellular association of CC chemokine receptor 5 and focal adhesion kinase in primary human CD4+ T cells by macrophage-tropic HIV envelope.

Binding of HIV-1 envelope glycoproteins to the surface of a CD4+ cell transduces intracellular signals through the primary envelope receptor, CD4, and/or the envelope coreceptor, a seven-transmembrane chemokine receptor. Macrophage-tropic strains of HIV-1 preferentially use CCR5 as an entry coreceptor, whereas T cell-tropic strains use CXC chemokine receptor-4 for entry. Intracellular signals transduced by HIV-1 envelope may have immunopathogenic consequences, including anergy, syncytium formation, apoptosis, and inappropriate cell trafficking. We demonstrate here that a recombinant envelope protein derived from an M-tropic isolate of HIV-1 can transduce CD4-dependent as well as CCR5-dependent intracellular signals in primary human CD4+ T cells. Novel HIV-induced intracellular signals that were identified include tyrosine phosphorylation of focal adhesion kinase (FAK) and CCR5, which are involved in cell adhesion and chemotaxis, respectively. HIV envelope-induced cellular association of FAK and CCR5 was also demonstrated, suggesting that ligation of CD4 and CCR5 leads to the formation of an activation complex composed of FAK and CCR5. Activation of this signaling pathway by HIV-1 envelope may be an important pathogenic mechanism of dysregulated cellular activation and trafficking during HIV infection.     

Ligation of the T cell receptor complex results in phosphorylation of Smad2 in T lymphocytes

TGF-beta modulates immune responses by regulating T cell function. The Smad family of proteins has been recently shown to transduce signals for the TGF-beta superfamily and Smad2 mediates TGF-beta signaling. Here, we showed that TGF-beta phosphorylated Smad2 and induced interaction between Smad2 and Smad4 in primary T cells and the Jurkat T cell line. Interestingly, ligation of the T cell receptor (TCR)/CD3 complex with anti-CD3 mAb also phosphorylated Smad2, but failed to induce interaction between Smad2 and Smad4 in the Jurkat T cell line. Phosphorylation of Smad2 via the TCR/CD3 complex was not abrogated by treatment with neutralizing antibody against TGF-beta. Furthermore, PD98059, a MEK inhibitor, suppressed Smad2 phosphorylation by stimulation with anti-CD3 mAb in Jurkat T cell line. These findings indicated that not only TGF-beta but also stimulation via the TCR/CD3 complex phosphorylated Smad2 through mitogen-activated protein (MAP) kinase cascades, suggesting that Smad2 may function in both TGF-beta- and TCR/CD3 complex-mediated signaling pathways in T cells.     

Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases.

Integrin-mediated cell adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation of intracellular proteins. Among these are the focal adhesion proteins p130cas (Cas) and focal adhesion kinase (FAK). Here we identify the kinase(s) mediating integrin-induced Cas phosphorylation and characterize protein-protein interactions mediated by phosphorylated Cas. We found that expression of a constitutively active FAK in fibroblasts results in a consecutive tyrosine phosphorylation of Cas. This effect required the autophosphorylation site of FAK, which is a binding site for Src family kinases. Integrin-mediated phosphorylation of Cas was not, however, compromised in fibroblasts lacking FAK. In contrast, adhesion-induced tyrosine phosphorylation of Cas was reduced in cells lacking Src, whereas enhanced phosphorylation of Cas was observed Csk- cells, in which Src kinases are activated. These results suggest that Src kinases are responsible for the integrin-mediated tyrosine phosphorylation of Cas. FAK seems not to be necessary for phosphorylation of Cas, but when autophosphorylated, FAK may recruit Src family kinases to phosphorylate Cas. Cas was found to form complexes with Src homology 2 (SH2) domain-containing signaling molecules, such as the SH2/SH3 adapter protein Crk, following integrin-induced tyrosine phosphorylation. Guanine nucleotide exchange factors C3G and Sos were found in the Cas-Crk complex upon integrin ligand binding. These observations suggest that Cas serves as a docking protein and may transduce signals to downstream signaling pathways following integrin-mediated cell adhesion.