Tyrosine Phosphorylation Of Phospholipase C-gamma Research Articles

Peptidoglycan and lipopolysaccharide activate PLCγ2, leading to enhanced cytokine production in macrophages and dendritic cells

In macrophages and monocytes, microbial components trigger the production of pro-inflammatory cytokine through Toll-like receptors (TLRs). Although major TLR signaling pathways are mediated by serine/threonine kinases, including TAK1, IKK and MAP kinases, tyrosine phosphorylation of intracellular proteins by TLR ligands has been suggested in a number of reports. Here, we demonstrated that peptidoglycan (PGN) of a Gram-positive bacterial cell wall component, a TLR2 ligand and lipopoysaccharide (LPS) of a Gram-positive bacterial component, a TLR4 ligand induced tyrosine phosphorylation of phospholipase Cgamma-2 (PLCgamma2), leading to intracellular free Ca2+ mobilization in bone marrow-derived macrophages (BMMphi) and bone marrow-derived dendritic cells (BMDC). PGN- and LPS-induced Ca2+ mobilization was not observed in BMDC from PLCgamma2 knockout mice. Thus, PLCgamma2 is essential for TLR2 and TLR4-mediated Ca2+ flux. In PLCgamma2-knockdown cells, PGN-induced IkappaB-alpha phosphorylation and p38 activation were reduced. Moreover, PLCgamma2 was necessary for the full production of TNF-alpha and IL-6. These data indicate that the PLCgamma2 pathway plays an important role in bacterial ligands-induced activation of macrophages and dendritic cells.

Human cytomegalovirus induces cellular tyrosine kinase signaling and promotes glioma cell invasiveness

Given our previous findings that human cytomegalovirus (HCMV) nucleic acids and proteins are expressed in human malignant glioma in vivo, we investigated cellular signaling events associated with HCMV infection of human glioma and astroglial cells. HCMV infection caused rapid activation of the phosphatidylinositol-3 kinase (PI-3K) effector AKT kinase in human astro-glial and fibroblast cells, and induced tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Co-immunoprecipitation experiments revealed association of the p85 regulatory subunit of PI-3K with a high-molecular weight protein phosphorylated on tyrosine, following short-term exposure to HCMV. In contrast to a previous report, we were unable to confirm the identity of this high-molecular weight protein as being the epidermal growth factor receptor (EGFR). Stimulation of glioma and fibroblast cell lines over-expressing EGFR with HCMV (whole virus) or soluble glycoprotein B did not induce tyrosine phosphorylation of the receptor, as did the genuine ligand, EGF. Furthermore, we found that expression levels of the human ErbB1-4 receptors were not rate-limiting for HCMV infection. Dispensability of EGFR function during early HCMV infection was substantiated by demonstration of viral immediate early gene expression in cells lacking the EGFR gene, indicating that HCMV may promote oncogenic signaling pathways independently of EGFR activation. Among non-receptor cellular kinases, HCMV infection induced phosphorylation of focal adhesion kinase (FAK) Tyr397, which is indispensable for integrin-mediated cell migration and invasion. HCMV-induced FAK activation was paralleled by increased extracellular matrix-dependent migration of human malignant glioma but not normal astro-glial cells, suggesting that HCMV can selectively augment glioma cell invasiveness.

Bruton's Tyrosine Kinase and Phospholipase Cγ2 Mediate Chemokine-Controlled B Cell Migration and Homing

Control of integrin-mediated adhesion and migration by chemokines plays a critical role in B cell development, differentiation, and function; however, the underlying signaling mechanisms are poorly defined. Here we show that the chemokine SDF-1 induced activation of Bruton's tyrosine kinase (Btk) and that integrin-mediated adhesion and migration in response to SDF-1 or CXCL13, as well as in vivo homing to lymphoid organs, was impaired in Btk-deficient (pre-)B cells. Furthermore, SDF-1 induced tyrosine phosphorylation of Phospholipase Cgamma2 (PLCgamma2), which, unlike activation of the migration regulatory GTPases Rac or Rap1, was mediated by Btk. PLCgamma2-deficient B cells also exhibited impaired SDF-1-controlled migration. These results reveal that Btk and PLCgamma2 mediate chemokine-controlled migration, thereby providing insights into the control of B cell homeostasis, trafficking, and function, as well as into the pathogenesis of the immunodeficiency disease X-linked agammaglobulinemia (XLA).

SHP Protein Tyrosine Phosphatase Expression in Rat Uterine Tissue

Enhanced tyrosine phosphorylation of phospholipase C-gamma1 (PLCgamma1) is associated with increased spontaneous contractile activity. PLCgamma1 phosphorylation is regulated by cellular protein tyrosine kinases and tyrosine phosphatases (PTPs). The studies in this report were undertaken to characterize the expression of two PTPs known to bind to PLCgamma1: Src-homology phosphatase type-1 (SHP-1) and type-2 (SHP-2). Uterine and other tissues were obtained from non-pregnant (estrus) and pregnant (gestational day 12 through day 1 postpartum) Sprague-Dawley rats. PTP activity in myometrial homogenates was determined using an in vitro fluorometric PTP assay with and without bpV(phen) (a nonselective PTP inhibitor), or PTP-Inhibitor 1 (PTP-I1, a SHP selective inhibitor). Western blots were performed using polyclonal antibodies to SHP-1 and SHP-2. Immunoprecipitation studies were performed to demonstrate an association between PLCgamma1 and the SHP proteins. The in vitro PTP assays demonstrated comparable enzyme activity in myometrium from estrus and pregnant animals. BpV(phen) produced a 93% reduction in PTP activity (P <.05); similarly, PTP-I1 produced an 86% reduction in enzyme activity (P <.05). Western blots confirmed robust expression of both SHP-1 and SHP-2 protein in rat uterus. SHP-1 expression decreased significantly at the end of gestation; in contrast, SHP-2 levels remained stable. Immunoprecipitation studies confirmed an association between the SHP proteins and PLCgamma1. These studies have demonstrated that SHP-1 and SHP-2 are expressed in rat myometrium and appear to be responsible for the PTP activity in this tissue, thereby providing a molecular mechanism for the modulation of PLCgamma1 phosphotyrosine levels in the rat uterus.

Vascular Endothelial Growth Factor (VEGF)-D and VEGF-A Differentially Regulate KDR-mediated Signaling and Biological Function in Vascular Endothelial Cells

Vascular endothelial growth factor (VEGF)-D binds to VEGF receptors (VEGFR) VEGFR2/KDR and VEGFR3/Flt4, but the signaling mechanisms mediating its biological activities in endothelial cells are poorly understood. Here we investigated the mechanism of action of VEGF-D, and we compared the signaling pathways and biological responses induced by VEGF-D and VEGF-A in endothelial cells. VEGF-D induced KDR and phospholipase C-gamma tyrosine phosphorylation more slowly and less effectively than VEGF-A at early times but had a more sustained effect and was as effective as VEGF-A after 60 min. VEGF-D activated extracellular signal-regulated protein kinases 1 and 2 with similar efficacy but slower kinetics compared with VEGF-A, and this effect was blocked by inhibitors of protein kinase C and mitogen-activated protein kinase kinase. In contrast to VEGF-A, VEGF-D weakly stimulated prostacyclin production and gene expression, had little effect on cell proliferation, and stimulated a smaller and more transient increase in intracellular [Ca(2+)]. VEGF-D induced strong but more transient phosphatidylinositol 3-kinase (PI3K)-mediated Akt activation and increased PI3K-dependent endothelial nitric-oxide synthase phosphorylation and cell survival more weakly. VEGF-D stimulated chemotaxis via a PI3K/Akt- and endothelial nitric-oxide synthase-dependent pathway, enhanced protein kinase C- and PI3K-dependent endothelial tubulogenesis, and stimulated angiogenesis in a mouse sponge implant model less effectively than VEGF-A. VEGF-D-induced signaling and biological effects were blocked by the KDR inhibitor SU5614. The finding that differential KDR activation by VEGF-A and VEGF-D has distinct consequences for endothelial signaling and function has important implications for understanding how multiple ligands for the same VEGF receptors can generate ligand-specific biological responses.

Impaired signaling via the high-affinity IgE receptor in Wiskott-Aldrich syndrome protein-deficient mast cells.

Wiskott-Aldrich syndrome protein (WASP) is the product of the gene deficient in boys with X-linked Wiskott-Aldrich syndrome. We assessed the role of WASP in signaling through the high-affinity IgE receptor (FcepsilonRI) using WASP-deficient mice. IgE-dependent degranulation and cytokine secretion were markedly diminished in bone marrow-derived mast cells from WASP-deficient mice. Upstream signaling events that include FcepsilonRI-triggered total protein tyrosine phosphorylation, and protein tyrosine phosphorylation of FcepsilonRIbeta and Syk were not affected by WASP deficiency. However, tyrosine phosphorylation of phospholipase Cgamma and Ca(2+) mobilization were diminished. IgE-dependent activation of c-Jun N-terminal kinase, cell spreading and redistribution of cellular F-actin in mast cells were reduced in the absence of WASP. We conclude that WASP regulates FcepsilonRI-mediated granule exocytosis, cytokine production and cytoskeletal changes in mast cells.

Src Kinase Mediates the Regulation of Phospholipase C-γ Activity by Glycosphingolipids

Glucosylceramide-based glycosphingolipids have been previously demonstrated to regulate negatively the formation of inositol 1,4,5-trisphosphate by phospholipase C-gamma1. In the present study, the depletion of endogenous glucosylceramide by D-t-EtDO-P4 in cultured ECV304 cells induced autophosphorylation of Src kinase at tyrosine residue 418 within the catalytic loop and dephosphorylation of Src kinase at tyrosine residues 529 within the carboxyl-terminal regulatory region. Phosphotransferase activities of Src kinase were also induced in the glucosylceramide-depleted cells. c-Src kinase activity and phosphorylations at Src Tyr-418 and epidermal growth factor (EGF) receptor Tyr-1068 were significantly enhanced by bradykinin in response to 100 nm D-t-EtDO-P4 compared with control cells. The phosphorylation and dephosphorylation on Tyr-418 and Tyr-529 residues of c-Src were reversed by treatment of 4-amino-5-(4-chlorophenyl)-7-t-butyl(pyrazolo)[3,4-d]pyrimidine (PP2), an inhibitor of Src kinase, in control cells. Glucosylceramide-depleted cells resisted treatment with PP2, and both phosphorylation of Tyr-418 and dephosphorylation of Tyr-529 induced by depletion of glucosylceramide were maintained. Compared with untreated cells, tyrosine phosphorylation of phospholipase C-gamma1 was enhanced by EGF stimulation in glucosylceramide-depleted cells, associated with enhanced tyrosine phosphorylation of the EGF receptor at Tyr-1068 and Tyr-1086 stimulated by EGF. The Src inhibitor, PP2, significantly blocked EGF-induced tyrosine phosphorylation of phospholipase C-gamma1 in control cells, whereas in glucosylceramide-depleted cells, suppression of Src kinase activity by PP2 toward EGF-induced tyrosine phosphorylation of phospholipase C-gamma1 was less significant. Thus the activation of Src kinase by depletion of glucosylceramide-based glycosphingolipids in cultured ECV304 cells is a critical up-stream event in the activation of phospholipase C-gamma1.

Rosmarinic acid inhibits Ca2+-dependent pathways of T-cell antigen receptor-mediated signaling by inhibiting the PLC-γ1 and Itk activity

Rosmarinic acid (RosA) is a hydroxylated compound frequently found in herbal plants and is mostly responsible for anti-inflammatory and antioxidative activity. Previously, we observed that RosA inhibited T-cell antigen receptor (TCR)- induced interleukin 2 (IL-2) expression and subsequent T-cell proliferation in vitro. In this study, we investigated in detail inhibitory mechanism of RosA on TCR signaling, which ultimately activates IL-2 promoter by activating transcription factors, such as nuclear factor of activated T cells (NF-AT) and activating protein-1 (AP-1). Interestingly, RosA inhibited NF-AT activation but not AP-1, suggesting that RosA inhibits Ca(2+)-dependent signaling pathways only. Signaling events upstream of NF-AT activation, such as the generation of inositol 1,4,5-triphosphate and Ca(2+) mobilization, and tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1) were strongly inhibited by RosA. Tyrosine phosphorylation of PLC-gamma 1 is largely dependent on 3 kinds of protein tyrosine kinases (PTKs), ie, Lck, ZAP-70, and Itk. We found that RosA efficiently inhibited TCR-induced tyrosine phosphorylation and subsequent activation of Itk but did not inhibit Lck or ZAP-70. ZAP-70-dependent signaling pathways such as the tyrosine phosphorylation of LAT and SLP-76 and serine/threonine phosphorylation of mitogen-activated protein kinases (MAPKs) were intact in the presence of RosA, confirming that RosA suppresses TCR signaling in a ZAP-70-independent manner. Therefore, we conclude that RosA inhibits TCR signaling leading to Ca(2+) mobilization and NF-AT activation by blocking membrane-proximal events, specifically, the tyrosine phosphorylation of inducible T cells kinase (Itk) and PLC-gamma 1.

Tyrosine phosphatase CD45 regulates hydrogen peroxide-induced calcium mobilization in B cells.

By taking advantage of established CD45-deficient DT40 cells, the roles of CD45 in oxidative stress signaling were investigated. Using p-nitrophenyl phosphate as substrate, it was found that CD45 constituted nearly 40% of the total protein-tyrosine phosphatase activity. Almost 90% of the phosphatase activity was rapidly inactivated upon hydrogen peroxide treatment. Hydrogen peroxide-induced tyrosine phosphorylation of cellular proteins and c-Jun N-terminal kinase activation were markedly enhanced in CD45-deficient cells relative to that in its parental cells. In comparison, hydrogen peroxide-induced inositol 1,4,5-trisphosphate production and Ca(2+) mobilization were impaired in CD45-deficient DT40 cells. However, hydrogen peroxide-induced tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2), phosphatidylinositol 3-kinase activity precipitated by anti-phosphotyrosine antibody, and activation of Bruton's tyrosine kinase appeared intact in CD45-deficient DT40 cells. This suggests that CD45 mediates the ability of hydrogen peroxide-activated PLCgamma2 to hydrolyze its substrate via a mechanism independent of both tyrosine phosphorylation of PLCgamma2 and phosphatidylinositol 3-kinase, as well as activation of Bruton's tyrosine kinase. Taken together, our observations demonstrated that, in addition to its negative regulatory or phosphatase activity, CD45 has a positive role in oxidative stress signaling.

Regulation of Phospholipase C-γ Activity by Glycosphingolipids

Glycosphingolipid-enriched domains are hot spots for cell signaling within plasma membranes and are characterized by the enrichment of glycosphingolipids. A role for glucosylceramide-based glycosphingolipids in phospholipase C-mediated inositol 1,4,5-trisphosphate formation has been previously documented. These earlier studies utilized a first generation glucosylceramide synthase inhibitor to deplete cells of their glycosphingolipids. Recently, more active and specific glucosylceramide synthase inhibitors, including d-threo-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidinopropanol (d-t-EtDO-P4), have been designed. d-t-EtDO-P4 has the advantage of blocking glucosylceramide synthase at low nanomolar concentrations but does not cause secondary elevations in cell ceramide levels. In the present study, d-t-EtDO-P4 depleted cellular glucosylceramide and lactosylceramide in cultured ECV304 cells at nanomolar concentrations without obvious cellular toxicity. The expression of several signaling proteins was evaluated in glycosphingolipid-depleted ECV304 cells to study the role of glycosphingolipids in phospholipase C-mediated signaling. No difference was observed in the cellular expression of phospholipase C-gamma between controls and glycolipid-depleted cells. Western blot analysis, however, revealed that depletion of endogenous glycosphingolipids in cultured ECV304 cells with d-t-EtDO-P4 induced tyrosine phosphorylation of phospholipase C-gamma in a concentration-dependent manner with maximum induction at 100 nm. The phosphorylation of phospholipase C-gamma induced by d-t-EtDO-P4 was abolished by exogenously added glucosylceramide, consistent with a specific glycosphingolipid-phospholipase C-gamma interaction. The phospholipase C-gamma phosphorylation was maximally enhanced by bradykinin when cells were exposed to 100 nm d-t-EtDO-P4. The measurement of cellular activity of phospholipase C-gamma, by myo-inositol 1,4,5-trisphosphate radioreceptor assay, demonstrated that depletion of glucosylceramide-based glycosphingolipids in cultured ECV304 cells with d-t-EtDO-P4 resulted in significantly increased formation of inositol 1,4,5-trisphosphate above base line, and an increased sensitivity of phospholipase C-gamma to bradykinin stimulation. Thus, the activation of phospholipase C-gamma is negatively regulated by membrane glycosphingolipids in ECV304 cells.

Glycoprotein IIb-IIIa-dependent aggregation by glycoprotein Ibalpha is reinforced by a Src family kinase inhibitor (PP1)-sensitive signalling pathway.

It has been proposed that the receptor for von Willebrand factor (vWF), glycoprotein (GP)Ib-IX-V, signals through the same pathway as the collagen receptor, GPVI, namely via Src kinases, the Fc receptor (FcR) gamma-chain and Syk, leading to tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2). The aim of the present study was to assess the functional significance of this pathway in platelet activation by GPIb-IX-V. In washed platelets, vWF/ristocetin and vWF/botrocetin stimulate weak tyrosine phosphorylation of the FcR gamma-chain, Syk and PLCgamma2, but not the adaptor LAT (linker for activation of T-cells), which is localized to glycolipid-enriched membrane domains. Increases in tyrosine phosphorylation were blocked by the Src family kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1). Under the same conditions, neither stimulus induced activation of PLCgamma2 nor functional responses, such as Ca(2+) elevation, secretion or GPIIb-IIIa-dependent aggregation. In contrast, in platelet-rich plasma (PRP), threshold concentrations of ristocetin or asialo-vWF stimulated GPIb-dependent biphasic aggregation, in which the second phase was blocked by PP1. Importantly, a significant component of the initial phase and the complete second phase of aggregation was blocked by GPIIb-IIIa receptor antagonists in PRP. Higher concentrations of ristocetin stimulated GPIIb-IIIa-independent agglutination in PRP. These results demonstrate that GPIb-IX-V initiates activation of GPIIb-IIIa in PRP through an undefined pathway that is reinforced by a PP1-sensitive pathway. In contrast, activation of GPIbalpha in washed platelets does not promote functional responses.

Zap-70-independent Ca(2+) mobilization and Erk activation in Jurkat T cells in response to T-cell antigen receptor ligation.

The tyrosine kinase ZAP-70 has been implicated as a critical intermediary between T-cell antigen receptor (TCR) stimulation and Erk activation on the basis of the ability of dominant negative ZAP-70 to inhibit TCR-stimulated Erk activation, and the reported inability of anti-CD3 antibodies to activate Erk in ZAP-70-negative Jurkat cells. However, Erk is activated in T cells receiving a partial agonist signal, despite failing to activate ZAP-70. This discrepancy led us to reanalyze the ZAP-70-negative Jurkat T-cell line P116 for its ability to support Erk activation in response to TCR/CD3 stimulation. Erk was activated by CD3 cross-linking in P116 cells. However, this response required a higher concentration of anti-CD3 antibody and was delayed and transient compared to that in Jurkat T cells. Activation of Raf-1 and MEK-1 was coincident with Erk activation. Remarkably, the time course of Ras activation was comparable in the two cell lines, despite proceeding in the absence of LAT tyrosine phosphorylation in the P116 cells. CD3 stimulation of P116 cells also induced tyrosine phosphorylation of phospholipase C-gamma1 (PLCgamma1) and increased the intracellular Ca(2+) concentration. Protein kinase C (PKC) inhibitors blocked CD3-stimulated Erk activation in P116 cells, while parental Jurkat cells were refractory to PKC inhibition. The physiologic relevance of these signaling events is further supported by the finding of PLCgamma1 tyrosine phosphorylation, Erk activation, and CD69 upregulation in P116 cells on stimulation with superantigen and antigen-presenting cells. These results demonstrate the existence of two pathways leading to TCR-stimulated Erk activation in Jurkat T cells: a ZAP-70-independent pathway requiring PKC and a ZAP-70-dependent pathway that is PKC independent.

Nitric oxide activation of TrkB through peroxynitrite

NIH-3T3 cells stably transfected with TrkB, the receptor for brain-derived neurotrophic factor (BDNF), were used to study the effects of NO and peroxynitrite on TrkB. 3-Morpholinosydnonimine (SIN-1), a donor of NO and O2- which immediately react to form peroxynitrite, induced TrkB tyrosine phosphorylation in a dose-dependent relationship from 2 to 40 mM. TrkB phosphorylation by SIN-1 was blocked by superoxide dismutase, which converts O2 to H2O2 and prevents its reaction with NO to form peroxynitrite, and by K252a, an inhibitor of TrkB phosphorylation by BDNF. Treatment with NO or O2- alone did not activate TrkB. Treatment directly with 1-4 mM peroxynitrite resulted in a dose-dependent increase in tyrosine phosphorylation of TrkB. SIN-1 treatment induced tyrosine phosphorylation of phospholipase C-gamma1 (PLC-gamma1) and induced its binding with activated TrkB, similar to that seen with BDNF downstream signaling pathways. These studies demonstrate activation of TrkB through peroxynitrite.

Signal transduction and transforming properties of the TEL-TRKC fusions associated with t(12;15)(p13;q25) in congenital fibrosarcoma and acute myelogenous leukemia.

The TEL-TRKC fusion is expressed as a consequence of t(12;15)(p13;q25), and is associated with two human cancers: congenital fibrosarcoma and acute myelogenous leukemia (AML). We report that the T/T(F) and T/T(L) fusion variants associated with congenital fibrosarcoma and AML, respectively, are constitutively tyrosine phosphorylated, and confer factor-independent growth to the murine hematopoietic cell line Ba/F3. Retroviral transduction of T/T(L) causes a rapidly fatal myeloproliferative disease in a murine bone marrow transplant (BMT) model, whereas T/T(F) causes a long-latency, pre-B-cell lymphoblastic lymphoma. TEL-TRKC variants are potent activators of the MAP kinase pathway, but neither variant activates Stat5 or other Stat family members. T/T(L), but not T/T(F), induces tyrosine phosphorylation of phospholipase Cgamma (PLCgamma), phosphoinositol-3 kinase and SHC. However, mutation analysis demonstrates that PLCgamma tyrosine phos phorylation by T/T(L) is dispensable for induction of the myeloproliferative phenotype by T/T(L). Collectively, these data demonstrate that the TEL-TRKC fusion variants are oncoproteins that activate the MAP kinase pathway, and do not require activation of either PLCgamma or Stat5 for efficient induction of a myeloproliferative phenotype in the murine BMT model.

Utilization of Distinct Signaling Pathways by Receptors for Vascular Endothelial Cell Growth Factor and Other Mitogens in the Induction of Endothelial Cell Proliferation

This study was initiated to identify signaling proteins used by the receptors for vascular endothelial cell growth factor KDR/Flk1, and Flt1. Two-hybrid cloning and immunoprecipitation from human umbilical vein endothelial cells (HUVEC) showed that KDR binds to and promotes the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Neither placental growth factor, which activates Flt1, epidermal growth factor (EGF), or fibroblast growth factor (FGF) induced tyrosine phosphorylation of PLCgamma, indicating that KDR is uniquely important to PLCgamma activation in HUVEC. By signaling through KDR, VEGF promoted the tyrosine phosphorylation of focal adhesion kinase, induced activation of Akt, protein kinase Cepsilon (PKCepsilon), mitogen-activated protein kinase (MAPK), and promoted thymidine incorporation into DNA. VEGF activates PLCgamma, PKCepsilon, and phosphatidylinositol 3-kinase independently of one another. MEK, PLCgamma, and to a lesser extent PKC, are in the pathway through which KDR activates MAPK. PLCgamma or PKC inhibitors did not affect FGF- or EGF-mediated MAPK activation. MAPK/ERK kinase inhibition diminished VEGF-, FGF-, and EGF-promoted thymidine incorporation into DNA. However, blockade of PKC diminished thymidine incorporation into DNA induced by VEGF but not FGF or EGF. Signaling through KDR/Flk1 activates signaling pathways not utilized by other mitogens to induce proliferation of HUVEC.

Involvement of CD2 and CD3 in galectin-1 induced signaling in human Jurkat T-cells.

Galectin-1 (gal-1) a member of the mammalian beta-galactoside-binding proteins recognizes preferentially Galbeta1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. In the present work, gal-1 has been identified to be a ligand for the CD3-complex as well as for CD2 as detected by affinity chromatography of Jurkat T-cell lysates on gal-1 agarose and by binding of the biotinylated lectin to CD3 and CD2 immunoprecipitates on blots. In CD45(+)Jurkat E6.1 cells, the lectin stimulates a sustained increase in the intracytoplasmic calcium concentration ([Ca(2+)](i)) consisting of both the release of calcium from intracellular stores and the calcium influx from the extracellular space. This effect of gal-1 on [Ca(2+)](i)is completely inhibited by lactose at 10 mM and was absent in CD45(-)Jurkat J45.01 cells. Preincubation of Jurkat E6.1 cells with cholera toxin or with the protein tyrosine kinase inhibitor herbimycin A reduced the gal-1 induced calcium response whereas the increase in [Ca(2+)](i)stimulated by CD2 or CD3 monoclonal antibodies (mAbs) was completely inhibited. Depolarization of E6.1 cells in a high-potassium buffer, a standard method to activate voltage-operated calcium channels, was without effect on [Ca(2+)](i). Membrane depolarization with gramicidin or by a high-potassium buffer was without effects on the lectin-mediated calcium release from intracellular stores but inhibited the gal-1 induced receptor-operated calcium influx. In Jurkat E6.1 cells the lectin stimulates the transient generation of inositol-1,4,5-trisphosphate and the tyrosine phosphorylation of phospholipase Cgamma1. The results suggest that the ligation of CD2 and CD3 by gal-1 induces early events in T-cell activation comparable with that elicited by CD2 or CD3 mAbs.

Pp60v-src reactivation inhibits serum-induced accumulation of inositol phosphates and phosphatidylethanol in tsNRK.

In tsRSV-infected NRK (tsNRK) cells, pp60(v-src) reactivation by temperature-shift from a nonpermissive temperature, 39 C, to a permissive one, 32 degrees C, induced the production of inositol phosphates (IPt) and phosphatidylethanol (PEt). This was accompanied by an increase in membrane-associated protein kinase C (PKC) activity in the absence of exogenous growth factors. However, with serum-stimulation, the amounts of IPt and PEt at 32 degrees C were less than those at 39 degrees C. Pretreatment with PKC inhibitors, Ro-31-8220 and staurosporine, enhanced the accumulation of IPt but not of PEt at 32 degrees C. The tyrosine phosphorylation of phospholipase Cgamma1 (PLCgamma1) was increased either by serum or by pp60(v-src) reactivation. These results suggest that serum transduces its signal through PLCgamma1 mediation, and that pp60(v-src), possibly through the PKC mediation, negatively affects serum-induced PLCgamma1 activation.

Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line.

The adaptor molecule LAT (linker for activation of T cells) is a palmitoylated integral membrane protein that localizes to the glycolipid-enriched microdomains in the plasma membrane. Upon TCR engagement, LAT becomes phosphorylated on multiple tyrosine residues and then binds several critical signaling molecules. Here, we describe the generation and characterization of a LAT-deficient cell line. Using this cell line, we demonstrate that LAT is required for TCR-mediated Ca2+ mobilization and optimal tyrosine phosphorylation of phospholipase C-gamma1, Vav and SLP-76. LAT is also required for Erk activation, CD69 up-regulation, and AP- and NFAT-mediated gene transcription. We also demonstrate, by reconstituting this cell line with LAT mutants, that the LAT transmembrane domain and palmitoylation at Cys26, but not Cys29, are required for LAT function and TCR signaling. These studies provide further evidence for the crucial role of the LAT molecule, and demonstrate the use of a LAT-deficient cell line for the analysis of LAT structure and function.

Fetal hemorrhage and platelet dysfunction in SLP-76-deficient mice.

The adapter protein SLP-76 is expressed in T lymphocytes and hematopoietic cells of the myeloid lineage, and is known to be a substrate of the protein tyrosine kinases that are activated after ligation of the T-cell antigen receptor. Transient overexpression of SLP-76 in a T-cell line potentiates transcriptional activation after T-cell receptor ligation, while loss of SLP-76 expression abrogates several T-cell receptor-dependent signaling pathways. Mutant mice that lack SLP-76 manifest a severe block at an early stage of thymocyte development, implicating SLP-76 in signaling events that promote thymocyte maturation. While it is clear that SLP-76 plays a key role in development and activation of T lymphocytes, relatively little is understood regarding its role in transducing signals initiated after receptor ligation in other hematopoietic cell types. In this report, we describe fetal hemorrhage and perinatal mortality in SLP-76-deficient mice. Although megakaryocyte and platelet development proceeds normally in the absence of SLP-76, collagen-induced platelet aggregation and granule release is markedly impaired. Furthermore, treatment of SLP-76-deficient platelets with collagen fails to elicit tyrosine phosphorylation of phospholipase C-gamma2 (PLC-gamma2), suggesting that SLP-76 functions upstream of PLC-gamma2 activation. These data provide one potential mechanism for the fetal hemorrhage observed in SLP-76-deficient mice and reveal that SLP-76 expression is required for optimal receptor-mediated signal transduction in platelets as well as T lymphocytes.

Dual effects of heparin on VEGF binding to VEGF receptor-1 and transduction of biological responses.

Vascular endothelial growth factor (VEGF) regulates blood vessel formation by binding to the receptor tyrosine kinases VEGF receptor-1 (Flt-1) or VEGF receptor-2 (KDR) and to the structurally unrelated neuropilins. As exon 7-containing isoforms of VEGF bind to heparin, angiogenesis may be modulated by heparin/heparan sulfate. We analyzed the effect of heparin on VEGF(165)-binding and activation of VEGF receptor-1 in porcine aortic endothelial cells, which lack expression of VEGF receptor-2 and neuropilins. Heparin decreased binding of (125)I-VEGF to 50% at 5 microg/ml and cross-linking of (125)I-VEGF to VEGF receptor-1 on intact cells was similarly decreased. Schatchard analyses showed that the affinity for binding of (125)I-VEGF to VEGF receptor-1 was decreased in the presence of heparin. In contrast, VEGF receptor-1 kinase activity was elevated when cells were treated simultaneously with VEGF and heparin. In accordance, VEGF-induced tyrosine phosphorylation of phospholipase Cgamma (PLCgamma) and DNA synthesis were augmented by heparin. However, basal PLCgamma tyrosine phosphorylation and DNA synthesis levels were to some extent increased by incubation of cells with heparin alone. We conclude that although heparin decreases binding of VEGF to VEGF receptor-1, the remaining binding results in more efficient kinase activation. Taken together, there is no loss of VEGF/VEGF receptor-1 function in the presence of heparin.