Phosphorylation Of Phospholipase C-gamma Research Articles

423 Gentian violet induces pro-apoptotic and anti-proliferative effects in cutaneous T-cell lymphoma (CTCL): Preclinical studies

Previously, we showed that triggering the extrinsic apoptotic pathway by up-regulating activation-induced cell death (AICD) is a very effective way to kill CTCL cells in-vitro and ex-vivo (J Invest Dermatol. 2015; 135: 861-8). To identify novel small molecules that induce extrinsic apoptosis in CTCL, we used CTCL line HH in an ELISA assay to detect cleaved caspase 8 induced by 1710 unique compounds in the NIH Clinical Collection, Selleck Kinase Inhibitor Library and Prestwick Chemical Library. As assessed by Annexin V/PI flow cytometry of HH, the non-prescription topical antimicrobial remedy, gentian violet (GV), induced more total apoptosis than mechlorethamine (FDA-approved for topical therapy of CTCL). Furthermore, GV induced roughly 4-6X greater apoptosis in CTCL lines (MyLa, SeAx, Hut78, HH, SZ4) than in normal keratinocytes, suggesting a favorable topical toxicity profile. In addition to increasing caspase 8, GV also upregulated death receptors DR4, DR5 and ligands TRAIL, FASL. This is consistent with induction of extrinsic apoptosis via the TRAIL and FAS pathways. Increased phosphorylation of phospholipase C-gamma 1, Ca++ influx and ROS were also detected in CTCL lines, indicating the mechanism of FASL upregulation involves key elements of the AICD pathway. Methotrexate significantly increased GV apoptosis in Sezary blood cells and all CTCL lines tested except FAS-null SeAx, suggesting the role of FAS in this process. In ex-vivo studies, 1μM GV induced up to 90% CTCL apoptosis in Sezary blood cells. GV also reduced expression of anti-apoptotic MCL-1 and pro-proliferative NFkB components while increasing IkB levels that promote NFkB catabolism. Furthermore, GV inhibited cell proliferation and induced cell cycle arrest. Although purple at neutral pH, GV can be rendered colorless by altering its pH. These preclinical findings broaden our knowledge of the anti-neoplastic effects of GV and provide a rationale for clinical studies of its utility as a novel inexpensive topical therapy for CTCL.

Mechanisms of carvacrol-induced expression of type I collagen gene

Skin aging is accompanied by wrinkle formation and appears to be principally related to decreases in the levels of type I collagen, the primary component of the dermal layer of skin. To investigate the effect of carvacrol on collagen gene expression and its mechanisms of action. To elucidate the effect of carvacrol on collagen expression and its mechanism, several experiments were performed in human dermal fibroblasts. Collagen production, small interference RNA, Ca(2+) mobilization, COL1A2/AP-1 luciferase reporter assays and Western blots for proteins that are involved in collagen gene expression were used in this study. Carvacrol activated both the human COL1A2 promoter activity and the synthesis of human type I procollagen. Additionally, we attempted to characterize the mechanism of action of carvacrol in type I procollagen synthesis. In a human COL1A2 promoter luciferase assay, the small interference RNA for SP-1 did not reduce the carvacrol-induced promoter activation. Also, Smad 2 phosphorylation was not induced by carvacrol. However, in the AP-1 (activator protein-1) luciferase reporter assay and in the Western blot analysis for mitogen-activated protein kinases (MAPKs), carvacrol induced the activation of the AP-1 promoter and the phosphorylation of JNK and ERK1/2 (p42/44 MAPK), but did not induce the phosphorylation of p38 MAPK. Carvacrol also induced intracellular Ca(2+) mobilization and phosphorylation of phospholipase Cgamma1 (PLCgamma1), a molecule upstream of Ca(2+). In addition, PAO, a PLCgamma1 inhibitor, attenuated the carvacrol-induced production of collagen. Our study suggests that the PLCgamma1 signaling pathway may be involved in the carvacrol-induced expression of the type I collagen gene.

Selinidin Suppresses IgE-Mediated Mast Cell Activation by Inhibiting Multiple Steps of Fc.EPSILON.RI Signaling

IgE-mediated mast cell activation is critical for development of allergic inflammation. We have recently found that selinidin, one of the coumarin derivatives isolated from Angelica keiskei, attenuates mast cell degranulation following engagement of the high-affinity receptor for IgE (Fc epsilonRI) with IgE and antigen. In the present study, we investigated the effects of selinidin on intracellular signaling and mast cell activation employing bone marrow-derived mast cells. Here, we report that selinidin attenuates the release of beta-hexosaminidase, synthesis of leukotriene C4, and production of tumor necrosis factor-alpha without affecting IgE-Fc epsilonRI binding. Furthermore, biochemical analyses of the Fc epsilonRI-mediated signaling pathway demonstrated that selinidin decreases phosphorylation of phospholipase C-gamma1, p38 mitogen-activated protein kinase, and IkappaB-alpha upon FcepsilonRI stimulation. These results suggest that this compound suppresses IgE-mediated mast cell activation by inhibiting multiple steps of FcepsilonRI-dependent signaling pathways and would be beneficial for the prevention of allergic inflammation.

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.

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.

Zebrafish G protein γ2 is required for VEGF signaling during angiogenesis

Vascular endothelial growth factor (VEGF) is a major mediator of pathologic angiogenesis, a process necessary for the formation of new blood vessels to support tumor growth. Historically, VEGF has been thought to signal via receptor tyrosine kinases, which are not typically considered to be G protein dependent. Here, we show that targeted knockdown of the G protein gng2 gene (Ggamma2) blocks the normal angiogenic process in developing zebrafish embryos. Moreover, loss of gng2 function inhibits the ability of VEGF to promote the angiogenic sprouting of blood vessels by attenuating VEGF induced phosphorylation of phospholipase C-gamma1 (PLCgamma1) and serine/threonine kinase (AKT). Collectively, these results demonstrate a novel interaction between Ggamma2- and VEGF-dependent pathways to regulate the angiogenic process in a whole-animal model. Blocking VEGF function using a humanized anti-VEGF antibody has emerged as a promising treatment for colorectal, non-small lung cell, and breast cancers. However, this treatment may cause considerable side effects. Our findings provide a new opportunity for cotargeting G protein- and VEGF-dependent pathways to synergistically block pathologic angiogenesis, which may lead to a safer and more efficacious therapeutic regimen to fight cancer.

The phosphorylation of phospholipase C-gamma1, Raf-1, MEK, and ERK1/2 induced by a conserved retroviral peptide

A synthetic 17-amino acid peptide (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane proteins has been found to exhibit suppressive properties for numerous immune functions. It has been shown that CKS-17 causes an imbalance of human types 1 and 2 cytokines and inhibition of the immune responses of lymphocytes, monocytes, and macrophages. CKS-17 induced increased intracellular levels of cAMP, which plays an important role in regulation of cytokine biosynthesis. In this study, using a Jurkat T-cell line and Western blot analysis, CKS-17 induced phosphorylation of PLC-gamma1, Raf-1, MEK and ERK1/2. Using a PLC selective inhibitor U73122 or PLC-gamma1-deficient Jurkat cell line, phosphorylation induced by CKS-17 of ERK1/2, PLC-gamma1, or Raf-1, respectively, were undetectable or significantly reduced. Reintroduction of PLC-gamma1 into the PLC-gamma1-deficient Jurkat cells restored the phosphorylation of ERK1/2 and PLC-gamma1 induced by CKS-17. Further, pretreatment of Jurkat cells with PKC inhibitors blocks the phosphorylation of Raf-1, MEK, and ERK1/2 induced by CKS-17. These results indicate that CKS-17 induces the PLC-gamma1-PKC-Raf-1-MEK-ERK1/2 signaling pathway.

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.

The Phospholipase Cγ1-dependent Pathway of FcϵRI-mediated Mast Cell Activation Is Regulated Independently of Phosphatidylinositol 3-Kinase

Mast cell degranulation following Fc epsilon RI aggregation is generally believed to be dependent on phosphatidylinositide 3-kinase (PI 3-kinase)-mediated phospholipase C (PLC)gamma activation. Here we report evidence that the PLC gamma 1-dependent pathway of Fc epsilon RI-mediated activation of mast cells is independent of PI 3-kinase activation. In primary cultures of human mast cells, Fc epsilon RI aggregation induced a rapid translocation and phosphorylation of PLC gamma 1, and subsequent inositol trisphosphate (IP3) production, which preceded PI 3-kinase-related signals. In addition, although PI 3-kinase-mediated responses were completely inhibited by wortmannin, even at high concentrations, this PI 3-kinase inhibitor had no effect on parameters of Fc epsilon RI-mediated PLC gamma activation, and had little effect on the initial increase in intracellular calcium levels that correlated with PLC gamma activation. Wortmannin, however, did produce a partial (approximately 50%) concentration-dependent inhibition of Fc epsilon RI-mediated degranulation in human mast cells and a partial inhibition of the later calcium response at higher concentrations. Further studies, conducted in mast cells derived from the bone marrow of mice deficient in the p85 alpha and p85 beta subunits of PI 3-kinase, also revealed no defects in Fc epsilon RI-mediated PLC gamma 1 activation. These data are consistent with the conclusion that the PLC gamma-dependent component of Fc epsilon RI-mediated calcium flux leading to degranulation of mast cells is independent of PI 3-kinase. However, PI 3-kinase may contribute to the later phase of Fc epsilon RI-mediated degranulation in human mast cells.

Reconstitution of Src-dependent Phospholipase Cγ Phosphorylation and Transient Calcium Release by Using Membrane Rafts and Cell-free Extracts from Xenopus Eggs

We reported previously that egg membrane rafts serve as a subcellular microdomain for sperm-dependent tyrosine kinase signaling in Xenopus fertilization. Moreover, we demonstrated that raft-associated Src tyrosine kinase was activated by sperm in vitro. Here we show that egg rafts incubated with sperm or hydrogen peroxide (H2O2) can promote Src-dependent phosphorylation of phospholipase Cgamma (PLCgamma) and transient calcium release in the extracts of unfertilized Xenopus eggs. In vivo egg activation by sperm or H2O2 also promotes tyrosine phosphorylation and raft-translocalization of PLCgamma. Immunodepletion of PLCgamma from the egg extracts inhibits the raft-dependent calcium release. Rafts prepared from H2O2-activated eggs also promote Src-dependent dephosphorylation of p42 mitogen-activated protein kinase and cell cycle transition from metaphase II to interphase in egg extracts. PLCgamma phosphorylation and calcium release in egg extracts can be promoted by rafts prepared from COS-7 cells expressing the Xenopus Src gene. These results demonstrate that the signaling events elicited by fertilization in Xenopus eggs can be reconstituted in vitro. The development of such experimental platforms will allow us to dissect the molecular mechanism of sperm-dependent activation of raft-associated Src and subsequent up-regulation of PLCgamma and egg activation machinery in Xenopus eggs.

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.

C-terminal fragment of tetanus toxin heavy chain activates Akt and MEK/ERK signalling pathways in a Trk receptor-dependent manner in cultured cortical neurons.

Previous publications from our group [Gil, Chaib, Pelliccioni and Aguilera (2000) FEBS Lett. 481, 177-182; Gil, Chaib, Blasi and Aguilera (2001) Biochem. J. 356, 97-103] have reported the activation, in rat brain synaptosomes, of several phosphoproteins, such as neurotrophin tyrosine kinase (Trk) A receptor, phospholipase Cgamma-1, protein kinase C (PKC) isoforms and extracellular-signal-regulated kinases 1 and 2 (ERK-1/2). In the present study, we examined, by means of phospho-specific antibodies, the activation of the signalling cascades involving neurotrophin Trk receptor, Akt kinase and ERK pathway, in cultured cortical neurons from foetal rat brain, by tetanus toxin (TeTx) as well as by the C-terminal part of its heavy chain (H(C)-TeTx). TeTx and H(C)-TeTx induce fast and transient phosphorylation of Trk receptor at Tyr(674) and Tyr(675), but not at Tyr(490), although the potency of TeTx in this action was higher when compared with H(C)-TeTx action. Moreover, H(C)-TeTx and TeTx also induced phosphorylation of Akt (at Ser(473) and Thr(308)) and of ERK-1/2 (Thr(202)/Tyr(204)), in a time- and concentration-dependent manner. The detection of TeTx- and H(C)-TeTx-induced phosphorylation at Ser(9) of glycogen synthase kinase 3beta confirms Akt activation. In the extended analysis of the ERK pathway, phosphorylation of the Raf, mitogen-activated protein kinase kinase (MEK)-1/2 and p90Rsk kinases and phosphorylation of the transcription factor cAMP-response-element-binding protein were detected. The use of tyrphostin AG879, an inhibitor of Trk receptors, demonstrates their necessary participation in the H(C)-TeTx-induced activation of Akt and ERK pathways, as well as in the phosphorylation of phospholipase Cgamma-1. Furthermore, both pathways are totally dependent on phosphatidylinositol 3-kinase action, and they are independent of PKC action, as assessed using wortmannin and Ro-31-8220 as inhibitors. The activation of PKC isoforms was determined by their translocation from the cytosolic compartment to the membranous compartment, showing a clear H(C)-TeTx-induced translocation of PKC-alpha and -beta, but not of PKC- epsilon.

Suppression of early T-cell-receptor-triggered cellular activation by the Janus kinase 3 inhibitor WHI-P-154.

Therapeutic targeting of Janus kinase 3 (JAK3) has received particular attention, because it is associated with the common gamma signaling of cytokine receptors and thus vitally influences T-cell growth and survival. Recent evidence, however, indicates a critical role for JAK3 in signaling linked to the T-cell antigen receptor. In this study we investigated whether targeting JAK3 with a rationally designed inhibitor affects early T-cell activation events. T cells were stimulated by CD3 and CD28 cross-linking, and interleukin (IL)-2 production, activation marker expression, increase of free intracellular Ca2+ concentration, activation of the extracellular-related kinase, and nuclear translocation of transcription factors were evaluated. We found that JAK3 inhibitor treatment dramatically impaired T-cell-receptor (TCR)-induced IL-2 production, surface activation marker expression (CD69, CD154), and homotypic T-cell aggregation. Accordingly, mRNA production of IL-2, interferon-gamma, and IL-10 was profoundly inhibited. Molecular analysis revealed that TCR-triggered phosphorylation of phospholipase C-gamma1, increase in cytoplasmic Ca2+ concentration, and activation of extracellular-related kinase were markedly reduced by the JAK3 inhibitor, resulting in substantially decreased DNA binding of nuclear factor of activated T cells and alkaline phosphatase-1 and subsequent IL-2 promoter activation. Remarkably, on TCR-independent stimulation, IL-2 production, CD69 expression, and blast formation were completely insensitive to JAK3 inhibitor treatment. These data indicate that pharmacologic targeting of JAK3 uncouples early TCR-triggered signaling from essential downstream events, which may have important implications for the use of such compounds in T-cell-mediated disorders such as allograft rejection or graft-versus-host disease.

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.

Vav1 and Ly-GDI Two Regulators of Rho GTPases, Function Cooperatively as Signal Transducers in T Cell Antigen Receptor-induced Pathways

The Rho family GTPases are pivotal for T cell signaling; however, the regulation of these proteins is not fully known. One well studied regulator of Rho GTPases is Vav1; a hematopoietic cell-specific guanine nucleotide exchange factor critical for signaling in T cells, including stimulation of the nuclear factor of activated T cells (NFAT). Surprisingly, Vav1 associates with Ly-GDI, a hematopoietic cell-specific guanine nucleotide dissociation inhibitor of Rac. Here, we studied the functional significance of the interaction between Vav1 and Ly-GDI in T cells. Upon organization of the immunological synapse, both Ly-GDI and Vav1 relocalize to T cell extensions in contact with the antigen-presenting cell. Ly-GDI is phosphorylated on tyrosine residues following T cell receptor stimulation, and it associates with the Src homology 2 region of an adapter protein, Shc. In addition, the interaction between Ly-GDI and Vav1 requires tyrosine phosphorylation. Overexpression of Ly-GDI alone is inhibitory to NFAT stimulation and calcium mobilization. However, when co-expressed with Vav1, Ly-GDI enhances Vav1 induction of NFAT activation, phospholipase Cgamma phosphorylation, and calcium mobilization. Moreover, Ly-GDI does not alter the regulation of these phenomena when coexpressed with oncogenic Vav1. Since oncogenic Vav1 does not bind Ly-GDI, this suggests that the functional cooperativity of Ly-GDI and Vav1 is dependent upon their association. Thus, our data suggest that the interaction of Vav1 and Ly-GDI creates a fine tuning mechanism for the regulation of intracellular signaling pathways leading to NFAT stimulation.

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.

The IL-15R alpha chain signals through association with Syk in human B cells.

The alpha-chain of the IL-15R (IL-15Ralpha) serves as the specific, high-affinity receptor for IL-15. It is expressed by lymphoid and nonlymphoid cells, including B cell lymphoma lines. In this study, we have further explored IL-15Ralpha-mediated signaling in activated primary B cells and in Raji cells, a human B-lymphoblastoid cell line which expresses the IL-15Ralpha and IL-2Rgamma chains, but lacks the IL-2Rbeta chain. Stimulation of Raji cells with IL-15 induces their proliferation and rescues them from C2-ceramide-induced apoptosis. By immunoprecipitation and Western blotting, we show that treatment of Raji cells and activated primary B cells with IL-15 induces coprecipitation of Syk kinase with the IL-15Ralpha chain. Upon association, the activated Syk kinase phosphorylates the IL-15Ralpha chain as well as phospholipase Cgamma, which coprecipitates with Syk. Furthermore, transfection of Raji cells with stem-loop Syk antisense oligonucleotides prevents IL-15Ralpha and phospholipase Cgamma phosphorylation as well as the inhibition of apoptosis by IL-15. Mutation of a defined region of the intracellular signaling portion of IL-15Ralpha (Tyr227) abrogates both the IL-15Ralpha/Syk association and IL-15Ralpha phosphorylation. Taken together, this suggests that Syk kinase physically and functionally associates with the IL-15Ralpha chain in B cells and that Syk plays a key role in mediating IL-15-induced signal transduction, thus accounting for the distinct functional consequences of IL-15 vs IL-2 binding to B cells.

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.

Schiff base-mediated co-stimulation primes the T-cell-receptor-dependent calcium signalling pathway in CD4 T cells.

In addition to macromolecular interactions that provide co-stimulation during antigen-presenting cell (APC) and CD4+ T-cell conjugation, covalent chemical events between specialized ligands have been implicated in T-cell co-stimulation. These take the form of transient Schiff base formation between carbonyls and amines expressed on APC and T-cell surfaces. Small Schiff base-forming molecules, such as tucaresol, can substitute for the physiological donor of carbonyl groups and provide co-stimulation to T cells, thereby functioning as orally active immunopotentiatory drugs. The Schiff base co-stimulatory pathway in T cells has been partially characterized in terms of changes in Na+ and K+ transport, and activation of the mitogen activated protein kinase (MAPK) ERK2. In the present study, the effects of Schiff base co-stimulation by tucaresol on the T-cell receptor (TCR)-dependent pathway leading to Ca2+ release were investigated. Schiff base co-stimulation by tucaresol was found to prime for enhanced TCR-dependent phospholipase C-gamma phosphorylation, inositol 1,4,5-triphosphate production, and Ca2+ mobilization that correlated with functional enhancement of interleukin-2 production in primary T cells. The effects on Ca2+ occurred comparably in Jurkat and primary CD4+ T cells responding to anti-CD3 monoclonal antibody. Enhancement of the Ca2+ response required a 10-min priming period and was prevented by prior covalent ligation of cell-surface free amino groups by sulpho-N-hydroxy succinimido-biotin; clofilium-mediated inhibition of tucaresol-induced changes in intracellular K+; and selective inhibition of the MAPK pathway. The data are consistent with a priming mechanism in which late co-stimulation-triggered events exert a positive influence on early TCR-triggered events. In additional studies of murine T cells expressing trans-gene TCRs, tucaresol was likewise shown to prime for enhanced Ca2+ mobilization in response to physiological TCR-engagement by MHC-peptide complexes.

Protein tyrosine phosphatase CD148-mediated inhibition of T-cell receptor signal transduction is associated with reduced LAT and phospholipase Cgamma1 phosphorylation.

In this study, we investigate the role of the receptor-like protein tyrosine phosphatase CD148 in T-cell activation. Overexpression of CD148 in the Jurkat T-cell line inhibited activation of the transcription factor nuclear factor of activated T cells following T-cell receptor (TCR) stimulation but not following stimulation through a heterologously expressed G protein-coupled receptor, the human muscarinic receptor subtype 1. Using a tetracycline-inducible expression system, we show that the TCR-mediated activation of both the Ras and calcium pathways was inhibited by expression of CD148 at levels that approximate those found in activated primary T cells. These effects were dependent on the phosphatase activity of CD148. Analysis of TCR-induced protein tyrosine phosphorylation demonstrated that most phosphoproteins were unaffected by CD148 expression. However, phospholipase Cgamma1 (PLCgamma1) and LAT were strikingly hypophosphorylated in CD148-expressing cells following TCR stimulation, whereas the phosphorylation levels of Slp-76 and Itk were modestly reduced. Based on these results, we propose that CD148 negatively regulates TCR signaling by interfering with the phosphorylation and function of PLCgamma1 and LAT.