Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Stimulation of the T lymphocyte antigen receptor-CD3 complex (TCR-CD3) causes T cell activation by a process associated with increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity. Evidence exists suggesting that GTP-binding (G) proteins, particularly the pertussis toxin (PT)-sensitive Gi proteins, participate in this signal transduction pathway. To clarify the role of Gi proteins in TCR-CD3 signaling, and to investigate other possible functions of Gi molecules in T cells, we expressed the S1 subunit of PT in the thymocytes of transgenic mice using the lymphocyte-specific lck promoter. Transgenic thymocytes contained S1 activity and exhibited profound depletion of Gi protein PT substrates in a manner suggesting their inactivation by S1 in vivo. Nevertheless, treatment of transgenic thymocytes with mitogenic stimuli provoked normal increases in intracellular free Ca2+ concentrations and IL-2 secretion, indicating that Gi proteins are not required for T cell activation. These normal signaling responses notwithstanding, mature thymocytes accumulated in lck-PT mice and did not appear in secondary lymphoid organs or in the circulation. Viewed in the context of the known features of Bordetella pertussis infection, our results suggest that a PT-sensitive signaling process, probably involving Gi proteins, regulates thymocyte emigration.
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PMID:Dissection of thymocyte signaling pathways by in vivo expression of pertussis toxin ADP-ribosyltransferase. 212 51

Lymphocytes are shown to express a limited number of a unique category of membrane Ag, such as Thy-1, Ly-6, Ly-31, and Qa-2, that are covalently linked to the membrane phosphatidylinositol (PI). We have identified a new glycosyl-phosphatidylinositol (GPI)-anchored lymphocyte Ag, B7, by using a mAb and have determined the primary structure by cDNA cloning. B7 Ag was expressed on the majority, if not all, of the mature lymphocytes of both T and B lineages, including strongly CD3+ thymocytes, most splenic T cells, and approximately 60% of splenic IgM+ B cells, whereas the expression of B7 Ag on bone marrow cells was negligible. The expression of B7 Ag was nearly completely abolished with as little as 2 mU of PI-specific phospholipase C per ml, which did not completely eliminate Ly-6C and Thy-1 expression. Unlike the expression of other GPI-linked lymphocyte Ag, the expression of B7 was rapidly down-regulated upon the activation of T cells by mitogens or IL-2 both in vitro and in vivo. Immunoprecipitation analysis revealed that B7 Ag was an approximately 12-kDa protein. With a CDM8 expression vector, a cDNA encoding B7 Ag was cloned, and it was confirmed that the B7 Ag on cDNA-transfected cells was indeed PI-specific phospholipase C sensitive. The B7 cDNA contained an open reading frame of 222 bp including a typical N-terminal leader sequence and a characteristic sequence at the C terminus encoding hydrophobic amino acids. A computer search revealed no significant homology to any known molecule at both DNA and amino acid sequence levels. Northern blot analysis indicated that the B7 transcript was expressed on lymphohematopoietic tissues, including thymus, spleen, and bone marrow, but not on other organs, such as liver, kidney, and brain. The results indicated that B7 Ag is a new member of the GPI-anchored proteins which is selectively expressed on mature resting but not activated lymphocytes.
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PMID:Identification and gene cloning of a new phosphatidylinositol-linked antigen expressed on mature lymphocytes. Down-regulation by lymphocyte activation. 214 7

The CD28 homodimer is thought to function as a signal transducing receptor during activation of T cells. Evidence is presented that the degree of aggregation of CD28 on the cell surface regulates two distinct CD28-associated signals. Binding of bivalent CD28 monoclonal antibody (MoAb) 9.3 upregulates lymphokine production by messenger RNA (mRNA) stabilization, without direct initiation of lymphokine mRNA transcription. This signal was not dependent on inositol phospholipid production or activation of a protein tyrosine kinase (PTK). In contrast, further crosslinking of CD28 on the cell surface rapidly induced formation of large amounts of inositol trisphosphate (InsP3) and increased cytoplasmic calcium concentration [( Ca2+]i), but did not stimulate PTK. CD28 crosslinking directly activated a subset of resting T cells, since CD25 (interleukin [IL]-2 receptor alpha chain) mRNA was rapidly induced in purified T cells, and proliferation, even without addition of exogenous IL-2, was sometimes observed. CD25 expression was detected on the cell surface of approximately 20% of CD4+ T cells. The degree of CD28 aggregation required for activation was investigated by preparing soluble 9.3 x 9.3 conjugates ranging in size from approximately 300 Kd to greater than 1,000 Kd, and comparing their function in T-cell proliferation assays with phorbol-12-myristate-13-acetate (PMA), anti-CD3, or IL-2. There was a correlation between conjugate size and proliferation with IL-2, whereas costimulation with PMA or CD3 was optimized at a lower degree of CD28 aggregation. The inositol phospholipid (InsP) generation and increase in [Ca2+]i after CD28 receptor aggregation appeared to proceed through a pathway different from the CD3/T-cell receptor (TCR) pathway since it was enhanced by pretreatment with PMA, while the InsP and [Ca2+]i signal from crosslinking CD3 was suppressed by PMA. Furthermore, the proliferation response to CD28 aggregation was resistant to inhibition by CD3 modulation. Thus, CD28 aggregation appears to trigger a phospholipase C activation pathway that differs from the CD3/TCR-linked pathway.
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PMID:CD28 ligation in T-cell activation: evidence for two signal transduction pathways. 215 82

Engagement of the TCR (CD3-Ti) by Ag/MHC, CD3 mAb, or lectin mitogen stimulates the very early tyrosine phosphorylation of several cellular substrates including TCR-zeta. The T cell specific protein-tyrosine kinase (PTK), p56lck, has been implicated in the tyrosine phosphorylation of TCR-zeta. However, the significance of this event with regard to CD3-Ti signal transduction remains unclear. Herein, we have investigated the effect of the selective PTK inhibitor genistein (4',5,7-trihydroxyisoflavone) on cellular events associated with activation via CD3-Ti triggering. Genistein inhibited the T cell PTK, p56lck, in a dose-dependent fashion with an ID50 = 40 microM. Genistein also inhibited CD3 mAb or PHA-induced TCR-zeta chain phosphorylation in intact peripheral blood T cells. Genistein blocked the expression of IL-2 and IL-2R (CD25) in T cells stimulated with PHA/PMA or CD3 mAb/PMA, but did not inhibit the de novo expression of the CD69 early activation Ag, which is induced primarily by a PKC-dependent pathway. IL-2 and CD25 expression induced by calcium ionophore A23187 and PMA was largely refractory to inhibition by genistein, suggesting an effect of the drug on calcium-dependent pathways stimulated via CD3-Ti triggering. In this last regard, genistein partially inhibited the CD3 mAb-induced rise in [Ca2+]i but did not inhibit PHA- or CD3 mAb-induced phosphatidylinositol hydrolysis. Consequently, protein-tyrosine phosphorylation does not appear to be a prerequisite for CD3-Ti-mediated activation of phosphatidylinositol-specific phospholipase C activity and PIP2 hydrolysis. An alternative role for PTK in CD3-Ti signal transduction is suggested.
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PMID:Differential inhibition of T cell receptor signal transduction and early activation events by a selective inhibitor of protein-tyrosine kinase. 217 80

Interaction of specific ligands with TCR initiates a cascade of biochemical events which leads to expression of high affinity IL-2R and subsequent IL-2 secretion. Activation of phospholipase C (PL-C) is considered to be a key event in the initiation of this cascade. However, in addition to this PL-C-dependent pathway, PL-C-independent pathways have been hypothesized. Identification of the steps constituting these PL-C-independent pathways has been difficult because activation of PL-C and the subsequent cascade of events mask the effects of such pathways. Specific inhibitors for PL-C, or mutants defective in, the PL-C pathway would facilitate delineation of alternative activation pathways. We have identified a murine pork insulin/IAd-specific T cell hybridoma, B8P3.11, in which perturbation of the B8P3.11 TCR by either Ag in association with Ia, anti-CD3 antibodies, or a mitogenic lectin does not induce increases in myo-inositol 1,4,5-triphosphate production or cytosolic free calcium, yet it does lead to IL-2 secretion. Treatment of B8P3.11 with pertussis toxin, at concentrations which ADP-ribosylate GTP-binding proteins, inhibits IL-2 secretion. Thus, signal transduction resulting in IL-2 secretion by B8P3.11 likely involves a G protein. In contrast, TCR/ligand interaction activates the PL-C-dependent pathway in LBRM 331A5, a T cell lymphoma. Furthermore, pertussis toxin treatment, which blocks IL-2 secretion by B8P3.11, does not alter IL-2 secretion by LBRM 331A5. However, similar pertussis toxin substrates are present in both cells. Therefore, B8P3.11 T cells should help to elucidate PL-C-independent activation pathways.
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PMID:IL-2 secretion is pertussis toxin sensitive in a T lymphocyte hybridoma. 252 28

IL-2 pretreatment of cloned Th lymphocytes has been demonstrated to render these cells unresponsive to subsequent stimulation through the TCR. These cells remain unresponsive for up to 7 days after removal from IL-2. Cells rendered unresponsive to Ag by pretreatment with IL-2 also demonstrated reduced increases in intracellular calcium ([Ca2+]i) after stimulation, hence this unresponsiveness is believed to result from absence of sufficient [Ca2+]i for activation of lymphokine genes. We have confirmed these observations, and demonstrate that only that portion of the [Ca2+]i increase derived from extracellular sources is inhibited in IL-2 pretreated cells. Further, inositol degradation and diacylglycerol production after stimulation are observed to be markedly reduced in cells rendered unresponsive by IL-2 pretreatment, suggesting that signal transduction leading to cleavage of phosphatidylinositol 4,5-bisphosphate after Ag receptor engagement is incomplete in these cells. However, treatment of IL-2 pretreated cells with AlF4- results in both production of inositol phosphates as well as increased intracellular calcium, suggesting that phospholipase C remains active in these cells. It appears that chronic IL-2 exposure regulates Th activation by inhibiting the signal transduction which follows engagement of the TCR.
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PMID:Pretreatment of cloned helper T lymphocytes with IL-2 induces unresponsiveness to antigen and concanavalin A, associated with decreased inositol phosphate and diacylglycerol production. 252 65

Since our previous experiments suggested that glycosylation-inhibiting factor (GIF) is a phosphorylated derivative of a phospholipase inhibitory protein, we determined whether other well-known phospholipase inhibitors may have similar biological activities. The results showed that phospholipase A2 (PLA2) inhibitors, such as recombinant human lipocortin I and ONO-RS-082, could switch T cell hybridoma 12H5 cells from the formation of glycosylated IgE-binding factors (IgE-BF) to the formation of unglycosylated IgE-BF, whereas neomycin, a phospholipase C inhibitor, failed to affect the nature of IgE-BF formed by the cells. The minimum concentrations of lipocortin I and ONO-RS-082 required for switching the 12H5 cells to the formation of unglycosylated IgE-BF were comparable to or less than IC50 of the inhibitors for PLA2. The ability of partially purified GIF to switch the 12H5 cells to the formation of unglycosylated IgE-BF was markedly enhanced by treatment of the preparation with alkaline phosphatase. It was also found that lipocortin I and ONO-RS-082, but not neomycin, facilitated the generation of GIF-producing T cells. When spleen cells of ovalbumin (OVA)-primed BDF1 mice were stimulated with homologous antigen and the activated T cells were propagated by recombinant IL-2 in the presence of GIF, lipocortin I, or ONO-RS-082, T cells obtained in the cultures constitutively produced their own GIF. Antigenic stimulation of the T cells induced the formation of unglycosylated IgE-BF and GIF with an affinity for OVA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of phospholipase A2 inhibitors on mouse T lymphocytes. I. Phospholipase A2 inhibitors exert similar immunological activities as glycosylation inhibiting factor. 253 36

T cells of autoimmune-prone mice homozygous for the lpr mutation respond poorly to mitogens in terms of proliferation and of IL-2 production. In a previous study, we have correlated this deficient activation with the inability of mitogens to stimulate hydrolysis of phosphatidylinositol 4,5-bisphosphate in lpr T cells, although these cells bind mitogen and express the TCR/CD3 complex. In order to determine whether activation-deficient lpr T cells contain functional GTP-binding (G) protein(s) and phospholipase C, we examined the effects of the G protein activating agent sodium fluoride plus Al+3 (AlF-4). AlF-4 stimulated phosphatidylinositol turnover, a response characteristic of TCR/CD3 occupancy, in mature L3T4+ and Ly2+ T cells. Second, and more important, AlF-4 stimulated the same biochemical events in L3T4-, Ly2- (double-negative) T cells from the normal thymus or from the enlarged lymph nodes of autoimmune-prone mice homozygous for the lpr mutation. However, these double-negative T cells were unresponsive to receptor-active ligands such as T cell mitogens or anti-CD3-epsilon mAb, despite their ability to bind these ligands. These findings suggest that activation-deficient double negative T cells express the receptors, G protein(s) and effector enzymes necessary for second messenger formation and further suggest that the failure of these cells to generate the relevant second messengers in response to mitogens or anti-CD3-epsilon antibody may be due to inefficient coupling of the TCR/CD3 complex to G proteins.
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PMID:Stimulation of PIP2 hydrolysis by aluminum fluoride in resting T cell subsets of normal and autoimmune-prone lpr mice. 254 77

Human peripheral blood T cells were stimulated to proliferate when cultured with submitogenic doses of PMA and goat antibodies to 5'-nucleotidase (5'-NT). The degree of proliferation, as measured by [3H]TdR incorporation on day 3, was similar to that achieved by stimulation with PHA. Anti-5'-NT antibodies had no effect on PHA-induced proliferation. Maximal stimulation was achieved with 0.6 to 1.0 ng/ml of PMA and 125 micrograms/ml of IgG isolated from a goat anti-5'-NT antiserum. Both intact IgG and F(ab')2 fragments were stimulatory. IL-2R expression and IL-2 secretion were also induced by anti-5'-NT antibodies and PMA. Anti-5'-NT-induced proliferation was inhibited greater than 95% by a murine anti-IL-2 receptor mAb and required less than 0.3% monocytes. Similar results have been obtained with a murine mAb specific for 5'-NT. As expected, anti-5'-NT antibodies and PMA did not induce the proliferation of ecto-5'-NT-T cells isolated by cell sorting. Pretreatment of total T cells with phosphatidylinositol-specific phospholipase C removed an average of 89% of the 5'-NT activity from the cell surface and also inhibited by 83% the ability of the cells to proliferate in response to anti-5'-NT antibodies and PMA. Thus, the activation signal provided by anti-5'-NT antibodies is apparently transduced, in large part, by a form of the enzyme that is attached to the membrane via glycosyl-phosphatidylinositol linkage. These data suggest that 5'-NT may play a role in lymphocyte activation as has been proposed for other glycosyl-phosphatidylinositol-anchored lymphocyte surface proteins.
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PMID:Antibodies to 5'-nucleotidase (CD73), a glycosyl-phosphatidylinositol-anchored protein, cause human peripheral blood T cells to proliferate. 255 May 43

The catalytic subunit of cholera toxin (CT) can chemically modify the alpha polypeptides of certain G-binding proteins and thus alter their function. In order to study the involvement of CT-sensitive G proteins in T cell activation, we have utilized CT in an in vitro system in which purified, resting human peripheral T cells are activated by anti-CD3 antibodies and rIL-2. Perturbation of the TCR/CD3 molecular complex by anti-CD3 antibodies causes changes in membrane phospholipids and induces a rise in cytoplasmic Ca2+. These events, however, are insufficient to allow progression into cellular proliferation and addition of IL-2 is required. Under these conditions, treatment of cells with a low concentration of CT (2 ng/ml) causes a significant inhibition of the anti-CD3-induced calcium event as well as the anti-CD3 plus IL-2-stimulated proliferation. Under our experimental conditions, inhibition of both proliferation and intracellular Ca2+ elevation by CT requires the involvement of the TCR/CD3 complex. This is supported by the observation that the toxin does not inhibit either the proliferation triggered by ionomycin and PMA or the Ca2+ influx induced by the ionophore. These data suggest that in TCR/CD3-mediated T cell activation CT acts at a point between TCR/CD3 perturbation and the generation of intracellular Ca2+. In view of the ability of CT to activate the alpha subunit of the G protein that stimulates adenyl cyclase (G alpha s), it is possible that the effect of CT on T cells is secondary to intracellular elevation of cAMP. However, measurement of cAMP levels both early after CT addition and at later time points, when proliferation is maximal, reveals lack of cyclic nucleotide accumulation. The presented data are consistent with the interpretation that the CT-mediated inhibition is caused by the modification of a G-binding protein that is either directly or indirectly associated with triggering of T cells via the TCR/CD3 molecular complex. The data also suggest that this protein is not G alpha s and it probably represents an as yet unidentified moiety or one of the several G proteins that have been recently described as regulators of phospholipase C activation.
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PMID:Cholera toxin inhibits resting human T cell activation via a cAMP-independent pathway. 255 13


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