G-proteins are not directly involved in the CD3-antigen-mediated production of inositol phosphates in HPB-ALL T-leukaemia cells expressing phospholipase C isoforms γ1 and β3

The possible involvement of G-proteins in T cell antigen-receptor complex (TCR)-mediated inositol phosphate production was investigated in HPB-ALL T-cells, which were found to express the phospholipase C gamma 1 and beta 3 isoforms. Cross-linking the CD3 antigen on streptolysin-O-permeabilized cells stimulated a dose-dependent increase in inositol phosphate production, as did addition of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) or vanadate, a phosphotyrosine phosphatase inhibitor. It was possible, therefore, that the CD3-antigen-mediated production of inositol phosphates was either via a G-protein-dependent mechanism or by stimulation of protein tyrosine phosphorylation. The CD3-induced inositol phosphate production was potentiated by addition of vanadate, but not by addition of GTP[S]. Guanosine 5′-[beta-thio]diphosphate (GDP[S]) inhibited the rise in inositol phosphates induced by GTP[S], vanadate or cross-linking the CD3 antigen. The increase in protein tyrosine phosphorylation stimulated by vanadate or the OKT3 monoclonal antibody was not observed in the presence of GDP[S], showing that in permeabilized HPB-ALL cells, GDP[S] inhibits the actions of tyrosine kinases as well as G-protein function. Addition of either ADP[S] or phenylarsine oxide inhibited CD3- and vanadate-mediated increases in both tyrosine phosphorylation and inositol phosphate production, but did not inhibit GTP[S]-stimulated inositol phosphate production. On the other hand, pretreatment of cells with phorbol 12,13-dibutyrate inhibited subsequent GTP[S]-stimulated inositol phosphate production but did not inhibit significantly inositol phosphate production stimulated by either OKT3 F(ab')2 fragments or vanadate. Our results are consistent with the CD3 antigen stimulating inositol phosphate production by increasing the level of protein tyrosine phosphorylation, but not by activating a G-protein.

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The B cell antigen receptor of class IgD induces a stronger and more prolonged protein tyrosine phosphorylation than that of class IgM.

Journal of Experimental Medicine ◽

10.1084/jem.181.3.1005 ◽

1995 ◽

Vol 181 (3) ◽

pp. 1005-1014 ◽

Cited By ~ 42

Author(s):

K M Kim ◽

M Reth

Keyword(s):

B Cell ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Antigen Receptor ◽

Cross Linking ◽

Protein Tyrosine Phosphorylation ◽

Antigen Receptors ◽

Antigen Specificity ◽

Protein Tyrosine ◽

Substrate Phosphorylation

Most mature B lymphocytes coexpress two classes of antigen receptor, immunoglobulin (Ig)M and IgD. The differences in the signal transduction from the two receptors are still a matter of controversy. We have analyzed B cell lines expressing IgM or IgD antigen receptors with the same antigen specificity. Cross-linking of these receptors with either antigen, or class-specific antibodies, results in the activation of protein tyrosine kinases and the phosphorylation of the same substrate proteins. The kinetic and the intensity of phosphorylation, however, was quite different between the two receptors when they were cross-linked by antigen. In membrane IgM-expressing cells, the substrate phosphorylation reached a maximum after 1 minute and diminished after 60 minutes whereas, in the membrane IgD-expressing cells, the substrate phosphorylation increased further over time, reached its maximum at 60 minutes, and persisted longer than 240 minutes after exposure to antigen. As a result, the intensity of protein tyrosine phosphorylation induced by cross-linking of membrane IgD was stronger than that induced by membrane IgM. Studies of chimeric receptors demonstrate that only the membrane-proximal C domain and/or the transmembrane part of membrane-bound IgD molecule is required for the long-lasting substrate phosphorylation. Together, these data suggest that the signal emission from the two receptors is controlled differently.

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Relationships between the degree of cross-linking of surface immunoglobulin and the associated inositol 1,4,5-trisphosphate and Ca2+ signals in human B cells

Biochemical Journal ◽

10.1042/bj2840447 ◽

1992 ◽

Vol 284 (2) ◽

pp. 447-455 ◽

Cited By ~ 16

Author(s):

F M McConnell ◽

S B Shears ◽

P J L Lane ◽

M S Scheibel ◽

E A Clark

Keyword(s):

B Cells ◽

Tyrosine Kinase ◽

Phospholipase C ◽

Inositol Phosphates ◽

Inositol Phosphate ◽

Cross Linking ◽

Immunoglobulin Receptor ◽

Surface Immunoglobulin ◽

Human B Cells ◽

Inositol 1,4,5 Trisphosphate

Cross-linking of surface immunoglobulin (Ig) receptors on human B cells leads to the activation of a tyrosine kinase. The activated tyrosine kinase subsequently phosphorylates a number of substrates, including phospholipase C-gamma. This enzyme breaks down phosphoinositol bisphosphate to form two intracellular messengers, diacylglycerol and inositol 1,4,5-trisphosphate, leading to the activation of protein kinase C and the release of intracellular Ca2+ respectively. We have used h.p.l.c. and flow cytometry to measure accurately the inositol phosphate turnover and Ca2+ release in anti-Ig-stimulated human B cells. In particular, we have examined the effect of dose of the cross-linking antibody on the two responses. The identity of putative messenger inositol phosphates has been verified by structural analysis, and the amounts of both inositol phosphates and Ca2+ present have been quantified. In the Ramos Burkitt lymphoma, which is very sensitive to stimulus through its Ig receptors, both inositol phosphate production and Ca2+ release were found to be related to the dose of anti-Ig antibody applied. This suggests that phospholipase C-mediated signal transduction in human B cells converts the degree of cross-linking of the immunoglobulin receptor quantitatively into intracellular signals.

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Lysophosphatidic acid induces protein tyrosine phosphorylation in the absence of phospholipase C activation in human platelets

Platelets ◽

10.1080/09537109777456 ◽

1997 ◽

Vol 8 (2) ◽

pp. 181-188

Author(s):

M. Torti ◽

E. Tolnai Festetics ◽

A. Bertoni ◽

R. Moratti ◽

C. Balduini ◽

...

Keyword(s):

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Lysophosphatidic Acid ◽

Human Platelets ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine

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Platelet-derived growth factor increases the in vivo activity of phospholipase C-gamma 1 and phospholipase C-gamma 2

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.2018-2025.1991 ◽

1991 ◽

Vol 11 (4) ◽

pp. 2018-2025

Author(s):

L Sultzman ◽

C Ellis ◽

L L Lin ◽

T Pawson ◽

J Knopf

Keyword(s):

Growth Factor ◽

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Inositol Phosphates ◽

Inositol Phosphate ◽

Cell Surface Receptor ◽

Platelet Derived Growth Factor ◽

Surface Receptor ◽

Phospholipase C Gamma

Upon binding to its cell surface receptor, platelet-derived growth factor (PDGF) causes the tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1) and stimulates the production of diacylglycerol and inositol 1,4,5-triphosphate. We showed that following stimulation by PDGF, rat-2 cells overexpressing PLC-gamma 1 display an increase in the levels of both tyrosine-phosphorylated PLC-gamma 1 and inositol phosphates compared with the parental rat-2 cells. This increased responsiveness to PDGF is a direct effect of PLC-gamma 1 overexpression, as a cell line expressing similar levels of an enzymatically inactive point mutant of PLC-gamma 1, PLC-gamma 1 335Q, did not show elevated inositol phosphate production in response to PDGF. Hematopoietic cells express PLC-gamma 2, a PLC isoform that is closely related to PLC-gamma 1. When rat-2 cells overexpressing PLC-gamma 2 were treated with PDGF, an increase in both the tyrosine phosphorylation and the in vivo activity of PLC-gamma 2 was observed. Aluminum fluoride (AIF4-), a universal activator of PLC linked to G-proteins, did not produce an increase in the levels of inositol phosphates in either of the overexpressing cell lines compared with parental rat-2 cells, demonstrating that PLC-gamma isoforms respond specifically to a receptor with tyrosine kinase activity.

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Development of muscarinic-cholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells. Evidence for a switch in guanine-nucleotide-binding protein coupling

Biochemical Journal ◽

10.1042/bj2710443 ◽

1990 ◽

Vol 271 (2) ◽

pp. 443-448 ◽

Cited By ~ 5

Author(s):

J V Barnett ◽

S M Shamah ◽

B Lassegue ◽

K K Griendling ◽

J B Galper

Keyword(s):

Phospholipase C ◽

G Proteins ◽

Pertussis Toxin ◽

Inositol Phosphate ◽

In Ovo ◽

Atrial Cells ◽

Muscarinic Stimulation ◽

Inositol Phosphate Production ◽

Guanine Nucleotide Binding ◽

Stimulation Of

We have demonstrated that muscarinic stimulation of inositol phosphate production in cultured atrial cells from chicks at 14 days in ovo is partially sensitive to inhibition by pertussis toxin. In these cells, muscarinic agonist binding is coupled to phospholipase C activity via at least two guanine-nucleotide-binding proteins (G-proteins), one sensitive to pertussis toxin and the other (Gp) insensitive to pertussis toxin [Barnett, Shamah, Lassegue, Griendling & Galper (1990) Biochem. J. 271, 437-442]. In the current study we demonstrate that during embryonic development of the chick heart, muscarinic stimulation of inositol phosphate production decreases by 50% between days 5 and 14 in ovo in cells cultured from both atrium and ventricle. In atrial cells, however, pertussis toxin-sensitive muscarinic stimulation of inositol phosphate production increased from undetectable levels at day 5 in ovo to 40% of total stimulation at day 12 in ovo. Muscarinic stimulation of inositol phosphate production in the ventricle did not become sensitive to pertussis toxin at any age studied. In permeabilized atrial cells from embryonic chicks at 5 days in ovo, guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated InsP1 levels by 40 +/- 10% (mean +/- S.E.M., n = 3), InsP2 levels by 117 +/- 18% and InsP3 levels by 51 +/- 8%, suggesting that at day 5 in ovo all of the muscarinic-stimulated inositol phosphate production was coupled to phospholipase C via Gp. H.p.l.c. analysis demonstrated that, in spite of these changes in coupling of phospholipase C to different G-proteins, no changes could be demonstrated in the isomers of InsP3 produced in response to carbamylcholine at both days 5 and 14 in ovo. These data demonstrate that embryonic development of the chick atrium is associated with a switch in coupling of muscarinic receptors to phospholipase C from Gp to a pertussis toxin substrate. This developmental switch in coupling of G-proteins may be related to possible developmental switches in levels of muscarinic receptor isoforms or switches in the subtype of phospholipase C.

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Phospholipase C-β1 is regulated by a pertussis toxin-insensitive G-protein

Biochemical Journal ◽

10.1042/bj2800753 ◽

1991 ◽

Vol 280 (3) ◽

pp. 753-760 ◽

Cited By ~ 13

Author(s):

T F J Martin ◽

J E Lewis ◽

J A Kowalchyk

Keyword(s):

Phospholipase C ◽

G Protein ◽

G Proteins ◽

Pertussis Toxin ◽

Gh3 Cells ◽

Phospholipid Vesicles ◽

Protein Tyrosine Phosphorylation ◽

Gtp Binding ◽

Gamma Subunits ◽

Gh3 Cell

Regulation of phospholipase C (PLC) by receptors is mediated either through protein tyrosine phosphorylation or by activation of GTP-binding proteins (Gp). For the latter, pertussis toxin (PT)-sensitive and -insensitive pathways have been described, indicating PLC regulation by at least two types of G-proteins. The identity of PLC isoenzymes which are regulated by either type of Gp remains to be determined. Thyrotropin-releasing hormone stimulates a PLC in GH3 cells via a PT-insensitive Gp. Reconstitution methods for the assay of the GH3-cell Gp were developed. Previously, the membrane PLC was found to be reversibly extracted from membranes by high salt and to be activated by guanosine 5′-[gamma-thio]triphosphate (GTP[S]) only when membrane-associated, suggesting that Gp was retained in salt-extracted membranes. In the present work, Gp was cholate-solubilized from PLC-deficient membranes and incorporated into phospholipid vesicles, which were found to confer GTP[S]- and AlF4(-)-stimulated activity on a solubilized membrane PLC. The reconstitution provided a direct assay for the GH3-cell Gp which was shown to be distinct from Gi, Go and Gs proteins by immunodepletion studies. Incorporation of G-protein beta-gamma subunits into phospholipid vesicles with Gp inhibited GTP[S]-stimulated activity in the reconstitution. The results indicated that Gp is a heterotrimeric G-protein with the properties expected for the PT-insensitive GH3-cell Gp protein. PLC-beta 1 was fully purified and shown to be regulated by Gp in the reconstitution. In contrast, PT-sensitive G-proteins failed to affect the activity of PLC-beta 1. The results indicate (1) that a PT-insensitive Gp regulates PLC-beta 1 and (2) that PT-sensitive and -insensitive pathways of PLC regulation employ different PLC isoenzymes as well as different G-proteins.

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Lysophosphatidic acid induces protein tyrosine phosphorylation in the absence of phospholipase C activation in human platelets

Platelets ◽

10.3109/09537109709169335 ◽

1997 ◽

Vol 8 (2) ◽

pp. 181-187

Author(s):

M. Torti ◽

E. Tolnai Festetics ◽

A. Bertoni ◽

R. Moratti ◽

C. Balduini ◽

...

Keyword(s):

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Lysophosphatidic Acid ◽

Human Platelets ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine

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Stimulation of Fc gamma RIIIA results in phospholipase C-gamma 1 tyrosine phosphorylation and p56lck activation.

Journal of Experimental Medicine ◽

10.1084/jem.176.6.1745 ◽

1992 ◽

Vol 176 (6) ◽

pp. 1745-1750 ◽

Cited By ~ 70

Author(s):

L Azzoni ◽

M Kamoun ◽

T W Salcedo ◽

P Kanakaraj ◽

B Perussia

Keyword(s):

T Cells ◽

Tyrosine Kinase ◽

Nk Cells ◽

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine ◽

Tyrosine Residues ◽

Stimulation Of

Binding of ligand to the alpha subunit of Fc gamma RIIIA(CD16), expressed at the natural killer (NK) cell membrane in association with homo or heterodimers of proteins of the zeta family, results in phosphorylation of several proteins on tyrosine residues. We have analyzed the role of protein tyrosine phosphorylation in the regulation of molecular events induced upon stimulation of Fc gamma RIIIA in NK cells and in T cells expressing the Fc gamma RIII alpha chain in association with endogenous zeta 2 homodimers and devoid of other (CD3, CD2) transducing molecules. Our data indicate that treatment of these cells with protein tyrosine kinase inhibitors prevents not only Fc gamma RIIIA-induced protein tyrosine phosphorylation but also phosphatidylinositol 4,5 diphosphate hydrolysis and increased intracellular Ca2+ concentration, indicating a primary role of tyrosine kinase(s) in the induction of these early activation events. Occupancy of Fc gamma RIIIA by ligand results in phospholipase C (PLC)-gamma 1 tyrosine phosphorylation in NK cells and in Fc gamma RIIIA-transfected CD3-/CD2- T cells, and induces functional activation of p56lck in Fc gamma RIIIA alpha/zeta 2-transfected T cells, suggesting the possibility that the receptor-induced PLC-gamma 1 activation occurs upon phosphorylation of its tyrosine residues mediated by this kinase and is, at least in part, responsible for the signal transduction mediated via CD16 upon ligand binding.

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