scholarly journals A Collagen-Like Peptide Stimulates Tyrosine Phosphorylation of syk and Phospholipase Cγ2 in Platelets Independent of the Integrin α2β1

Blood ◽  
1997 ◽  
Vol 89 (4) ◽  
pp. 1235-1242 ◽  
Author(s):  
Judith Asselin ◽  
Jonathan M. Gibbins ◽  
Marcus Achison ◽  
Young Han Lee ◽  
Laurence F. Morton ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Protein Tyrosine Phosphorylation ◽  
Direct Role ◽  
Amino Acid Code ◽  
Helical Peptide ◽  
Platelet Receptor ◽  
Dependent Pathway

AbstractActivation of platelets by collagen is mediated through a tyrosine kinase-dependent pathway that is associated with phosphorylation of the Fc receptor γ chain, the tyrosine kinase syk, and phospholipase Cγ2 (PLCγ2). We recently described a collagen-related triple-helical peptide (CRP) with the sequence GCP*(GPP*)GCP*G (single letter amino acid code: P* = hydroxyproline; Morton et al, Biochem J 306:337, 1995). The cross-linked peptide is a potent stimulus of platelet activation but, unlike collagen, does not support α2β1-mediated, Mg2+-dependent adhesion, suggesting that its action is independent of the integrin α2β1 . This finding suggests the existence of a platelet receptor other than α2β1 that underlies activation. In the present study, we show that CRP stimulates tyrosine phosphorylation of the same pattern of proteins in platelets as collagen, including syk and PLCγ2. Protein tyrosine phosphorylation induced by CRP is not altered in the absence of Mg2+ or the presence of monoclonal antibodies (MoAbs) to the integrin α2β1 (MoAb 6F1 and MoAb 13), conditions that prevent the interaction of collagen with the integrin. In contrast, phosphorylation of syk and PLCγ2 by collagen is partially reduced by MoAb 6F1 and MoAb 13 or by removal of Mg2+. This may reflect a direct role of α2β1 in collagen-induced signaling events or an indirect role in which the integrin facilitates the binding of collagen to its signaling receptor. The results show an α2β1-independent pathway of platelet activation by CRP that involves phosphorylation of syk and PLCγ2. This pathway appears to contribute to platelet activation by collagen.

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

1992 ◽  
Vol 176 (6) ◽  
pp. 1745-1750 ◽  
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.

scholarly journals 436. Investigation of the role of SRC in capacitation associated tyrosine phosphorylation of human spermatozoa

2008 ◽  
Vol 20 (9) ◽  
pp. 116
Author(s):  
L. A. Mitchell ◽  
B. Nixon ◽  
M. A. Baker ◽  
R. J. Aitken
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Signal Transduction Pathway ◽  
Human Sperm ◽  
Complex Signal ◽  
Phosphorylation Cascade ◽  
A Site ◽  
Signalling Cascade ◽  
Mammalian Spermatozoa

Capacitation is a pre-requisite for mammalian spermatozoa allowing them to gain the ability to fertilise an oocyte. A fundamental part of this mechanism is a dramatic increase in tyrosine phosphorylation. Implicated in this process in the mouse is a unique cAMP/PKA-mediated pathway involving a PKA-activated tyrosine kinase suggested to be pp60c-src (SRC). The Src kinases examined were predominantly expressed in the human sperm tail, a site compatible with a role in mediating the capacitation-associated tyrosine phosphorylation cascade. Co-immunoprecipitation revealed that PKA-c could be isolated from sperm and this interaction was restricted to capacitated cells, suggesting PKA-mediated activation of SRC forms an integral part of the signalling cascade assembled during capacitation. Upon activation, SRC undergoes autophosphorylation of Y416 and thus phosphorylation of this residue indicates the presence of active SRC kinase. The phosphorylation status of SRC was compared using both 2D-immunoblotting and immunocytochemical studies, both revealing a significant increase in SRC activation during capacitation. Furthermore, suppression of PKA and SRC through application of SU6656, or H89, a PKA inhibitor, led to a dramatic decrease in tyrosine phosphorylation and SRC activity. In conclusion, this study has provided evidence for the involvement of non-receptor tyrosine kinase, SRC, in regulating tyrosine phosphorylation associated with capacitation. Inhibition of SRC did not completely suppress tyrosine phosphorylation suggesting this complex signal transduction pathway exhibits a degree of functional redundancy.

Mitogenic signaling of thrombin in mesangial cells: role of tyrosine phosphorylation

1994 ◽  
Vol 267 (4) ◽  
pp. F528-F536 ◽  
Author(s):  
G. Grandaliano ◽  
G. G. Choudhury ◽  
P. Biswas ◽  
H. E. Abboud
Keyword(s):  
Gene Expression ◽  
Dna Synthesis ◽  
Tyrosine Phosphorylation ◽  
Mesangial Cells ◽  
Cellular Proteins ◽  
Chain Gene ◽  
Protein Tyrosine Phosphorylation ◽  
Glomerular Mesangial Cells ◽  
Human Glomerular Mesangial Cells

Thrombin elicits multiple biological effects on a variety of cells. We have previously shown that thrombin is a potent mitogen for human glomerular mesangial cells. This mitogenic effect of thrombin is associated with activation of phospholipase C (PLC) and induction of platelet-derived growth factor (PDGF) gene expression. The thrombin receptor, which belongs to the guanine nucleotide binding protein (G protein)-coupled receptor family, has recently been shown to induce rapid tyrosine phosphorylation of cellular proteins. In the present study, we investigated the role of protein-tyrosine phosphorylation in mediating the cellular responses elicited by thrombin in human glomerular mesangial cells. Amino acid labeling followed by immunoprecipitation with phosphotyrosine antibodies demonstrate that thrombin stimulates tyrosine phosphorylation of a set of cellular proteins. Treatment of mesangial cells with thrombin followed by immunoblotting with phosphotyrosine antibodies showed three major bands of tyrosine-phosphorylated proteins approximately 130, 70, and 44-42 kDa. Phosphorylation of these proteins was inhibited by two tyrosine kinase inhibitors, herbimycin A and genistein. Both compounds inhibited DNA synthesis and PDGF B-chain gene expression but had no effect on inositol phosphates production or increases in cytosolic calcium in response to thrombin. These data demonstrate that protein-tyrosine phosphorylation is not required for thrombin-induced PLC activation with inositol phosphate formation and subsequent intracellular calcium release, but it is an absolute requirement for thrombin-induced DNA synthesis and PDGF B-chain gene expression.

scholarly journals Internalization of the granulocyte-macrophage colony-stimulating factor receptor is not required for induction of protein tyrosine phosphorylation in human myeloid cells

Blood ◽  
1991 ◽  
Vol 78 (8) ◽  
pp. 1928-1935 ◽  
Author(s):  
K Okuda ◽  
B Druker ◽  
Y Kanakura ◽  
M Koenigsmann ◽  
JD Griffin
Keyword(s):  
Signal Transduction ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Receptor Internalization ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF) exerts its biologic activities through binding to specific high-affinity cell surface receptors. After binding, the ligand/receptor complex is rapidly internalized in most hematopoietic cells. Using a human factor- dependent cell line, MO7, and normal human neutrophils, we found that the internalization is exquisitely temperature-dependent, such that ligand/receptor internalization does not detectably occur at 4 degrees C. Activation of the GM-CSF receptor has previously been shown to stimulate a number of postreceptor signal transduction pathways, including activation of a tyrosine kinase and activation of the serine/threonine kinase, Raf-1. The GM-CSF-stimulated increase in tyrosine kinase activity occurs rapidly at both 4 degrees C and 37 degrees C, and therefore is likely to be independent of receptor internalization. At 37 degrees C, the protein tyrosine phosphorylation was transient in MO7 cells, with maximum phosphorylation observed after 5 to 15 minutes, followed by a rapid decline. At 4 degrees C, the protein tyrosine phosphorylation of the same substrates was greater than at 37 degrees C, and no decline in substrate phosphorylation was observed for at least 90 minutes. In contrast to tyrosine phosphorylation, the activation and hyper-phosphorylation of Raf-1 observed at 37 degrees C in both MO7 cells and neutrophils was markedly diminished at 4 degrees C. These results indicate that at least one postreceptor signal transduction mechanism, activation of a tyrosine kinase, does not require ligand/receptor internalization, and indicate that receptor internalization may be a consequence, rather than the initiator, of signal transduction.

Role of tyrosine kinase pathways in ETB receptor activation of NHE3

1996 ◽  
Vol 271 (3) ◽  
pp. C763-C771 ◽  
Author(s):  
T. S. Chu ◽  
H. Tsuganezawa ◽  
Y. Peng ◽  
A. Cano ◽  
M. Yanagisawa ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Integral Membrane Protein ◽  
Receptor Activation ◽  
Kinase C ◽  
Focal Adhesion Proteins ◽  
Etb Receptor

Endothelin-1 (ET-1) binding to ETB receptors increases the activity of the apical membrane Na+/H+ antiporter (NHE3) of renal proximal tubule and cultured OKP cells. In OKPETB6 cells, a clonal cell line of OKP cells that overexpresses ETB receptors, ET-1-induced increases in Na+/H+ antiporter activity are mediated 50% by Ca2(+)-dependent pathways and 50% by tyrosine kinase pathways. ET-1 induces tyrosine phosphorylation of proteins of 68, 110, 125, 130, and 210 kDa. ET-1-induced tyrosine phosphorylation is mediated by the ETB receptor and is not dependent on increases in cell Ca2+ or protein kinase C. The 68-, 110-, 125-, and 130-kDa phosphoproteins are cytosolic, whereas the 210-kDa phosphoprotein is an integral membrane protein. Immunoprecipitation studies showed that the 68-kDa protein is paxillin and the 125-kDa protein is p125FAK (focal adhesion kinase). Cytochalasin D, which disrupts focal adhesions, prevented ET-1-induced tyrosine phosphorylation of paxillin, p110, p125FAK, and p130 but did not prevent tyrosine phosphorylation of p210 and did not prevent ET-1-induced increases in Na+/H+ antiporter activity. Thus 50% of ETB receptor-induced Na+/H+ antiporter activation is mediated by tyrosine kinase pathways, possibly involving p210. ETB receptor activation also induces tyrosine phosphorylation of focal adhesion proteins, but this is not required for antiporter activation.

Protein tyrosine phosphorylation mediates TNF-induced endothelial-neutrophil adhesion in vitro

1998 ◽  
Vol 274 (2) ◽  
pp. H513-H519 ◽  
Author(s):  
Susan A. Kelly ◽  
Pascal J. Goldschmidt-Clermont ◽  
Emily E. Milliken ◽  
Toshiyuki Arai ◽  
Elise H. Smith ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Adhesion Molecules ◽  
Neutrophil Adhesion ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Tnf Α ◽  
Dose Dependent

Proinflammatory cytokines initiate the vascular inflammatory response via the upregulation of adhesion molecules on the luminal endothelial surface. We investigated directly the role of protein tyrosine phosphorylation in the upregulation of the endothelial adhesion molecules, intercellular adhesion molecule 1 (ICAM-1) and E-selectin, and the consequent adhesion of neutrophils, after tumor necrosis factor (TNF)-α-stimulation of human aortic endothelial cells in vitro. Time- and dose-dependent TNF-α-stimulated ICAM-1 and E-selectin upregulation and neutrophil adhesion each were suppressed by tyrosine kinase inhibitors, including genistein (200 μM), but not genistin, its isoflavone analog without tyrosine kinase inhibitory activity. Tyrphostin AG 126, a synthetic selective tyrosine kinase inhibitor, also suppressed ICAM-1 and E-selectin upregulation and neutrophil adhesion, each in a dose-dependent manner, whereas tyrphostin AG 1288 had no effect. Tyrosine phosphorylation of two proteins (85 and 145 kDa in the cytoskeleton fraction) found minutes after TNF-α-stimulation was also inhibited by genistein. These findings suggest that, in endothelial cells, TNF-α upregulates ICAM-1 and E-selectin expression and consequent neutrophil adhesion via protein tyrosine phosphorylation.

A New Role for FcγRIIA in the Potentiation of Human Platelet Activation Induced by Weak Stimulation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1648-1648
Author(s):  
Ilaria Canobbio ◽  
Lucia Stefanini ◽  
Gianni F. Guidetti ◽  
Cesare Balduini ◽  
Mauro Torti
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Lipid Rafts ◽  
Platelet Activation ◽  
Human Platelets ◽  
G Protein Coupled Receptors ◽  
Weak Stimulation ◽  
High Concentrations ◽  
G Protein Coupled

Abstract The low affinity receptor for immunoglobulin G, FcγRIIA, is expressed in human platelets, mediates heparin-associated thrombocytopenia, and participates in platelet activation induced by von Willebrand factor. Activation of FcγRIIA occurs upon clustering of the receptor induced by immunocomplexes, and consists in the phosphorylation of two tyrosine residues within the ITAM, typically promoted by an associated Src kinase. The phosphorylated receptor acts as a docking site for SH2 domain-containing signaling proteins, including the tyrosine kinase Syk. This event initiates an intracellular tyrosine kinase-based signaling cascade that eventually leads to phosphorylation and activation of phospholipase C (PLC) γ2, and elicits cellular responses. To date, very little is known on the possible involvement of FcγRIIA in platelet activation induced by soluble agonists. We have found that stimulation of platelets with agonists acting on G-protein-coupled receptors resulted in Src-kinase-mediated tyrosine phosphorylation of FcγRIIA. Treatment of platelets with the blocking monoclonal antibody IV.3 against FcγRIIA, but not with control IgG, inhibited platelet aggregation induced by TRAP1, TRAP4, the thromboxane A2 analogue U46619, and low concentrations of thrombin. By contrast, platelet aggregation induced by high doses of thrombin was unaffected by blockade of FcγRIIA. We also found that the anti-FcγRIIA monoclonal antibody IV.3 inhibited pleckstrin phosphorylation and calcium mobilization induced by low, but not high, concentrations of thrombin. Thrombin- and U46619-induced tyrosine phosphorylation of Syk and PLCγ2, which represent substrates typically involved in FcγRIIA-mediated signaling, was clearly reduced by incubation with anti-FcγRIIA antibody IV.3. Morever, we were able to demonstrated that platelet stimulation by thrombin induced the association of FcγRIIA with Syk. Signaling through immunoreceptor typically takes places in characteristic membrane microdomains called lipid rafts. Upon stimulation with thrombin, FcγRIIA relocated in lipid rafts, and thrombin-induced tyrosine phosphorylation of FcγRIIA occurred within these membrane domains. Controlled disruption of lipid rafts by depleting membrane cholesterol prevented tyrosine phosphorylation of FcγRIIA, and impaired platelet aggregation induced by U46619 or by low, but not high, concentrations of thrombin. These results indicate that FcγRIIA can be activated in human platelets downstream G-protein-coupled receptors, and initiates a tyrosine kinase-based signaling pathway that significantly contributes to platelet activation and aggregation in response to weak stimulation.

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