Protein-tyrosine Kinase p72 syk Is Activated by Platelet Activating Factor in Platelets

1994 ◽  
Vol 72 (06) ◽  
pp. 937-941 ◽  
Author(s):  
Karim Rezaul ◽  
Shigeru Yanagi ◽  
Kiyonao Sada ◽  
Takanobu Taniguchi ◽  
Hirohei Yamamura
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Platelet Activating Factor ◽  
Kinase Assay ◽  
Potential Candidate ◽  
Protein Tyrosine ◽  
Induced Aggregation

SummaryIt has been demonstrated that activation of platelets by platelet-activating factor (PAF) results in a dramatic increase in tyrosine phosphorylation of several cellular proteins. We report here that p72 syk is a potential candidate for the protein-tyrosine phosphorylation following PAF stimulation in porcine platelets. Immunoprecipitation kinase assay revealed that PAF stimulation resulted in a rapid activation of p72 syk which peaked at 10 s. The level of activation was found to be dose dependent and could be completely inhibited by the PAF receptor antagonist, CV3988. Phosphorylation at the tyrosine residues of p72 syk coincided with activation of yllsyk. Pretreatment of platelets with aspirin and apyrase did not affect PAF induced activation of p72 syk .Furthermore, genistein, a potent protein-tyrosine-kinase inhibitor, diminished PAF-induced p72 syk activation and Ca2+ mobilization as well as platelet aggregation. These results suggest that p72 syk may play a critical role in PAF-induced aggregation, possibly through regulation of Ca2+ mobilization.

scholarly journals Transfection of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 cells.

1996 ◽  
Vol 184 (1) ◽  
pp. 71-79 ◽  
Author(s):  
J Zhang ◽  
E H Berenstein ◽  
R L Evans ◽  
R P Siraganian
Keyword(s):  
Tyrosine Kinase ◽  
Histamine Release ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Immunoglobulin E ◽  
Critical Role ◽  
Protein Tyrosine ◽  
High Affinity ◽  
Phospholipase C Gamma

Aggregation of the high affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells results in rapid tyrosine phosphorylation and activation of Syk, a cytoplasmic protein tyrosine kinase. To examine the role of Syk in the Fc epsilon RI signaling pathway, we identified a variant of RBL-2H3 cells that has no detectable Syk by immunoblotting and by in vitro kinase reactions. In these Syk-deficient TB1A2 cells, aggregation of Fc epsilon RI induced no histamine release and no detectable increase in total cellular protein tyrosine phosphorylation. However, stimulation of these cells with the calcium ionophore did induce degranulation. Fc epsilon RI aggregation induced tyrosine phosphorylation of the beta and gamma subunits of the receptor, but no increase in the tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2 and no detectable increase in intracellular free Ca2+ concentration. By transfection, cloned lines were established with stable expression of Syk. In these reconstituted cells, Fc epsilon RI aggregation induced tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2, an increase in intracellular free Ca2+ and histamine release. These results demonstrate that Syk plays a critical role in the early Fc epsilon RI-mediated signaling events. It further demonstrates that Syk activation occurs downstream of receptor phosphorylation, but upstream of most of the Fc epsilon RI-mediated protein tyrosine phosphorylations.

scholarly journals Tyrosine Phosphorylation and p72syk Activation by an Anti-Glycoprotein Ib Monoclonal Antibody

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1590-1598 ◽  
Author(s):  
Mutsumasa Yanabu ◽  
Yukio Ozaki ◽  
Shosaku Nomura ◽  
Tetsuya Miyake ◽  
Yasuhiko Miyazaki ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Platelet Rich Plasma ◽  
Platelet Activating Factor ◽  
Protein Tyrosine Phosphorylation ◽  
Fab Fragment ◽  
Glycoprotein Ib ◽  
Protein Tyrosine

Abstract NNKY5-5, an IgG monoclonal antibody directed against the von Willebrand factor-binding domain of glycoprotein (GP) Ibα, induced weak but irreversible aggregation (or association) of platelets in citrate-anticoagulated platelet-rich plasma. This phenomenon was defined as small aggregate formation (SAF ). Platelets in hirudin-anticoagulated plasma or washed platelets showed little response to NNKY5-5 alone, but the antibody potentiated aggregation induced by low concentrations of adenosine diphosphate or platelet-activating factor. NNKY5-5 did not induce granule release or intracellular Ca2+ mobilization. However, NNKY5-5 caused tyrosine phosphorylation of a 64-kD protein and activation of a tyrosine kinase, p72syk. An anti-FcγII receptor antibody had no effect on SAF, suggesting that NNKY5-5 activated platelets by interacting with glycoprotein Ib. Fab′ fragments of NNKY5-5 did not induce SAF, but potentiated aggregation induced by other agonists. The Fab′ fragment of NNKY5-5 induced the activation of p72syk, suggesting that such activation was independent of the FcγII receptor. Cross-linking of the receptor-bound Fab′ fragment of NNKY5-5 with a secondary antibody induced SAF. GRGDS peptide, chelation of extracellular Ca2+, and an anti-GPIIb/IIIa antibody inhibited NNKY5-5-induced SAF, but had no effect on 64-kD protein tyrosine phosphorylation or p72syk activations. Various inhibitors, including aspirin and protein kinase C, had no effect on SAF, protein tyrosine phosphorylation, or p72syk activation. In contrast, tyrphostin 47, a potent tyrosine kinase inhibitor, inhibited NNKY5-5–induced SAF as well as tyrosine phosphorylation and p72syk activation. Our findings suggest that binding of NNKY5-5 to GPIb potentiates platelet aggregation by facilitating the interaction between fibrinogen and GPIIb/IIIa through a mechanism associated with p72syk activation and tyrosine phosphorylation of a 64-kD protein.

scholarly journals Tyrosine Phosphorylation and p72syk Activation by an Anti-Glycoprotein Ib Monoclonal Antibody

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1590-1598 ◽  
Author(s):  
Mutsumasa Yanabu ◽  
Yukio Ozaki ◽  
Shosaku Nomura ◽  
Tetsuya Miyake ◽  
Yasuhiko Miyazaki ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Platelet Rich Plasma ◽  
Platelet Activating Factor ◽  
Protein Tyrosine Phosphorylation ◽  
Fab Fragment ◽  
Glycoprotein Ib ◽  
Protein Tyrosine

NNKY5-5, an IgG monoclonal antibody directed against the von Willebrand factor-binding domain of glycoprotein (GP) Ibα, induced weak but irreversible aggregation (or association) of platelets in citrate-anticoagulated platelet-rich plasma. This phenomenon was defined as small aggregate formation (SAF ). Platelets in hirudin-anticoagulated plasma or washed platelets showed little response to NNKY5-5 alone, but the antibody potentiated aggregation induced by low concentrations of adenosine diphosphate or platelet-activating factor. NNKY5-5 did not induce granule release or intracellular Ca2+ mobilization. However, NNKY5-5 caused tyrosine phosphorylation of a 64-kD protein and activation of a tyrosine kinase, p72syk. An anti-FcγII receptor antibody had no effect on SAF, suggesting that NNKY5-5 activated platelets by interacting with glycoprotein Ib. Fab′ fragments of NNKY5-5 did not induce SAF, but potentiated aggregation induced by other agonists. The Fab′ fragment of NNKY5-5 induced the activation of p72syk, suggesting that such activation was independent of the FcγII receptor. Cross-linking of the receptor-bound Fab′ fragment of NNKY5-5 with a secondary antibody induced SAF. GRGDS peptide, chelation of extracellular Ca2+, and an anti-GPIIb/IIIa antibody inhibited NNKY5-5-induced SAF, but had no effect on 64-kD protein tyrosine phosphorylation or p72syk activations. Various inhibitors, including aspirin and protein kinase C, had no effect on SAF, protein tyrosine phosphorylation, or p72syk activation. In contrast, tyrphostin 47, a potent tyrosine kinase inhibitor, inhibited NNKY5-5–induced SAF as well as tyrosine phosphorylation and p72syk activation. Our findings suggest that binding of NNKY5-5 to GPIb potentiates platelet aggregation by facilitating the interaction between fibrinogen and GPIIb/IIIa through a mechanism associated with p72syk activation and tyrosine phosphorylation of a 64-kD protein.

scholarly journals Specific tyrosine phosphorylation induced in Schistosoma mansoni miracidia by haemolymph from schistosome susceptible, but not resistant, Biomphalaria glabrata

Parasitology ◽  
2007 ◽  
Vol 135 (3) ◽  
pp. 337-345 ◽  
Author(s):  
A. J. WALKER ◽  
D. ROLLINSON
Keyword(s):  
Tyrosine Kinase ◽  
Schistosoma Mansoni ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Fold Increase ◽  
Biomphalaria Glabrata ◽  
Snail Host ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine

SUMMARYMolecular interplay during snail-schistosome interactions is poorly understood and there is much to discover concerning the effect of snail host molecules on molecular processes in schistosomes. Using the Biomphalaria glabrata – Schistosoma mansoni host-parasite system, the effects of exposure to haemolymph, derived from schistosome-resistant and susceptible snail strains, on protein tyrosine phosphorylation in miracidia have been investigated. Western blotting revealed several tyrosine phosphorylated proteins in this larval stage. Exposure of miracidia to haemolymph from susceptible snails for 60 min resulted in a striking, 5-fold, increase in the tyrosine phosphorylation of a 56 kDa (p56) S. mansoni protein. In contrast, haemolymph from resistant snails had little effect on protein tyrosine phosphorylation levels in miracidia. Confocal microscopy revealed that tyrosine phosphorylation was predominantly associated with proteins present in the tegument. Finally, treatment of miracidia with the tyrosine kinase inhibitor genistein significantly impaired their development into primary sporocysts. The results open avenues for research that focus on the potential importance of phospho-p56 to the outcome of schistosome infection in snails, and the significance of protein tyrosine kinase-mediated signalling events to the transformation of S. mansoni larvae.

scholarly journals Role of Protein Tyrosine Kinase p53/56lyn in Diminished Lipopolysaccharide Priming of Formylmethionylleucyl- phenylalanine-Induced Superoxide Production in Human Newborn Neutrophils

2004 ◽  
Vol 72 (11) ◽  
pp. 6455-6462 ◽  
Author(s):  
Sen Rong Yan ◽  
David M. Byers ◽  
Robert Bortolussi
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Polymorphonuclear Neutrophils ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine ◽  
Adult Cells

ABSTRACT Human newborns are more susceptible than adults to bacterial infection. With gram-negative bacteria, this may be due to a diminished response of newborn leukocytes to lipopolysaccharide (LPS). Since protein tyrosine kinase inhibition abolishes LPS priming in adult cells, we hypothesized that protein tyrosine kinases may have a critical role in LPS priming of polymorphonuclear neutrophils (PMNs) and that newborn PMNs may have altered protein tyrosine kinase activities. In the present study, we investigated the role of src family protein tyrosine kinases in the LPS response of newborn PMNs compared to adult cells. In a respiratory assay, the LPS-primed increase in formylmethionylleucylphenylalanine (fMLP)-triggered O2 − release by adult PMNs was greatly decreased by PP1 [4-amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine], a src kinase inhibitor, to the level of untreated newborn PMNs, in which LPS failed to prime. LPS activated the src-like kinases p59hck (HCK) and p58fgr (FGR) in both adult and newborn PMNs but increased the activation of p53/56 lyn (LYN) only in adult cells. In newborn PMNs, LYN was highly phosphorylated independent of LPS. We evaluated subcellular fractions of PMNs and found that the phosphorylated form of LYN was mainly in the Triton-extractable, cytosolic fraction in adult PMNs, while in newborn cells it was located mainly in Triton-insoluble, granule- and membrane-associated fractions. In contrast, the phosphorylated mitogen-activated protein kinases ERK1/2 and p38 were mainly detected in the cytosol in both adult and newborn PMNs. These data indicate a role for LYN in the regulation of LPS priming. The trapping of phosphorylated LYN in the membrane-granule fraction in newborn PMNs may contribute to the deficiency of newborn cells in responding to LPS stimulation.

Activation of c-Src in cells bearing v-Crk and its suppression by Csk

1992 ◽  
Vol 12 (10) ◽  
pp. 4706-4713
Author(s):  
H Sabe ◽  
M Okada ◽  
H Nakagawa ◽  
H Hanafusa
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Src Kinase ◽  
Cellular Protein ◽  
Morphological Transformation ◽  
Protein Tyrosine ◽  
In Cells

The protein product of the CT10 virus, p47gag-crk (v-Crk), which contains Src homology region 2 (SH2) and 3 (SH3) domains but lacks a kinase domain, is believed to cause an increase in cellular protein tyrosine phosphorylation. A candidate tyrosine kinase, Csk (C-terminal Src kinase), has been implicated in c-Src Tyr-527 phosphorylation, which negatively regulates the protein tyrosine kinase of pp60c-src (c-Src). To investigate how c-Src kinase activity is regulated in vivo, we first looked at whether v-Crk can activate c-Src kinase. We found that cooverexpression of v-Crk and c-Src caused elevation of c-Src kinase activity, resulting in an increase of tyrosine phosphorylation of cellular proteins and morphological transformation of rat 3Y1 fibroblasts. v-Crk and c-Src complexes were not detected, although v-Crk bound to a variety of tyrosine-phosphorylated proteins in cells overexpressing v-Crk and c-Src. Overexpression of Csk in these transformed cells caused reversion to normal phenotypes and also reduced the level of c-Src kinase activity. However, Csk did not cause reversion of cells transformed by v-Src or c-Src527F, in which Tyr-527 was changed to Phe. These results strongly suggest that Csk acts on Tyr-527 of c-Src and suppresses c-Src kinase activity in vivo. Because Csk can suppress transformation by cooverexpression of v-Crk and c-Src, we suggest that v-Crk causes activation of c-Src in vivo by altering the phosphorylation state of Tyr-527.

Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK

1993 ◽  
Vol 13 (2) ◽  
pp. 785-791
Author(s):  
M D Schaller ◽  
C A Borgman ◽  
J T Parsons
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Protein Tyrosine Kinase ◽  
Signal Transduction Pathway ◽  
Focal Adhesions ◽  
Cellular Adhesion ◽  
Protein Tyrosine ◽  
Intracellular Signals ◽  
Embryo Cells

Integrins play a central role in cellular adhesion and anchorage of the cytoskeleton and participate in the generation of intracellular signals, including tyrosine phosphorylation. We have recently isolated a cDNA encoding a unique, focal adhesion-associated protein tyrosine kinase (FAK) that is a component of an integrin-mediated signal transduction pathway. Here we report the isolation of cDNAs encoding the C-terminal, noncatalytic domain of the FAK kinase, termed FRNK (FAK-related nonkinase). Both the FAK- and FRNK-encoded polypeptides, pp125FAK and p41/p43FRNK, are expressed in normal chicken embryo cells. pp125FAK and p41/p43FRNK were localized to focal adhesions, suggesting that pp125FAK is directed to the focal adhesions by sequences within its C-terminal domain. We also show that the fibronectin-dependent increase in tyrosine phosphorylation of pp125FAK is accompanied by a concomitant posttranslational modification of p41FRNK.

The GPI-anchored adhesion molecule F3 induces tyrosine phosphorylation: involvement of the FNIII repeats

1996 ◽  
Vol 109 (3) ◽  
pp. 699-704 ◽  
Author(s):  
M. Cervello ◽  
V. Matranga ◽  
P. Durbec ◽  
G. Rougon ◽  
S. Gomez
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Cho Cells ◽  
Intracellular Signaling ◽  
Protein Tyrosine Kinase ◽  
Cross Linking ◽  
Protein Tyrosine ◽  
Intracellular Signaling Pathway ◽  
Type Iii ◽  
Cell Cell

The glycosyl-phosphatidylinositol (GPI)-anchored F3 molecule, a member of the Ig superfamily made up of Ig and FNIII-like domains, is involved in cell-cell adhesion, neuronal pathfinding and fasciculation. Little is known about the mechanism(s) that governs the F3-mediated cell-cell recognition. In particular, it is not known whether F3 transduces signals across the membrane. Here we show that in F3-transfected CHO cells (1A cells) an increase in tyrosine phosphorylation occurs during F3-mediated aggregation. Moreover, under aggregation conditions F3 immunoprecipitated from 32P-metabolically labeled 1A cells associated with three major phosphorylated proteins. Interestingly, genistein inhibited the F3-mediated aggregation. Increased tyrosine phosphorylation was also observed using antibody-mediated F3-cross-linking. Furthermore, F3 expressed both in 1A cells and in post-natal mouse cerebellum forms non-covalent soluble complexes with protein tyrosine kinase(s). In cerebellum the F3-associated kinase was identified as fyn. By contrast, a truncated F3 protein, expressed in CHO cells, from which all the FN type III repeats have been deleted, does not associate with a kinase. Cross-linking of the F3-truncated form does not induce modulation of tyrosine phosphorylation. Taken together these data demonstrate that F3 is a molecule that transduces signals through both association with protein tyrosine kinase and modulation of protein tyrosine phosphorylation. The presence of FN type III domains is essential for the activation of the intracellular signaling pathway.

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