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

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.

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

Blood ◽

10.1182/blood.v89.5.1590.1590_1590_1598 ◽

1997 ◽

Vol 89 (5) ◽

pp. 1590-1598 ◽

Cited By ~ 3

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.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648987 ◽

1994 ◽

Vol 72 (06) ◽

pp. 937-941 ◽

Cited By ~ 7

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.

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Bradykinin stimulates the ERK→Elk-1→Fos/AP-1 pathway in mesangial cells

AJP Renal Physiology ◽

10.1152/ajprenal.1998.275.3.f343 ◽

1998 ◽

Vol 275 (3) ◽

pp. F343-F352 ◽

Cited By ~ 16

Author(s):

Samir S. El-Dahr ◽

Susana Dipp ◽

William H. Baricos

Keyword(s):

Transcription Factor ◽

Tyrosine Kinase ◽

Dna Synthesis ◽

Tyrosine Phosphorylation ◽

Kinase Inhibitor ◽

Mesangial Cells ◽

Fold Increase ◽

Antisense Oligodeoxynucleotide ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine

Among its diverse biological actions, the vasoactive peptide bradykinin (BK) induces the transcription factor AP-1 and proliferation of mesangial cells (S. S. El-Dahr, S. Dipp, I. V. Yosipiv, and W. H. Baricos. Kidney Int. 50: 1850–1855, 1996). In the present study, we examined the role of protein tyrosine phosphorylation and the mitogen-activated protein kinases, ERK1/2,in mediating BK-induced AP-1 and DNA replication in cultured rat mesangial cells. BK (10−9 to 10−7 M) stimulated a rapid increase in tyrosine phosphorylation of multiple proteins with an estimated molecular mass of 120–130, 90–95, and 44–42 kDa. Immunoblots using antibodies specific for ERK or tyrosine-phosphorylated ERK revealed a shifting of p42 ERK2 to a higher molecular weight that correlated temporally with an increase in tyrosine-phosphorylated ERK2. Genistein, a specific tyrosine kinase inhibitor, prevented the phosphorylation of ERK2 by BK. In-gel kinase assays indicated that BK-induced tyrosine phosphorylation of ERK2 is accompanied by fourfold activation of its phosphotransferase activity toward the substrate PHAS-I ( P < 0.05). Furthermore, BK stimulated a 2.5-fold increase ( P < 0.05) in phosphorylation of Elk-1, a transcription factor required for growth factor-induced c-fos transcription. In accord with the stimulation of Elk-1 phosphorylation, BK induced c-fos gene expression and the production of Fos/AP-1 complexes. In addition, thymidine incorporation into DNA increased twofold ( P < 0.05) following BK stimulation. Each of these effects was blocked by tyrosine kinase inhibition with genistein or herbimycin A. Similarly, antisense oligodeoxynucleotide targeting of ERK1/2 mRNA inhibited BK-stimulated DNA synthesis. In contrast, protein kinase C inhibition or depletion had no effect on BK-induced c-fos mRNA, AP-1-DNA binding activity, or DNA synthesis. Collectively, these data demonstrate that BK activates the ERK→Elk-1→AP-1 pathway and that BK mitogenic signaling is critically dependent on protein tyrosine phosphorylation.

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

Parasitology ◽

10.1017/s0031182007003964 ◽

2007 ◽

Vol 135 (3) ◽

pp. 337-345 ◽

Cited By ~ 7

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.

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Tyrosine phosphorylation induced by cross-linking of Fc γ-receptor type II in human neutrophils

Biochemical Journal ◽

10.1042/bj3060489 ◽

1995 ◽

Vol 306 (2) ◽

pp. 489-495 ◽

Cited By ~ 14

Author(s):

L Liang ◽

C K Huang

Keyword(s):

Tyrosine Phosphorylation ◽

Pertussis Toxin ◽

Protein Tyrosine Kinase ◽

Kinase Inhibitor ◽

Human Neutrophils ◽

Cross Linking ◽

Type Ii ◽

Protein Tyrosine Phosphorylation ◽

Fab Fragment ◽

Protein Tyrosine

Neutrophils express several receptors for the Fc region of IgG molecules. Specific cross-linking of the type II receptor (Fc gamma RII) can be achieved by treating neutrophils with the Fab fragment of a specific monoclonal antibody IV.3 against the receptor followed by goat anti-mouse IgG F(ab′)2 fragment. Such treatment initiates a number of neutrophil responses including the release of O2-. and increased protein tyrosine phosphorylation. The increase in tyrosine phosphorylation is rapid and transient and correlates with O2-. release. Both responses are inhibited by pretreatment of neutrophils with a protein tyrosine kinase inhibitor, genistein. The increase in protein tyrosine phosphorylation is not inhibited by pretreatment of neutrophils with pertussis toxin or an intracellular Ca2+ chelator, but is enhanced by a phosphoprotein phosphatase inhibitor, okadaic acid. The activity of a neutrophil Ca2+/calmodulin-dependent protein kinase II (CAMPKII) is also stimulated by cross-linking Fc gamma RII. The increase in CAMPKII activity is inhibited by pretreatment with either genistein or Ca2+ chelator. The results suggest that the increase in protein tyrosine phosphorylation induced by cross-linking of Fc gamma RII requires neither pertussis-toxin-sensitive G-proteins nor a rise in intracellular Ca2+ but can be regulated by protein phosphatases. Furthermore, protein tyrosine phosphorylation may be an early signal functionally linked to Fc gamma RII-mediated signal transduction leading to CAMPKII activation and O2-. release in human neutrophils.

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Protein tyrosine phosphorylation and regulation of the receptor for platelet-activating factor in rat Kupffer cells. Effect of sodium vanadate

Biochemical Journal ◽

10.1042/bj2880777 ◽

1992 ◽

Vol 288 (3) ◽

pp. 777-784 ◽

Cited By ~ 12

Author(s):

W Chao ◽

H Liu ◽

D J Hanahan ◽

M S Olson

Keyword(s):

Tyrosine Phosphorylation ◽

Kupffer Cells ◽

Kinase Inhibitor ◽

Platelet Activating Factor ◽

Surface Expression ◽

Pge2 Production ◽

Protein Tyrosine Phosphorylation ◽

Sodium Vanadate ◽

Protein Tyrosine ◽

Pg E2

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC) and sodium vanadate (a phosphotyrosine phosphatase inhibitor) induced a time- and concentration-dependent increase in phosphotyrosine in several proteins and stimulated prostaglandin (PG) E2 production in cultured rat Kupffer cells. In addition, vanadate induced a decrease in the surface expression of AGEPC receptors and, as a consequence, inhibited AGEPC-stimulated PGE2 production. The vanadate-induced decrease in the surface expression of AGEPC receptors was time- and concentration-dependent and was partially prevented by genistein, a putative tyrosine kinase inhibitor. Upon removal of vanadate from the culture medium and re-incubation of cells in vanadate-free medium, the surface AGEPC receptors were restored within 7 h after the removal of vanadate. Both AGEPC- and vanadate-stimulated PGE2 formation was attenuated by genistein. Thus the present investigation demonstrates that both AGEPC and sodium vanadate stimulate tyrosine phosphorylation of cellular proteins, and vanadate induces a decrease in the number of the surface AGEPC receptors. These results suggest that protein tyrosine phosphorylation may play a role, directly or indirectly, in the regulation of surface expression of AGEPC receptors as well as in PGE2 production in response to vanadate and AGEPC.

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

Blood ◽

10.1182/blood.v78.8.1928.1928 ◽

1991 ◽

Vol 78 (8) ◽

pp. 1928-1935 ◽

Cited By ~ 16

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.

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

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1998.274.2.h513 ◽

1998 ◽

Vol 274 (2) ◽

pp. H513-H519 ◽

Cited By ~ 2

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.

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