Stimulation of protein tyrosine phosphorylation by NMDA receptor activation

Glutamate triggers neuronal degeneration after ischemia-reperfusion in the brain. However, the details of intracellular signal transduction that propagates cell death remain unknown. The present work investigated whether protein tyrosine phosphorylation mediates neuronal death in the ischemic brain. Transient forebrain ischemia for 5-10 min in Mongolian gerbils or intoxication with the glutamate analogue kainic acid (12 mg/kg) in Sprague-Dawley rats caused neuronal death selectively in the hippocampus 2-4 days or 1 day later, respectively. Under these conditions, 160-, 115-, 105-, 92-, and 85-kDa proteins showed a significant increase in tyrosyl residue phosphorylation selectively in the hippocampus 3-12 h after ischemia or 4-8 h after kainic acid-induced seizures. Tyrosine kinases, including pp60c-src, were activated without a change of tyrosine phosphatases. Administration of radicicol, a selective inhibitor of tyrosine kinases, attenuated stimulation of tyrosine phosphorylation and hippocampal degeneration after ischemia or kainic acid injection. The results suggest that protein tyrosine phosphorylation might propagate delayed neuronal death in the mature hippocampus through glutamate overload after ischemia-reperfusion.

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Protein tyrosine phosphorylation in rabbit peritoneal neutrophils

Biochemical Journal ◽

10.1042/bj2690431 ◽

1990 ◽

Vol 269 (2) ◽

pp. 431-436 ◽

Cited By ~ 42

Author(s):

C K Huang ◽

V Bonak ◽

G R Laramee ◽

J E Casnellie

Keyword(s):

Tyrosine Phosphorylation ◽

Kinase Inhibitor ◽

Leukotriene B4 ◽

Early Event ◽

Ionophore A23187 ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine ◽

Group A ◽

Group B ◽

Stimulation Of

Protein tyrosine phosphorylation in rabbit peritoneal neutrophils was examined by immunoblotting with antibodies specific for phosphotyrosine. Stimulation of the neutrophils with chemotactic factor fMet-Leu-Phe (10 nM) caused rapid increases of tyrosine phosphorylation of several proteins with apparent molecular masses of (Group A) 54-58 kDa and 100-125 kDa and (Group B) 36-41 kDa. Stimulation of Group A proteins was observed by fMet-Leu-Phe (10 nM, maximum at 20 s) and A23187 (1 microM, 1 min). Stimulation of Group B proteins was observed by fMet-Leu-Phe (ED50 0.15 nM, 1 min), leukotriene B4 (ED50 0.15 nM, 1 min), phorbol 12-myristate 13-acetate (PMA) (ED50 25 ng/ml, 10 min) and partially by ionophore A23187 (1 microM, 1 min). Pretreatment of the cell with the protein kinase inhibitor H-7 (25 microM, 5 min) and PMA (0.1 microgram/ml, 3 min) partially inhibited the fMet-Leu-Phe effect. However, pretreatment of the cells with quin 2/AM (20 microM, 10 min) completely inhibited the fMet-Leu-Phe effect. The results indicate that rapid regulation of tyrosine phosphorylation is an early event occurring in stimulated neutrophils. Furthermore the effect of fMet-Leu-Phe on tyrosine phosphorylation may require Ca2+ mobilization and may partially require the activity of H-7-sensitive protein kinases.

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Protein tyrosine phosphorylation, cellular Ca2+, and Ca2+ sensitivity for contraction of smooth muscle

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y94-207 ◽

1994 ◽

Vol 72 (11) ◽

pp. 1434-1439 ◽

Cited By ~ 55

Author(s):

Joseph Di Salvo ◽

Gabrielle Pfitzer ◽

Lori A. Semenchuk

Keyword(s):

Smooth Muscle ◽

Tyrosine Phosphorylation ◽

Vascular Smooth Muscle Cells ◽

Adrenergic Receptors ◽

Kinase Activity ◽

Tyrosine Phosphatase ◽

Protein Tyrosine Phosphorylation ◽

Tyrosine Kinase Activity ◽

Protein Tyrosine ◽

Stimulation Of

Our studies are guided by the novel hypothesis that protein tyrosine phosphorylation is an important mechanism for regulating contraction of smooth muscle. Several lines of evidence are reviewed which suggest that enhanced tyrosine phosphorylation participates in mechanisms that regulate cytosolic Ca2+ and Ca2+ sensitivity for contraction. First, vanadate-induced contraction of guinea-pig taenia coli is functionally linked to enhanced protein tyrosine phosphorylation of at least three substrates, apparently resulting from vanadate-mediated inhibition of protein tyrosine phosphatase activity. Second, vanadate-induced contraction is dependent on extracellular Ca2+. Third, increases in cytosolic Ca2+ resulting from stimulation of αi-adrenergic receptors in cultured canine vascular smooth muscle cells are associated with enhanced tyrosine phosphorylation and are inhibited by genistein, a potent inhibitor of tyrosine kinase activity. Fourth, genistein markedly and reversibly suppresses Ca2+ sensitivity for contraction in ileal longitudinal smooth muscle permeabilized with staphylococcal α-toxin. Moreover, the same or similar substrates (e.g., 42–45, 70, 80–85, 95, 100, 110, 116, and 205 kDa) are tyrosine phosphorylated in response to Ca2+ or stimulation of muscarinic or αi-adrenergic receptors. Collectively, these data strongly suggest that tyrosine phosphorylation is an important mechanism for regulation of smooth muscle contraction.Key words: actin–myosin interaction, calcium sensitivity, genistein, permeabilized smooth muscle, tyrosine kinase activity, tyrosine phosphatase activity, vascular smooth muscle cells, vanadate.

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Hyaluronan and the hyaluronan receptor RHAMM promote focal adhesion turnover and transient tyrosine kinase activity.

The Journal of Cell Biology ◽

10.1083/jcb.126.2.575 ◽

1994 ◽

Vol 126 (2) ◽

pp. 575-588 ◽

Cited By ~ 153

Author(s):

C L Hall ◽

C Wang ◽

L A Lange ◽

E A Turley

Keyword(s):

Tyrosine Kinase ◽

Cell Motility ◽

Tyrosine Phosphorylation ◽

Focal Adhesion ◽

Peptide Sequence ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine ◽

Cell Locomotion ◽

Focal Adhesion Turnover ◽

Stimulation Of

The molecular mechanisms whereby hyaluronan (HA) stimulates cell motility was investigated in a C-H-ras transformed 10T 1/2 fibroblast cell line (C3). A significant (p < 0.001) stimulation of C3 cell motility with HA (10 ng/ml) was accompanied by an increase in protein tyrosine phosphorylation as detected by anti-phosphotyrosine antibodies using immunoblot analysis and immunofluorescence staining of cells. Tyrosine phosphorylation of several proteins was found to be both rapid and transient with phosphorylation occurring within 1 min of HA addition and dissipating below control levels 10-15 min later. These responses were also elicited by an antibody generated against a peptide sequence within the HA receptor RHAMM. Treatment of cells with tyrosine kinase inhibitors (genistein, 10 micrograms/ml or herbimycin A, 0.5 micrograms/ml) or microinjection of anti-phosphotyrosine antibodies inhibited the transient protein tyrosine phosphorylation in response to HA as well as prevented HA stimulation of cell motility. To determine a link between HA-stimulated tyrosine phosphorylation and the resulting cell locomotion, cytoskeletal reorganization was examined in C3 cells plated on fibronectin and treated with HA or anti-RHAMM antibody. These agents caused a rapid assembly and disassembly of focal adhesions as revealed by immunofluorescent localization of vinculin. The time course with which HA and antibody induced focal adhesion turnover exactly paralleled the induction of transient protein tyrosine phosphorylation. In addition, phosphotyrosine staining colocalized with vinculin within structures in the lamellapodia of these cells. Notably, the focal adhesion kinase, pp125FAK, was rapidly phosphorylated and dephosphorylated after HA stimulation. These results suggest that HA stimulates locomotion via a rapid and transient protein tyrosine kinase signaling event mediated by RHAMM. They also provide a possible molecular basis for focal adhesion turnover, a process that is critical for cell locomotion.

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Bradykinin B2 receptor-induced and inositol tetrakisphosphate-evoked Ca2+ entry is sensitive to a protein tyrosine phosphorylation inhibitor in ras-transformed NIH/3T3 fibroblasts

Biochemical Journal ◽

10.1042/bj3190649 ◽

1996 ◽

Vol 319 (2) ◽

pp. 649-656 ◽

Cited By ~ 16

Author(s):

Minako HASHII ◽

Shigeru NAKASHIMA ◽

Shigeru YOKOYAMA ◽

Koh-ichi ENOMOTO ◽

Yoshio MINABE ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Receptor Activation ◽

Signal Pathways ◽

Protein Tyrosine Phosphorylation ◽

Membrane Hyperpolarization ◽

Bradykinin B2 Receptor ◽

Protein Tyrosine ◽

3T3 Fibroblasts ◽

B2 Receptors ◽

Nih 3T3

Signal transduction from mouse bradykinin B2 receptors to calcium influx was studied in ras-transformed NIH/3T3 (DT) fibroblasts. DT cells were preloaded with fura-2 and whole-cell voltage-clamped. Activation of B2 receptors resulted in a decrease of cellular fluorescence at the excitation wavelength of 340, or 360 nm after MnCl2 application, in both the presence and absence of external Ca2+ in DT cells, at a holding potential of -40 mV. This Mn2+ entry through the Ca2+ influx pathway increased with membrane hyperpolarization. Internal application of inositol 1,3,4,5-tetrakisphosphate (InsP4), but not of inositol 1,4,5-trisphosphate, mimicked membrane potential-dependent Mn2+ entry. Bradykinin- and InsP4-induced Ca2+ influx was blocked by 10–100 µM genistein, a tyrosine kinase inhibitor. B2 receptor activation induced time-dependent tyrosine phosphorylation of mitogen-activated protein kinase and 120 kDa protein, which was dose-dependently inhibited by genistein. Bradykinin was unable to induce Ca2+ oscillations in genistein-treated DT cells. Our results show that bradykinin-induced Ca2+ influx and oscillations depend upon protein tyrosine phosphorylation. The results suggest that two bradykinin B2 receptor-activated signal pathways, protein tyrosine phosphorylation and formation of InsP4, merge at the Ca2+ influx process in ras-transformed NIH/3T3 fibroblasts.

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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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