Melanoma cell spreading on fibronectin induced by 12(S)-HETE involves both protein kinase C- and protein tyrosine kinase-dependent focal adhesion formation and tyrosine phosphorylation of focal adhesion kinase (pp125FAK)

1995 ◽  
Vol 165 (2) ◽  
pp. 291-306 ◽  
Author(s):  
Dean G. Tang ◽  
Mark Tarrien ◽  
Philip Dobrzynski ◽  
Kenneth V. Honn
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Protein Tyrosine Kinase ◽  
Adhesion Formation ◽  
Cell Spreading ◽  
Kinase C ◽  
Focal Adhesion Formation

Effects of Anesthetic Agents on Focal Adhesion Kinase (pp125FAK) Tyrosine Phosphorylation in Rat Hippocampal Slices

2004 ◽  
Vol 101 (2) ◽  
pp. 344-353 ◽  
Author(s):  
Souhayl Dahmani ◽  
Antoine Tesnière ◽  
Danielle Rouelle ◽  
Madeleine Toutant ◽  
Jean-Marie Desmonts ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Receptor Antagonist ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Hippocampal Slices ◽  
Anesthetic Agents ◽  
Kinase C

Background Tyrosine protein kinase proteins exert a prominent control on signaling pathways and may couple rapid events, such as action potential and neurotransmitter release, to long-lasting changes in synaptic strength and survival. Whether anesthetics modulate tyrosine kinase activity remains unknown. The aim of the current study was therefore to examine the effects of intravenous and volatile anesthetics on the phosphorylation of focal adhesion kinase (ppFAK), a functionally important nonreceptor tyrosine kinase, in the rat hippocampus. Methods Phosphorylation of ppFAK was examined in hippocampal slices by immunoblotting with both antiphosphotyrosine and specific anti-ppFAK antibodies. Experiments were performed in the absence (control) or presence of various concentrations of pharmacologic or anesthetic agents or both. Results Clinically relevant concentrations of thiopental, propofol, etomidate, isoflurane, sevoflurane, and desflurane induced a concentration-related increase in tyrosine phosphorylation. In contrast, ketamine (up to 100 microm) and the nonimmobilizer F6 (1,2-dichlorohexafluorocyclobutane, 25 microm) did not significantly affect ppFAK phosphorylation. The anesthetic-induced increase in ppFAK phosphorylation was blocked by GF 109203X, RO 318220, and chelerythrin (100 microm), three structurally distinct inhibitors of protein kinase C and U 73122 (50 microm), an inhibitor of phospholipase C. The propofol- and isoflurane-induced increase in ppFAK phosphorylation was reversible and showed nonadditivity of effects with phorbol 12-myristate 13-acetate (an activator of protein kinase C, 0.1 microm). In contrast, ketamine (up to 100 microm), MK801 (10 microm, an N-methyl-d-aspartate receptor antagonist), bicuculline (10 microm, a gamma-aminobutyric acid type A receptor antagonist), and dantrolene (30 microm, an inhibitor of the ryanodine receptor) were ineffective in blocking anesthetic-induced activation of tyrosine phosphorylation. Conclusion Except for ketamine, anesthetic agents markedly increase tyrosine phosphorylation of ppFAK in the rat hippocampus, most likely via the phospholipase C-protein kinase C pathway, whereas the nonimmobilizer F6 does not. These results suggest that ppFAK represents a target for anesthetic action in the brain.

Protein kinase C involvement in focal adhesion formation

1992 ◽  
Vol 101 (2) ◽  
pp. 277-290 ◽  
Author(s):  
A. Woods ◽  
J.R. Couchman
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Focal Adhesion ◽  
Kinase Inhibitors ◽  
Phorbol Esters ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Stress Fibers ◽  
Kinase C ◽  
Focal Adhesion Formation

Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form. Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h and then treated with kinase inhibitors H7 and HA1004 for 2h, IRM indicated a reduction in focal adhesion formation at concentrations where protein kinase C (PKC) should be inhibited. In contrast, focal adhesions formed normally at concentrations of these inhibitors where cyclic AMP- or cyclic GMP-dependent kinases should be inactivated. Inhibition of PKC, but not that of cyclic AMP- or cyclic GMP-dependent kinases, also prevented the formation of stress fibers and induced a dispersal of talin and vinculin, but not integrin beta 1 subunits, from small condensations present at 1h. Consistent with the reduction in focal adhesion formation when PKC was inhibited, activation of PKC by 30 minutes of treatment with phorbol esters induced focal adhesion formation in cells spread for 3h on substrata composed of the cell-binding (RGD-containing) fragment of fibronectin, while untreated cells or those treated with inactive phorbol esters did not form these structures.

Involvement of proline-rich tyrosine kinase 2 in platelet activation: tyrosine phosphorylation mostly dependent on αIIbβ3 integrin and protein kinase C, translocation to the cytoskeleton and association with Shc through Grb2

2000 ◽  
Vol 347 (2) ◽  
pp. 561-569 ◽  
Author(s):  
Tsukasa OHMORI ◽  
Yutaka YATOMI ◽  
Naoki ASAZUMA ◽  
Kaneo SATOH ◽  
Yukio OZAKI
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Human Platelets ◽  
Sh2 Domain ◽  
Kinase C ◽  
Tyrosine Kinase 2 ◽  
Αiibβ3 Integrin

Proline-rich tyrosine kinase 2 (Pyk2) (also known as RAFTK, CAKβ or CADTK) has been identified as a member of the focal adhesion kinase (FAK) family of protein-tyrosine kinases and it has been suggested that the mode of Pyk2 activation is distinct from that of FAK. In the present study we investigated the mode of Pyk2 activation in human platelets. When platelets were stimulated with thrombin, Pyk2, as well as FAK, was markedly tyrosine-phosphorylated, in a manner mostly dependent on αIIbβ3 integrin-mediated aggregation. The residual Pyk2 tyrosine phosphorylation observed in the absence of platelet aggregation was completely abolished by pretreatment with BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,Nʹ,Nʹ-tetra-acetic acid acetoxymethyl ester]. The Pyk2 phosphorylation was inhibited by protein kinase C (PKC) inhibitors at concentrations that inhibited platelet aggregation. In contrast, direct activation of PKC with the active phorbol ester PMA induced the tyrosine phosphorylation of Pyk2 and FAK but only when platelets were fully aggregated with the exogenous addition of fibrinogen (the ligand for αIIbβ3 integrin). Furthermore, PMA-induced Pyk2 (and FAK) tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. The activation of the von Willebrand factor (vWF)--glycoprotein Ib pathway with botrocetin together with vWF failed to induce Pyk2 (and FAK) tyrosine phosphorylation. Most Pyk2 and FAK was present in the cytosol and membrane skeleton fractions in unstimulated platelets. When platelets were stimulated with thrombin, both Pyk2 and FAK were translocated to the cytoskeleton in an aggregation-dependent manner. In immunoprecipitation studies, Pyk2, as well as FAK, seemed to associate with Shc through Grb2. With the use of glutathione S-transferase fusion proteins containing Shc-SH2, Grb2-SH2, and Grb2 N-terminal and C-terminal SH3 domains, it was implied that the proline-rich region of Pyk2 (and FAK) binds to the N-terminal SH3 domain of Grb2 and that the phosphotyrosine residue of Shc binds to the SH2 domain of Grb2. Although Pyk2 and FAK have been reported to be differentially regulated in many cell types, our results suggest that, in human platelets, the mode of Pyk2 activation is mostly similar to that of FAK, in terms of αIIbβ3 integrin-dependent and PKC-dependent tyrosine phosphorylation. Furthermore, Pyk2, as well as FAK, might have one or more important roles in post-aggregation tyrosine phosphorylation events, in association with the cytoskeleton and through interaction with adapter proteins including Grb2 and Shc.

scholarly journals The expression of COX-2 in VEGF-treated endothelial cells is mediated through protein tyrosine kinase

2002 ◽  
Vol 11 (1) ◽  
pp. 17-22 ◽  
Author(s):  
Pravit Akarasereenont ◽  
Kitirat Techatraisak ◽  
Athiwat Thaworn ◽  
Sirikul Chotewuttakorn
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Kinase C ◽  
Protein Kinase C Inhibitor ◽  
Cox 2 ◽  
Cox 1

Cyclooxygenase (COX), existing as the COX-1 and COX-2 isoforms, converts arachidonic acid to prostaglandin H2, which is then further metabolized to various prostaglandins. Vascular endothelial growth factor (VEGF) has been shown to play important roles in inflammation and is upregulated by the prostaglandin E series through COX-2 in several cell types. Here, we have investigated the effects of VEGF on the COX isoform expressed in human umbilical vein endothelial cells (HUVEC). The signalling mechanism of the COX isoform expressed in endothelial cells activated with VEGF will be also investigated using the tyrosine kinase inhibitor, genistein, and protein kinase C inhibitor, staurosporine. The activity of COX2 was assessed by measuring the production of 6-keto-prostaglandin F1α in the presence of exogenous arachidonic acids (10 μM, 10 min) by enzyme immunoassay. The expression of COX isoform protein was detected by immunoblot using specific antibodies. Untreated HUVEC contained no COX-2 protein. In HUVEC treated with VEGF (0.01-50 ng/ml), COX-2 protein, but not COX-1, and COX activity were increased in a dose-dependent manner. Interestingly, the increased COX-2 protein and activity in response to VEGF (10 ng/ml) was inhibited by the tyrosine kinase inhibitor, genistein (0.05-5 μg/ml), but not by the protein kinase C inhibitor, staurosporine (0.1-10 ng/ml). Thus, the induction of COX-2 by VEGF in endothelial cells was mediated through protein tyrosine kinase, and the uses of specific COX-2 inhibitors in these conditions, in which VEGF was involved, might have a role.

Platelet P-selectin expression: requirement for protein kinase C, but not protein tyrosine kinase or phosphoinositide 3-kinase

2003 ◽  
Vol 89 (06) ◽  
pp. 1016-1023 ◽  
Author(s):  
Danielle Libersan ◽  
Yahye Merhi
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Human Platelet ◽  
The Novel ◽  
Protein Tyrosine ◽  
Kinase C ◽  
Activated Platelets ◽  
Phosphoinositide 3 Kinase

SummaryP-selectin is translocated from the α-granules to the surface of activated platelets where it participates in thrombosis and inflammation. We investigated the signaling pathways involved in thrombin-induced human platelet P-selectin expression. Assessed by flow cytometry, inhibition of protein kinase C (PKC) with chelerythrine reduced P-selectin expression by 66%, platelet/neutrophil binding, GPIIb/IIIa activation and aggregation (p<0.05). Gö 6976, an inhibitor of the conventional PKCs (α and β), did not alter P-selectin expression. However, rottlerin inhibited by 50% its expression (p<0.05), but only at doses that interfere with the novel (є, η) and atypical (ζ) PKCs. Inhibition of protein tyrosine kinase (PTK) and phosphoinositide 3-kinase (PI3-K) did not significantly affect P-selectin expression. In conclusion, thrombin-induced P-selectin expression is PKC-sensitive, but PTK and PI3-K-insensitive. The novel є and η and atypical ζ, but not the conventional α and β and the novel θ PKCs, may be involved in this process.

Endothelin-1–Induced Interleukin-8 Production in Human Brain-Derived Endothelial Cells Is Mediated by the Protein Kinase C and Protein Tyrosine Kinase Pathways

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1291-1299 ◽  
Author(s):  
R. Zidovetzki ◽  
P. Chen ◽  
M. Chen ◽  
F.M. Hofman
Keyword(s):  
Signal Transduction ◽  
Endothelial Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Protein Tyrosine ◽  
Kinase C ◽  
Eta Receptor ◽  
Specific Inhibitors

We have previously demonstrated that endothelin-1 (Et-1) induces human central nervous system-derived endothelial cells (CNS-EC) to produce and secrete the chemokine interleukin 8 (IL-8). In the present study, we use specific inhibitors and activators to elucidate the signal transduction pathways involved in this process. Et-1–induced IL-8 production was blocked by ETA receptor antagonist BQ610, but not by ETB receptor antagonist BQ788, demonstrating that CNS-EC activation is initiated by Et-1 binding to the ETA receptor. IL-8 mRNA expression is blocked by the protein kinase C inhibitor bisindolylmaleimide or protein tyrosine kinase inhibitors, genestein and geldanamycin, establishing the involvement of the protein kinase C and protein tyrosine kinase pathways in the activation process. The transcription factor, NF-κB, is involved in Et-1 activation as determined by specific inhibitors of translocation and direct analysis of DNA-binding proteins. Neither inhibition nor activation of cAMP-dependent protein kinase affected IL-8 production in the absence or presence of Et-1. Similarly, no effect was observed upon inhibition of protein phosphatases by okadaic acid. Thus, the signal transduction process induced by Et-1 in CNS-EC, leading to increased mRNA IL-8 expression, is initiated by Et-1 binding to ETA receptor followed by subsequent activation of protein kinase C, protein tyrosine kinase, and NF-κB. Because increased expression of Et-1 is associated with hypertension and stroke and IL-8 is likely to be involved in the accumulation of neutrophils causing tissue damage in ischemic/reperfusion injury, identification of the mechanism involved in the Et-1–induced increase in IL-8 production may have significant therapeutic value.

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