scholarly journals Sphingosine 1-phosphate stimulates Rho-mediated tyrosine phosphorylation of focal adhesion kinase and paxillin in Swiss 3T3 fibroblasts

1997 ◽  
Vol 324 (2) ◽  
pp. 481-488 ◽  
Author(s):  
Fang WANG ◽  
Catherine D. NOBES ◽  
Alan HALL ◽  
Sarah SPIEGEL
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Stress Fibre ◽  
3T3 Fibroblasts ◽  
Fibre Formation ◽  
Sphingosine 1 Phosphate ◽  
Swiss 3T3 Fibroblasts ◽  
Stress Fibre Formation ◽  
Stimulation Of

Sphingosine 1-phosphate (SPP), a sphingolipid second messenger implicated in the mitogenic action of platelet-derived growth factor [Olivera, A. and Spiegel, S. (1993) Nature (London) 365, 557–560], induced rapid reorganization of the actin cytoskeleton resulting in stress-fibre formation. SPP also induced transient tyrosine phosphorylation of focal adhesion kinase (p125FAK), a cytosolic tyrosine kinase that localizes in focal adhesions, and of the cytoskeleton-associated protein paxillin. Exoenzyme C3 transferase, which ADP-ribosylates Rho (a Ras-related small GTP binding protein) on asparagine-41 and renders it biologically inactive, inhibited both stress-fibre formation and protein tyrosine phosphorylation induced by SPP. Thus Rho may be an upstream regulator of both stress-fibre formation and tyrosine phosphorylation of p125FAK and paxillin. Pretreatment with PMA, an activator of protein kinase C (PKC), inhibited the stimulation of stress-fibre formation induced by 1-oleoyl-lysophosphatidic acid (LPA) but not that by SPP. Similarly, PMA also decreased LPA-induced tyrosine phosphorylation of p125FAK and paxillin without abrogating the response to SPP. Thus PKC is involved in LPA- but not SPP-dependent signalling. The polyanionic drug suramin, a broad-specificity inhibitor of ligand–receptor interactions, did not inhibit either the mitogenic effect of SPP or its stimulation of tyrosine phosphorylation of p125FAK. However, suramin markedly inhibited these responses induced by LPA. These results suggest that in contrast with LPA, SPP may be acting intracellularly in Swiss 3T3 fibroblasts to stimulate tyrosine phosphorylation of p125FAK and paxillin and cell growth.

scholarly journals Focal adhesion kinase (p125FAK) and paxillin are substrates for sphingomyelinase-induced tyrosine phosphorylation in Swiss 3T3 fibroblasts

1996 ◽  
Vol 315 (3) ◽  
pp. 1035-1040 ◽  
Author(s):  
Takehiko SASAKI ◽  
Kaoru HAZEKI ◽  
Osamu HAZEKI ◽  
Michio UI ◽  
Toshiaki KATADA
Keyword(s):  
Tyrosine Kinase ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Kinase Activity ◽  
Tyrosine Kinase Activity ◽  
3T3 Fibroblasts ◽  
Kinase Cascade ◽  
Swiss 3T3 ◽  
Swiss 3T3 Fibroblasts

We examined the effect of sphingomyelinase on tyrosine phosphorylation of intracellular proteins in mouse Swiss 3T3 fibroblasts. Incubation of the cells with bacterial sphingomyelinase resulted in the elevation of tyrosine phosphorylation of multiple cellular proteins of 190, 130, 120, 97 and 70 kDa within minutes. The 120 and 70 kDa tyrosine-phosphorylated peptides were identified as p125 focal adhesion kinase (p125FAK) and paxillin respectively by the use of specific antibodies against the proteins. Tyrosine kinase activity associated with anti-p125FAK immunoprecipitate was stimulated by incubation of cells with sphingomyelinase. Cytochalasin D, which selectively disrupts the network of actin filaments, inhibited sphingomyelinase-induced tyrosine phosphorylation of p125FAK and elevation of tyrosine kinase activity in the anti-p125FAK immunoprecipitates. Sphingomyelinase-induced phosphorylation of p125FAK was not inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. This was in sharp contrast with a wortmannin-sensitive phosphorylation of p125FAK observed in platelet-derived growth factor (PGDF)-stimulated cells. Thus hydrolysis of sphingomyelin is considered to regulate the tyrosine kinase cascade including p125FAK and paxillin by a mechanism distinct from PDGF.

scholarly journals Stimulation of actin stress fibre formation mediated by activation of phospholipase D

1996 ◽  
Vol 6 (5) ◽  
pp. 588-597 ◽  
Author(s):  
Michael J. Cross ◽  
Sally Roberts ◽  
Anne J. Ridley ◽  
Matthew N. Hodgkin ◽  
Allison Stewart ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Stress Fibre ◽  
Actin Stress Fibre ◽  
Fibre Formation ◽  
Stress Fibre Formation ◽  
Stimulation Of

scholarly journals The roles of multiple pathways in regulating bombesin-stimulated phospholipase D activity in Swiss 3T3 fibroblasts

1995 ◽  
Vol 306 (1) ◽  
pp. 115-122 ◽  
Author(s):  
C P Briscoe ◽  
A Martin ◽  
M Cross ◽  
M J O Wakelam
Keyword(s):  
Tyrosine Phosphorylation ◽  
Phospholipase D ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Protein Tyrosine ◽  
Permeabilized Cells ◽  
3T3 Fibroblasts ◽  
Swiss 3T3 ◽  
Swiss 3T3 Fibroblasts ◽  
Stimulation Of

The regulation of bombesin-stimulated phospholipase D (PLD) activity in Swiss 3T3 fibroblasts was examined. Increasing protein-tyrosine phosphorylation by using pervanadate to inhibit tyrosine phosphatases was found to stimulate protein kinase C (PKC)-independent [3H]phosphatidylbutanol ([3H]PtdBut) accumulation within 5 min, which continued to increase up to 30 min. The stimulation of PLD activity in response to submaximal [bombesin] could be decreased by approx. 50% by the tyrosine kinase inhibitor genistein, whereas pretreatment with genistein and the PKC inhibitor Ro-31-8220 completely abolished the generation of [3H]PtdBut in response to a maximal concentration of bombesin. The addition of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) into permeabilized cells resulted in an increase in [3H]PtdBut, which was abolished by depletion of cellular ATP. The additional presence of 30 microM GTP[S] did not increase the stimulation of PLD activity by any [bombesin] tested, whereas it was synergistic with that stimulated in response to phorbol 12-myristate 13-acetate. These findings suggest that bombesin-stimulated PLD activity is indirectly regulated by G-proteins, possibly through a kinase intermediate. Furthermore, activation of protein tyrosine kinases is proposed to account for the PKC-independent arm of bombesin-stimulated PLD activity. No evidence was obtained for a form of PLD directly regulated by tyrosine phosphorylation.

Proline-rich Tyrosine Kinase 2 and Focal Adhesion Kinase Are Involved in Different Phases of Platelet Activation by vWF

2002 ◽  
Vol 87 (03) ◽  
pp. 509-517 ◽  
Author(s):  
Ilaria Canobbio ◽  
Paolo Lova ◽  
Fabiola Sinigaglia ◽  
Cesare Balduini ◽  
Mauro Torti
Keyword(s):  
Tyrosine Kinase ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Tyrosine Kinases ◽  
Human Platelets ◽  
Glycoprotein Ib ◽  
Tyrosine Kinase 2 ◽  
Calcium Chelation ◽  
Stimulation Of

SummaryStimulation of human platelets with von Willebrand factor (vWF) induces the rapid tyrosine phosphorylation of several proteins, but very little is known on the tyrosine kinases involved in this process. In the present work, we investigated and compared the activation of two related tyrosine kinases expressed in platelets: the proline-rich tyrosine kinase 2 (Pyk2) and the focal adhesion kinase (FAK). Both kinases were tyrosine phosphorylated upon vWF interaction with glycoprotein Ib-IX-V complex, but with different mechanisms. Tyrosine phosphorylation of FAK was totally dependent on thromboxane A2 production, and was inhibited by the integrin αIIbβ3 antagonist RGDS peptide. Moreover, chelation of intracellular calcium or inhibition of protein kinase C (PKC) totally blocked vWF-induced tyrosine phosphorylation of FAK, indicating that this event is downstream phospholipase A2 and phospholipase C activation. By contrast, tyrosine phosphorylation of Pyk2 was only partially reduced by aspirin and RGDS, and was not affected by either calcium chelation or PKC inhibition, suggesting that activation of this kinase does not require phospholipase-mediated signalling. Both FAK and Pyk2 translocated to the cytoskeleton upon vWF stimulation of human platelets by a mechanism depending on agonist-induced actin polymerisation. Prevention of cytoskeletal relocation of Pyk2 and FAK by cytochalasin D totally blocked vWFinduced tyrosine phosphorylation of both kinases. Finally, phosphorylation of Pyk2 induced by vWF, but not by thrombin, was inhibited by piceatannol, suggesting that this kinase lies downstream Syk. These results demonstrate that both Pyk2 and FAK are involved in platelet stimulation by vWF, but indicate that only Pyk2 may play a role in the early signal transduction events activated by ligand binding to glycoprotein Ib-IX-V.

scholarly journals The simulation of stress fibre and focal adhesion development in cells on patterned substrates

2007 ◽  
Vol 5 (22) ◽  
pp. 507-524 ◽  
Author(s):  
Amit Pathak ◽  
Vikram S Deshpande ◽  
Robert M McMeeking ◽  
Anthony G Evans
Keyword(s):  
Focal Adhesion ◽  
Coupled Modelling ◽  
Stress Fibre ◽  
Fibre Formation ◽  
Highly Nonlinear ◽  
Pattern Shape ◽  
High Concentrations ◽  
Stress Dependent ◽  
Kinetics Of ◽  
Stress Fibre Formation

The remodelling of the cytoskeleton and focal adhesion (FA) distributions for cells on substrates with micro-patterned ligand patches is investigated using a bio-chemo-mechanical model. We investigate the effect of ligand pattern shape on the cytoskeletal arrangements and FA distributions for cells having approximately the same area. The cytoskeleton model accounts for the dynamic rearrangement of the actin/myosin stress fibres. It entails the highly nonlinear interactions between signalling, the kinetics of tension-dependent stress-fibre formation/dissolution and stress-dependent contractility. This model is coupled with another model that governs FA formation and accounts for the mechano-sensitivity of the adhesions from thermodynamic considerations. This coupled modelling scheme is shown to capture a variety of key experimental observations including: (i) the formation of high concentrations of stress fibres and FAs at the periphery of circular and triangular, convex-shaped ligand patterns; (ii) the development of high FA concentrations along the edges of the V-, T-, Y- and U-shaped concave ligand patterns; and (iii) the formation of highly aligned stress fibres along the non-adhered edges of cells on the concave ligand patterns. When appropriately calibrated, the model also accurately predicts the radii of curvature of the non-adhered edges of cells on the concave-shaped ligand patterns.

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