p125FAK Phosphorylation Research Articles

Cholecystokinin-stimulated tyrosine phosphorylation of p125FAK and paxillin is mediated by phospholipase C-dependent and -independent mechanisms and requires the integrity of the actin cytoskeleton and participation of p21rho.

Recent studies show that the effects of some oncogenes, integrins, growth factors and neuropeptides are mediated by tyrosine phosphorylation of the cytosolic kinase p125 focal adhesion kinase (p125(FAK)) and the cytoskeletal protein paxillin. Recently we demonstrated that cholecystokinin (CCK) C-terminal octapeptide (CCK-8) causes tyrosine phosphorylation of p125(FAK) and paxillin in rat pancreatic acini. The present study was aimed at examining whether protein kinase C (PKC) activation, calcium mobilization, cytoskeletal organization and small G-protein p21(rho) activation play a role in mediating the stimulation of tyrosine phosphorylation by CCK-8 in acini. CCK-8-stimulated phosphorylation of p125(FAK) and paxillin reached a maximum within 2.5 min. The CCK-8 dose response for causing changes in the cytosolic calcium concentration ([Ca2+]i) was similar to that for p125(FAK) and paxillin phosphorylation, and both were to the left of that for receptor occupation and inositol phosphate production. PMA increased tyrosine phosphorylation of both proteins. The calcium ionophore A23187 caused only 25% of the maximal stimulation caused by CCK-8. GF109203X, a PKC inhibitor, completely inhibited phosphorylation with PMA but had no effect on the response to CCK-8. Depletion of [Ca2+]i by thapsigargin had no effect on CCK-8-stimulated phosphorylation. Pretreatment with both GF109203X and thapsigargin decreased CCK-8-stimulated phosphorylation of both proteins by 50%. Cytochalasin D, but not colchicine, completely inhibited CCK-8- and PMA-induced p125(FAK) and paxillin phosphorylation. Treatment with Clostridium botulinum C3 transferase, which inactivates p21(rho), caused significant inhibition of CCK-8-stimulated p125(FAK) and paxillin phosphorylation. These results demonstrate that, in pancreatic acini, CCK-8 causes rapid p125(FAK) and paxillin phosphorylation that is mediated by both phospholipase C-dependent and -independent mechanisms. For this tyrosine phosphorylation to occur, the integrity of the actin, but not the microtubule, cytoskeleton is essential as well as the activation of p21(rho).

Dissection of Pathways Implicated in Integrin-mediated Actin Cytoskeleton Assembly: INVOLVEMENT OF PROTEIN KINASE C, RHO GTPase, AND TYROSINE PHOSPHORYLATION

A panel of antibodies to the alphaIIbbeta3 integrin was used to promote adhesion of Chinese hamster ovary cells transfected with the alphaIIbbeta3 fibrinogen receptor. While some alphaIIbbeta3 antibodies were not able to induce p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, all the antibodies equally support cell adhesion but not spreading and assembly of actin stress fibers. Absence of stress fibers was also obtained by plating on antibodies directed to the hamster beta1 integrin. In contrast, cells plated on matrix proteins spread organizing actin stress fibers. Treatment with phorbol esters phorbol 12-myristate 13-acetate (PMA) induced cells to spread on antibodies-coated dishes but not to organize actin in stress fibers. The combination of PMA and cytotoxic necrotizing factor 1 (CNF1), a specific Rho activator, induced cell spreading and organization of stress fibers. PMA or the combination of PMA and CNF1 also increases tyrosine phosphorylation of p125FAK in response to antibodies that were otherwise unable to trigger this response. These data show that: 1) matrix proteins and antibodies differ in their ability to induce integrin-dependent actin cytoskeleton organization (while matrix induced stress fibers formation, antibodies did not); 2) p125FAK tyrosine phosphorylation is insufficient per se to trigger actin stress fibers formation since antibodies that activate p125FAK tyrosine phosphorylation did not lead to actin stress fibers assembly; and 3) the inability of anti-integrin antibodies to trigger stress fibers organization is overcome by concomitant activation of the protein kinase C (PKC) and Rho pathways; PKC activation leads to cell spreading and Rho activation is required to organize actin stress fibers.

Regulation of cell spreading during differentiation in the muscarinic M5 receptor tumor-suppressor model.

Activation of the muscarinic receptor in Chinese hamster ovary (CHO) cells results in a reversal of the malignant phenotype for which spreading into a bipolar, fibroblastic morphology is a marker. The process of morphologic change requires multiple events, including alterations in adhesions to substrates and cytoskeletal re-arrangement. In this report, we demonstrate the calcium-dependent involvement of p125FAK in this cellular shape change using an inhibitor of ligand-induced calcium influx, carboxyamido-triazole (CAI). p125FAK becomes tyrosine-phosphorylated after exposure to the agonist carbachol (CC), reaching maximal phosphorylation prior to initiation of cellular shape change at 1 hr into CC exposure (386 +/- 103%). Phosphorylation remained elevated through the shape change (4-12 hr). CHOm5 cell exposure to the Ca2+-mobilizing agents maitotoxin and ionomycin also resulted in p125FAK phosphorylation. Inhibition of Ca2+ influx with CAI, an inhibitor of ligand-induced Ca2+ influx, had little effect on CC-induced phosphorylation but partially inhibited ionomycin-mediated p125FAK phosphorylation. While the intracellular Ca2+ chelator BAPTA failed to prevent CC-induced p125FAK tyrosine phosphorylation, it inhibited phosphorylation due to ionomycin. CC induced Ca2+-independent binding of phosphorylated p125FAK selectively to the C-terminal SH2 domain of phosphatidylinositol-3'-kinase (PI3K). Further, CC, maitotoxin and ionomycin induced in vitro kinase activity of p125FAK for the exogenous substrate poly(Glu4Tyr1). Kinase activity stimulated by all 3 agonists was inhibited by preincubation with either CAI or BAPTA. Our results indicate that increasing intracellular Ca2+ can stimulate both p125FAK autophosphorylation and kinase activity; however, p125FAK phosphorylation in response to CC also may be induced through a Ca2+-independent pathway.

DIFFERENTIAL EFFECTS OF THE TYROSINE KINASE INHIBITORS ON COLLAGEN TYPE 1-INDUCED PLATELET AGGREGATION AND ADHESION TO THIS PROTEIN

Herbimycin A, lavendustin A, and methyl 2,5-dihydroxycinnamate were used to study the role of protein tyrosine kinases in collagen-platelet interaction. All three compounds produced a concentration dependent inhibition of platelet aggregation induced by collagen type I, characterized by values of IC 50 equaled to 0.9, 10.0, and 5.0 μM, respectively. This effect was accompanied by strong inhibition of phosphorylation of p125 FAK, p90, p72 syk, p60 c-src, and p56 lyn. In the absence of the inhibitors, phosphorylation of these proteins is evoked by aggregation of platelets. In addition to the anti-aggregatory effect, the tyrosine kinase inhibitors reduced adhesion of platelets to collagen although to much lower extent than aggregation. Platelets which adhered to collagen showed also the presence of phosphorylated p125 FAK, p90, p72 syk, p60 c-src, and p56 lyn. Of these proteins, the extent of phosphorylation of p90 was particularly high. Adhesion of platelets was associated with inhibition of phosphorylation of p125 FAK, p60 c-src, and p56 lyn only when high concentration of lavendustin A and methyl 2,5-dihydroxycinnamate were used. Herbimycin A did not affect adhesion-evoked protein tyrosine phosphorylation. Phosphorylation of p90 and p72 syk was not affected by inhibitors. This study indicates that collagen type I can induce different transmembrane signalling dependent upon whether platelet aggregates formation or adhesion of platelets to this protein occurs. © 1997 Elsevier Science Ltd

Effect of gastrin-releasing peptide receptor number on receptor affinity, coupling, degradation, and modulation.

The relationship between receptor number and agonist-induced intracellular responses has been well studied in receptors coupled to adenylate cyclase; however, for receptors coupled to phospholipase C (PLC), very little is known about the effect of receptor number on receptor-mediated processes. To explore this issue, we investigated the effect of the number of receptors for gastrin-releasing peptide (GRP) on ligand affinity and on the ability to activate intracellular messengers [PLC, tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK)] and cause receptor modulation (internalization, desensitization, down-regulation) and ligand degradation. Three BALB 3T3 cell lines were made that stably expressed the gastrin-releasing peptide receptor (GRP-R) with receptor numbers varying by 280-fold (GRP-R-Low, GRP-R-Med, and GRP-R-Hi). Each cell line had the same affinity for agonist. The efficacy for bombesin to increase [3H]inositol phosphates but not tyrosine phosphorylation of p125FAK correlated well with receptor number. In contrast, the EC50 value for [3H]inositol phosphate generation for bombesin was the same in each cell line. Receptor number did not alter internalization. In the absence of protease inhibitors, there was an inverse correlation between receptor number and receptor down-regulation and desensitization. However, with protease inhibitors present, GRP-R-Med and GRP-R-Hi down-regulated significantly less than the GRP-R-Low. Similarly, GRP-R-Low desensitized significantly more than GRP-R-Med or GRP-R-Hi. GRP-R-Hi caused significantly greater ligand degradation than GRP-R-Low, and protease inhibitors completely inhibited degradation by GRP-R-Low and inhibited degradation by 70% for GRP-R-Hi. In conclusion, we show that for the PLC-coupled GRP-R, receptor number had little or no effect on binding affinity, potency for activating PLC, tyrosine phosphorylation of p125FAK, or extent of receptor internalization. In contrast, receptor number had an effect on ligand degradation, down-regulation, desensitization, and efficacy of PLC activation without altering the efficacy of tyrosine phosphorylation of p125FAK. These results demonstrate that the effect of receptor number differs for the different functions mediated by the GRP receptor and differs from that reported for adenylate cyclase-coupled receptors such as receptors mediating the action of adrenergic agents, secretin, and opioids.

Role of tyrosine specific phosphorylation of cellular proteins, especially EGF receptor and p125FAK in human lung cancer cells

To clarify the role of tyrosine phosphorylation of cellular proteins in human lung cancer cells, phosphotyrosine (PTYR)-containing proteins in lung cancer cell lines and in paired tissues resected from cancerous and normal lungs were studied by immunoblotting with an anti-PTYR antibody. We found that the profiles of protein phosphorylation were very similar among those cell lines which had different histological features, The major PTYR-containing proteins (180–190 KDa, 120–130 KD, and 95–100 KDa) were detected in lung cancer cell lines. The expression of EGF receptor (EGF-r) (p185) and c-erb B2 protein, and tyrosine phosphorylation of p125FAK were examined in cancerous lung tissues and normal lung tissues. In surgical specimens, approximately half of the samples of lung cancer tissues showed clear elevation of tyrosine phosphorylation. In these cancerous tissues, no clear amplification of EGF-r and c-erb B2 protein expression was observed. However, elevation of tyrosine phosphorylation of p125FAK was observed in cancerous lung tissues but not in normal lung tissues, and its phosphorylation was closely correlated with the nodal involvement of cancer and disease-free survival time. These results suggested that the intracellular signaling pathway via tyrosine phosphorylation plays a role in the generation and immortalization of lung cancer, and assessment of tyrosine phosphorylation of cellular proteins, especially p125FAK, may be available clinically as a prognostic factor.

Focal Adhesion and Stress Fiber Formation Is Regulated by Tyrosine Phosphatase Activity

Tyrosine phosphorylation of cytoskeletal proteins plays an important role in the regulation of focal adhesions and stress fiber organization. In the present study we examined the role of tyrosine phosphatases in this process using p125FAK and paxillin as substrates. We show that tyrosine phosphatase activity in Swiss 3T3 cells was markedly increased when actin stress fibers were disassembled by cell detachment from the substratum, by serum starvation, or by cytochalasin D treatment. This activity was blocked by phenylarsine oxide, an inhibitor of a specific class of tyrosine phosphatases characterized by two vicinal thiol groups in the active site. Phenylarsine oxide treatment of serum-starved cells induced increased tyrosine phosphorylation of p125FAK and paxillin in a dose-dependent manner and induced assembly of focal adhesions and actin stress fibers, showing that inhibition of one or more phenylarsine oxide-sensitive tyrosine phosphatases is a sufficient stimulus for triggering focal adhesion and actin stress fiber formation in adherent cells.

Dissociation of mitogen-activated protein kinase activation from p125 focal adhesion kinase tyrosine phosphorylation in Swiss 3T3 cells stimulated by bombesin, lysophosphatidic acid, and platelet-derived growth factor.

The experiments presented here were designed to examine the contribution of p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation to the activation of the mitogen-activated protein kinase cascade induced by bombesin, lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF) in Swiss 3T3 cells. We found that tyrosine phosphorylation of p125FAK in response to these growth factors is completely abolished in cells treated with cytochalasin D or in cells that were suspended in serum-free medium for 30 min. In marked contrast, the activation of p42mapk by these factors was independent of the integrity of the actin cytoskeleton and of the interaction of the cells with the extracellular matrix. The protein kinase C inhibitor GF 109203X and down-regulation of protein kinase C by prolonged pretreatment of cells with phorbol esters blocked bombesin-stimulated activation of p42mapk, p90rsk, and MAPK kinase-1 but did not prevent bombesin-induced tyrosine phosphorylation of p125FAK. Furthermore, LPA-induced p42mapk activation involved a pertussis toxin-sensitive guanylate nucleotide-binding protein, whereas tyrosine phosphorylation of p125FAK in response to LPA was not prevented by pretreatment with pertussis toxin. Finally, PDGF induced maximum p42mapk activation at concentrations (30 ng/ml) that failed to induce tyrosine phosphorylation of p125FAK. Thus, our results demonstrate that p42mapk activation in response to bombesin, LPA, and PDGF can be dissociated from p125FAK tyrosine phosphorylation in Swiss 3T3 cells.

Beta1 integrin-mediated activation of focal adhesion kinase and its association with Fyn and Zap-70 in human NK cells.

Adhesive interactions mediated by cell surface receptors have been shown to induce signal transduction pathways that regulate changes in cellular function. We have reported recently that fibronectin (FN) receptors, alpha4beta1 and alpha5beta1 integrins, on NK cells transduce transmembrane signals leading to tyrosine phosphorylation of 60-, 70-, and 120-kDa proteins. In the current study, we have identified a 120-kDa phosphoprotein as the focal adhesion kinase (p125FAK), a structurally unique nonreceptor protein tyrosine kinase that localizes to focal adhesions. Activity of p125FAK was induced by adhesion of NK cells to plastic-immobilized FN, by cross-linking of cell surface-bound FN or FN fragments, FN120 or FN40, with anti-FN mAb, or by cross-linking of alpha4beta1 or alpha5beta1 integrins with alpha-chain-specific Abs. We also observed that enhanced in vitro kinase activity was associated with immunoprecipitates of alpha4beta1 or alpha5beta1 integrins from lysates of FN-adherent NK cells as compared with BSA-treated NK cells. In addition to p125FAK activity, FN-induced kinase activity was also found to be mediated by Fyn, Lyn, and Zap-70, as assessed by in vitro phosphorylation of the immunoprecipitated kinases in the presence of [gamma-32P]ATP. Clustering of FN receptors on NK cells by agonists such as immobilized FN or alpha4- or alpha5-specific Abs also induced association of Fyn and Zap-70 with p125FAK. Our observations indicate that activation and phosphorylation of p125FAK as well as Zap-70 and certain kinases of the src family play an important role in formation of active signaling complexes in response to triggering via beta1 integrins on NK cells. These results also suggest the existence of cross-talk or points of convergence between the beta1 integrin-mediated and other receptor-signaling pathways.

Focal adhesion kinase (p125FAK) and paxillin are substrates for sphingomyelinase-induced tyrosine phosphorylation in 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.

Requirement for Phosphatidylinositol 3′-Kinase Activity in Platelet-derived Growth Factor-stimulated Tyrosine Phosphorylation of p125 Focal Adhesion Kinase and Paxillin

The role of phosphatidylinositol 3'-kinase (PI 3'-kinase) activity in platelet-derived growth factor (PDGF)-stimulated tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin has been examined. The tyrosine phosphorylation of p125FAK and paxillin in response to PDGF was markedly inhibited by wortmannin in a dose-dependent manner. PDGF-stimulated PI 3'-kinase activity, membrane ruffle formation, and tyrosine phosphorylation of p125FAK and paxillin were all inhibited by the same low concentrations of wortmannin (>90% inhibition at 40nM). In contrast, tyrosine phosphorylation of p125FAK and paxillin in response to bombesin, endothelin, and phorbol 12,13-dibutyrate was not inhibited by wortmannin in these cells. Furthermore, LY294002, an inhibitor of PI 3'-kinase structurally unrelated to wortmannin, also inhibited PDGF-stimulated p125FAK tyrosine phosphorylation. PDGF was shown to stimulate the tyrosine phosphorylation of p125FAK in porcine aortic endothelial (PAE) cells transfected with the wild type PDGF-beta receptors, but not in PAE cells transfected with PDGF-beta receptors in which the PI 3'-kinase binding sites (Tyr-740/751) were replaced by phenylalanine. PDGF-stimulated, PI 3'-kinase-dependent tyrosine phosphorylation of p125FAK was not inhibited by rapamycin, and thus it was dissociated from the activation of p70 S6 kinase, previously identified as a molecular downstream target of PI 3'-kinase. Thus, we have identified a PI 3'-kinase-dependent signal transduction pathway in the action of PDGF, which leads to the phosphorylation of p125FAK and paxillin.

Tyrosine phosphorylation of focal adhesion kinase (p125FAK): regulation by cAMP and thrombin in mesangial cells.

Stress fibers, composed of actin filaments, converge upon and associate with a number of proteins, including focal adhesion kinase (p125FAK), and integrin receptors to form areas of close contact between cells and the extracellular matrix referred to as focal adhesions. Treatment of mesangial cells with cAMP-elevating agents causes a loss of focal adhesions, fragmentation of stress fibers, and decreased tyrosine phosphorylation of p125FAK. Thrombin reverses these effects of cAMP, and this model can be used to address some of the cellular mechanisms involved in regulating the loss and formation of focal adhesions. This study reports the effects of cAMP and thrombin on mesangial cell shape, distribution of actin, formation of stress fibers, and tyrosine phosphorylation of p125FAK. cAMP-treated cells display a condensed cell body with slender processes that traverse the area formerly covered by the cell. Addition of thrombin to these cells restores actin filaments (stress fibers) and increases tyrosine phosphorylation of p125FAK, and the cells resume a flattened morphology, even in the continued presence of cAMP-elevating agents. Peptides that mimic the tethered ligand portion of the thrombin receptor have the same effects on cell morphology and stress fiber formation as thrombin. In selected experiments, agents that disrupt either stress fibers (cytochalasin D) or microtubules (nocodazole; Sigma Chemical, St. Louis, MO) were used to examine the role of these cytoskeletal elements in thrombin-induced restoration of focal adhesions. Cytochalasin D blocked the ability of thrombin to restore focal adhesions and phosphorylate p125FAK. The effects of nocodazole, an agent that destabilizes microtubules (but which has no known receptor), are very similar to those of thrombin. The findings discussed in this study indicate that thrombin can modulate the formation of focal adhesions. The organization of stress fibers and microtubules is apparently intimately related to the phosphorylation of p125FAK and can be modulated by soluble receptor agonists such as thrombin or via altered polymerization of microtubules.

Pasteurella multocida Toxin, a Potent Intracellularly Acting Mitogen, Induces p125FAK@ and Paxillin Tyrosine Phosphorylation, Actin Stress Fiber Formation, and Focal Contact Assembly in Swiss 3T3 Cells

Treatment of Swiss 3T3 cells with recombinant Pasteurella multocida toxin (rPMT), a potent intracellularly acting mitogen, stimulated tyrosine phosphorylation of multiple substrates including bands of M(r) 110,000-130,000 and M(r) 70,000-80,000. Tyrosine phosphorylation induced by rPMT occurred after a pronounced lag period (1 h) and was blocked by either lysosomotrophic agents or incubation at 22 degrees C. Focal adhesion kinase (p125FAK) and paxillin are prominent substrates for rPMT-stimulated tyrosine phosphorylation. Tyrosine phosphorylation by rPMT could be dissociated from both protein kinase C activation and the mobilization of calcium from intracellular stores. rPMT stimulated striking actin stress fiber formation and focal adhesion assembly in Swiss 3T3 cells. Cytochalasin D, which disrupts the actin cytoskeleton, completely inhibited rPMT-induced tyrosine phosphorylation. In addition, tyrosine phosphorylation of p125FAK and paxillin in response to rPMT was completely abolished when cells were subsequently treated with platelet-derived growth factor at a concentration (30 ng/ml) that disrupted the actin cytoskeleton. Our results demonstrate for the first time that rPMT, a bacterial toxin, induces tyrosine phosphorylation of p125FAK and paxillin and promotes actin stress fiber formation and focal adhesion assembly in Swiss 3T3 cells.

Neuropeptides stimulate tyrosine phosphorylation and tyrosine kinase activity in small cell lung cancer cell lines.

Stimulation of small cell lung cancer (SCLC) cells with neuropeptides bombesin, bradykinin, gastrin, and neurotensin resulted in increased tyrosine kinase activity and tyrosine phosphorylation of a number of polypeptides including a p120 kDa polypeptide identified by immunoblotting as focal adhesion kinase (p125FAK). The neuropeptides stimulated a rapid, concentration-dependent phosphorylation of p125FAK (EC50 of 1 nM, 5 nM, and 2 nM for bombesin, bradykinin, and gastrin, respectively), which was receptor mediated and inhibited by both specific and broad-spectrum neuropeptide receptor antagonists. Specific inhibition of protein tyrosine kinase activity by tyrphostin-25 inhibited both basal and neuropeptide-stimulated SCLC cell growth. These results identify a novel neuropeptide-stimulated growth signaling event in SCLC cells.

P125FAK Tyrosine Phosphorylation and Focal Adhesion Assembly: Studies with Phosphotyrosine Phosphatase Inhibitors

p125FAK is a major tyrosine kinase phosphorylated in response to integrin-dependent adhesion. In this study we use vanadate and phenylarsine oxide (PAO), known inhibitors of phosphotyrosine phosphatases (PTPases), as a tool to artificially modulate p125FAK phosphorylation in human endothelial and in Chinese hamster ovary cells. Vanadate treatment strongly upregulates in a dose-dependent manner the level of tyrosine phosphorylation of several proteins in adherent cells. PAO induces a more restricted profile of tyrosine-phosphorylated proteins, increasing primarily a broad band of 120-140 kDa. Maximal stimulation of p125FAK tyrosine phosphorylation is reached at 10 μM PAO. In contrast, in vanadate-treated cells the p125FAK tyrosine phosphorylation shows a biphasic curve, being increased at high doses of vanadate (100 μM) and downregulated at low doses (25 μM). Immunofluorescence analysis of cells treated with PTPase inhibitors showed a direct correlation between the level of p125FAK tyrosine phosphorylation and the assembly of focal adhesions and actin stress fibers. Downregulation of p125FAK tyrosine phosphorylation is observed by treating cells with cytochalasin D (CD), a drug known to rapidly disrupt the actin cytoskeleton. When PTPase inhibitors are added in combination to CD, the level of tyrosine phosphorylation of p125FAK remains high and focal adhesions and actin stress fibers are preserved from the CD-mediated disruption. Based on these data we suggest that assembly of actin cytoskeleton plays an important role in inhibiting PTPases involved in p125FAK tyrosine phosphorylation.

Endothelin-1 stimulates tyrosine phosphorylation of p125 focal adhesion kinase in mesangial cells.

Endothelin-1 (ET-1) is known to induce the contraction and proliferation of glomerular mesangial cells. Because ET-1 was found to stimulate the tyrosine phosphorylation of unidentified cellular proteins in cultured mesangial cells, protein tyrosine kinase might serve as one of the important signals leading to various functions of ET-1. Focal adhesion kinase (p125FAK) is a newly identified cytoplasmic protein tyrosine kinase that is activated by the phosphorylation of its own tyrosine residue. Because p125FAK was found to play a role in the signal transduction of not only integrins but also various neurotransmitters, including bombesin, endothelin, and vasopressin in Swiss 3T3 cells and Rat-1 fibroblasts, whether ET-1 could stimulate the tyrosine phosphorylation of p125FAK in glomerular mesangial cells was examined. ET-1 stimulated the tyrosine phosphorylation of p125FAK by threefold to fourfold in cultured mesangial cells. This effect of ET-1 was detected at 1 min and reached a maximum within 5 min and was blocked by BQ-123, an antagonist for ETA receptor. A23187, a calcium ionophore, failed to stimulate the tyrosine phosphorylation of p125FAK, and ET-1 was able to stimulate the tyrosine phosphorylation of p125FAK, even in a calcium-free medium. The activation of protein kinase C (PKC) by phorbol 12, 13-dibutyrate resulted in a stimulation of the tyrosine phosphorylation of p125FAK, and an inhibition of PKC by calphostin C or staurosporine significantly reduced the effect of ET-1. Furthermore, prolonged treatment of the cells with phorbol 12, 13-dibutyrate markedly inhibited the ET-1-induced tyrosine phosphorylation of p125FAK. These results indicate that p125FAK might play a role in a signal transduction system of ET-1 in glomerular mesangial cells and that the ET-1-induced tyrosine phosphorylation of p125FAK is largely dependent on the PKC pathway.

Sphingosylphosphorylcholine Rapidly Induces Tyrosine Phosphorylation of p125FAK and Paxillin, Rearrangement of the Actin Cytoskeleton and Focal Contact Assembly: REQUIREMENT OF p21rho IN THE SIGNALING PATHWAY

Sphingosylphosphorylcholine (SPC), a potent mitogen for Swiss 3T3 cells, rapidly induced tyrosine phosphorylation of multiple substrates including bands of M(r) 110,000-130,000 and M(r) 70,000-80,000 in Swiss 3T3 cells. Focal adhesion kinase (p125FAK) and paxillin were identified as prominent substrates for SPC-stimulated tyrosine phosphorylation. An increase in tyrosine phosphorylation of p125FAK was detected as soon as 30 s after SPC stimulation, reaching a maximum after 2.5 min. SPC induced tyrosine phosphorylation of p125FAK in a concentration-dependent fashion; a half-maximum effect occurred at 250 nM. Tyrosine phosphorylation of p125FAK induced by SPC could be dissociated from both protein kinase C activation and Ca2+ mobilization from intracellular stores. SPC induced a unique pattern of reorganization of the actin cytoskeleton with a rapid appearance of actin microspikes at the plasma membrane that was followed by the formation of actin stress fibers. This pattern of cytoskeletal changes was clearly distinguishable from that induced by bombesin and 1-oleoyl-lysophosphatidic acid. Formation of microspikes and actin stress fibers were accompanied by striking assembly of focal adhesion plaques. Cytochalasin D, which disrupts the network of actin microfilaments, completely prevented SPC-induced tyrosine phosphorylation of p125FAK. In addition, tyrosine phosphorylation of p125FAK was markedly inhibited in the presence of platelet-derived growth factor at a concentration (30 ng/ml) that disrupts actin stress fibers. Finally, microinjection of Clostridium botulinum C3 exoenzyme, which inactivates p21rho, prevented SPC-induced formation of actin stress fibers, focal adhesion assembly, and tyrosine phosphorylation. Thus, p21rho is upstream of both cytoskeletal reorganization and tyrosine phosphorylation in SPC-treated cells.

Signalling mechanisms in the regulation of vascular cell migration

The migration of arterial vascular smooth muscle cells (VSMC) is thought to play a central role in atherogenesis and restenosis. The migration of several other cell types, including monocytes, T-lymphocytes and endothelial cells is also involved in the development of the mature atherosclerotic lesion. Several defined growth factors, cytokines and extracellular matrix components which are released at the sites of lesions have been implicated in the regulation of migration of VSMC and other lesion-associated cells. Platelet-derived growth factor BB-homodimer of PDGF (PDGF-BB) is strongly implicated in neo-intima formation in vivo and is the most potent known chemoattractant for VSMC in vitro. Dynamic interactions between cell surface adhesive receptors (integrins) for ECM components, organisation of the actin cytoskeleton and the turnover of focal adhesions are all key processes in cell locomotion and migration. The signal transduction pathways which mediate the chemotactic effects of PDGF-BB and other migration factors on VSMC are unknown, but several classes of cellular components are implicated including components associated with focal adhesions, small GTP-binding proteins of the rho family, and certain substrates of the PDGF beta-receptor. Tyrosine phosphorylation of the novel focal adhesion-associated protein tyrosine kinase, p125 focal adhesion kinase (p125FAK), is regulated by integrins and by several factors which alter actin cytoskeletal organisation. Recent findings suggest that tyrosine phosphorylation of p125FAK and other focal adhesion-associated proteins may be implicated in the chemotactic response of VSMC to PDGF-BB. The migratory response to PDGF-BB may be dependent on both ligand isoform bio-availability and on receptor-isotype expression as well as on down-stream signalling events. Ultimately, cell migration in vivo will be determined by a complex array of diverse extracellular molecules organised in intercellular paracrine/autocrine networks as well as multiple interacting intracellular signal transduction pathways.

Possible involvement of focal adhesion kinase, p125FAK, in osteoclastic bone resorption.

Involvement of tyrosine phosphorylation in osteoclastic bone resorption was examined using osteoclast-like multinucleated cells prepared from co-cultures of mouse osteoblastic cells and bone marrow cells in the presence of 1 alpha,25-dihydroxyvitamin D3. When osteoclast-like cells were plated on culture dishes in the presence of 10% fetal bovine serum, they were sharply stained in their peripheral region by anti-phosphotyrosine antibody. Western blot analysis revealed that 115- to 130-kD proteins were tyrosine-phosphorylated in osteoclast-like cells. Using immunoprecipitation and immunoblotting, one of the proteins with 115-130 kD was identified as focal adhesion kinase (p125FAK), a tyrosine kinase, which is localized in focal adhesions. Immunostaining with anti-p125FAK antibody revealed that p125FAK was mainly localized at the periphery of osteoclast-like cells. Herbimycin A, a tyrosine kinase inhibitor, not only suppressed tyrosine phosphorylation of p125FAK but also changed the intracellular localization of p125FAK and disrupted a ringed structure of F-actin-containing podosomes in osteoclast-like cells. Antisense oligodeoxynucleotides to p125FAK inhibited dentine resorption by osteoclast-like cells, whereas sense oligodeoxynucleotides did not. These results suggest that p125FAK is involved in osteoclastic bone resorption and that tyrosine phosphorylation of p125FAK is critical for regulating osteoclast function.

Guanosine 5′-3-O-(Thio)triphosphate Stimulates Tyrosine Phosphorylation of p125FAK and Paxillin in Permeabilized Swiss 3T3 Cells: ROLE OF p21rho

Addition of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to streptolysin O-permeabilized Swiss 3T3 cells induced tyrosine phosphorylation of M(r) 110,000-130,000 and 70,000-80,000 bands. Specifically, GTP gamma S stimulated tyrosine phosphorylation of both focal adhesion kinase (p125FAK) and paxillin. GTP gamma S induced tyrosine phosphorylation was dose-dependent (EC50 of 2.5 microM) and reached maximum levels after 1.5 min for the M(r) 110,000-130,000 band and 2 min for the M(r) 70,000-80,000 paxillin band. Guanosine 5'-O-(2-thiodiphosphate) inhibited GTP gamma S-induced tyrosine phosphorylation with an IC50 of 100 microM. Protein kinase C did not mediate GTP gamma S-induced tyrosine phosphorylation. Varying the Ca2+ concentration from 0 to 6 microM did not increase tyrosine phosphorylation above basal levels and did not affect the ability of GTP gamma S to induce tyrosine phosphorylation. GTP gamma S was able to stimulate tyrosine phosphorylation in the presence of nanomolar concentrations of Mg2+. Furthermore, 30 microM AlF4- only weakly induced tyrosine phosphorylation in permeabilized cells. Pretreatment with the Clostridium botulinum C3 exoenzyme which inactivates p21rho, markedly reduced the ability of GTP gamma S to stimulate tyrosine phosphorylation of M(r) 110,000-130,000 and 70,000-80,000 bands including p125FAK and paxillin in permeabilized Swiss 3T3 cells. Furthermore, a peptide of p21rho (p21rho17-44) inhibited GTP gamma S-induced tyrosine phosphorylation in a dose-dependent manner (IC50 1 microM). This peptide also inhibited tyrosine phosphorylation of p125FAK and paxillin. In contrast, 20 microM p21ras17-44 peptide failed to inhibit GTP gamma S-induced tyrosine phosphorylation. Using permeabilized cells, our findings demonstrate that GTP gamma S stimulates tyrosine phosphorylation of p125FAK and paxillin and that a functional p21rho is implicated in this process.