P125 Fak Research Articles

The Src family kinase, Lyn, is activated in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters and growth factors which stimulate its association with numerous other signaling molecules

Src family kinases (SFK) play a central signaling role for growth factors, cytokines, G-protein-coupled receptors and other stimuli. SFKs play important roles in pancreatic acinar cell secretion, endocytosis, growth, cytoskeletal integrity and apoptosis, although little is known of the specific SFKs involved. In this study we demonstrate the SFK, Lyn, is present in rat pancreatic acini and investigate its activation/signaling. Ca 2+-mobilizing agents, cAMP-mobilizing agents and pancreatic growth factors activated Lyn. CCK, a physiological regulator of pancreatic function, rapidly activated Lyn. The specific SFK inhibitor, PP2, decreased Lyn activation; however, the inactive analogue, PP3, had no effect. Inhibition of CCK-stimulated changes in [Ca 2+] i decreased Lyn activation by 55%; GFX, a PKC inhibitor by 36%; and the combination by 95%. CCK activation of Lyn required stimulation of high and low affinity CCK A receptor states. CCK stimulated an association of Lyn with PKC-δ, Shc, p125 FAK and PYK2 as well as with their autophosphorylated forms, but not with Cbl, p85, p130 CAS or ERK 1/2. These results show Lyn is activated by diverse pancreatic stimulants. CCK's activation of Lyn is likely an important mediator of its ability to cause tyrosine phosphorylation of numerous important cellular mediators such as p125 FAK, PYK2, PKC-δ and Shc, which play central roles in CCK's effects on acinar cell function.

Rottlerin inhibits stimulated enzymatic secretion and several intracellular signaling transduction pathways in pancreatic acinar cells by a non-PKC-δ-dependent mechanism

Protein kinase C-δ (PKC-δ) becomes activated in pancreatic acini in response to cholecystokinin (CCK) and plays a pivotal role in the exocrine pancreatic secretion. Rottlerin, a polyphenolic compound, has been widely used as a potent and specific PKC-δ inhibitor. However, some recent studies showed that rottlerin was not effective in inhibiting PKCδ activity in vitro and that may display unspecific effects. The aims of this work were to investigate the specificity of rottlerin as an inhibitor of PKC-δ activity in intact cells and to elucidate the biochemical causes of its unspecificity. Preincubation of pancreatic acini with rottlerin (6 μM) inhibited CCK-stimulated translocation, tyrosine phosphorylation (TyrP) and activation of PKC-δ in pancreatic acini in a time-dependent manner. Rottlerin inhibited amylase secretion stimulated by both PKC-dependent pathways (CCK, bombesin, carbachol, TPA) and also by PKC-independent pathways (secretin, VIP, cAMP analogue). CCK-stimulation of MAPK activation and p125 FAK TyrP which are mediated by PKC-dependent and -independent pathways were also inhibited by rottlerin. Moreover, rottlerin rapidly depleted ATP content in pancreatic acini in a similar way as the mitochondrial uncouplers CCCP and FCCP. All studied inhibitory effects of rottlerin in pancreatic acini were mimicked by FCCP (agonists-stimulated amylase secretion, p125 FAK TyrP, MAPK activation and PKC-δ TyrP and translocation). Finally, rottlerin as well as FCCP display a potent inhibitory effect on the activation of other PKC isoforms present in pancreatic acini. Our results suggest that rottlerin effects in pancreatic acini are not due to a specific PKC-δ blockade, but likely due to its negative effect on acini energy resulting in ATP depletion. Therefore, to study the role of PKC-δ in cellular processes using rottlerin it is essential to keep in mind that may deplete ATP levels and inhibit different PKC isoforms. Our results give reasons for a more careful choice of rottlerin for PKC-δ investigation.

Mechanisms of blood pressure variability-induced cardiac hypertrophy and dysfunction in mice with impaired baroreflex

Enhanced blood pressure variability contributes to left ventricular hypertrophy and end-organ damage, even in the absence of hypertension. We hypothesized that the greater number of high-blood pressure episodes associated with enhanced blood pressure variability causes cardiac hypertrophy and dysfunction by activation of mechanosensitive and autocrine pathways. Normotensive mice were subjected to sinoaortic baroreceptor denervation (SAD) or sham surgery. Twelve weeks later, blood pressure variability was doubled in SAD compared with sham-operated mice. Blood pressure did not differ. Cardiac hypertrophy was reflected in greater heart/body weight ratios, larger myocyte cross-sectional areas, and greater left ventricular collagen deposition. Furthermore, left ventricular atrial and brain natriuretic peptide mRNA expression was greater in SAD than in sham-operated mice. SAD had higher left ventricular end-diastolic pressures and lower myocardial contractility indexes, indicating cardiac dysfunction. Cardiac protein content of phosphorylated p125 focal adhesion kinase (p125 FAK) and phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) was greater in SAD than in sham-operated mice, indicating activation of mechanosensitive pathways of cardiac hypertrophy. Furthermore, enhanced cardiac renin and transforming growth factor-beta1 (TGFbeta1) protein content indicates activation of autocrine pathways of cardiac hypertrophy. Adrenal tyrosine hydroxylase protein content and the number of renin-positive glomeruli were not different, suggesting that sympathetic activation and the systemic renin-angiotensin system did not contribute to cardiac hypertrophy. In conclusion, more frequent blood pressure rises in subjects with high blood pressure variability activate mechanosensitive and autocrine pathways leading to cardiac hypertrophy and dysfunction even in the absence of hypertension.

Redox modulation of tyrosine phosphorylation-dependent neutrophil adherence to endothelial cells

Reactive oxygen species (ROS) are now well known to be involved in an increased interaction between neutrophils and endothelial cells. Previously, we have shown that the increased adhesion of neutrophils to ROS-stimulated endothelial cells involves an increase in tyrosine phosphorylation of the focal adhesion kinase, p125 FAK, and several cytoskeleton proteins. This review article focuses on the involvement of adhesion molecules in the increased adhesion of neutrophils to ROS-stimulated endothelial cells, on the oxygen species responsible for this adhesion, and on the intracellular signaling pathway leading to the modification of the cytoskeleton by ROS. The evidence from our laboratory and others describing these events is summarized. Finally, the future perspectives that need to be explored in order to inhibit or reduce the ROS-mediated adhesion of neutrophils to endothelial cells are addressed.

Activation of p38 and Smads mediates BMP-2 effects on human trabecular bone-derived osteoblasts

The bone morphogenetic proteins (BMPs) are potent osteoinductive factors that accelerate osteoblast maturation, accompanied by increased cell-substrate adhesion. BMP-2 treatment of osteoblastic cells increases phosphorylation of the cytoplasmic BMP-2 signaling molecules, Smad1 and Smad5. We have previously reported that BMP-2 treatment increase cytoskeletal organization of human trabecular bone-derived osteoblast-like cells (osteoblasts), which is also accompanied by an activation of the focal adhesion kinase p125 FAK. We report here that activation of p125 FAK occurs with the same kinetics as the phosphorylation of Smad1, suggesting that BMP-2 initiates cross-talk between Smad signaling and the adhesion-mediated signaling pathway. As an adjunct to these effects, we examined activation of mitogen-activated protein (MAP) kinase family members in response to focal adhesion contact formation. Although phosphorylated forms of all three kinases were apparent, only SAPK2α/p38 (p38) was activated in response to BMP-2 treatment. Inhibition of p38 kinase activity suppressed BMP-2 induced Smad1 phosphorylation, as well as its translocation to the nucleus, suggesting the integration of p38 activation with Smad1 signaling. Finally, inhibition of p38 in osteoblasts also led to the complete abrogation of BMP-2 induced osteocalcin gene expression and matrix mineralization. These findings suggest that BMP-2 must activate p38 in order to mediate osteogenic differentiation and maturation.

KR-31372 inhibits KDR/Flk-1 tyrosine phosphorylation via K +ATP channel opening in its antiangiogenic effect

The aim of this study was to identify the signaling pathway of the antiangiogenesis by (2 R,3 R,4 S)- Nʺ-cyano- N-(6-nitro-3,4-dihydro-hydroxy-2-methyl-2-dimethoxymethyl 2 H-1-benzopyran-4yl)- N′-benzylguanidine (KR-31372). KR-31372 inhibited the in vitro basal tube formation using Matrigel-coated plate and in vivo neovascularizations in mice induced by Matrigel containing vascular endothelial growth factor (VEGF 165, 5 ng/ml). VEGF 165 markedly increased cell proliferation using 5-bromo-2′-deoxyuridine incorporation and chemotactic migration using transwell chamber in human umbilical vein endothelial cells, those of which were significantly suppressed by pretreatment with KR-31372 and levcromakalim concentration dependently. The suppression of all these variables were strongly antagonized by glibenclamide, ATP-sensitive K + channel blocker. KR-31372 (10 −6–10 −4 M) and levcromakalim (10 −5 M) concentration-dependently suppressed the VEGF 165-induced increases in KDR/Flk-1 tyrosine phosphorylation as well as the extracellular signal-related kinase 1/2 (ERK1/2), p38 MAK and p125 FAK tyrosine phosphorylation. These variables were significantly antagonized by glibenclamide. In conclusion, KR-31372 significantly inhibited the KDR/Flk-1 tyrosine phosphorylation-linked ERK1/2, p38 MAPK and p125 FAK tyrosine phosphorylation via mediation of K + ATP channel opening, thereby resulting in antiangiogenesis.

Interaction of the CC-chemokine RANTES with glycosaminoglycans activates a p44/p42 mitogen-activated protein kinase-dependent signaling pathway and enhances human immunodeficiency virus type 1 infectivity.

The interaction of the CC-chemokine RANTES with its cell surface receptors transduces multiple intracellular signals: low concentrations of RANTES (1 to 10 nM) stimulate G-protein-coupled receptor (GPCR) activity, and higher concentrations (1 microM) activate a phosphotyrosine kinase (PTK)-dependent pathway. Here, we show that the higher RANTES concentrations induce rapid tyrosine phosphorylation of multiple proteins. Several src-family kinases (Fyn, Hck, Src) are activated, as is the focal adhesion kinase p125 FAK and, eventually, members of the p44/p42 mitogen-activated protein kinase (MAPK) family. This PTK signaling pathway can be activated independently of known seven-transmembrane GPCRs for RANTES because it occurs in cells that lack any such RANTES receptors. Instead, activation of the PTK signaling pathway is dependent on the expression of glycosaminoglycans (GAGs) on the cell surface, in that it could not be activated by RANTES in GAG-deficient cells. We have previously demonstrated that RANTES can both enhance and inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1). Here we show that activation of both PTK and MAPK is involved in the enhancement of HIV-1 infectivity caused by RANTES in cells that lack GPCRs for RANTES but which express GAGs.

Bombesin and gastrin releasing peptide increase tyrosine phosphorylation of focal adhesion kinase and paxillin in non-small cell lung cancer cells

The effects of some oncogenes, growth factors and neuropeptides are mediated by tyrosine phosphorylation of focal adhesion kinase (p125 FAK) and paxillin cytoskeletal proteins. In this study the ability of bombesin/gastrin releasing peptide (BB/GRP) to stimulate tyrosine phosphorylation of p125 FAK and paxillin in non-small cell lung cancer (NSCLC) H1299 cells was investigated. BB, 100 nM caused increased p125 FAK and paxillin tyrosine phosphorylation maximally after 1 min. The effect of BB on p125 FAK and paxillin tyrosine phosphorylation was concentration-dependent being half maximal at 4–8 nM. Also, 100 nM GRP, GRP 14–27 but not GRP 1–16 increased p125 FAK and paxillin tyrosine phosphorylation indicating that the C-terminal of GRP is essential. BW2258U89, a GRP receptor antagonist, caused a dose-dependent inhibition of BB-stimulated p125 FAK and paxillin tyrosine phosphorylation with an IC 50 value of 3 μM. Cytochalasin D (0.3 μM), which inhibits actin polymerization, reduced the ability of BB to stimulate tyrosine phosphorylation of p125 FAK and paxillin. Genistein (50 μM) and H-7 (50 μM), which are kinase inhibitors, reduced the tyrosine phosphorylation of p125 FAK and paxillin stimulated by BB. Also, treatment of NCI-H1299 cells with FAK antisense resulted in decreased FAK tyrosine kinase activity and proliferation. These results suggest that p125 FAK is an important enzyme for NSCLC proliferation.

A role for phosphoinositides in tyrosine phosphorylation of p125 focal adhesion kinase in rat pancreatic acini

Previous studies have shown that different agonists increase tyrosine phosphorylation of the focal adhesion related proteins p125 FAK, p130 Cas, and paxillin in different cell types and that tyrosine phosphorylation depends on the integrity of the actin cytoskeleton. Because phosphoinositides are important for the maintenance of the cytoskeleton, the role of phosphoinositides in the tyrosine phosphorylation of these proteins in response to occupancy of m3 muscarinic and CCK A receptors has been investigated in pancreatic acini. Addition of carbachol or CCK-8 to pancreatic acini resulted in rapid increases in the tyrosine phosphorylation of p125 FAK, p130 Cas, and paxillin. Pretreatment of pancreatic acini with LY294002 or wortmannin resulted in a concentration-dependent inhibition of tyrosine phosphorylation of p125 FAK, p130 Cas, and paxillin stimulated by carbachol or CCK-8. Carbachol- or CCK-8-stimulated tyrosine phosphorylation of these proteins was not inhibited by rapamycin, PD 98059 or SB 203580, and thus it was dissociated from the activation of p70 S6 or MAP kinases. These results indicate that m3 muscarinic and CCK A receptor-mediated increase in p125 FAK, p130 Cas, and paxillin tyrosine phosphorylation in pancreatic acini depends on the ability of these cells to synthesise phosphoinositides.

Phosphorylation of p125 FAK and paxillin focal adhesion proteins in src-transformed cells with different metastatic capacity

Hamster fibroblasts transformed by Rous sarcoma virus (RSV) display different metastatic potentials that are associated with specific structural features of the v-src oncoprotein. This diverse metastatic activity could be due to various tyrosine phosphorylation levels of specific src protein substrates. To check this hypothesis, phosphorylation of the FAK and paxillin proteins, involved in signal transduction pathways and known as src protein substrates, was tested. It was shown that FAK and paxillin are hyperphosphorylated in the high metastatic cell lines as compared with the phosphotyrosine level of these proteins found in the low metastatic cell lines. In addition, our data confirm that v-src protein plays a direct role in paxillin phosphorylation.

Platelet-activating factor stimulation of p125 FAK and p130 Cas tyrosine phosphorylation in brain

The effect of platelet-activating factor (PAF) on protein tyrosine phosphorylation was studied in rat brain slices. PAF induced a time- and concentration-dependent increase in tyrosine phosphorylation of a doublet of approximately 125 kDa. These proteins were identified by immunoprecipitation as p125 FAK and p130 Cas, using monoclonal antibodies. This effect was mediated by PAF receptors, as shown by its inhibition by the action of a PAF antagonist. The tyrosine phosphorylation evoked by PAF was dependent, at least in part, on external calcium. The involvement of protein kinase C was demonstrated by the synergistic effect of TPA on PAF-stimulated tyrosine phosphorylation. The finding that PAF stimulates tyrosine phosphorylation of both focal adhesion protein p125 FAK and p130 Cas suggests that PAF might modulate the integrin mediated signal transduction in the brain.

Endothelin stimulates tyrosine phosphorylation of p125 FAK and p130 Cas in rat cerebral cortex

Stimulation of rat cerebral cortex with endothelin-1 (ET-1) caused an increase in the tyrosine phosphorylation of several proteins. Two of these phosphoproteins were identified by the immunoprecipitation assays as being the focal adhesion kinase p125 FAK and crk-associated substrate p130 Cas. This effect was time- and dose-dependent, with an EC 50 value of 3.9×10 −8 M. In addition, the cerebral cortex ET receptor subtype involved in this action was determined by using BQ-123 and BQ-788, which are ET A and ET B receptor antagonists respectively. Our results indicate that the ET-1 effect on protein tyrosine phosphorylation occurred through ET B receptors. The requirement for extracellular Ca 2+ on ET-1 action was also studied. ET-1-stimulated tyrosine phosphorylation of both p125 FAK and p130 Cas was abolished in the absence of external Ca 2+ or in the presence of nimodipine, a Ca 2+ channel-blocker. These results suggest that the ET-1-stimulated protein tyrosine phosphorylation was secondary to Ca 2+ influx through the dihydropyridine Ca 2+-channel. In slices where protein kinase C was inhibited, ET-1-stimulated tyrosine phosphorylation of both proteins was reduced. These results indicate that ET-1 modulates the tyrosine phosphorylation of specific proteins, which may be involved in adhesion processes in the brain.

Mechanism of BMP-2 stimulated adhesion of osteoblastic cells to titanium alloy

Cell adhesion is dependent on many factors, including the repertoire of extracellular matrix (ECM) proteins and their receptors, eg integrins, synthesized by the cell, the composition of the ECM adsorbed to the surface, and the intrinsic chemistry of the surface. Factors that govern bone cell, ie osteoblast, adhesion and ECM elaboration significantly influence its re-modeling into mature bone, and ultimately its ability to integrate with biomaterials used for orthopedic prostheses. In this study, we have investigated how treatment with bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-β (TGF-β) superfamily that promotes ectopic bone formation, modulates the organization and expression of osteoblastic cell proteins. Specifically, we analyzed how BMP-2 treatment affects cytoskeletal components, ECM, and α5, and β1 integrin receptor subunits in osteoblastic cells plated on Ti6A14V, a titanium alloy widely used for orthopedic implants that interacts with bone cells in vitro and in vivo. Osteoblastic cells were pre-treated with BMP-2 for 12 h prior to plating; BMP-2 treatment stimulated adhesion and proliferation of osteoblastic cells and this adhesive advantage was reflected in enhanced long-term matrix mineralization in the BMP-2 pre-treated cultures. Confocal laser scanning microscopic analysis of BMP-2 treated cells showed that enhanced cytoskeletal organization and focal contact formation occurred. These changes were accompanied by a concomitant increase in the spatial organization of fibronectin, whereas vitronectin, collagen type I, osteopontin, and osteocalcin showed little change. The changes in ECM organization correlated with increased fibronectin, α5 and β1 integrin subunit, and focal adhesion kinase (p125 FAK) expression, as well as increased p125 FAK phosphorylation. By confocal microscopy, the α5 integrin subunit was more concentrated in lamellipodia after BMP-2 treatment. These results demonstrate that BMP-2 significantly altered osteoblastic cytoskeletal and ECM organization and enhanced expression of fibronectin and of specific integrin receptor subunits, with concomitant changes in the levels and phosphorylation of p125 FAK. These effects may contribute to downstream cellular responses important for bone cell function, and growth.

Gastrin stimulates the formation of a p60 Src/p125 FAK complex upstream of the phosphatidylinositol 3-kinase signaling pathway

The molecular events whereby gastrin occupancy of G/CCK B receptors leads to phosphatidylinositol (PI) 3-kinase activation have been examined. We report here that this peptide promotes the association between two non-receptor tyrosine kinases, p60 Src and p125 FAK, and elicits a parallel increase in tyrosine phosphorylation and activity of both kinases. Gastrin-induced PI 3-kinase activity was coprecipitated with p60 Src and p125 FAK and was inhibited by herbimycin A, the selective Src inhibitor PP-2 or cytochalasin D, which disrupts the actin cytoskeleton and prevents p125 FAK activity. These results indicate, for the first time, that a p60 Src/p125 FAK complex acts upstream of the gastrin-stimulated PI 3-kinase pathway.

EGF stimulates tyrosine phosphorylation of focal adhesion kinase (p125 FAK) and paxillin in rat pancreatic acini by a phospholipase C-independent process that depends on phosphatidylinositol 3-kinase, the small GTP-binding protein, p21 rho, and the integrity of the actin cytoskeleton

Epidermal growth factor (EGF) is a potent mitogen in many cell types including pancreatic cells. Recent studies show that the effects of some growth factors on growth and cell migration are mediated by tyrosine phosphorylation of the cytosolic tyrosine kinase p125 focal adhesion kinase (p125 FAK) and the cytoskeletal protein, paxillin. The aim of the present study was to determine whether EGF activates this pathway in rat pancreatic acini and causes tyrosine phosphorylation of each of these proteins, and to examine the intracellular pathways involved. Treatment of pancreatic acini with EGF induced a rapid, concentration-dependent increase in p125 FAK and paxillin tyrosine phosphorylation. Depletion of the intracellular calcium pool or inhibition of PKC activation had no effect on the response to EGF. However, inhibition of the phosphatidylinositol 3-kinase (PI3-kinase) or inactivation of p21 rho inhibited EGF-stimulated phosphorylation of p125 FAK and paxillin by more than 70%. Finally, cytochalasin D, a selective disrupter of the actin filament network, completely inhibited EGF-stimulated tyrosine phosphorylation of both proteins. All these treatments did not modify EGF receptor autophosphorylation in response to EGF. These results identify p125 FAK and paxillin as components of the intracellular pathways stimulated after EGF receptor occupation in rat pancreatic acini. Activation of this cascade requires activation of PI3-kinase and participation of p21 rho, but not PKC activation and calcium mobilization.

Reactive oxygen species activate focal adhesion kinase, paxillin and P130CAS tyrosine phosphorylation in endothelial cells

Reactive oxygen species (ROS), particularly hydroxyl radical (HO ), increase neutrophil adherence to hypoxanthine-xanthine oxidase (HX-XO)-treated human umbilical vein endothelial cells (HUVEC) in culture. This adherence is inhibited by the tyrosine kinase inhibitors genistein (30 μM) and herbimycin A (0.9 μM), suggesting the involvement of tyrosine kinase. Phosphorylation of several HUVEC proteins in the range of 120–130 and 70 kDa was found to depend on the XO concentration and stimulation time. This phosphorylation was inhibited by the antioxidants dimethylthiourea (DMTU, 0.75 to 7.5 mM) and pentoxifylline (Ptx, 0.1 mM), and by the iron chelators desferrioxamine (DF, 1 mM) and hydroxybenzyl ethylene diamine (HBED, 0.5 mM), suggesting the involvement of HO . Three tyrosine-phosphorylated proteins, focal adhesion kinase (p125 FAK), paxillin (PAX) and p130cas were isolated and characterized by immunoprecipitation and western blotting. Antioxidants and iron chelators reduced their phosphorylation. HUVEC treated with ROS for 15 min showed actin stress fiber formation. Cytochalasin D (5 μM) inhibited tyrosine phosphorylation and PMN-HUVEC adherence, showing the importance of cytoskeleton integrity in these two functions. In conclusion, HO , which is involved in increased PMN-HUVEC adhesion, also increases tyrosine phosphorylation on three major cytoskeleton proteins which seem to play a role in this adhesion.

Are tyrosine phosphorylation of p125 FAK and paxillin or the small GTP binding protein, Rho, needed for CCK-stimulated pancreatic amylase secretion?

Studies of a possible role of tyrosine phosphorylation in the secretory process in rat pancreatic acinar cells provide conflicting conclusions. Recent studies show that tyrosine phosphorylation of the focal adhesion kinase, p125 FAK and the cytoskeletal protein, paxillin, may mediate a number of cellular changes and this phosphorylation is dependent on the activation of the small GTP binding protein, p21 Rho (Rho). In this work we have investigated the role of tyrosine phosphorylation of each of these proteins and of the activation of Rho in pancreatic enzyme secretion. Pretreatment with genistein, a tyrosine kinase inhibitor, decreased CCK-8-stimulated tyrosine phosphorylation of p125 FAK and paxillin and CCK-8-stimulated amylase secretion by more than 60%, raising the possibility that tyrosine phosphorylation of these two proteins could be important in the ability of CCK-8 to stimulate amylase release. However, genistein did not alter the amylase release stimulated by TPA but inhibited TPA-stimulated p125 FAK and paxillin tyrosine phosphorylation by 70%. Pretreatment with C3 transferase, which specifically inactivates Rho, causes a decrease in CCK-8-induced maximal amylase release by 33%. Moreover, C3 transferase pretreatment causes a 48% and a 38% decrease in the tyrosine phosphorylation of p125 FAK and paxillin by CCK-8, respectively. Pretreatment with different concentrations of cytochalasin D, an actin cytoskeleton assembly inhibitor, completely inhibited CCK-8-stimulated tyrosine phosphorylation of p125 FAK and paxillin without having any effect on either the potency or efficacy of CCK-8 at stimulating amylase release. Furthermore, cytochalasin D completely inhibited TPA-stimulated tyrosine phosphorylation of both proteins without affecting TPA-stimulated amylase release. These results show that tyrosine phosphorylation of p125 FAK and paxillin is not required for CCK-8 stimulation of enzyme secretion. However, our results suggest Rho is involved in the CCK-8 stimulation of amylase release by a parallel pathway to its involvement in the CCK-8-stimulated tyrosine phosphorylation of p125 FAK and paxillin.

Integrin-mediated signal transduction in cells lacking focal adhesion kinase p125 FAK

We have previously shown that integrin-dependent tyrosine phosphorylation of p130 Cas (Cas) could be induced in a mouse fibroblast cell line that does not express focal adhesion kinase p125 FAK (FAK). By analyzing FAK-deficient (FAK−/−) cells transiently expressing Cas mutant proteins, we demonstrate here that the Src homology 3 (SH3) domain of Cas is indispensable for adhesion-mediated Cas phosphorylation in this mutant cell line. While the FAK directly binds to Cas-SH3, our findings imply that SH3-binding molecule(s) other than FAK might regulate Cas phosphorylation, at least in FAK−/− cells. In this regard, we observed that FAK−/− cells expressed cell adhesion kinase β (CAKβ), a protein tyrosine kinase of the FAK subfamily. CAKβ expressed by FAK−/− cells was associated in vivo with Cas in a Cas-SH3-dependent manner. Moreover, integrin stimulation induces tyrosine phosphorylation of CAKβ in FAK−/− cells. Thus, our results suggest that CAKβ contributes to integrin-mediated signal transduction in place of FAK in FAK-deficient cells.

HIV-1 Tat induces tyrosine phosphorylation of p125FAK and its association with phosphoinositide 3-kinase in PC12 cells.

To evaluate the signal transduction potential of HIV-1 Tat in a neuronal cell model. The tyrosine phosphorylation levels of the focal adhesion kinase p125FAK and its association with phosphoinositide 3-kinase (PI 3-K) were evaluated in serum-starved rat pheochromocytoma PC12 cells, either treated with low concentrations (0.1-1 nM) of extracellular HIV-1 Tat protein or stably transfected with Tat cDNA. Extracellular Tat induced a rapid increase of p125FAK tyrosine phosphorylation and p125FAK-associated PI 3-K activity. By using recombinant mutated Tat proteins, it was found that deletion of amino acids 73-86 encoded by the second exon of the tat gene resulted in a significant decrease of the ability of Tat to induce p125FAK tyrosine phosphorylation. Paradoxically, mutations in the basic region encoded by the first exon of tat, which is essential for nuclear localization and HIV-1 LTR transactivation, increased the ability of Tat to stimulate p125FAK tyrosine phosphorylation. Moreover, in comparison with cells transfected with a control vector, PC12 cells stably transfected with tat cDNA showed greater amounts of p125FAK protein, an increase in p125FAK tyrosine phosphorylation and higher levels of p125FAK-associated PI 3-K activity. The addition of anti-Tat neutralizing antibody to tat-transfected PC12 cells in culture blocked both the p125FAK tyrosine phosphorylation and its association with PI 3-K but did not affect the total amount of p125FAK. HIV-1 Tat protein enhanced both the expression and the functionality of p1 25FAK in PC12 neuronal cells. Whereas the first event required intracellular Tat, the increased p125FAK phosphorylation was strictly dependent upon extracellular Tat.

CCK causes rapid tyrosine phosphorylation of p125 FAK focal adhesion kinase and paxillin in rat pancreatic acini

Recent studies show CCK stimulates tyrosine phosphorylation (TYR PHOSP) of a number of proteins and evidence from the pancreas and other cellular systems suggest this could be important in mediating some of CCK's growth and secretory effects. In other tissues various neuropeptides such as bombesin can cause tyrosine phosphorylation of p125 focal adhesion kinase (p125 FAK) and paxillin which are important in mediating their growth effects. The purpose of the present study was to determine the effects of CCK in rat pancreatic acini on the TYR PHOSP of these latter proteins. In dispersed rat pancreatic acini, cell lysates were incubated with an anti-phosphotyrosine mAb (PY20) which was immunoprecipitated and then analyzed by Western blotting with anti-phosphotyrosine mAb (4G10), anti-p125 FAK mAb or anti-paxillin mAb. CCK-8 at 5 min increased TYR PHOSP of five proteins of molecular weight >60 000 including a broad M r 110–130 000 and M r 70–80 000. An increase in TYR PHOSP of both p125 FAK and paxillin was detected within 1 min of adding CCK and reached a maximum at 2.5 min with a 9.1±1.9-fold increase for p125 FAK and 3.6±0.6-fold for paxillin. CCK-8 caused a half-maximal increase in TYR PHOSP of p125 FAK at 0.1 nM and paxillin at 0.03 nM. CCK-JMV also stimulated an increase in TYR PHOSP of both proteins, but was only 50% as efficacious as CCK-8. CCK-JMV caused a half-maximal increase at 10 nM and maximal at 1 μM for both proteins. To investigate whether the low affinity CCK receptor state also caused TYR PHOSP of both proteins, increasing concentrations of CCK-JMV were added to a maximally effective CCK-8 concentration (1 nM). Detectable inhibition of CCK-8-stimulated TYR PHOSP occurred with 1 μM CCK-JMV and with 3 μM CCK-JMV the CCK-8-stimulated response was inhibited 50% and was the same as that seen with CCK-JMV alone. These studies demonstrate that in rat pancreatic acini, CCK causes rapid TYR PHOSP of both p125 FAK and paxillin. This stimulation is mediated by both the high affinity and low affinity CCK receptor states. This phosphorylation of these proteins could be important in mediating CCK's effect on the cytoskeleton or growth effects as shown for a number of other agents (oncogenes, neuropeptides, integrins).