Activation of m3 muscarinic receptors induces rapid tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin in rat pancreatic acini

Tyrosine phosphorylation plays a key role in transmembrane and cytoplasmic signal transduction mechanisms stimulated by oncogenes, integrins, growth factors, neuropeptides, and bioactive lipids. Moreover, recent studies show that stimulation of odd-numbered muscarinic receptors increases the tyrosine phosphorylation of several proteins in different cellular types. The present study was aimed at examining whether activation of m3 muscarinic receptors in rat pancreatic acini evokes tyrosine phosphorylation of p125(FAK), and its substrates, p130(cas) and paxillin. Results show that stimulation of pancreatic acini with carbachol resulted in a rapid and transient increase in tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin. Tyrosine phosphorylation of these proteins occurred in a time- and concentration-dependent manner. Simultaneous blockage of both PKC activation and increases in [Ca(2+)](i) partially decreased p125(FAK), p130(cas), and paxillin tyrosine phosphorylation stimulated by carbachol. Pretreatment of pancreatic acini with Clostridium botulinum C3 transferase, which specifically inactivates p21(rho), partially inhibited carbachol-induced p125(FAK), p130(cas), and paxillin tyrosine phosphorylation. In contrast, this treatment had no effect on amylase release stimulated by carbachol. Cytochalasin D, which disrupts actin microfilaments network, completely inhibited carbachol stimulated tyrosine phosphorylation of these proteins without having significant effects in carbachol-stimulated amylase secretion. These results dissociate tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin from amylase secretion after m3 muscarinic receptors occupation in rat pancreatic acini. Taken together, these data suggest that (a) activation of m3 muscarinic receptors in rat pancreatic acini increases tyrosine phosphorylation of p125(FAK) and its substrates, p130(cas) and paxillin by diacylglycerol-activated PKC- and calcium- dependent, and independent pathways, (b) these responses require activation of p21(rho) and an intact actin cytoskeleton, and (c) p125(FAK), p130(cas), and paxillin are unlikely related to secretion in rat pancreatic acinar cells.     

Src family kinase involvement in muscarinic receptor-induced tyrosine phosphorylation in differentiated SH-SY5Y cells

Muscarinic receptor-mediated changes in protein tyrosine phosphorylation were examined in differentiated human neuroblastoma SH-SY5Y cells. Treatment of differentiated cells with 1 mM carbachol caused rapid increases in the tyrosine phosphorylation of focal adhesion kinase (FAK), Cas, and paxillin. The src family kinase-selective inhibitor PP1 reduced carbachol-stimulated tyrosine phosphorylation of FAK, Cas, and paxillin by 50 to 75%. In contrast, carbachol-stimulated activation of ERK1/2 was unaffected by PP1. Src family kinase activation by carbachol was further demonstrated by increased carbachol-induced tyrosine phosphorylation of the src-substrate, p120, and tyrosine phosphorylation of the src family kinase activation-associated autophosphorylation site. Site-specific FAK phosphotyrosine antibodies were used to determine that the carbachol-stimulated increase in the autophosphorylation of FAK was unaffected by pretreatment with PP1, whereas the carbachol-stimulated increase in the src family kinase-mediated phosphotyrosine of FAK was completely blocked by pretreatment with PP1. In SH-SY5Y cell lines stably overexpressing Fyn, the phosphotyrosine immunoreactivity of FAK was 625% that of control cells. Thus, muscarinic receptors activate protein tyrosine phosphorylation in differentiated cells, and the tyrosine phosphorylation of FAK, Cas, and paxillin, but not ERK1/2, is mediated by a src family tyrosine kinase activated in response to stimulation of muscarinic receptors.     

Cholecystokinin activates PYK2/CAKbeta by a phospholipase C-dependent mechanism and its association with the mitogen-activated protein kinase signaling pathway in pancreatic acinar cells.

PYK2/CAKbeta is a recently described cytoplasmic tyrosine kinase related to p125 focal adhesion kinase (p125(FAK)) that can be activated by a number of stimuli including growth factors, lipids, and some G protein-coupled receptors. Studies suggest PYK2/CAKbeta may be important for coupling various G protein-coupled receptors to the mitogen-activated protein kinase (MAPK) cascade. The hormone neurotransmitter cholecystokinin (CCK) is known to activate both phospholipase C-dependent cascades and MAPK signaling pathways; however, the relationship between these remain unclear. In rat pancreatic acini, CCK-8 (10 nM) rapidly stimulated tyrosine phosphorylation and activation of PYK2/CAKbeta by both activation of high affinity and low affinity CCK(A) receptor states. Blockage of CCK-stimulated increases in protein kinase C activity or CCK-stimulated increases in [Ca(2+)](i), inhibited by 40-50% PYK2/CAKbeta but not p125(FAK) tyrosine phosphorylation. Simultaneous blockage of both phospholipase C cascades inhibited PYK2/CAKbeta tyrosine phosphorylation completely and p125(FAK) tyrosine phosphorylation by 50%. CCK-8 stimulated a rapid increase in PYK2/CAKbeta kinase activity assessed by both an in vitro kinase assay and autophosphorylation. Total PYK2/CAKbeta under basal conditions was largely localized (77 +/- 7%) in the membrane fraction, whereas total p125(FAK) was largely localized (86 +/- 3%) in the cytosolic fraction. With CCK stimulation, both p125(FAK) and PYK2/CAKbeta translocated to the plasma membrane. Moreover CCK stimulation causes a rapid formation of both PYK2/CAKbeta-Grb2 and PYK2/CAKbeta-Crk complexes. These results demonstrate that PYK2/CAKbeta and p125(FAK) are regulated differently by CCK(A) receptor stimulation and that PYK2/CAKbeta is probably an important mediator of downstream signals by CCK-8, especially in its ability to activate the MAPK signaling pathway, which possibly mediates CCK growth effects in normal and neoplastic tissues.     

Low-affinity CCK-1 receptors inhibit bombesin-stimulated secretion in rat pancreatic acini--implication of the actin cytoskeleton

EXPERIMENTAL OBJECTIVES: Stimulation of low-affinity CCK-1 receptors on pancreatic acini leads to inhibition of enzyme secretion. We studied signal transduction mechanisms to identify potential causes for the reduced secretion. RESULTS: Co-stimulation experiments with CCK, CCK-JMV-180, and bombesin revealed an inhibition of bombesin-stimulated enzyme secretion by low-affinity CCK-1 receptors. Binding of 125I-gastrin-releasing peptide (the mammalian analogue of bombesin) to acini after CCK preincubation was not altered. After a short preincubation of acini with high concentrations of CCK, intracellular calcium remained responsive to bombesin. In contrast to bombesin or CCK at concentrations of 10(-10) M or lower, high concentrations of CCK caused a strong activation of p125 focal adhesion kinase (p125(FAK)) and a marked reorganisation of the actin cytoskeleton. CONCLUSIONS: Inhibitory mechanisms triggered by low-affinity CCK-1 receptors interrupt enzyme secretion from pancreatic acini at late stages in the signal transduction cascades since bombesin receptor binding and early signalling events remained intact after CCK preincubation. A reorganisation of the actin cytoskeleton is suggested to be the mechanism by which low-affinity CCK-1 receptors actively interrupt enzyme secretion stimulated by other receptors.     

Cytoskeletal and Phosphoinositide Requirements for Muscarinic Receptor Signaling to Focal Adhesion Kinase and Paxillin

Abstract: The mechanism whereby agonist occupancy of muscarinic cholinergic receptors elicits an increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin has been examined. Addition of oxotremorine-M to SH-SY5Y neuroblastoma cells resulted in rapid increases in the phosphorylation of FAK ( t _1/2 = 2 min) and paxillin that were independent of integrin-extracellular matrix interactions, cell attachment, and the production of phosphoinositide-derived second messengers. In contrast, the increased tyrosine phosphorylations of FAK and paxillin were inhibited by inclusion of either cytochalasin D or mevastatin, agents that disrupt the cytoskeleton. Furthermore, phosphorylation of FAK and paxillin could be prevented by addition of either wortmannin or LY-294002, under conditions in which the synthesis of phosphatidylinositol 4-phosphate was markedly attenuated. These results indicate that muscarinic receptor-mediated increases in the tyrosine phosphorylation of FAK and paxillin in SH-SY5Y neuroblastoma cells depend on both the maintenance of an actin cytoskeleton and the ability of these cells to synthesize phosphoinositides.     

Tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin does not require extracellular signal-regulated kinase activation in Swiss 3T3 cells stimulated by bombesin or platelet-derived growth factor

The experiments presented here were designed to examine the contribution of the extracellular signal-regulated mitogen-activated protein kinases (ERKs) to the tyrosine phosphorylation of the focal adhesion proteins p125(Fak), p130(Cas), and paxillin induced by G protein-coupled receptors (GPCRs) and tyrosine kinase receptors in Swiss 3T3 cells. Stimulation of these cells with bombesin, lysophosphatidic acid (LPA), endothelin, and platelet-derived growth factor (PDGF) led to a marked increase in the tyrosine phosphorylation of these focal adhesion proteins and in ERK activation. Exposure of the cells to two structurally unrelated mitogen-activated protein kinase or ERK kinase (MEK) inhibitors, PD98059 and U0126, completely abrogated ERK activation but did not prevent tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin. Furthermore, different dose-response relationships were obtained for tyrosine phosphorylation of focal adhesion proteins and for ERK activation in response to PDGF. Putative upstream events in the activation of focal adhesion proteins including actin cytoskeletal reorganization and myosin light chain (MLC) phosphorylation were also not prevented by inhibition of ERK activation. Thus, our results demonstrate that the activation of the ERK pathway is not necessary for the increase of the tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin induced by either GPCRs or tyrosine kinase receptors in Swiss 3T3 cells.     

Outside-in signaling pathway linked to CD146 engagement in human endothelial cells

CD146 (S-Endo 1 Ag or MUC18) is a transmembrane glycoprotein expressed on endothelial cells on the whole vascular tree. CD146 is located at the intercellular junction where it plays a role in the cohesion of the endothelial monolayer. CD146 engagement initiates an outside-in signaling pathway involving the protein tyrosine kinases FYN and FAK as well as paxillin. Here we report that CD146 engagement by its specific monoclonal antibody in human umbilical vein endothelial cells induces a Ca(2+) influx that is sensitive to thapsigargin and EGTA treatment, indicating that CD146 engagement initiates a store-operated calcium mobilization. In addition, biochemical and pharmacological analysis revealed that CD146 engagement initiates the tyrosine phosphorylation of phospholipase C-gamma, Pyk2, and p130(Cas). Pharmacological inhibition of Ca(2+) flux with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethyl ester and EGTA indicated that an increase in Ca(2+) is required for Pyk2 and p130(Cas) tyrosine phosphorylation. Moreover, a complex association was observed between Pyk2, p130(Cas), and paxillin. These results indicate that CD146 is coupled to a FYN-dependent pathway that triggers Ca(2+) flux via phospholipase C-gamma activation leading subsequently to the tyrosine phosphorylation of downstream targets such as Pyk2, p130(Cas), FAK, and paxillin. In addition to its role in cell-cell adhesion, CD146 is a signaling molecule involved in the dynamics of actin cytoskeleton rearrangement.     

Potential role of Gab1 and phospholipase C-gamma in osmotic shock-induced glucose uptake in 3T3-L1 adipocytes.

Osmotic shock induces GLUT4 translocation and glucose uptake through a mechanism independent of PI 3-kinase, but dependent on tyrosine phosphorylation of cellular proteins. To identify the tyrosine phosphorylated proteins required for osmotic shock-stimulated glucose uptake, we examined tyrosine phosphorylation of candidate proteins, and found that the 60-80kDa species including paxillin and the 120-130kDa species including p130Cas, PYK2, FAK and Gab1 were tyrosine-phosphorylated in response to osmotic shock. Inhibition of actin polymerization by cytochalasin D significantly decreased the tyrosine phosphorylation of paxillin, p130Cas, PYK2 and FAK but not Gab1, but had no effect on 2-deoxyglucose (DOG) uptake, suggesting a role for Gab1 in osmotic shock-induced glucose transport. Also, we found that osmotic shock increases the association of phospholipase C-gamma (PLC-gamma) with Gab1 and stimulates tyrosine phosphorylation of PLC-gamma itself. The PLC inhibitor, U73122, inhibited osmotic shock-induced 2-DOG uptake. These results suggest that tyrosine phosphorylation of Gab1 and subsequent recruitment and activation of PLC-gamma may play a role in osmotic shock-induced glucose transport.     

Enhanced proliferation and differentiation of rat hepatocytes cultured with bone marrow stromal cells

Treatment of intact Swiss 3T3 cells with calyculin-A, an inhibitor of myosin light chain (MLC) phosphatase, induces tyrosine phosphorylation of p125(Fak) in a sharply concentration- and time-dependent manner. Maximal stimulation was 4.2 +/- 2.1-fold (n = 14). The stimulatory effect of calyculin-A was observed at low nanomolar concentrations (<10 nM); at higher concentrations (>10 nM) tyrosine phosphorylation of p125(Fak) was strikingly decreased. Calyculin-A induced tyrosine phosphorylation of p125(Fak) through a protein kinase C- and Ca(2+)-independent pathway. Exposure to either cytochalasin-D or latrunculin-A, which disrupt actin organization by different mechanisms, abolished tyrosine phosphorylation of p125(Fak) in response to calyculin-A. Treatment with high concentrations of platelet-derived growth factor (20 ng/ml) which also disrupt actin stress fibers, completely inhibited tyrosine phosphorylation of p125(Fak) in response to calyculin-A. This agent also induced tyrosine phosphorylation of the focal adhesion-associated proteins p130(Cas) and paxillin. These tyrosine phosphorylation events were associated with a striking increase in the assembly of focal adhesions. The Rho kinase (ROK) inhibitor HA1077 that blocked focal adhesion formation by bombesin, had no effect on the focal adhesion assembly induced by calyculin-A. Thus, calyculin-A induces transient focal adhesion assembly and tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin, acting downstream of ROK.     

Activation of transforming G protein-coupled receptors induces rapid tyrosine phosphorylation of cellular proteins, including p125FAK and the p130 v-src substrate.

We have used the family of human muscarinic receptors (mAChRs) as a model for receptors coupled to G proteins and have shown that genes for certain mAChR subtypes can behave as potent agonist-dependent oncogenes. Furthermore, transforming mAChRs can transduce mitogenic signals in transfected NIH 3T3 cells. In this study, we show that in cells expressing ml mAChRs, the cholinergic agonist carbachol, induces a rapid and dose-dependent increase in tyrosine phosphorylation of cellular proteins which are different from those induced by PDGF. Interestingly, carbachol, but not PDGF, induces an increase in tyrosine phosphorylation of the p125FAK and p130 v-src substrates. Thus, growth promoting pathways activated by receptors coupled to G proteins might involve tyrosine phosphorylation of a small set of cellular proteins previously identified as substrates for oncogene-encoded tyrosine kinases.     

Focal adhesion kinase as well as p130Cas and paxillin is crucially involved in the enhanced malignant properties under expression of ganglioside GD3 in melanoma cells

Mass spectrometry analysis of immunoprecipitates from serum-treated GD3-expressing melanoma cells with PY20 (anti-phosphotyrosine antibody) revealed that focal adhesion kinase (FAK) is more strongly activated in GD3-expressing cells than in GD3-negative cells. Involvement of FAK in the increased proliferation and invasion in GD3-expressing melanomas was demonstrated by siRNA-mediated knockdown. Also, it was shown that FAK is located up-stream of p130Cas and paxillin in the enhanced signaling pathway. GD3 expression enhanced the association of FAK with p130Cas after treatment with fetal calf serum. Thus, focal adhesion kinase as well as p130Cas and paxillin should be a crucial molecule undergoing stronger tyrosine phosphorylation in GD3-expressing melanoma cells. Molecules linking GD3 and FAK such as integrins in the enhanced signaling pathway remain to be investigated.     

The Src kinase Yes is activated in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters, but not pancreatic growth factors, which stimulate its association with numerous other signaling molecules

For growth factors, cytokines, G-protein-coupled receptors and numerous other stimuli, the Src Family of kinases (SFK) play a central signaling role. SFKs also play an important role in pancreatic acinar cell function including metabolism, secretion, endocytosis, growth and cytoskeletal integrity, although the specific SFKs involved are not fully known. In the present study we used specific antibodies for the SFK, Yes, to determine its presence, activation by pancreatic secretagogues or growth factors, and interaction with cellular signaling cascades mediated by CCK in which Yes participates in to cause acinar cell responses. Yes was identified in acini and secretagogues known to activate phospholipase C (PLC) [CCK, carbachol, bombesin] as well as post-receptor stimulants activating PKC [TPA] or mobilizing cellular calcium [thapsigargin/calcium ionophore (A23187)] each activated Yes. Secretin, which activates adenylate cyclase did not stimulate Yes, nor did pancreatic growth factors. CCK activation of Yes required both high- and low-affinity CCK(1)-receptor states. TPA-/CCK-stimulated Yes activation was completely inhibited by thapsigargin and the PKC inhibitor, GF109203X. CCK/TPA stimulated the association of Yes with focal adhesion kinases (Pyk2, FAK) and its autophosphorylated forms (pY397FAK, pY402Pyk2). Moreover, CCK/TPA stimulated Yes interacted with a number of other signaling proteins, including Shc, PKD, p130(Cas), PI3K and PTEN. This study demonstrates that in rat pancreatic acini, the SFK member Yes is expressed and activated by CCK and other gastrointestinal hormones/neurotransmitters. Because its activation results in the direct activation of many cellular signaling cascades that have been shown to mediate CCK's effect in acinar cell function our results suggest that it is one of the important pancreatic SFKs mediating these effects.     

Cholecystokinin rapidly stimulates CrkII function in vivo in rat pancreatic acini. Formation of CrkII-protein complexes.

Crk belongs to a family of adapter proteins whose structure allows interaction with tyrosine-phosphorylated proteins and is therefore an important modulator of downstream signals, representing a convergence of the actions of numerous stimuli. Recently, it was demonstrated that cholecystokinin (CCK) induced tyrosine phosphorylation of proteins related to fiber stress formation in rat pancreatic acini. Here, we investigated whether CCK receptor activation signals through CrkII and forms complexes with tyrosine-phosphorylated proteins in rat pancreatic acini. We demonstrated that CCK promoted the transient formation of CrkII-paxillin and CrkII-p130Cas complexes with maximal effect at 1 min. Additionally, CCK decreased the electrophoretic mobility of CrkII. This decrease was time- and concentration-dependent and inversely related with its function. Carbachol and bombesin also decreased CrkII electrophoretic mobility, whereas epidermal growth factor, vasoactive intestinal peptide, secretin or pituitary adenylate cyclase-activating polypeptide had no effect. CCK-induced CrkII electrophoretic shift was dependent on the Src family of tyrosine kinases and occurred in the intact animal, suggesting a physiological role of CrkII mediating CCK actions in the exocrine pancreas in vivo.     

Gastrointestinal hormones cause rapid c-Met receptor down-regulation by a novel mechanism involving clathrin-mediated endocytosis and a lysosome-dependent mechanism

The activated c-Met receptor has potent effects on normal tissues and tumors. c-Met levels are regulated by hepatocyte growth factor (HGF); however, it is unknown if they can be regulated by gastrointestinal (GI) hormones. c-Met is found in many GI tissues/tumors that possess GI hormone receptors. We studied the effect of GI hormones on c-Met in rat pancreatic acini, which possess both receptors. CCK-8, carbachol, and bombesin, but not VIP/secretin, decreased c-Met. CCK-8 caused rapid and potent c-Met down-regulation and abolished HGF-induced c-Met and Gab1 tyrosine phosphorylation, while stimulating c-Met serine phosphorylation. The effect of cholecystokinin (CCK) was also seen in intact acini using immunofluorescence, in a biotinylated fraction representing membrane proteins, in single acinar cells, in Panc-1 tumor cells, and in vivo in rats injected with CCK. CCK-8 did not decrease cell viability or overall responsiveness. GF109203X, thapsigargin, or their combination partially reversed the effect of CCK-8. In contrast to HGF-induced c-Met down-regulation, the effect of CCK was decreased by a lysosome inhibitor (concanamycin) but not the proteasome inhibitor lactacystin. Inhibitors of clathrin-mediated endocytosis blocked the effect of CCK. HGF but not CCK-8 caused c-Met ubiquitination. These results show CCK and other GI hormones can cause rapid c-Met down-regulation, which occurs by a novel mechanism. These results could be important for c-Met regulation in normal as well as in neoplastic tissue in the GI tract.     

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

Protein kinase C-delta (PKC-delta) 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-delta inhibitor. However, some recent studies showed that rottlerin was not effective in inhibiting PKCdelta 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-delta activity in intact cells and to elucidate the biochemical causes of its unspecificity. Preincubation of pancreatic acini with rottlerin (6 microM) inhibited CCK-stimulated translocation, tyrosine phosphorylation (TyrP) and activation of PKC-delta 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-delta 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-delta blockade, but likely due to its negative effect on acini energy resulting in ATP depletion. Therefore, to study the role of PKC-delta 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-delta investigation.     

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-delta, 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-delta and Shc, which play central roles in CCK's effects on acinar cell function.     

Beta 2 integrin-dependent tyrosine phosphorylation of paxillin in human neutrophils treated with tumor necrosis factor

The focal adhesion protein paxillin undergoes tyrosine phosphorylation in response to signals mediated by integrins, neuropeptides and oncogene products, possibly via activation of the focal adhesion- associated kinase, p125FAK. In the present work, tumor necrosis factor- alpha (TNF) stimulated tyrosine phosphorylation of paxillin in human neutrophils. Cell adhesion and participation of the beta 2 integrin CD18 were necessary, but not sufficient, for the response. Adherent neutrophils also tyrosine phosphorylated paxillin in response to phorbol ester, formylmethionyl-leucyl-phenylalanine and opsonized bacteria. In contrast, p125FAK was constitutively tyrosine phosphorylated in a manner unaffected by adherence and/or TNF. Thus, cytokines and microbial products are among the stimuli that can induce the tyrosine phosphorylation of paxillin, and kinases other than p125FAK may be responsible. This is the first identification of paxillin and p125FAK in human cells and neutrophils, and one of the few identifications of a specific protein that undergoes tyrosine phosphorylation in response to any agonist in neutrophils or in response to TNF in any cell.     

PKC-regulated myogenesis is associated with increased tyrosine phosphorylation of FAK,Cas, and paxillin,formation of Cas-CRK complex,and JNK activation

Previous reports suggest that PKC plays an important role in regulating myogenesis. However, the regulatory signaling pathways are not fully understood. We examined the effects of PKC downregulation on signaling events during skeletal muscle differentiation. We found that downregulation of PKC results in increased myogenesis in C2C12 cells as measured by creatine kinase activity and myogenin expression. We showed that, during differentiation, downregulation of PKC expression results in increased tyrosine phosphorylation of FAK, Cas, and paxillin, concomitant with enhanced Cas-CrkII complex formation, which leads to activation of JNK2. But in proliferated muscle cells, PKC inhibition results in FAK and Cas tyrosine dephosphorylation. Further, disruption of actin cytoskeleton by cytochalasin D prevents the activation of FAK and Cas as well as the formation of Cas-CrkII complex stimulated by PKC downregulation during muscle cell differentiation. Finally, we observed that PKC downregulation increases the tyrosine phosphorylation of focal adhesion associated proteins. Based on the above data, we propose that PKC downregulation results in enhanced tyrosine phosphorylation of FAK, Cas, and paxillin, thus promoting the establishment of Cas-CrkII complex, leading to activation of JNK and that these interactions are dependent upon the integrity of actin cytoskeleton during muscle cell differentiation. Data presented here significantly contribute to elucidating the regulatory role of PKC in myogenesis possibly through integrin signaling pathway.     

Protein tyrosine phosphatase-dependent proteolysis of focal adhesion complexes in endothelial cell apoptosis

Adenosine and/or homocysteine causes endothelial cell apoptosis, a mechanism requiring protein tyrosine phosphatase (PTPase) activity. We investigated the role of focal adhesion contact disruption in adenosine-homocysteine endothelial cell apoptosis. Analysis of focal adhesion kinase (FAK), paxillin, and vinculin demonstrated disruption of focal adhesion complexes after 4 h of treatment with adenosine-homocysteine followed by caspase-induced proteolysis of FAK, paxillin, and p130(CAS). No significant changes were noted in tyrosine phosphorylation of FAK or paxillin. Pretreatment with the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone prevented adenosine-homocysteine-induced DNA fragmentation and FAK, paxillin, and p130(CAS) proteolysis. Asp-Glu-Val-Asp-ase activity was detectable in endothelial cells after 4 h of treatment with adenosine-homocysteine. The PTPase inhibitor sodium orthovanadate did not prevent endothelial cell retraction or FAK, paxillin, or vinculin redistribution. Sodium orthovanadate did block adenosine-homocysteine-induced FAK, paxillin, and p130(CAS) proteolysis and Asp-Glu-Val-Asp-ase activity. Thus disruption of focal adhesion contacts and caspase-induced degradation of focal adhesion contact proteins occurs in adenosine-homocysteine endothelial cell apoptosis. Focal adhesion contact disruption induced by adenosine-homocysteine is independent of PTPase or caspase activation. These studies demonstrate that disruption of focal adhesion contacts is an early, but not an irrevocable, event in endothelial cell apoptosis.