Protein kinase C-mediated tyrosine phosphorylation of paxillin and focal adhesion kinase requires cytoskeletal integrity and is uncoupled to mitogen-activated protein kinase activation in human hepatoma cells

Treatment of cultured human hepatoma HepG2 cells with the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), results in an increase in tyrosine phosphorylation of several proteins, including the focal adhesion kinase (FAK) and paxillin using anti-phosphotyrosine Western blotting and immunoprecipitation. However, when cells are in suspension or in the presence of cytochalasin D which disrupts the intracellular network of actin microfilaments, TPA loses its ability to stimulate tyrosine phosphorylation of FAK and paxillin but it still activates mitogen-activated protein kinase (MAPK) and induces PKC translocation from cytosol to the membrane in HepG2 cells. On the other hand, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, blocks TPA-induced MAPK activation but has no effect on TPA-induced tyrosine phosphorylation. Our findings suggest that TPA-induced tyrosine phosphorylation of FAK and paxillin in human hepatoma cells is PKC dependent and requires the integrity of the cell cytoskeleton but is uncoupled to the signal transduction pathway of PKC leading to the translocation of PKC and MAPK activation.     

Angiotensin II induces focal adhesion kinase/paxillin phosphorylation and cell migration in human umbilical vein endothelial cells

In the present study, we demonstrated that Ang II provokes a transitory enhancement of focal adhesion kinase (FAK) and paxillin phosphorylation in human umbilical endothelial cells (HUVEC). Moreover, Ang II induces a time- and dose-dependent augmentation in cell migration, but does not affect HUVEC proliferation. The effect of Ang II on FAK and paxillin phosphorylation was markedly attenuated in cells pretreated with wortmannin and LY294002, indicating that phosphoinositide 3-kinase (PI3K) plays an important role in regulating FAK activation. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific inhibitor PP2 for Src family kinases, demonstrating the involvement of protein tyrosine kinases, and particularly Src family of tyrosine kinases, in the downstream signalling pathway of Ang II receptors. Furthermore, FAK and paxillin phosphorylation was markedly blocked after treatment of HUVEC with AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) phosphorylation. Pretreatment of cells with inhibitors of PI3K, Src family tyrosine kinases, and EGFR also decreased HUVEC migration. In conclusion, these results suggest that Ang II mediates an increase in FAK and paxillin phosphorylation and induces HUVEC migration through signal transduction pathways dependent on PI3K and Src tyrosine kinase activation and EGFR transactivation.     

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.     

Growth factors stimulate kidney proximal tubule cell migration independent of augmented tyrosine phosphorylation of focal adhesion kinase

Migration of human proximal tubule cells (HKC-5) was stimulated by epidermal growth factor (EGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1). Integrin signaling via phosphorylation of focal adhesion kinase (FAK) appears to play a central role in cell migration. Once stimulated, FAK undergoes autophosphorylation at tyrosine (Y) 397, followed by phosphorylation of several sites including Y576/Y577 which increases FAK's kinase activity, as well as at Y407, Y861, and Y925. EGF, HGF, and IGF-1 stimulate FAK phosphorylation in various cells. We showed that endothelin stimulated phosphorylation of Y397 in fibroblasts but not HKC-5 cells. After EGF stimulation, HKC-5 cells showed no change in tyrosine phosphorylation at FAK Y397, 407, 576, 861, or 925. Similarly, HGF and IGF-1 did not stimulate the phosphorylation of FAK Y397 in HKC-5 cells. Further, after inhibition of FAK expression by siRNA, cell migration was similar to cells treated with non-target siRNA and responded to EGF with increased migration. Thus, in proximal tubule cells, stimulation of cell migration by growth factors was independent of augmented FAK tyrosine phosphorylation.     

Glutamate induces focal adhesion kinase tyrosine phosphorylation and actin rearrangement in heterologous mGluR1-expressing CHO cells via calcium/calmodulin signaling

Group 1 metabotropic glutamate receptors (mGluR1 and mGluR5) stimulate phospholipase C (PLC) and lead to mobilization of intracellular Ca(2+) and activation of protein kinase C (PKC). In this investigation, using heterologous receptor-expressing Chinese hamster ovary (CHO) cells, we showed that stimulation of mGluR1 or mGluR5 with glutamate rapidly increases tyrosine phosphorylation of focal adhesion kinase (FAK) (maximum at 1-3 min) in a dose-dependent manner (half-maximal responses at approximately 2 microM). In mGluR1-expressing cells, the glutamate-induced increase of FAK tyrosine phosphorylation was blocked by not only the PLC inhibitor, U73122, but also depletion of intracellular Ca(2+) and effectively abrogated by calmodulin (CaM) inhibitors, calmidazolium and fluphenazine. However, neither the PKC inhibitor, GF109203X, nor the CaM kinase II inhibitor, KN-62, inhibited glutamate-stimulated FAK tyrosine phosphorylation. Stimulation of mGluR1 caused a marked increase in actin stress fiber formation. Importantly, this actin rearrangement was prevented by the CaM inhibitor, but not by the PKC inhibitor and is thus in a good agreement with the signaling cascade of the mGluR1-FAK pathway. These results suggest that the Ca(2+)/CaM signaling and its downstream FAK tyrosine phosphorylation play an important role in cellular function of mGluR1.     

Myofibroblast differentiation by transforming growth factor-beta1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase

Myofibroblast differentiation and activation by transforming growth factor-beta1 (TGF-beta1) is a critical event in the pathogenesis of human fibrotic diseases, but regulatory mechanisms for this effect are unclear. In this report, we demonstrate that stable expression of the myofibroblast phenotype requires both TGF-beta1 and adhesion-dependent signals. TGF-beta1-induced myofibroblast differentiation of lung fibroblasts is blocked in non-adherent cells despite the preservation of TGF-beta receptor(s)-mediated signaling of Smad2 phosphorylation. TGF-beta1 induces tyrosine phosphorylation of focal adhesion kinase (FAK) including that of its autophosphorylation site, Tyr-397, an effect that is dependent on cell adhesion and is delayed relative to early Smad signaling. Pharmacologic inhibition of FAK or expression of kinase-deficient FAK, mutated by substituting Tyr-397 with Phe, inhibit TGF-beta1-induced alpha-smooth muscle actin expression, stress fiber formation, and cellular hypertrophy. Basal expression of alpha-smooth muscle actin is elevated in cells grown on fibronectin-coated dishes but is decreased on laminin and poly-d-lysine, a non-integrin binding polypeptide. TGF-beta1 up-regulates expression of integrins and fibronectin, an effect that is associated with autophosphorylation/activation of FAK. Thus, a safer and more effective therapeutic strategy for fibrotic diseases characterized by persistent myofibroblast activation may be to target this integrin/FAK pathway while not interfering with tumor-suppressive functions of TGF-beta1/Smad signaling.     

蛋白激酶PRKX对人肝癌细胞粘附和迁移能力的影响

目的观察蛋白激酶PRKX对人肝癌细胞SMMC-7721粘附和迁移能力的影响。方法采用脂质体转染的方法,将PRKX表达质粒转染到SMMC-7721细胞中,蛋白印迹方法鉴定转染前后PRKX蛋白的表达。细胞-基质粘附实验测定对照组和PRKX转染组SMMC-7721细胞的粘附能力。细胞迁移实验测定对照组和PRKX转染组SMMC-7721细胞的迁移能力。结果 SMMC-7721细胞转染组PRKX蛋白的表达增加,SMMC-7721细胞转染组的粘附能力和迁移能力均较对照组增加。结论 PRKX可增加人肝癌细胞SMMC-7721的粘附和迁移能力。     

The effect of epidermal growth factor receptor variant III on glioma cell migration by stimulating ERK phosphorylation through the focal adhesion kinase signaling pathway

Epidermal growth factor receptor variant III (EGFRvIII), the most common EGFR mutation, is associated with cell migration of glioblastoma multiforme (GBM) cases; however, the mechanism has not been elucidated. In this study, we found that the EGFRvIII-promoted glioma cell migration was closely linked to high levels of tyrosine phosphorylation in focal adhesion kinase (FAK) Y397. We also demonstrated that EGFRvIII formed a complex with FAK, resulting in enhanced tyrosine phosphorylation levels of FAK Y397 and EGFR Y1068. After knockdown of FAK expression via anti-FAK shRNA, the U87ΔEGFR cell migration was significantly inhibited, accompanying with the reduced phosphorylation levels of extracellular signal-regulated kinase (ERK1/2). Furthermore, the role of ERK1/2 in FAK-regulated cell migration was confirmed. Taken together, our results suggest that FAK and its downstream molecule ERK were involved in EGFRvIII-promoted glioma cell migration in U87ΔEGFR cells.     

Changes in protein tyrosine phosphorylation during mannose and senescence induced cell death in rice

In mammals protein tyrosine phosphorylation plays an important role in the activation of apoptosis. However, tyrosine phosphorylation associated with cell death has not been examined in plants. We monitored changes in tyrosine phosphorylation during cell death in rice (Oryza sativa L.) suspension cultures. Cell death was induced in the cell cultures by mannose treatment or by allowing the cultures to senescence. We have demonstrated that both mannose and senescence induced DNA fragmentation in rice suspension cells. In the presence of mannose, the tyrosine phosphorylation patterns of mannose treated and non-treated cell proteins are basically the same, except the tyrosine phosphorylation intensity is considerably different. In aged suspension-cultured cells, the occurrence of DNA fragmentation was detected. In addition, the tyrosine phosphorylation pattern was changed. These results suggest that protein tyrosine phosphorylation may have a role in distinct signal transduction pathways responding to mannose and senescence. The expression of a gene that encodes mitogen-activated protein kinase (MAPK), OsMAPK2, is up-regulated during mannose treatment, suggesting the possible involvement of rice MAPK in pathways associated with rice cell death induced by >d-mannose.     

Transforming growth factor-beta1 induces the non-classical secretion of peroxiredoxin-I in A549 cells

Recent studies found that peroxiredoxin-I (Prx-I) is secreted from A549 cells although it does not contain a signal peptide and is known to be a cytosolic protein. Transforming growth factor-beta1 (TGF-beta1) treatment dramatically enhanced Prx-I secretion from A549 cells, and this effect was not inhibited by brefeldin A. Further investigation revealed that A549 cells constitutively secrete TGF-beta1. Furin, a TGF-beta1-converting enzyme, was also highly activated in A549 cells. Ectopic expression of alpha(1)-antitrypsin Portland (alpha(1)-PDX), a potent furin inhibitor, blocked both TGF-beta1 activation and Prx-I secretion. Our findings collectively suggest that non-classical secretion of Prx-I is induced by TGF-beta1, which is constitutively activated by furin in A549 cells.     

Transforming growth factor-beta1 enhanced vascular endothelial growth factor synthesis in mesenchymal stem cells

Angiogenesis is essential for transplantation of mesenchymal stem cells (MSCs). Vascular endothelial growth factor (VEGF) is one of the most potent angiogenic factors identified to date. Elevated VEGF levels in MSCs correlate with the potential of MSCs transplantation. As an indirect angiogenic agent, transforming growth factor-β1 (TGF-β1) plays a pivotal role in the regulation of vasculogenesis and angiogenesis. However, the effect of TGF-β1 on VEGF synthesis in MSCs is still unknown. Besides, the intracellular signaling mechanism by which TGF-β1 stimulates this process remains poorly understood. In this article, we demonstrated that exposure of MSCs to TGF-β1 stimulated the synthesis of VEGF. Meanwhile, TGF-β1 stimulated the phosphorylation of Akt and extracellular signal-regulated kinase 1/2 (ERK1/2). Moreover, Ly 294002, a specific inhibitor of phosphatidylinositol-3-kinase (PI3K)/Akt significantly attenuated the VEGF synthesis stimulated by TGF-β1. Additionally, U0126, a specific inhibitor of ERK1/2, also significantly attenuated the TGF-β1-stimulated VEGF synthesis. These results indicated that TGF-β1 enhanced VEGF synthesis in MSCs, and the Akt and ERK1/2 activation were involved in this process.     

Glutamate-stimulated activation of DNA synthesis via mitogen-activated protein kinase in primary astrocytes: involvement of protein kinase C and related adhesion focal tyrosine kinase

Glutamate is the major excitatory neurotransmitter in the CNS. Although its role in neurons has been studied extensively, little is known about its function in astrocytes. We studied the effects of glutamate on signaling pathways in primary astrocytes. We found that the tyrosine kinase related adhesion focal tyrosine kinase (RAFTK) is tyrosine phosphorylated in response to glutamate in a time- and dose-dependent manner. This phosphorylation was pertussis toxin (PTX) sensitive and could be attenuated by the depletion of Ca2+ from intracellular stores. RAFTK tyrosine phosphorylation was mediated primarily by class I/II metabotropic glutamate receptors and depends on protein kinase C (PKC) activation. Glutamate treatment of primary astrocytes also results in a significant increase in the activity of the mitogen-activated protein kinases [extracellular signal-related kinases 1/2 (ERK1/2)]. Like RAFTK phosphorylation, ERK1/2 activation is PTX sensitive and can be attenuated by the depletion of intracellular Ca2+ and by PKC inhibition, suggesting that RAFTK might mediate the glutamate-dependent activation of ERK1/2. Furthermore, we demonstrated that glutamate stimulation of primary astrocytes leads to a significant increase in DNA synthesis. Glutamate-stimulated DNA synthesis is PTX sensitive and can be inhibited by the MAP kinase kinase inhibitor PD98059, suggesting that in primary astrocytes, glutamate might signal via RAFTK and MAP kinase to promote DNA synthesis and cell proliferation.     

Focal adhesion kinase affects the sensitivity of human hepatocellular carcinoma cell line SMMC-7721 to tumor necrosis factor-alpha/cycloheximide-induced apoptosis by regulating protein kinase B levels.

Most cell lines are resistant to tumor necrosis factor-alpha (TNF-alpha) cytotoxicity and require cotreatment of TNF-alpha with cycloheximide (Chx) to undergo apoptosis. Recently, the serine/threonine protein kinase, protein kinase B has been demonstrated to protect cells from apoptosis induced by TNF-alpha. In this study, we have shown that the human hepatocellular carcinoma cell line, SMMC-7721, was insensitive to TNF-alpha cytotoxicity and underwent apoptosis quickly in the presence of TNF-alpha and Chx. PKB levels decreased during TNF-alpha/Chx-induced apoptosis. No significant change in PKB levels was found in the presence of TNF-alpha or Chx alone. It seemed that the level of PKB closely correlated with apoptosis. The protein level of focal adhesion kinase (FAK) was reduced by 66% by transfecting FAK antisense cDNA recombinant vector into SMMC-7721 cells. We determined the apoptosis-induced effect of TNF-alpha/Chx on the FAK antisense cDNA transfectant cells. The results indicated that the percentage of apoptotic cells was enhanced at lower doses of TNF-alpha (10, 20 or 50 U.mL(-1)) and decreased at a higher dose of TNF-alpha (1000 U.mL(-1)) in the transfected cells as compared to the control. Correspondingly, in the FAK antisense cDNA transfectant cells treated with lower doses of TNF-alpha in presence of 10 microg.mL(-1) Chx, the PKB level was lower, but in the FAK antisense cDNA transfectants treated with higher doses of TNF-alpha in presence of 10 microg.mL(-1) Chx, the PKB level was higher. In response to TNF-alpha alone, FAK antisense cDNA transfectants showed a decrease in the level of PKB. However, in the case of TNF-alpha cotreated with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PtdIns3K), the FAK antisense cDNA transfectants produced significantly less amounts of PKB than the control. It seemed that FAK could stimulate PKB levels through a pathway not involving PtdIns3K. These results suggest that FAK can affect the sensitivity of SMMC-7721 cells to TNF-alpha/Chx-induced apoptosis in a biphasic manner by regulating PKB levels.     

AMP-activated protein kinase phosphorylation in brain is dependent on method of killing and tissue preparation

AMP-activated protein kinase (AMPK) is activated when the catalytic α subunit is phosphorylated on Thr172 and therefore, phosphorylation of the α subunit is used as a measure of activation. However, measurement of α subunit of AMPK (α-AMPK) phosphorylation in vivo can be technically challenging. To determine the most accurate method for measuring α-AMPK phosphorylation in the mouse brain, we compared different methods of killing and tissue preparation. We found that freeze/thawing samples after homogenization on ice dramatically increased α-AMPK phosphorylation in mice killed by cervical dislocation. Killing of mice by focused microwave irradiation, which rapidly heats the brain and causes enzymatic inactivation, prevented the freeze/thaw-induced increase in α-AMPK phosphorylation and similar levels of phosphorylation were observed compared with mice killed with cervical dislocation without freeze/thawing of samples. Sonication of samples in hot 1% sodium dodecyl sulfate blocked the freeze/thaw-induced increase in α-AMPK phosphorylation, but phosphorylation was higher in mice killed by cervical dislocation compared with mice killed by focused microwave irradiation. These results demonstrate that α-AMPK phosphorylation is dependent on method of killing and tissue preparation and that α-AMPK phosphorylation can increase in a manner that does not reflect biological alterations.     

Nuclear-factor-kappaB (NF-kappaB) and radical oxygen species play contrary roles in transforming growth factor-beta1 (TGF-beta1)-induced apoptosis in hepatocellular carcinoma (HCC) cells

Nuclear-Factor-κB (NF-κΒ can counteract transforming growth factor-β1 (TGF-β1)-induced apoptosis in malignant hepatocytes through up-regulation of its downstream genes, such as X-linked inhibitor of apoptosis protein (XIAP). Reports have demonstrated that TGF-β1 can induce oxidative stress, and c-Jun N-terminal Kinase1 (JNK1) is indispensable for TGF-β1-induced apoptosis pathway, but the relationship between radical oxygen species (ROS) and the activation of JNKs is still unclear. In the present study, we found that ROS can induce JNK activation in TGF-β1 mediated apoptosis in hepatocytes. The inhibitors of hydrogen peroxide and superoxide, which were produced by mitochondria under stress, could inhibit the phosphorylation of c-Jun in XIAP knockdown cells. In conclusion, it is the first time to show that both NF-κB and antioxidants can counteract TGF-β1-induced apoptosis in hepatic cell death through JNK1 pathway.     

CXC chemokine ligand 12/Stromal cell-derived factor-1 regulates cell adhesion in human colon cancer cells by induction of intercellular adhesion molecule-1

Background

Gene therapy and viral therapy are used for cancer therapy for many years, but the results are less than satisfactory. Our aim was to construct a new recombinant adenovirus which is more efficient to kill hepatocarcinoma cells but more safe to normal cells.

Methods

By using the Cancer Targeting Gene-Viro-Therapy strategy, Apoptin, a promising cancer therapeutic gene was inserted into the double-regulated oncolytic adenovirus AD55 in which E1A gene was driven by alpha fetoprotein promoter along with a 55 kDa deletion in E1B gene to form AD55-Apoptin. The anti-tumor effects and safety were examined by western blotting, virus yield assay, real time polymerase chain reaction, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, Hoechst33342 staining, Fluorescence-activated cell sorting, xenograft tumor model, Immunohistochemical assay, liver function analysis and Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling assay.

Results

The recombinant virus AD55-Apoptin has more significant antitumor effect for hepatocelluar carcinoma cell lines (in vitro) than that of AD55 and even ONYX-015 but no or little impair on normal cell lines. Furthermore, it also shows an obvious in vivo antitumor effect on the Huh-7 liver carcinoma xenograft in nude mice with bigger beginning tumor volume till about 425 mm3 but has no any damage on the function of liver. The induction of apoptosis is involved in AD55-Apoptin induced antitumor effects.

Conclusion

The AD55-Apoptin can be a potential anti-hepatoma agent with remarkable antitumor efficacy as well as higher safety in cancer targeting gene-viro-therapy system.     

Advanced glycation end-product-inhibited cell proliferation and protein expression of beta-catenin and cyclin D1 are dependent on glycogen synthase kinase 3beta in LLC-PK1 cells

Glycogen synthase kinase 3β (GSK3β) is increased by high glucose in mesangial cells. Thus, we studied the role of GSK3β in advanced glycation end-product (AGE)-induced effects in the proximal tubule-like LLC-PK1 cells. We found that AGE (100 μg/ml) time-dependently (8-48 h) increased phospho-GSK3β-Tyr216 (active GSK3β) and time-dependently (4-24 h) decreased phospho-GSK3β-Ser21/9 (inactive GSK3β) protein expression. Meanwhile, AGE (100 μg/ml) activated GSK3β kinase at 8-48 h. AGE (100 μg/ml) dose-dependently (75-100 μg/ml) decreased β-catenin protein expression but AGE did not decrease β-catenin protein expression until 48 h. SB216763 (a GSK3β inhibitor) and GSK3β shRNA attenuated AGE (100 μg/ml)-inhibited cell proliferation and protein expression of β-catenin and cyclin D1 at 48 h. SB216763 also attenuated AGE-induced type IV collagen. We conclude that AGE activates GSK3β in LLC-PK1 cells. AGE-inhibited β-catenin and cyclin D1 protein expression are dependent on GSK3β. Moreover, AGE-inhibited cell proliferation and AGE-induced type IV collagen protein expression are dependent on GSK3β.     

Activation of focal adhesion kinase in human lung cancer cells involves multiple and potentially parallel signaling events

Integrins are adhesion receptors that transmit signals bidirectionally across the plasma membrane. In our previous report we have shown that the squamous lung cancer cell line, Calu-1, binds to collagen type IV (Coll IV) through beta1-integrin and results in phosphorylation of focal adhesion kinase (FAK) (Ann Thorac Surg 2004; 78:450-457). Considering the critical role of FAK in cell migration, proliferation, and survival, here we investigated potential mechanisms of its activation and regulation in Calu-1 cells. We observed the phosphorylation of Tyr397 of FAK (the autophosphorylation site of FAK) and paxillin, the immediate downstream substrate of FAK following the adhesion of Calu-1 cells to Coll IV. FAK remains phosphorylated during proliferation either on Coll IV or on uncoated plates for 72 h, as determined by peroxivanadate treatment. Exposure of Calu-1 cells with 60 microM genistein, reduces FAK phosphorylation (7.6 fold) and cell proliferation. Extracellular signal regulated kinases (ERKs) were also phosphorylated after Coll IV attachment. Disruption of Calu-1 cell cytoskeleton integrity by 1-5 muM Cytochalasin D resulted in the inhibition of cell adhesion (50% to 75%, p<0.19 - 6.6 x 10(7)) and ERKs phosphorylation (2 fold) without any effect on FAK phosphorylation. Protein Kinase C inhibitor, Calphostin C at 100 and 250 nM concentrations did not block Coll IV induced FAK phosphorylation but activated the ERKs in a dose dependent manner. beta1-integrin is essential for Coll IV induced FAK activation, but it is not physically associated with FAK as determined by immunodetection assay. Collectively, this report defines the existence of multiple and potentially parallel Coll IV/beta1-integrin mediated signaling events in Calu-1 cells, which involve FAK, ERKs, and PKC.