Collagen IV regulates Caco-2 migration and ERK activation via alpha1beta1- and alpha2beta1-integrin-dependent Src kinase activation

Our previous work indicates intestinal epithelial cell ERK activation by collagen IV, a major component of the intestinal epithelial basement membrane, requires focal adhesion kinase (FAK) and suggests FAK and ERK may have important roles in regulating intestinal epithelial cell migration. We therefore sought to identify FAK downstream targets regulating intestinal epithelial cell spreading, migration, and ERK activation on collagen IV and the integrins involved. Both dominant-negative Src and Src inhibitor PP2 strongly inhibited collagen IV ERK activation in Caco-2 intestinal epithelial cells. Collagen IV stimulated Grb2 binding site FAK Y925 phosphorylation, which was inhibited by PP2 and required FAK Y397 autophosphorylation. Additionally, FAK Y925F expression blocked collagen IV ERK activation. alpha(1)beta(1)- Or alpha(2)beta(1)-integrin blockade with alpha(1)- or alpha(2)-integrin subunit antibodies indicated that either integrin can mediate adhesion, cell spreading, and FAK, Src, and ERK activation on collagen IV. Both dominant-negative Src and PP2 inhibited Caco-2 spreading on collagen IV. PP2 inhibited p130(Cas) tyrosine phosphorylation, but dominant-negative p130(Cas) did not inhibit cell spreading. PP2 inhibited Caco-2 migration on collagen IV much more strongly than the mitogen-activated protein kinase kinase inhibitor PD-98059, which completely inhibited collagen IV ERK activation. These results suggest a pathway for collagen IV ERK activation requiring Src phosphorylation of FAK Y925 not previously described for this matrix protein and suggest either alpha(1)beta(1)- or alpha(2)beta(1)-integrins can regulate Caco-2 spreading and ERK activation on collagen IV via Src. Additionally, these results suggest Src regulates Caco-2 migration on collagen IV primarily through ERK-independent pathways.     

Collagen IV-dependent ERK activation in human Caco-2 intestinal epithelial cells requires focal adhesion kinase

Integrin-initiated extracellular signal-regulated kinase (ERK) activation by matrix adhesion may require focal adhesion kinase (FAK) or be FAK-independent via caveolin and Shc. This remains controversial for fibroblast and endothelial cell adhesion to fibronectin and is less understood for other matrix proteins and cells. We investigated Caco-2 intestinal epithelial cell ERK activation by collagen I and IV, laminin, and fibronectin. Collagens or laminin, but not fibronectin, stimulated tyrosine phosphorylation of FAK, paxillin, and p130(cas) and activated ERK1/2. Shc, tyrosine-phosphorylated by matrix adhesion in many cells, was not phosphorylated in Caco-2 cells in response to any matrix. Caveolin expression did not affect Caco-2 Shc phosphorylation in response to fibronectin. FAK, ERK, and p130(cas) tyrosine phosphorylation were activated after 10-min adhesion to collagen IV. FAK activity increased for 45 min after collagen IV adhesion and persisted for 2 h, while p130(cas) phosphorylation increased only slightly after 10 min. ERK activity peaked at 10 min, declined after 30 min, and returned to base line after 1 h. Transfection with FAK-related nonkinase, but not substrate domain deleted p130(cas), strongly inhibited ERK2 activation in response to collagen IV, indicating Caco-2 ERK activation is at least partly regulated by FAK.     

Focal adhesion kinase protein levels in gut epithelial motility

Mucosal healing requires migration and proliferation. Most studies of focal adhesion kinase (FAK), a protein that regulates motility, proliferation, and apoptosis, have focused on rapid phosphorylation. We reported lower FAK protein levels in motile Caco-2 colon cancer cells and postulated that this reduction in FAK available for activation might impact cell migration and mucosal healing. Therefore, total and active FAK (FAK(397)) immunoreactivity was assessed at the migrating fronts of human Caco-2 and rat IEC-6 intestinal epithelial cells. Caco-2 and IEC-6 motility, quantitated as migration into linear or circular wounds, was examined following FAK protein inhibition by small interfering RNA (siRNA). FAK protein stability and mRNA expression were ascertained by cycloheximide decay, RT-PCR, and in situ hybridization in static and migrating Caco-2 cells. Cells at the migrating front of Caco-2 and IEC-6 monolayers exhibited lower immunostaining for both total and activated FAK than cells immediately behind the front. Western blot analysis also demonstrated diminished FAK protein levels in motile cells by >/=30% in both the differential density seeding and multiple scrape models. siRNA FAK protein inhibition enhanced motility in both the linear scrape (20% in Caco-2) and circular wound (16% in Caco-2 and 19% in IEC-6 cells) models. FAK protein degradation did not differ in motile and static Caco-2 cells and was unaffected by FAK(397) phosphorylation, but FAK mRNA was lower in migrating Caco-2 cells. Thus FAK protein abundance appears regulated at the mRNA level during gut epithelial cell motility and may influence epithelial cell migration coordinately with signals that modify FAK phosphorylation.     

Integrin and FAK-mediated MAPK activation is required for cyclic strain mitogenic effects in Caco-2 cells

Rhythmic strain stimulates Caco-2 proliferation. We asked whether mitogen-activated protein kinase (MAPK) activation mediates strain mitogenicity and characterized upstream signals regulating MAPK. Caco-2 cells were subjected to strain on collagen I-precoated membranes or antibodies to integrin subunits. Twenty-four hours of cyclic strain increased cell numbers compared with static conditions. MAPK-extracellular signal-regulated kinase (ERK) kinase inhibition (20 microM PD-98059) blocked strain mitogenicity. p38 Inhibition (10 microM SB-202190) did not. Strain rapidly and time-dependently activated focal adhesion kinase (FAK), paxillin, ERK1 and 2, and p38 on collagen. c-Jun NH(2)-terminal kinase (JNK)1 and 2 exhibited delayed activation. Similar activation occurred when Caco-2 cells were subjected to strain on a substrate of functional antibody to the alpha2-, alpha3-, alpha6-, or beta1-integrin subunits but not on a substrate of functional antibody to the alpha5-subunit. FAK inhibition by FAK397 transfection blocked ERK2 and JNK1 activation by in vitro kinase assays, but pharmacological protein kinase C inhibition did not block ERK1 or 2 activation by strain. Strain-induced ERK signals mediate strain's mitogenic effects and may require integrins and FAK activation.     

Role of RhoA and its effectors ROCK and mDia1 in the modulation of deformation-induced FAK, ERK, p38, and MLC motogenic signals in human Caco-2 intestinal epithelial cells

Repetitive deformation enhances intestinal epithelial migration across tissue fibronectin. We evaluated the contribution of RhoA and its effectors Rho-associated kinase (ROK/ROCK) and mammalian diaphanous formins (mDia1) to deformation-induced intestinal epithelial motility across fibronectin and the responsible focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), p38, and myosin light chain (MLC) signaling. We reduced RhoA, ROCK1, ROCK2, and mDia1 by smart-pool double-stranded short-interfering RNAs (siRNA) and pharmacologically inhibited RhoA, ROCK, and FAK in human Caco-2 intestinal epithelial monolayers on fibronectin-coated membranes subjected to 10% repetitive deformation at 10 cycles/min. Migration was measured by wound closure. Stimulation of migration by deformation was prevented by exoenzyme C3, Y27632, or selective RhoA, ROCK1, and ROCK2 or mDia1 siRNAs. RhoA, ROCK inhibition, or RhoA, ROCK1, ROCK2, mDia1, and FAK reduction by siRNA blocked deformation-induced nuclear ERK phosphorylation without preventing ERK phosphorylation in the cytoplasmic protein fraction. Furthermore, RhoA, ROCK inhibition or RhoA, ROCK1, ROCK2, and mDia1 reduction by siRNA also blocked strain-induced FAK-Tyr(925), p38, and MLC phosphorylation. These results suggest that RhoA, ROCK, mDia1, FAK, ERK, p38, and MLC all mediate the stimulation of intestinal epithelial migration by repetitive deformation. This pathway may be an important target for interventions to promote mechanotransduced mucosal healing during inflammation.     

Hepatocyte growth factor induces ERK-dependent paxillin phosphorylation and regulates paxillin-focal adhesion kinase association.

Hepatocyte growth factor (HGF) modulates cell adhesion, migration, and branching morphogenesis in cultured epithelial cells, events that require regulation of cell-matrix interactions. Using mIMCD-3 epithelial cells, we studied the effect of HGF on the focal adhesion proteins, focal adhesion kinase (FAK) and paxillin and their association. HGF was found to increase the tyrosine phosphorylation of paxillin and to a lesser degree FAK. In addition, HGF induced association of paxillin and activated ERK, correlating with a gel retardation of paxillin that was prevented with the ERK inhibitor U0126. The ability of activated ERK to phosphorylate and induce gel retardation of paxillin was confirmed in vitro in both full-length and amino-terminal paxillin. Several potential ERK phosphorylation sites in paxillin flank the paxillin-FAK association domains, so the ability of HGF to regulate paxillin-FAK association was examined. HGF induced an increase in paxillin-FAK association that was inhibited by pretreatment with U0126 and reproduced by in vitro phosphorylation of paxillin with ERK. The prevention of the FAK-paxillin association with U0126 correlated with an inhibition of the HGF-mediated FAK tyrosine phosphorylation and inhibition of HGF-dependent cell spreading and adhesion. An examination of cellular localization of FAK and paxillin demonstrated that HGF caused a condensation of focal adhesion complexes at the leading edges of cell processes and FAK-paxillin co-localization in these large complexes. Thus, these data suggest that HGF can induce serine/threonine phosphorylation of paxillin most probably mediated directly by ERK, resulting in the recruitment and activation of FAK and subsequent enhancement of cell spreading and adhesion.     

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.     

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.     

p130cas but not paxillin is essential for Caco-2 intestinal epithelial cell spreading and migration on collagen IV

We have previously observed that collagen IV regulates Caco-2 intestinal epithelial cell spreading and migration via Src kinase and stimulates Src-dependent tyrosine phosphorylation of p130cas. We observed that collagen IV also stimulated Src-dependent phosphorylation of both paxillin Tyr31 and paxillin Tyr118. Caco-2 transfection with paxillin or p130cas siRNAs inhibited expression of these proteins by more than 90% for at least 5 days after transfection. Although p130cas siRNA inhibited cell spreading on collagen IV by 33%, three different paxillin siRNAs did not inhibit cell spreading. p130cas siRNA did not affect Src Tyr416 or Src Tyr527 phosphorylation, FAK Tyr397 phosphorylation, or Src-dependent phosphorylation of FAK Tyr925, suggesting that p130cas did not inhibit cell spreading by altering FAK or Src activity. Rat p130cas expression after siRNA knock-out of endogenous human p130cas in Caco-2 cells reduced cell spreading inhibition by 71%. In contrast, expression of rat p130cas from which the Src-phosphorylated substrate domain was deleted did not rescue siRNA inhibition of cell spreading. Combined treatment with siRNAs to Crk and CrkL, which bind to the p130cas substrate domain, inhibited cell spreading by 54%. Both p130cas siRNA and the combined Crk/CrkL siRNAs strongly inhibited (52 and 46% inhibition, respectively) Caco-2 sheet migration on collagen IV and noticeably inhibited lamellipodial extension, whereas paxillin siRNA only inhibited migration by 18% and did not noticeably affect lamellipodial extension. These results suggest that Src may regulate Caco-2 migration on collagen IV via both p130cas and paxillin but that Src phosphorylation of p130cas is more important for this process.     

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.     

Collagen IV regulates Caco-2 cell spreading and p130Cas phosphorylation by FAK-dependent and FAK-independent pathways

We previously observed that collagen IV regulates Caco-2 intestinal epithelial cell spreading and migration via Src-dependent p130(Cas) phosphorylation and stimulates focal adhesion kinase (FAK). However, the role of FAK and the related kinase, Pyk2, in Caco-2 spreading and migration is unclear. FAK- or Pyk2-specific siRNAs reduced protein levels by 90%. However, when detached cells were replated on collagen IV neither individual nor combined FAK and Pyk2 siRNAs affected the cell spreading rate. As combined FAK and Pyk2 siRNAs increased p130(Cas) protein levels, we cotransfected cells with 1 nm p130(Cas) siRNA to partially reduce p130(Cas) protein to control levels. Although p130(Cas) Tyr(P)(249) phosphorylation was reduced by 60%, cell spreading was unaffected. Combined siRNA reduction of FAK, Pyk2 and p130(Cas) increased cell spreading by 20% compared to p130(Cas) siRNA alone, suggesting that FAK and Pyk2 negatively regulate spreading in addition to stimulating spreading via p130(Cas). FAK-binding mutant SH3 domain-deleted rat p130(Cas) was not phosphorylated after adhesion and, unlike full-length p130(Cas), did not restore spreading after human-specific p130(Cas) siRNA knockdown of endogenous p130(Cas). Together, these data suggest that FAK positively regulates Caco-2 spreading on collagen IV via p130(Cas) phosphorylation, but also suggests that FAK may negatively regulate spreading through other mechanisms and the presence of additional FAK-independent pathways regulating p130(Cas).     

Vascular endothelial growth factor regulates focal adhesion assembly in human brain microvascular endothelial cells through activation of the focal adhesion kinase and related adhesion focal tyrosine kinase

Vascular endothelial growth factor (VEGF) plays a significant role in blood-brain barrier breakdown and angiogenesis after brain injury. VEGF-induced endothelial cell migration is a key step in the angiogenic response and is mediated by an accelerated rate of focal adhesion complex assembly and disassembly. In this study, we identified the signaling mechanisms by which VEGF regulates human brain microvascular endothelial cell (HBMEC) integrity and assembly of focal adhesions, complexes comprised of scaffolding and signaling proteins organized by adhesion to the extracellular matrix. We found that VEGF treatment of HBMECs plated on laminin or fibronectin stimulated cytoskeletal organization and increased focal adhesion sites. Pretreating cells with VEGF antibodies or with the specific inhibitor SU-1498, which inhibits Flk-1/KDR receptor phosphorylation, blocked the ability of VEGF to stimulate focal adhesion assembly. VEGF induced the coupling of focal adhesion kinase (FAK) to integrin alphavbeta5 and tyrosine phosphorylation of the cytoskeletal components paxillin and p130cas. Additionally, FAK and related adhesion focal tyrosine kinase (RAFTK)/Pyk2 kinases were tyrosine-phosphorylated by VEGF and found to be important for focal adhesion sites. Overexpression of wild type RAFTK/Pyk2 increased cell spreading and the migration of HBMECs, whereas overexpression of catalytically inactive mutant RAFTK/Pyk2 markedly suppressed HBMEC spreading ( approximately 70%), adhesion ( approximately 82%), and migration ( approximately 65%). Furthermore, blocking of FAK by the dominant-interfering mutant FRNK (FAK-related non-kinase) significantly inhibited HBMEC spreading and migration and also disrupted focal adhesions. Thus, these studies define a mechanism for the regulatory role of VEGF in focal adhesion complex assembly in HBMECs via activation of FAK and RAFTK/Pyk2.     

Signaling through focal adhesion kinase

Integrin receptor binding to extracellular matrix proteins generates intracellular signals via enhanced tyrosine phosphorylation events that are important for cell growth, survival, and migration. This review will focus on the functions of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) and its role in linking integrin receptors to intracellular signaling pathways. FAK associates with several different signaling proteins such as Src-family PTKs, p130^Cas, Shc, Grb2, PI 3-kinase, and paxillin. This enables FAK to function within a network of integrin-stimulated signaling pathways leading to the activation of targets such as the ERK and JNK/mitogen-activated protein kinase pathways. Focus will be placed on the structural domains and sites of FAK tyrosine phosphorylation important for FAK-mediated signaling events and how these sites are conserved in the FAK-related PTK, Pyk2. We will review what is known about FAK activation by integrin receptor-mediated events and also non-integrin stimuli. In addition, we discuss the emergence of a consensus FAK substrate phosphorylation sequence. Emphasis will also be placed on the role of FAK in generating cell survival signals and the cleavage of FAK during caspase-mediated apoptosis. An in-depth discussion will be presented of integrin-stimulated signaling events occurring in the FAK knockout fibroblasts (FAK⁻) and how these cells exhibit deficits in cell migration. FAK re-expression in the FAK⁻ cells confirms the role of this PTK in the regulation of cell morphology and in promoting cell migration events. In addition, these results reinforce the potential role for FAK in promoting an invasive phenotype in human tumors.     

Low substratum rigidity of collagen gel promotes ERK phosphorylation via lipid raft to augment cell migration

Previous study demonstrated that low substratum rigidity down-regulates focal adhesion proteins. In this study we found that cells cultured on collagen gel exhibited higher migration capacity than those cultured on collagen gel-coated dishes. Low rigidity of collagen gel induced delayed but persistent phosphorylation of ERK1/2. Inhibition of collagen gel-induced ERK1/2 phosphorylation by MEK inhibitors and ERK2 kinase mutant induced a rounding up of the cells and prevented collagen gel-induced cell migration. Interestingly, phosphorylated ERK1/2 induced by low rigidity was present in focal adhesion sites and the lipid raft. MbetaCD (Methyl-beta-cyclodextrin), a lipid raft inhibitor, inhibited collagen gel-induced ERK1/2 phosphorylation, and cell migration. Overexpression of FAK C-terminal fragment (FRNK) in MDCK cells triggered ERK phosphorylation. Meanwhile, low substratum rigidity induced degradation of FAK into a 35 kDa C-terminal fragment. A calpain inhibitor that partially rescued FAK degradation also prevented low rigidity-induced ERK phosphorylation. However, MbetaCD did not prevent low rigidity-induced FAK degradation. Taken together, we demonstrate that the degradation product of FAK induced by collagen gel triggers activation of ERK1/2, which in turn facilitates cell spreading and migration through the lipid raft.     

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.     

Dissociation of focal adhesion kinase and paxillin tyrosine phosphorylation induced by bombesin and lysophosphatidic acid from epidermal growth factor receptor transactivation in Swiss 3T3 cells

Tyrosine phosphorylation of the nonreceptor tyrosine kinase p125 focal adhesion kinase (FAK) and the adapter protein paxillin is rapidly increased by multiple agonists, including bombesin (BOM) and lysophosphatidic acid (LPA), through heptahelical G protein-coupled receptors (GPCRs). The pathways involved remain incompletely understood. The experiments presented here were designed to test the role of epidermal growth factor receptor (EGFR) transactivation in the rapid increase of tyrosine phosphorylation of FAK and paxillin induced by GPCR agonists. Our results show that treatment with the selective EGFR tyrosine kinase inhibitor AG 1478, at concentrations that completely blocked the increase in tyrosine phosphorylation of these proteins induced by EGF, did not affect the stimulation of tyrosine phosphorylation of either FAK or paxillin induced by multiple GPCR agonists including LPA, BOM, vasopressin, bradykinin, and endothelin. Similar results were obtained when Swiss 3T3 cells were treated with another highly specific inhibitor of the EGF receptor kinase activity, PD-158780. Collectively, our results clearly dissociate EGFR transactivation from the tyrosine phosphorylation of FAK and paxillin induced by multiple GPCR agonists.     

CAIR-1/BAG-3 modulates cell adhesion and migration by downregulating activity of focal adhesion proteins

CAIR-1/BAG-3 is a stress and survival protein that has been shown to bind SH3 domain-containing proteins through its proline-rich (PXXP) domain. Because stress and survival pathways are active during invasion and metastasis, we hypothesized that CAIR-1 is a regulator of signaling pathways that modulate cell adhesion and migration. MDA-435 human breast carcinoma cells were stably transfected with full-length CAIR-1 (FL) or a proline-rich domain deleted mutant (dPXXP). FL cells migrated poorly through collagen IV-coated filters to serum (14% of control, p=0.0004), whereas migration of dPXXP cells was more robust (228%, p=0.00001). Adhesion to collagen IV-coated surfaces was reduced in FL cells and augmented in dPXXP cells (FL 64%, p=0.03; dPXXP 138%, p=0.01). Rhodamine-phalloidin staining highlighted more stress fibers and thicker filopodial protrusions in dPXXP cells. Fewer focal adhesions were also seen in FL cells. A reduction in tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin occurred in FL cells under these conditions. In contrast, increased FAK and paxillin phosphorylation was documented in dPXXP cells. Differential FAK phosphorylation occurred at the major autophosphorylation site Y(397) and Src phosphorylation site Y(861). Concordant with these findings, there was decreased interaction between FAK and its downstream partners p(130)Cas and Crk observed in FL cells but not in dPXXP cells. These results collectively indicate that CAIR-1 may negatively regulate adhesion, focal adhesion assembly, signaling, and migration via its PXXP domain.     

Regulation of neutrophil adhesion by pituitary growth hormone accompanies tyrosine phosphorylation of Jak2, p125FAK, and paxillin

Neutrophil adhesion is fundamentally important during the onset of inflammatory responses. The adhesion signaling pathways control neutrophil arrest and extravasation and influence neutrophil shape and function at sites of inflammation. In the present study the intracellular signaling pathways for the adhesion of human neutrophils by pituitary growth hormone (GH) were examined. Pituitary GH triggered the tyrosine phosphorylation of Janus kinase 2 (Jak2) and STAT3 in neutrophils. In addition, pituitary GH treatment resulted in the morphological changes and the tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin. Preincubation with genistein, a tyrosine kinase inhibitor, blocked the GH-stimulated adhesion and Jak2, STAT3, p125FAK, and paxillin phosphorylation. Confocal microscopy revealed that pituitary GH stimulates the focal localization of p125FAK, paxillin, phosphotyrosine, and filamentous actin filament into the membrane rufflings and uropods of human neutrophils. Immunoprecipitation experiments revealed a physical association of Jak2 with p125FAK via STAT3 in vivo. Also an in vitro kinase assay showed an augmentation of p125FAK autophosphorylation as a result of pituitary GH treatment. These results suggest that pituitary GH modulates neutrophil adhesion through tyrosine phosphorylation of Jak2, p125FAK, and paxillin and actin polymerization.     

ILK mediates the effects of strain on intestinal epithelial wound closure

The intestinal epithelium is subjected to repetitive deformation during normal gut function by peristalsis and villous motility. Such repetitive strain promotes intestinal epithelial migration across fibronectin in vitro, but signaling mediators for this are poorly understood. We hypothesized that integrin-linked kinase (ILK) mediates strain-stimulated migration in intestinal epithelial cells cultured on fibronectin. ILK kinase activity increased rapidly 5 min after strain induction in both Caco-2 and intestinal epithelial cell-6 (IEC-6) cells. Wound closure in response to strain was reduced in ILK small interfering RNA (siRNA)-transfected Caco-2 cell monolayers when compared with control siRNA-transfected Caco-2 cells. Pharmacological blockade of phosphatidylinositol-3 kinase (PI3K) or Src or reducing Src by siRNA prevented strain activation of ILK. ILK coimmunoprecipitated with focal adhesion kinase (FAK), and this association was decreased by mutation of FAK Tyr925 but not FAK Tyr397. Strain induction of FAK Tyr925 phosphorylation but not FAK Tyr397 or FAK Tyr576 phosphorylation was blocked in ILK siRNA-transfected cells. ILK-Src association was stimulated by strain and was blocked by the Src inhibitor PP2. Finally, ILK reduction by siRNA inhibited strain-induced phosphorylation of myosin light chain and Akt. These results suggest a strain-dependent signaling pathway in which ILK association with FAK and Src mediates the subsequent downstream strain-induced motogenic response and suggest that ILK induction by repetitive deformation may contribute to recovery from mucosal injury and restoration of the mucosal barrier in patients with prolonged ileus. ILK may therefore be an important target for intervention to maintain the mucosa in such patients.