Control of adhesion-dependent cell survival by focal adhesion kinase

The interactions of integrins with extracellular matrix proteins can activate focal adhesion kinase (FAK) and suppress apoptosis in normal epithelial and endothelial cells; this subset of apoptosis has been termed "anoikis." Here, we demonstrate that FAK plays a role in the suppression of anoikis. Constitutively activated forms of FAK rescued two established epithelial cell lines from anoikis. Both the major autophosphorylation site (Y397) and a site critical to the kinase activity (K454) of FAK were required for this effect. Activated FAK also transformed MDCK cells, by the criteria of anchorage-independent growth and tumor formation in nude mice. We provide evidence that this transformation resulted primarily from the cells' resistance to anoikis rather than from the activation of growth factor response pathways. These results indicate that FAK can regulate anoikis and that the conferral of anoikis resistance may suffice to transform certain epithelial cells.     

Insulin-like growth factor I prevents mannitol-induced degradation of focal adhesion kinase and Akt

In our laboratory, we are interested in hyperosmolarity-induced apoptosis in neuronal cells. We have shown that high concentrations of glucose or mannitol induce apoptotic cell death in dorsal root ganglia in culture and in SH-SY5Y and SH-EP human neuroblastoma cells. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that has a critical role for transmitting integrin-mediated-signals. In this study, we report that hyperosmolar treatment mediates FAK dephosphorylation and cleavage, which is prevented by insulin-like growth factor I (IGF-I) treatment. Mannitol treatment of SH-EP cells transfected with vector (SH-EP/pSFFV) results in concentration- and time-dependent dephosphorylation and degradation of FAK. Dephosphorylation and degradation of FAK are tightly correlated with apoptotic morphological changes, including the disruption of actin stress fibers, the loss of focal adhesion sites, membrane blebbing, and cell detachment. Treatment of SH-EP/pSFFV cells with IGF-I or transfection of IGF-I receptor prevents these changes. Treatment of cells with pharmacologic inhibitors of the mitogen-activated protein kinase or phosphatidylinositol 3-kinase pathways does not affect mannitol-induced FAK dephosphorylation and degradation. However, phosphatidylinositol 3-kinase is necessary for IGF-I-mediated protection against FAK alteration. Mannitol treatment also results in the degradation of Akt. Mannitol induces the activation of caspases-3 and -9 in a time course similar to the dephosphorylation and degradation of FAK. Treatment of the cells with ZVAD, a general caspase inhibitor, blocks the mannitol-induced FAK and Akt degradation as well as cell detachment and apoptosis. These results suggest that one of the pathways of mannitol-mediated apoptosis is through the degradation of FAK and Akt and that IGF-I protects the cells from apoptosis by blocking the activation of caspases, which may be responsible for the loss of FAK and Akt.     

Role of focal adhesion kinase in MAP kinase activation by insulin-like growth factor-I or insulin.

Integrin-induced focal adhesion kinase (FAK) phosphorylation as well as insulin-like growth factor-I (IGF-I) and insulin activate MAP kinase. Since IGF-I or insulin have been suggested to affect FAK phosphorylation, we analyzed the role of FAK in IGF-I- or insulin-induced MAP kinase activation. Although MAP kinase was stimulated by IGF-I or insulin, FAK tyrosine phosphorylation remained unchanged in fibroblasts expressing normal or transiently elevated levels of IGF-I and insulin receptors. Further analysis in FAK deficient fibroblasts suggested that FAK impedes MAP kinase activation by IGF-I or insulin.     

SRC-mediated phosphorylation of focal adhesion kinase couples actin and adhesion dynamics to survival signaling

Integrin-associated focal adhesions not only provide adhesive links between cellular actin and extracellular matrix but also are sites of signal transmission into the cell interior. Many cell responses signal through focal adhesion kinase (FAK), often by integrin-induced autophosphorylation of FAK or phosphorylation by Src family kinases. Here, we used an interfering FAK mutant (4-9F-FAK) to show that Src-dependent FAK phosphorylation is required for focal adhesion turnover and cell migration, by controlling assembly of a calpain 2/FAK/Src/p42ERK complex, calpain activation, and proteolysis of FAK. Expression of 4-9F-FAK in FAK-deficient fibroblasts also disrupts F-actin assembly associated with normal adhesion and spreading. In addition, we found that FAK's ability to regulate both assembly and disassembly of the actin and adhesion networks may be linked to regulation of the protease calpain. Surprisingly, we also found that the same interfering 4-9F-FAK mutant protein causes apoptosis of serum-deprived, transformed cells and suppresses anchorage-independent growth. These data show that Src-mediated phosphorylation of FAK acts as a pivotal regulator of both actin and adhesion dynamics and survival signaling, which, in turn, control apparently distinct processes such as cell migration and anchorage-independent growth. This also highlights that dynamic regulation of actin and adhesions (which include the integrin matrix receptors) is critical to signaling output and biological responses.     

Insulin-like growth factor-mediated muscle cell survival: central roles for Akt and cyclin-dependent kinase inhibitor p21

Polypeptide growth factors activate specific transmembrane receptors, leading to the induction of multiple intracellular signal transduction pathways which control cell function and fate. Recent studies have shown that growth factors promote cell survival by stimulating the serine-threonine protein kinase Akt, which appears to function primarily as an antiapoptotic agent by inactivating death-promoting molecules. We previously established C2 muscle cell lines lacking endogenous expression of insulin-like growth factor II (IGF-II). These cells underwent apoptotic death in low-serum differentiation medium but could be maintained as viable myoblasts by IGF analogues that activated the IGF-I receptor or by unrelated growth factors such as platelet-derived growth factor BB (PDGF-BB). Here we show that IGF-I promotes muscle cell survival through Akt-mediated induction of the cyclin-dependent kinase inhibitor p21. Treatment of myoblasts with IGF-I or transfection with an inducible Akt maintained muscle cell survival and enhanced production of p21, and ectopic expression of p21 was able to sustain viability in the absence of growth factors. Blocking of p21 protein accumulation through a specific p21 antisense cDNA prevented survival regulated by IGF-I or Akt but did not block muscle cell viability mediated by PDGF-BB. Our results define Akt as an intermediate and p21 as a critical effector of an IGF-controlled myoblast survival pathway that is active during early myogenic differentiation and show that growth factors are able to maintain cell viability by inducing expression of pro-survival molecules.     

Matrix survival signaling: from fibronectin via focal adhesion kinase to c-Jun NH(2)-terminal kinase

Most transformed cells have lost anchorage and serum dependence for growth and survival. Previously, we established that when serum is absent, fibronectin survival signals transduced by focal adhesion kinase (FAK), suppress p53-regulated apoptosis in primary fibroblasts and endothelial cells (Ilić et al. 1998. J. Cell Biol. 143:547–560). The present goals are to identify survival sequences in FAK and signaling molecules downstream of FAK required for anchorage-dependent survival of primary fibroblasts. We report that binding of the SH3 domain of p130Cas to proline-rich region 1 of FAK is required to support survival of fibroblasts on fibronectin when serum is withdrawn. The FAK–p130Cas complex activates c-Jun NH2-terminal kinase (JNK) via a Ras/Rac1/Pak1/MAPK kinase 4 (MKK4) pathway. Activated (phospho-) JNK colocalizes with FAK in focal adhesions of fibroblasts cultured on fibronectin, which supports their survival, but not in fibroblasts cultured on collagen, which does not. Cells often survive in the absence of extracellular matrix if serum factors are provided. In that case, we confirm work of others that survival signals are transduced by FAK, phosphatidylinositol 3′-kinase (PI3-kinase), and Akt/protein kinase B (PKB). However, when serum is absent, PI3-kinase and Akt/PKB are not involved in the fibronectin-FAK-JNK survival pathway documented herein. Thus, survival signals from extracellular matrix and serum are transduced by FAK via two distinct pathways.     

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.     

Synergistic effect of focal adhesion kinase overexpression and hepatocyte growth factor stimulation on cell transformation

Although an elevated level of focal adhesion kinase (FAK) has been observed in a variety of invasive human tumors, forced expression of FAK alone in cultured cells does not cause them to exhibit transformed phenotypes. Therefore, the role of FAK in oncogenic transformation remains unclear. In this study, we have demonstrated that FAK overexpression in Madin-Darby canine kidney epithelial cells rendered them susceptible to transformation by hepatocyte growth factor (HGF). Using various FAK mutants, we found that the simultaneous bindings of Src and p130(cas) were required for FAK to potentiate cell transformation. Expression of FAK-related nonkinase, kinase-deficient Src, or the Src homology 3 domain of p130(cas), which respectively serve as dominant negative versions of FAK, Src, and p130(cas), apparently reversed the transformed phenotypes of FAK-overexpressed cells upon HGF stimulation. Moreover, FAK overexpression was able to enhance HGF-elicited signals, leading to sustained activation of ERK, JNK, and AKT, which could be prevented by the expression of the Src homology 3 domain of p130(cas). Taken together, our results indicate that the synergistic effect of FAK overexpression and HGF stimulation leads to cell transformation and implicate a critical role of p130(cas) in this process.     

Role of Grb7 targeting to focal contacts and its phosphorylation by focal adhesion kinase in regulation of cell migration

We have previously described Grb7 association with focal adhesion kinase (FAK) and its possible roles in cell migration. In this paper, we investigated the mechanisms by which Grb7 and its association with FAK regulate cell migration. We found that deletion of the Grb7 SH2 domain eliminated partial Grb7 localization to focal contacts and its ability to stimulate cell migration. Replacement of the SH2 domain with the focal adhesion targeting sequence from FAK resulted in the focal contacts localization of the chimeric molecule and restored its activity to stimulate cell migration. We also found that Grb7 could be phosphorylated by FAK, which was dependent on the FAK kinase activity but not the presence of the Src family kinases. Cell adhesion also enhanced Grb7 phosphorylation in FAK+/+ cells but not FAK-/- cells, suggesting that Grb7 is a physiological substrate of FAK. Furthermore, both Grb7 and the chimeric molecule did not increase migration of FAK-/- cells, although the chimeric molecule was targeted to the focal contacts. Last, we showed that other Grb7 family members could not stimulate cell migration under similar experimental conditions. Together, these results demonstrate a role for Grb7 targeting to focal contacts and its phosphorylation by FAK in the regulation of cell migration.     

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.     

Contractility modulates cell adhesion strengthening through focal adhesion kinase and assembly of vinculin‐containing focal adhesions

Actin–myosin contractility modulates focal adhesion assembly, stress fiber formation, and cell migration. We analyzed the contributions of contractility to fibroblast adhesion strengthening using a hydrodynamic adhesion assay and micropatterned substrates to control cell shape and adhesive area. Serum addition resulted in adhesion strengthening to levels 30–40% higher than serum‐free cultures. Inhibition of myosin light chain kinase or Rho‐kinase blocked phosphorylation of myosin light chain to similar extents and eliminated the serum‐induced enhancements in strengthening. Blebbistatin‐induced inhibition of myosin II reduced serum‐induced adhesion strength to similar levels as those obtained by blocking myosin light chain phosphorylation. Reductions in adhesion strengthening by inhibitors of contractility correlated with loss of vinculin and talin from focal adhesions without changes in integrin binding. In vinculin‐null cells, inhibition of contractility did not alter adhesive force, whereas controls displayed a 20% reduction in adhesion strength, indicating that the effects of contractility on adhesive force are vinculin‐dependent. Furthermore, in cells expressing FAK, inhibitors of contractility reduced serum‐induced adhesion strengthening as well as eliminated focal adhesion assembly. In contrast, in the absence of FAK, these inhibitors did not alter adhesion strength or focal adhesion assembly. These results indicate that contractility modulates adhesion strengthening via FAK‐dependent, vinculin‐containing focal adhesion assembly. J. Cell. Physiol. 223:746–756, 2010. © 2010 Wiley‐Liss, Inc.     

Control of motile and invasive cell phenotypes by focal adhesion kinase

Cell motility is stimulated by extracellular stimuli and initiated by intracellular signaling proteins that localize to sites of cell contact with the extracellular matrix termed focal contacts. Focal adhesion kinase (FAK) is an intracellular protein-tyrosine kinase (PTK) that acts to regulate the cycle of focal contact formation and disassembly required for efficient cell movement. FAK is activated by a variety of cell surface receptors and transmits signals to a range of targets. Thus, FAK acts as an integrator of cell motility-associated signaling events. We will review the stimulatory and regulatory mechanisms of FAK activation, the different signaling connections of FAK that are mediated by a growing number of FAK-interacting proteins, and the modulation of FAK function by tyrosine and serine phosphorylation. We will also summarize findings with regard to FAK function in vertebrate and invertebrate development as well as recent insights into the mechanistic role(s) of FAK in promoting cell migration. As increased FAK expression and tyrosine phosphorylation have been correlated with the progression to an invasive cell phenotype, there is growing interest in elucidating the important FAK-related signaling connections promoting invasive tumor cell movement. To this end, we will discuss the effects of FAK inhibition via the dominant-negative expression of the FAK C-terminal domain termed FAK-related non-kinase (FRNK) and how these studies have uncovered a distinct role for FAK in promoting cell invasion that may differ from its role in promoting cell motility.     

Role of aggregation factor and cell type in sponge cell adhesion.

A pair of sponge species, Microciona prolifera and Halichondria bowerbanki, which lack mutual species specificity in their aggregation "factor", are useful in establishing the mechanisms of action of these factors. These sponges were dissociated both mechanically, which leaves the factor on the cell surface, and by Humphrey's (1963) method, which isolates the factor from the cells. The adhesive specificities which arose, in the various combinations tested, point to an intercellular factor bridge consisting of a single symmetrical unit. An analysis of most other workers' results is consistent with this interpretation. However, MacLennan and Dodd's (1967) results using other species would require a bridge consisting of two or more asymmetrical units. Differences were found in the specificity of adhesion of various types of cells within a single species. This presents a heretofore unconsidered problem in assesing the adhesive factor's mechanism of action. Three structurally distinct cell types were separated from a suspension of dissociated Microciona cells by velocity sedimentation. These cells differ greatly in adhesiveness. The differences in adhesion are correlated with numbers and positions of cells incorporated into aggregates. Such differences are considered in explaining the mechanism of action of the factors.     

Epidermal growth factor-induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase

Upregulated epidermal growth factor (EGF) receptor (EGFR) expression and EGFR-induced signaling have been correlated with progression to invasion and metastasis in a wide variety of carcinomas, but the mechanism behind this is not well understood. We show here that, in various human carcinoma cells that overexpress EGFR, EGF treatment induced rapid tyrosine dephosphorylation of focal adhesion kinase (FAK) associated with downregulation of its kinase activity. The downregulation of FAK activity was both required and sufficient for EGF-induced refractile morphological changes, detachment of cells from the extracellular matrix, and increased tumor cell motility, invasion, and metastasis. Tumor cells with downregulated FAK activity became less adherent to the extracellular matrix. However, once cells started reattaching, FAK activity was restored by activated integrin signaling. Moreover, this process of readhesion and spreading could not be abrogated by further EGF stimulation. Interruption of transforming growth factor alpha-EGFR autocrine regulation with an EGFR tyrosine kinase inhibitor led to a substantial increase in FAK tyrosine phosphorylation and inhibition of tumor cell invasion in vitro. Consistent with this, FAK tyrosine phosphorylation was reduced in cells from tumors growing in transplanted, athymic, nude mice, which have an intact autocrine regulation of the EGFR. We suggest that the dynamic regulation of FAK activity, initiated by EGF-induced downregulation of FAK leading to cell detachment and increased motility and invasion, followed by integrin-dependent reactivation during readhesion, plays a role in EGF-associated tumor invasion and metastasis.     

Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase

Imaging studies implicate microtubule targeting of focal adhesions in focal adhesion disassembly, although the molecular mechanism is unknown. Here, we develop a model system of focal adhesion disassembly based on the finding that microtubule regrowth after nocodazole washout induces disassembly of focal adhesions, and that this disassembly occurs independently of Rho and Rac, but depends on focal adhesion kinase (FAK) and dynamin. During disassembly, dynamin interacts with FAK and colocalizes with focal adhesions. Inhibition of dynamin prevents migration of cells with a focal adhesion phenotype. Our results show that focal adhesion disassembly involves microtubules, dynamin and FAK, and is not simply the reversal of focal adhesion formation.     

Nck-2 interacts with focal adhesion kinase and modulates cell motility

Nck-2 is a ubiquitously expressed adaptor protein comprising primarily three N-terminal SH3 domains and one C-terminal SH2 domain. We report here that Nck-2 interacts with focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase critically involved in the cellular control of motility. Using a mutational strategy, we have found that the formation of the Nck-2-FAK complex is mediated by interactions involving multiple SH2 and SH3 domains of Nck-2. The Nck-2 SH2 domain-mediated interaction with FAK is dependent on phosphorylation of Tyr397, a site that is involved in the regulation of cell motility. A fraction of Nck-2 co-localizes with FAK at cell periphery in spreading cells. Furthermore, overexpression of Nck-2 modestly decreased cell motility, whereas overexpression of a mutant form of Nck-2 containing the SH2 domain but lacking the SH3 domains significantly promoted cell motility. These results identify a novel interaction between Nck-2 and FAK and suggest a role of Nck-2 in the modulation of cell motility.     

Inhibition of focal adhesion kinase decreases tumor growth in human neuroblastoma

Neuroblastoma is the most common extracranial solid tumor of childhood. Focal adhesion kinase (FAK) is an intracellular kinase that regulates both cellular adhesion and apoptosis. FAK is overexpressed in a number of human tumors including neuroblastoma. Previously, we have shown that the MYCN oncogene, the primary adverse prognostic indicator in neuroblastoma, regulates the expression of FAK in neuroblastoma. In this study, we have examined the effects of FAK inhibition upon neuroblastoma using a small molecule [1,2,4,5-benzenetetraamine tetrahydrochloride (Y15)] to inhibit FAK expression and the phosphorylation of FAK at the Y397 site. Utilizing both non-isogenic and isogenic MYCN+/MYCN- neuroblastoma cell lines, we found that Y15 effectively diminished phosphorylation of the Y397 site of FAK. Treatment with Y15 resulted in increased detachment, decreased cell viability and increased apoptosis in the neuroblastoma cell lines. We also found that the cell lines with higher MYCN are more sensitive to Y15 treatment than their MYCN negative counterparts. In addition, we have shown that treatment with Y15 in vivo leads to less tumor growth in nude mouse xenograft models, again with the greatest effects seen in MYCN+ tumor xenografts. The results of the current study suggest that FAK and phosphorylation at the Y397 site plays a role in neuroblastoma cell survival, and that the FAK Y397 phosphorylation site is a potential therapeutic target for this childhood tumor.     

Src SH2 arginine 175 is required for cell motility: specific focal adhesion kinase targeting and focal adhesion assembly function

Src kinase is a crucial mediator of adhesion-related signaling and motility. Src binds to focal adhesion kinase (FAK) through its SH2 domain and subsequently activates it for phosphorylation of downstream substrates. In addition to this binding function, data suggested that the SH2 domain might also perform an important role in targeting Src to focal adhesions (FAs) to enable further substrate phosphorylations. To examine this, we engineered an R175L mutation in cSrc to prevent the interaction with FAK pY397. This constitutively open Src kinase mediated up-regulated substrate phosphorylation in SYF cells but was unable to promote malignant transformation. Significantly, SrcR175L cells also had a profound motility defect and an impaired FA generation capacity. Importantly, we were able to recapitulate wild-type motile behavior and FA formation by directing the kinase to FAs, clearly implicating the SH2 domain in recruitment to FAK and indicating that this targeting capacity, and not simply Src-FAK scaffolding, was critical for normal Src function.     

Spatial activation of ezrin by epidermal growth factor receptor and focal adhesion kinase co-ordinates epithelial cell migration

Epidermal growth factor receptor (EGFR) plays a critical role in the promotion of epithelial cell proliferation and migration. Previous studies have suggested a cooperative role between EGFR and integrin signalling pathways that enable efficient adhesion and migration but the mechanisms controlling this remain poorly defined. Here, we show that EGFR forms a complex with focal adhesion kinase in epithelial cells. Surprisingly, this complex enhances local Src activity at focal adhesions to promote phosphorylation of the cytoskeletal adaptor protein ezrin at Y478, leading to actomyosin contractility, suppression of focal adhesion dynamics and slower migration. We further demonstrate this regulation of Src is due to the suppression of PTP1B activity. Our data provide new insight into EGF-independent cooperation between EGFR and integrins and suggest transient interactions between these kinases at the leading edge of cells act to spatially control signalling to permit efficient motility.