Integrins on eggs: the betaC subunit is essential for formation of the cortical actin cytoskeleton in sea urchin eggs

Eggs of several metazoans have been demonstrated to express integrins; however, their function is unclear. Previous studies have shown that the betaC integrin subunit is expressed on unfertilized sea urchin eggs and proteolytically removed at fertilization. Here we report that the betaC subunit is reexpressed on the egg surface immediately after fertilization. Using morpholino antisense oligonucleotides to block translation, we show that without betaC expression, eggs undergo cleavage resulting in loosely adherent cells that fail to develop beyond a blastula. Without betaC containing integrins, the cortical actin network of the egg does not form, yet contractile rings appear. Coinjection of RNA encoding the betaC or chicken beta1 subunit, but lacking the morpholino target sequence, rescues the cortical actin network and normal embryos result. Coinjection of RNA encoding the betaC subunit lacking the cytoplasmic domain fails to rescue. These studies demonstrate that the cortical actin cytoskeleton is anchored by betaC integrins and contractile ring actin is not. We suggest that one important function of egg integrins is to organize the actin cortex.     

Generation of point‐mutant FAK knockin mice

Focal adhesion kinase is a non‐receptor protein tyrosine kinase with signaling functions downstream of integrins and growth factor receptors. In addition to its role in adhesion, migration, and proliferation it also has non‐kinase scaffolding functions in the nucleus. Focal adhesion kinase (FAK) activation involves the following: (1) ligand bound growth factors or clustered integrins activate FAK kinase domain; (2) FAK autophosphorylates tyrosine (Y) 397; (3) Src binds pY397 and phosphorylates FAK at various other sites including Y861; (4) downstream signaling of activated FAK elicits changes in cellular behavior. Although many studies have demonstrated roles for the kinase domain, Y397 and Y861 sites, in vitro much less is known about their functions in vivo. Here, we report the generation of a series of FAK‐mutant knockin mice where mutant FAK, either kinase dead, non‐phosphorylatable mutants Y397F and Y861F, or mutant Y397E—containing a phosphomimetic site that results in a constitutive active Y397, can be expressed in a Cre inducible fashion driven by the ROSA26 promoter. In future studies, intercrossing these mice with FAKflox/flox mice and inducible cre‐expressing mice will enable the in vivo study of mutant FAK function in the absence of endogenous FAK in a spatially and temporally regulated fashion within the whole organism. genesis 52:907–915, 2014. © 2014 Wiley Periodicals, Inc.     

Regulation of the Cell Cycle by Focal Adhesion Kinase

In this report, we have analyzed the potential role and mechanisms of integrin signaling through FAK in cell cycle regulation by using tetracycline-regulated expression of exogenous FAK and mutants. We have found that overexpression of wild-type FAK accelerated G1 to S phase transition. Conversely, overexpression of a dominant-negative FAK mutant ΔC14 inhibited cell cycle progression at G1 phase and this inhibition required the Y397 in ΔC14. Biochemical analyses indicated that FAK mutant ΔC14 was mislocalized and functioned as a dominant-negative mutant by competing with endogenous FAK in focal contacts for binding signaling molecules such as Src and Fyn, resulting in a decreases of Erk activation in cell adhesion. Consistent with this, we also observed inhibition of BrdU incorporation and Erk activation by FAK Y397F mutant and FRNK, but not FRNKΔC14, in transient transfection assays using primary human foreskin fibroblasts. Finally, we also found that ΔC14 blocked cyclin D1 upregulation and induced p21 expression, while wild-type FAK increased cyclin D1 expression and decreased p21 expression. Taken together, these results have identified FAK and its associated signaling pathways as a mediator of the cell cycle regulation by integrins.     

SRC catalytic but not scaffolding function is needed for integrin-regulated tyrosine phosphorylation, cell migration, and cell spreading

Src family kinases (SFKs) are crucial for signaling through a variety of cell surface receptors, including integrins. There is evidence that integrin activation induces focal adhesion kinase (FAK) autophosphorylation at Y397 and that Src binds to and is activated by FAK to carry out subsequent phosphorylation events. However, it has also been suggested that Src functions as a scaffolding molecule through its SH2 and SH3 domains and that its kinase activity is not necessary. To examine the role of SFKs in integrin signaling, we have expressed various Src molecules in fibroblasts lacking other SFKs. In cells plated on fibronectin, FAK could indeed autophosphorylate at Y397 independently of Src but with lower efficiency than when Src was present. This step was promoted by kinase-inactive Src, but Src kinase activity was required for full rescue. Src kinase activity was also required for phosphorylation of additional sites on FAK and for other integrin-directed functions, including cell migration and spreading on fibronectin. In contrast, Src mutations in the SH2 or SH3 domain greatly reduced binding to FAK, Cas, and paxillin but had little effect on tyrosine phosphorylation or biological assays. Furthermore, our indirect evidence indicates that Src kinase activity does not need to be regulated to promote cell migration and FAK phosphorylation. Although Src clearly plays important roles in integrin signaling, it was not concentrated in focal adhesions. These results indicate that the primary role of Src in integrin signaling is as a kinase. Indirect models for Src function are proposed.     

OipA plays a role in Helicobacter pylori-induced focal adhesion kinase activation and cytoskeletal re-organization

The initial signalling events leading to Helicobacter pylori infection associated changes in motility, cytoskeletal reorganization and elongation of gastric epithelial cells remain poorly understood. Because focal adhesion kinase (FAK) is known to play important roles in regulating actin cytoskeletal organization and cell motility we examined the effect of H. pylori in gastric epithelial cells co-cultured with H. pylori or its isogenic cag pathogenicity island (PAI) or oipA mutants. H. pylori induced FAK phosphorylation at distinct tyrosine residues in a dose- and time-dependent manner. Autophosphorylation of FAK Y397 was followed by phosphorylation of Src Y418 and resulted in phosphorylation of the five remaining FAK tyrosine sites. Phosphorylated FAK and Src activated Erk and induced actin stress fibre formation. FAK knock-down by FAK-siRNA inhibited H. pylori- mediated Erk phosphorylation and abolished stress fibre formation. Infection with oipA mutants reduced phosphorylation of Y397, Y576, Y577, Y861 and Y925, inhibited stress fibre formation and altered cell morphology. cag PAI mutants reduced phosphorylation of only FAK Y407 and had less effect on stress fibre formation than oipA mutants. We propose that activation of FAK and Src are responsible for H. pylori -induced induction of signalling pathways resulting in the changes in cell phenotype important for pathogenesis.     

pHocal adhesion kinase regulation is on a FERM foundation

Increases in intracellular pH (pHi) occur upon integrin receptor binding to matrix proteins and in tumor cells. In this issue, Choi et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201308034) show that pHi increase activates FAK by causing deprotonation of histidine 58 in its FERM (band 4.1, ezrin, radixin, moesin) homology domain, which exposes a region important for FAK autophosphorylation. This model of FAK activation could contribute to motility of tumor cells by promoting focal adhesion turnover.Integrins are transmembrane receptors for extracellular matrix that mediate physical cell attachment and also control cell shape, growth, and survival. Integrin signals are generated by the recruitment and activation of protein tyrosine kinases (PTKs) such as Src, Abl, Syk, and FAK that initiate protein phosphorylation signaling cascades. These sites of integrin signal initiation and cell attachment are generally termed focal adhesions. Despite identification of adhesion protein constituents, our understanding of the molecular mechanisms of PTK activation at focal adhesions remains rudimentary. FAK, Src, and Abl signaling contribute to tumor growth and metastasis, and small molecule drugs targeting these PTKs have been approved or are undergoing clinical trials. However, for FAK we really do not fully understand the contributing factors that lead to its elevated activation, and although FAK is an amplified gene in cancer, mutations that increase FAK activation are uncommon.In this issue, Choi et al. (2013) elucidate a novel connection between increased intracellular pH (pHi) and FAK activation. In the early 1990s, transient pHi elevation upon matrix binding was one of the first integrin-associated signals identified (Schwartz et al., 1991). Pharmacological inhibitors pointed to the importance of sodium–proton antiporters in mediating increased pHi. NHE-1 (sodium-hydrogen antiporter 1) is part of a larger family (NHE1-9) and a ubiquitously expressed transmembrane protein that actively extrudes protons from inside the cell to counter balance acidity and maintain cytosolic pHi (Malo and Fliegel, 2006). NHE-I can be found at focal adhesions (Grinstein et al., 1993) and can connect to the actin cytoskeleton via binding to ezrin (Denker et al., 2000). NHE-1 point mutations disrupting either ion translocation or its binding to ezrin prevent cell migration (Denker and Barber, 2002). NHE-1 overexpression in cancer cells elevates pHi and tumor progression (Webb et al., 2011).Also in the early 1990s, FAK was the first PTK shown to localize to focal adhesions and to be activated by integrins (Parsons, 2003). FAK and the closely related proline-rich tyrosine kinase 2 (Pyk2) share a common domain structure of an N-terminal FERM domain and an ∼40 amino acid linker domain containing an autophosphorylation site (Y397 in FAK) that serves as a Src homology 2 (SH2) binding site for Src-family PTKs. The FAK linker is followed by a central kinase domain, a scaffolding region containing two proline-rich motifs that are SH3 domain binding sites, and a C-terminal focal adhesion–targeting (FAT) domain (Fig. 1 A). It is the FAK FAT domain that binds to integrin-associated proteins (paxillin and talin) and facilitates FAK phosphorylation at Y397 via protein clustering (Toutant et al., 2002). However, the importance of the results of Choi et al. (2013) lies in the role of the FAK FERM domain in the intramolecular regulation of FAK Y397 autophosphorylation.Open in a separate windowFigure 1.Overview of FAK structure and activation. (A) FAK schematic. Depicted is the FAK N-terminal FERM domain comprised of three lobes (F1, F2, and F3), a linker domain, central kinase domain, and a C-terminal focal adhesion–targeting (FAT) domain. Shown are histidine (H) residue 58 and tyrosine (Y) residues 194, 397, 576/577, and 925, and proline-rich domains (Pro-1, Pro-2, and Pro-3) that are sites for SH3 domain binding. FERM plus linker (F+L), FERM-linker-kinase (F+L+K), and linker-kinase (L+K) are constructs used by Choi et al. (2013). (B) Model of growth factor–stimulated FAK activation. Upon binding of the FAK FERM F2 lobe to c-Met receptor and/or phosphatidylinositol 4,5-P₂ (PIP2) lipid, FAK undergoes conformational changes and Y194 phosphorylation, leading to FAK Y397 autophosphorylation in the linker region. Src binding to and phosphorylation of FAK within the kinase domain leads to full FAK activation. HGF, hepatocyte growth factor.FERM domains are typically comprised of three lobes (F1, F2, and F3) grouped in a cloverleaf-like structure (Frame et al., 2010). In an inactive conformation, the FAK FERM F2 lobe binds to and blocks the FAK kinase domain active site. The FAK FERM F1 lobe binds to and sequesters FAK Y397 in the linker region (Fig. 1 B). Point mutations of FAK in the F1 lobe, F2 lobe, or within the kinase domain can weaken these inhibitory intramolecular binding interactions and result in elevated FAK Y397 phosphorylation (Lietha et al., 2007). It has been hypothesized that the normal sequence of events for FAK activation starts with the binding of some “activating” factor to the FAK F2 lobe that would trigger FERM lobe displacement and allow FAK cis or trans auto-phosphorylation of Y397 (Fig. 1 B). Subsequent full FAK activation occurs via SH2 domain binding of Src to phosphorylated Y397, resulting in Src-mediated phosphorylation within the FAK kinase domain at Y576 and Y577 to promote catalytic activation and phosphorylation within the FAT domain at Y925 to promote Grb2 SH2 binding (Schaller, 2010). Recently, growth factor receptor phosphorylation of FAK at Y194 within the FERM F2 lobe was shown to promote FAK activation (Fig. 1 B; Chen et al., 2011). Conformational changes triggered by FAK kinase activity also regulate FAK FERM–mediated binding to targets such as VE-cadherin (Chen et al., 2012). Outside of FAK phosphorylation at Y194, and a potential role for lipid binding to FAK FERM (Cai et al., 2008), additional “initiators” of FERM conformational changes remain undefined.Choi et al. (2013) provide new and important insights in FERM-mediated FAK activation by changes in pHi. By using a chimeric paxillin construct fused to the pH biosensor pHluorin and to mCherry, Choi et al. (2013) showed that pHi increases within peripheral adhesions in mouse embryo fibroblasts (MEFs) spreading on fibronectin (FN) at 30 to 50 min after plating. Stable NHE-1 knockdown resulted in the lowering of pHi and the inhibition of FAK but not Src activation at 30 and 60 min during MEF spreading on FN. Interestingly, NHE-1 knockdown or pharmacological NHE-1 inhibition resulted in MEFs with a rounded morphology and an increased number of small focal adhesions. This adhesion and spreading phenotype is similar to that of FAK-null MEFs (Sieg et al., 1999).To determine if there was a connection between increased pHi and FAK activation, Choi et al. (2013) performed recombinant FAK in vitro phosphorylation assays. The FAK FERM-linker-kinase (F+L+K) fragment but not the linker-kinase (L+K) (Fig. 1 A) exhibited increased Y397 phosphorylation as a function of pH (pH 7.5 > pH 6.5). Intriguingly, regulation of FAK Y397 phosphorylation by pH was dependent upon the presence of the FAK FERM domain, which was shown by using the FERM-linker (F+L) as a substrate in trans (Fig. 1 A). These results support the hypothesis that exposure of the FAK linker region for phosphorylation is pH dependent (Fig. 2). Changes in amino acid protonation can be considered a post-translational modification, as ionic interactions contribute to secondary and tertiary protein structure (Schönichen et al., 2013). The pK_a of histidine is ∼6.5, and variations from this value depend upon the local protein environment. The challenge is to determine which sites functionally serve as pH sensors in vivo.Open in a separate windowFigure 2.Simplified model of FAK activation via histidine 58 (H58) deprotonation. The FAK FERM F1 lobe sequesters FAK Y397 in the linker region keeping FAK in an inactive and closed conformation. Integrin engagement at focal adhesions results in transient and local increases of pHi through NHE-1 activity. Changes in pHi result in H58 deprotonation within the FERM F1 lobe, leading to FAK conformational changes that expose the FAK linker region and enabling FAK Y397 autophosphorylation. Src binding to and phosphorylation of FAK within the kinase domain leads to full FAK activation.The FAK FERM domain contains seven histidine residues, three of which (H41, H58, and H75) are within the FERM F1 lobe. Mutation of these sites individually to alanine revealed that H58A mutation selectively enhanced F+L and F+L+K Y397 phosphorylation at pH 6.5. This approach allowed Choi et al. (2013) to conclude that H58 deprotonation confers some type of change within the F+L region to facilitate Y397 autophosphorylation (Fig. 2). Using the crystal structure of FAK F+L+K as a template, molecular dynamic simulations with H58 (neutral or positively charged) revealed conformational differences within residues of the linker region around Y397 despite the lack of direct binding to H58. Although Choi et al. (2013) did not identify key partner electrostatic interactions that contribute to conformational changes upon H58 deprotonation, recent studies by (Ritt et al. (2013) proposed that E466 within the FAK kinase domain may be important for this regulation within full-length FAK. However, this does not explain results from Choi et al. (2013) for the pH dependence of Y397 phosphorylation within F+L.Lastly, full-length H58A FAK exhibited elevated Y397 phosphorylation upon re-expression in FAK-null MEFs in combination with NHE-1 shRNA knockdown. H58A FAK promoted spreading and adhesion changes in both control and NHE-1 shRNA FAK-null MEFs, whereas wild-type FAK did not rescue FAK-null phenotypes in the absence of NHE-1 expression. Choi et al. (2013) note that pH-regulated FAK Y397 phosphorylation required combined integrin stimulation and elevated pH to activate FAK. In tumor cells, NHE-1 inhibition prevents elevated FAK Y397 phosphorylation and recent studies show that FAK can phosphorylate cortactin to promote adhesion turnover (Tomar et al., 2012). Cortactin tyrosine phosphorylation facilitates the recruitment of NHE-1 to tumor cell invadopodia (Magalhaes et al., 2011), leading to the pH-dependent release of actin-depolymerizing factor cofilin from cortactin (Frantz et al., 2008). As FAK activity promotes ovarian and breast tumor metastasis (Walsh et al., 2010; Ward et al., 2013), it is possible that FAK may serve as a pH-dependent sensor to initiate cell spreading.In the control of cell motility, NHE-1 is postulated to create pH nanodomains at focal adhesions to control protein–protein interactions (Ludwig et al., 2013). A simplistic model is that integrin clustering facilitates rapid FAK recruitment to focal adhesion where increases in pH trigger FERM conformational changes, release of the FAK linker region, and allow for FAK Y397 phosphorylation in cis or trans (Fig. 2). Adhesion turnover is increased at alkaline pHi, consistent with leading edge cell spreading and extension. At nascent adhesions FAK recruits talin (Lawson et al., 2012), and at alkaline pH FAK signaling activity may be enhanced over talin binding to filamentous actin needed for adhesion maturation (Srivastava et al., 2008). However, as pHi falls, pH sensor residues within the talin rod domain confer enhanced actin binding and this may be part of a signaling switch to promote a cycle of focal adhesion maturation. Additionally, pHi changes may alter phosphorylation site specificity by Src within the FAK FAT domain (Cable et al., 2012). Thus, pHi can affect FAK activity and FAK phosphorylation. Moreover, the FAK-related Pyk2 PTK is activated by acidic pHi within cells of the kidney (Li et al., 2004). Although the molecular mechanism is not known as to how Pyk2 is regulated by acidity, clearly there is much more to discover about the role of pHi changes at adhesions and invadopodia and how this may alter PTK activation in the control of cell movement and invasion.     

The mif gene is transcriptionally regulated by glucose in insulin-secreting cells

We have established that focal adhesion kinase (FAK)-transfected HL-60 (HL-60/FAK) cells were highly resistant to hydrogen peroxide and etoposide-induced apoptosis compared to vector-transfected cells. Mutagenesis study revealed that Y397 is required for anti-apoptotic activity in HL-60/FAK, since Y397F-mutated FAK (397FAK) lost anti-apoptotic function. Assuming that 397FAK functions as a dominant negative FAK, we introduced 397FAK cDNA into a human glioma cell line, T98G, using an adenoviral vector. We found that 397FAK induced marked apoptosis with significant FAK degradation. As PI3-kinase-Akt survival pathway was constitutively activated in T98G cells, we hypothesized that this pathway was shut off by 397FAK gene transfection. As expected, activation of PI3-kinase-Akt survival pathway was decreased by the 397FAK gene transfection. 397FAK activated mainly caspase-6 which induced degradation of transfected FAK as well as endogenous FAK. These results indicated that 397FAK induces apoptosis in T98G cells, by interrupting signals of FAK leading to the survival pathway in T98G glioma cells.     

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.     

Differential Effect of the Focal Adhesion Kinase Y397F Mutant on v-Src-Stimulated Cell Invasion and Tumor Growth

Summary Upon cell adhesion to extracellular matrix proteins, focal adhesion kinase (FAK) rapidly undergoes autophosphorylation on its Tyr-397 which consequently serves as a binding site for the Src homology 2 domains of the Src family protein kinases and several other intracellular signaling molecules. In this study, we have attempted to examine the effect of the FAK Y397F mutant on v-Src-stimulated cell transformation by establishing an inducible expression of the Y397F mutant in v-Src-transformed FAK-null (FAK^−/−) mouse embryo fibroblasts. We found that the FAK Y397F mutant had both positive and negative effects on v-Src-stimulated cell transformation; it promoted v-Src-stimulated invasion, but on the other hand it inhibited the v-Src-stimulated anchorage-independent cell growth in vitro and tumor formation in vivo . The positive effect of the Y397F mutant on v-Src-stimulated invasion was correlated with an increased expression of matrix metalloproteinase-2, both of which were inhibited by the specific phosphatidylinositol 3-kinase inhibitor wortmannin or a dominant negative mutant of AKT, suggesting a critical role for the phosphatidylinositol 3-kinase/AKT pathway in both events. However, the expression of the Y397F mutant rendered v-Src-transformed FAK^−/− cells susceptible to anoikis, correlated with suppression on v-Src-stimulated activation of ERK and AKT. In addition, under anoikis stress, the induction of the Y397F mutant in v-Src-transformed FAK^−/− cells selectively led to a decrease in the level of p130^Cas, but not other focal adhesion proteins such as talin, vinculin, and paxillin. These results suggest that FAK may increase the susceptibility of v-Src-transformed cells to anoikis by modulating the level of p130^Cas.     

Paradoxical roles of FAK in tumor cell migration and metastasis

Focal adhesion kinase (FAK), as a key mediator of signaling induced by integrins, plays an instrumental role in many cellular functions, including cell survival and proliferation. Many studies have reported that FAK is a positive regulator of normal cell migration and cancer cell metastasis. However, emerging evidence shows that FAK—under certain oncogenic signaling, such as that initiated by activated Ras and some growth factor receptor kinases—negatively regulates cancer cell migration. Activated Ras may promote tumor cell migration by dephosphorylation of FAK at Y397 and facilitation of focal adhesion turnover at the leading edge of cells.     

The cytolytic isolation of the cortex of the sea urchin egg

When the vitelline layer of sea urchin eggs (Lytechinus pictus) is disrupted by trypsin or dithiothreitol and the eggs are placed in an isosmotic medium devoid of Ca²⁺, cytolysis of the eggs occurs. During lysis the entire egg cortex peels off in one piece. Lysis is temperature and pH dependent and is inhibited by cytochalasin B. Cortices from unfertilized eggs contain seven major macromolecular components. A 42K-dalton component is believed to be actin, representing between 12 and 27% of the total protein. Cortices from fertilized eggs may contain between 50 and 65% actin. The actin appears to increase the strength of its attachment to the cortex after fertilization. This method of isolating the entire cortex may be useful for studying structural and enzymatic changes which may occur in the cortex during the cell cycle.     

The FAK–Arp2/3 interaction promotes leading edge advance and haptosensing by coupling nascent adhesions to lamellipodia actin

Cell migration is initiated in response to biochemical or physical cues in the environment that promote actin-mediated lamellipodial protrusion followed by the formation of nascent integrin adhesions (NAs) within the protrusion to drive leading edge advance. Although FAK is known to be required for cell migration through effects on focal adhesions, its role in NA formation and lamellipodial dynamics is unclear. Live-cell microscopy of FAK^−/− cells with expression of phosphorylation deficient or a FERM-domain mutant deficient in Arp2/3 binding revealed a requirement for FAK in promoting the dense formation, transient stabilization, and timely turnover of NA within lamellipodia to couple actin-driven protrusion to adhesion and advance of the leading edge. Phosphorylation on Y397 of FAK promotes dense NA formation but is dispensable for transient NA stabilization and leading edge advance. In contrast, transient NA stabilization and advance of the cell edge requires FAK–Arp2/3 interaction, which promotes Arp2/3 localization to NA and reduces FAK activity. Haptosensing of extracellular matrix (ECM) concentration during migration requires the interaction between FAK and Arp2/3, whereas FAK phosphorylation modulates mechanosensing of ECM stiffness during spreading. Taken together, our results show that mechanistically separable functions of FAK in NA are required for cells to distinguish distinct properties of their environment during migration.     

Identification of Tyr-397 as the primary site of tyrosine phosphorylation and pp60src association in the focal adhesion kinase, pp125FAK.

A number of cellular processes, such as proliferation, differentiation, and transformation, are regulated by cell-extracellular matrix interactions. Previous studies have identified a novel tyrosine kinase, the focal adhesion kinase p125FAK, as a component of cell adhesion plaques. p125FAK was identified as a 125-kDa tyrosine-phosphorylated protein in cells transformed by the v-src oncogene. p125FAK is an intracellular protein composed of three domains: a central domain with homology to protein tyrosine kinases, flanked by two noncatalytic domains of 400 amino acids which bear no significant homology to previously cloned proteins. p125FAK is believed to play an important regulatory role in cell adhesion because it localizes to cell adhesion plaques and because its phosphorylation on tyrosine residues is regulated by binding of cell surface integrins to the extracellular matrix. Recent studies have shown that Src, through its SH2 domain, stably associates with pp125FAK and that this association prevents dephosphorylation of pp125FAK in vitro by protein tyrosine phosphatases. In this report, we identify Tyr-397 as the primary in vivo and in vitro site of p125FAK tyrosine phosphorylation and association with Src. Substituting phenylalanine for tyrosine at position 397 significantly reduces p125FAK tyrosine phosphorylation and association with Src but does not abolish p125FAK kinase activity. In addition, p125FAK kinase is able to trans-phosphorylate Tyr-397 in vitro in a kinase-deficient p125FAK variant. Phosphorylation of Tyr-397 provides a site [Y(P)AEI] that fits the consensus sequence for the binding of Src.     

Focal adhesion kinase controls actin assembly via a FERM-mediated interaction with the Arp2/3 complex

Networks of actin filaments, controlled by the Arp2/3 complex, drive membrane protrusion during cell migration. How integrins signal to the Arp2/3 complex is not well understood. Here, we show that focal adhesion kinase (FAK) and the Arp2/3 complex associate and colocalize at transient structures formed early after adhesion. Nascent lamellipodia, which originate at these structures, do not form in FAK-deficient cells, or in cells in which FAK mutants cannot be autophosphorylated after integrin engagement. The FERM domain of FAK binds directly to Arp3 and can enhance Arp2/3-dependent actin polymerization. Critically, Arp2/3 is not bound when FAK is phosphorylated on Tyr 397. Interfering peptides and FERM-domain point mutants show that FAK binding to Arp2/3 controls protrusive lamellipodia formation and cell spreading. This establishes a new function for the FAK FERM domain in forming a phosphorylation-regulated complex with Arp2/3, linking integrin signalling directly with the actin polymerization machinery.     

Knock-in Mutation Reveals an Essential Role for Focal Adhesion Kinase Activity in Blood Vessel Morphogenesis and Cell Motility-Polarity but Not Cell Proliferation

Focal adhesion kinase (FAK) associates with both integrins and growth factor receptors in the control of cell motility and survival. Loss of FAK during mouse development results in lethality at embryonic day 8.5 (E8.5) and a block in cell proliferation. Because FAK serves as both a scaffold and signaling protein, gene knock-outs do not provide mechanistic insights in distinguishing between these modes of FAK function. To determine the role of FAK activity during development, a knock-in point mutation (lysine 454 to arginine (R454)) within the catalytic domain was introduced by homologous recombination. Homozygous FAK^R454/R454 mutation was lethal at E9.5 with defects in blood vessel formation as determined by lack of yolk sac primary capillary plexus formation and disorganized endothelial cell patterning in FAK^R454/R454 embryos. In contrast to the inability of embryonic FAK^−/− cells to proliferate ex vivo, primary FAK^R454/R454 mouse embryo fibroblasts (MEFs) were established from E8.5 embryos. R454 MEFs exhibited no difference in cell growth compared with normal MEFs, and R454 FAK localized to focal adhesions but was not phosphorylated at Tyr-397. In E8.5 embryos and primary MEFs, FAK R454 mutation resulted in decreased c-Src Tyr-416 phosphorylation. R454 MEFs exhibited enhanced focal adhesion formation, decreased migration, and defects in cell polarity. Within immortalized MEFs, FAK activity was required for fibronectin-stimulated FAK-p190RhoGAP association and p190RhoGAP tyrosine phosphorylation linked to decreased RhoA GTPase activity, focal adhesion turnover, and directional motility. Our results establish that intrinsic FAK activity is essential for developmental processes controlling blood vessel formation and cell motility-polarity but not cell proliferation. This work supports the use of FAK inhibitors to disrupt neovascularization.     

The alphaBbetaC integrin is expressed on the surface of the sea urchin egg and removed at fertilization

Integrins are expressed on the surface of some vertebrate eggs where they are thought to have a role in fertilization. The objective of this study is to determine if integrins are expressed on sea urchin eggs. The alphaB and betaC subunits were cloned using the homology polymerase chain reaction. Monoclonal and polyclonal antibodies were developed against bacterially expressed fragments of the extracellular domains of the betaC subunit and the alphaB subunit. As well, a monoclonal antibody was developed against a synthesized peptide corresponding to part of the cytoplasmic domain of betaC. Analysis of biotinylated egg cortex extracts immunoprecipitated with either anti-betaC or anti-alphaB yields bands of 130 and 225 kDa. Immunoblots confirm that betaC is part of the complex immunoprecipitated with anti-alphaB. Confocal immunofluorescence and immunogold electron microscopy show that betaC is present on the surface of the unfertilized egg at the tips of microvilli and in cortical granules. During the cortical reaction, immunoreactivity with antibodies to the extracellular domains of betaC and alphaB disappears from the egg surface, and microvillar casts on the fertilization envelope become immunoreactive. With antibodies to the cytoplasmic domain of betaC, immunoreactivity is lost from the surface of the egg, but the fertilization envelope does not immediately become immunoreactive. In immunoblots of egg cortex there are immunoreactive bands of the predicted sizes for alphaB and betaC. However, in fertilization envelopes, a second band that is slightly lower in molecular weight is also present. Eggs fertilized in the presence of soybean trypsin inhibitor have elongated microvilli that remain bound to the elevating fertilization envelope and immunoreactive to anti-betaC antibodies. Eggs fertilized in the presence of an ovoperoxidase inhibitor, 3-amino-1,2,4-triazole, have a patchy distribution of betaC immunoreactivity in fertilization envelopes. Together, these data suggest that alphaBbetaC integrins are expressed on the surface of unfertilized eggs and, during the cortical reaction, the extracellular domains are cleaved by proteases and cross-linked into the fertilization envelope by ovoperoxidase. The alphaBbetaC integrin receptors may have several potential functions prior to their removal at fertilization, including attachment of the vitelline envelope to the egg surface and anchoring the cortical cytoskeleton.     

Modulation of FAK and Src adhesion signaling occurs independently of adhesion complex composition

Integrin adhesion complexes (IACs) form mechanochemical connections between the extracellular matrix and actin cytoskeleton and mediate phenotypic responses via posttranslational modifications. Here, we investigate the modularity and robustness of the IAC network to pharmacological perturbation of the key IAC signaling components focal adhesion kinase (FAK) and Src. FAK inhibition using AZ13256675 blocked FAK^Y397 phosphorylation but did not alter IAC composition, as reported by mass spectrometry. IAC composition was also insensitive to Src inhibition using AZD0530 alone or in combination with FAK inhibition. In contrast, kinase inhibition substantially reduced phosphorylation within IACs, cell migration and proliferation. Furthermore using fluorescence recovery after photobleaching, we found that FAK inhibition increased the exchange rate of a phosphotyrosine (pY) reporter (dSH2) at IACs. These data demonstrate that kinase-dependent signal propagation through IACs is independent of gross changes in IAC composition. Together, these findings demonstrate a general separation between the composition of IACs and their ability to relay pY-dependent signals.     

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.     

Stretch-induced cell proliferation is mediated by FAK-MAPK pathway

Previously we reported that a uni-axial cyclic stretch treatment of rat 3Y1 fibroblasts induced focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation (Wang et al., 2001) [Wang, J.G., Miyazu, M., Matsushita, E., Sokabe, M., Naruse, K., 2001. Uni-axial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation. Biochem. Biophys. Res. Comm. 288, 356-361]. In the present study, we investigated whether stretch-induced MAPK activation leads to proliferation of fibroblasts. 3Y1 fibroblasts were subjected to a uni-axial cyclic stretch treatment (1 Hz, 120% in length) and the bromodeoxyuridine (BrdU) incorporation was measured to access cell proliferation. BrdU incorporation increased in a time-dependent manner and became significant within 6 hours. To investigate the involvement of FAK, we transiently expressed FAK mutants that lacked tyrosine phosphorylation site (s) (F397Y, F925Y, F397/925Y). Transient expression of wild-type FAK or mock vector did not inhibit the stretch-induced BrdU incorporation, however, the FAK mutants significantly blocked BrdU incorporation. Treatment of the cells with MAPK inhibitors, PD98059 or SB203580, blocked extracellular signal-regulated kinase (ERK) phosphorylation and p38 MAPK phosphorylation, respectively, and also blocked stretch-induced BrdU incorporation. These results suggest that the stretch-induced FAK activation followed by MAPK activation plays an important role in the stretch-induced proliferation of 3Y1 fibroblasts.     

Cytochalasin B blocks sperm incorporation but allows activation of the sea urchin egg

Cytochalasin B (CB) (2 × 10⁻⁶ M) prevents the incorporation of sperm into the eggs of Lytechinus pictus and Strongylocentrotus purpuratus as judged by light and transmission electron microscopy (TEM). At lower concentrations of CB (2 × 10⁻⁷ M), sperm are successfully incorporated into the egg, but their migration in the area of the egg cortex is impaired. The site of action of CB on the sperm may be on the initial rotation of the sperm nucleus in the cortex; the subsequent migration is not affected by CB. Although sperm incorporation is prevented at the higher CB concentrations, the eggs become activated—as judged by cortical reaction, increased protein synthesis and increased respiration. These findings raise the concept that egg activation by sperm could result from some pre-fusion event and hence that sperm-egg fusion would not be a prerequisite for the triggering of development. An alternative hypothesis is that fusion occurs between the acrosome process membrane and egg membrane, but since CB has destroyed the integrity of the cortex actin, the fusion bridge is so weak that it cannot be maintained without some contractile or cytoskeletal support by the cortex. The sperm may activate the CB-treated egg in the same manner as pricking with a microelectrode sometimes does.