Conformational Dynamics of the Focal Adhesion Targeting Domain Control Specific Functions of Focal Adhesion Kinase in Cells

Focal adhesion (FA) kinase (FAK) regulates cell survival and motility by transducing signals from membrane receptors. The C-terminal FA targeting (FAT) domain of FAK fulfils multiple functions, including recruitment to FAs through paxillin binding. Phosphorylation of FAT on Tyr⁹²⁵ facilitates FA disassembly and connects to the MAPK pathway through Grb2 association, but requires dissociation of the first helix (H1) of the four-helix bundle of FAT. We investigated the importance of H1 opening in cells by comparing the properties of FAK molecules containing wild-type or mutated FAT with impaired or facilitated H1 openings. These mutations did not alter the activation of FAK, but selectively affected its cellular functions, including self-association, Tyr⁹²⁵ phosphorylation, paxillin binding, and FA targeting and turnover. Phosphorylation of Tyr⁸⁶¹, located between the kinase and FAT domains, was also enhanced by the mutation that opened the FAT bundle. Similarly phosphorylation of Ser⁹¹⁰ by ERK in response to bombesin was increased by FAT opening. Although FAK molecules with the mutation favoring FAT opening were poorly recruited at FAs, they efficiently restored FA turnover and cell shape in FAK-deficient cells. In contrast, the mutation preventing H1 opening markedly impaired FAK function. Our data support the biological importance of conformational dynamics of the FAT domain and its functional interactions with other parts of the molecule.     

焦点粘着激酶的研究进展

焦点粘着激酶是依赖于整合素的细胞信号转导通路的基础性信号传递分子.通过磷酸化酪氨酸位点和富脯氨酸序列,活化的焦点粘着激酶与细胞骨架蛋白、Src族激酶、磷酸肌醇-3激酶、Graf以及多种衔接子蛋白相互作用,调节细胞的粘附、迁移、增殖和分化.     

Mechanism of Focal Adhesion Kinase Mechanosensing

Mechanosensing at focal adhesions regulates vital cellular processes. Here, we present results from molecular dynamics (MD) and mechano-biochemical network simulations that suggest a direct role of Focal Adhesion Kinase (FAK) as a mechano-sensor. Tensile forces, propagating from the membrane through the PIP₂ binding site of the FERM domain and from the cytoskeleton-anchored FAT domain, activate FAK by unlocking its central phosphorylation site (Tyr576/577) from the autoinhibitory FERM domain. Varying loading rates, pulling directions, and membrane PIP₂ concentrations corroborate the specific opening of the FERM-kinase domain interface, due to its remarkably lower mechanical stability compared to the individual alpha-helical domains and the PIP₂-FERM link. Analyzing downstream signaling networks provides further evidence for an intrinsic mechano-signaling role of FAK in broadcasting force signals through Ras to the nucleus. This distinguishes FAK from hitherto identified focal adhesion mechano-responsive molecules, allowing a new interpretation of cell stretching experiments.     

黏着斑激酶与肿瘤微环境

黏着斑激酶（focal adhesion kinase, FAK）是一种胞质非受体酪氨酸激酶。FAK和肿瘤密切相关,在多种癌细胞中高表达,促进癌细胞的发生、生长、存活、增殖、粘附、转移和侵袭以及血管生成等过程。肿瘤微环境包括肿瘤细胞、周围血管、免疫细胞、纤维母细胞、内皮细胞、信号分子和细胞外基质,它对癌症的发展和恶化具有重要作用。肿瘤细胞可以通过分泌细胞外信号影响微环境,使其有利于肿瘤生存和发展|肿瘤微环境中的基质细胞能通过产生趋化因子、基质降解酶和生长因子促进肿瘤侵袭和转移。本文综述肿瘤微环境在癌症发生发展过程中的作用及FAK在肿瘤微环境中的调控作用,为肿瘤疾病的治疗提供新思路。     

黏着斑激酶与细胞迁移

细胞迁移过程始于细胞前端板状伪足的形成、外周黏附的建立、细胞体的收缩和尾部的解离.黏着斑激酶是一种非受体酪氨酸蛋白激酶,通过其激酶活性和"脚手架"的功能在细胞迁移的各个过程中发挥关键作用.现重点介绍黏着斑激酶介导的信号转导通路及其在调控细胞迁移方面的研究进展.     

Induction of Apoptosis after Expression of PYK2, a Tyrosine Kinase Structurally Related to Focal Adhesion Kinase

Many cells (e.g., epithelial cells) require attachment to the extracellular matrix (ECM) to survive, a phenomenon known as anchorage-dependent cell survival. Disruption of the cell–ECM interactions mediated by the integrin receptors results in apoptosis. Focal adhesion kinase (FAK), a 125-kD protein tyrosine kinase activated by integrin engagement, appears to be involved in mediating cell attachment and survival. Proline-rich tyrosine kinase 2 (PYK2), also known as cellular adhesion kinase β (CAKβ) and related adhesion focal tyrosine kinase, is a second member of the FAK subfamily and is activated by an increase in intracellular calcium levels, or treatment with TNFα and UV light. However, the function of PYK2 remains largely unknown. In this study, we show that over-expression of PYK2, but not FAK, in rat and mouse fibroblasts leads to apoptotic cell death. Using a series of deletion mutants and chimeric fusion proteins of PYK2/FAK, we determined that the NH₂-terminal domain and tyrosine kinase activity of PYK2 were required for the efficient induction of apoptosis. Furthermore, the apoptosis mediated by PYK2 could be suppressed by over-expressing catalytically active v-Src, c-Src, phosphatidylinositol-3-kinase, or Akt/protein kinase B. In addition, it could also be suppressed by overexpressing an ICE or ICE-like proteinase inhibitor, crmA, but not Bcl2. Collectively, our results suggest that PYK2 and FAK, albeit highly homologous in primary structure, appear to have different functions; FAK is required for cell survival, whereas PYK2 induces apoptosis in fibroblasts.     

粘附斑激酶(FAK)及其信号通路研究进展

粘附斑激酶(focal adhesion kinase,FAK)是一类胞质非受体蛋白酪氨酸激酶,属于蛋白酪氨酸激酶(protein tyrosine kinase)超家族,因而也称为PTKⅡ.FAK在细胞信号转导中处于十分重要的位置,它是胞内外信号出入的中枢,介导多条信号通路.FAK可以整合来自整合素、生长因子以及机械刺激等的信号,激活胞内PI3K/Akt、Ras/MAPK等信号通路,调节细胞生长.FAK还与胚胎发育、肿瘤发生与迁移有关.     

Focal Adhesion Kinase Modulates Cell Adhesion Strengthening via Integrin Activation

Focal adhesion kinase (FAK) is an essential nonreceptor tyrosine kinase regulating cell migration, adhesive signaling, and mechanosensing. Using FAK-null cells expressing FAK under an inducible promoter, we demonstrate that FAK regulates the time-dependent generation of adhesive forces. During the early stages of adhesion, FAK expression in FAK-null cells enhances integrin activation to promote integrin binding and, hence, the adhesion strengthening rate. Importantly, FAK expression regulated integrin activation, and talin was required for the FAK-dependent effects. A role for FAK in integrin activation was confirmed in human fibroblasts with knocked-down FAK expression. The FAK autophosphorylation Y397 site was required for the enhancements in adhesion strengthening and integrin-binding responses. This work demonstrates a novel role for FAK in integrin activation and the time-dependent generation of cell–ECM forces.     

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.     

Focal Adhesion Kinase Signaling and the Aggressive Melanoma Phenotype

Focal adhesion kinase (FAK) mediates myriad cellular functions and has been found to be over-expressed in numerous human cancers. We recently explored the role of FAK in promoting the aggressive phenotype of melanoma cells, characterized by increased invasion, migration, and vasculogenic mimicry (VM) potential. We found FAK to be phosphorylated on its key tyrosine residues (397 and 576) in aggressive melanoma cells cultured on a three-dimensional type 1 collagen matrix in vitro, as well as in radial and vertical growth phase melanomas in situ. Furthermore, expressing FAK related non-kinase (FRNK) in melanoma cells directly resulted in the inhibition of the aggressive phenotype, as demonstrated by decreased invasion, migration, and VM potential, in part by blocking an Erk1/2 mediated signaling pathway. Additional data indicated that increased FAK activity may promote cellular proliferation and anchorage independent growth of aggressive melanoma. Together these observations implicate FAK as a promoter of the aggressive melanoma phenotype, thereby identifying a rational target for therapeutic intervention of malignant melanoma.     

The Focal Adhesion: A Regulated Component of Aortic Stiffness

Increased aortic stiffness is an acknowledged predictor and cause of cardiovascular disease. The sources and mechanisms of vascular stiffness are not well understood, although the extracellular matrix (ECM) has been assumed to be a major component. We tested here the hypothesis that the focal adhesions (FAs) connecting the cortical cytoskeleton of vascular smooth muscle cells (VSMCs) to the matrix in the aortic wall are a component of aortic stiffness and that this component is dynamically regulated. First, we examined a model system in which magnetic tweezers could be used to monitor cellular cortical stiffness, serum-starved A7r5 aortic smooth muscle cells. Lysophosphatidic acid (LPA), an activator of myosin that increases cell contractility, increased cortical stiffness. A small molecule inhibitor of Src-dependent FA recycling, PP2, was found to significantly inhibit LPA-induced increases in cortical stiffness, as well as tension-induced increases in FA size. To directly test the applicability of these results to force and stiffness development at the level of vascular tissue, we monitored mouse aorta ring stiffness with small sinusoidal length oscillations during agonist-induced contraction. The alpha-agonist phenylephrine, which also increases myosin activation and contractility, increased tissue stress and stiffness in a PP2- and FAK inhibitor 14-attenuated manner. Subsequent phosphotyrosine screening and follow-up with phosphosite-specific antibodies confirmed that the effects of PP2 and FAK inhibitor 14 in vascular tissue involve FA proteins, including FAK, CAS, and paxillin. Thus, in the present study we identify, for the first time, the FA of the VSMC, in particular the FAK-Src signaling complex, as a significant subcellular regulator of aortic stiffness and stress.     

黏着斑激酶与肿瘤发生、发展及预后的关系

黏着斑激酶(focal adhesion kinase,FAK)作为一种非受体蛋白酪氨酸激酶,因其与肿瘤间的密切联系而备受关注。多年研究发现,FAK在许多肿瘤中过表达,通过多条信号通路调控细胞侵袭、迁移、增殖及凋亡等,从而参与肿瘤的发生、发展进程。研究表明FAK可以作为肿瘤预后因子,是潜在的抗肿瘤治疗靶点。本文对FAK与肿瘤进程的关系作一综述,以期更好地认识FAK在肿瘤发生、发展过程中所起的作用,为相关研究者提供资料参考。     

Myosin 3A Kinase Activity Is Regulated by Phosphorylation of the Kinase Domain Activation Loop

Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.     

Targeting Focal Adhesion Kinase Suppresses the Malignant Phenotype in Rhabdomyosarcoma Cells

Despite the tremendous advances in the treatment of childhood solid tumors, rhabdomyosarcoma (RMS) continues to provide a therapeutic challenge. Children with metastatic or relapsed disease have a disease-free survival rate under 30%. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumorigenesis. Signaling pathways both upstream and downstream to FAK have been found to be important in sarcoma tumorigenesis, leading us to hypothesize that FAK would be present in RMS and would impact cellular survival. In the current study, we showed that FAK was present and phosphorylated in pediatric alveolar and embryonal RMS tumor specimens and cell lines. We also examined the effects of FAK inhibition upon two RMS cell lines utilizing parallel approaches including RNAi and small molecule inhibitors. FAK inhibition resulted in decreased cellular survival, invasion, and migration and increased apoptosis. Furthermore, small molecule inhibition of FAK led to decreased tumor growth in a nude mouse RMS xenograft model. The findings from this study will help to further our understanding of the regulation of tumorigenesis in RMS and may provide desperately needed novel therapeutic strategies for these difficult-to-treat tumors.     

Autophosphorylation-independent and -dependent Functions of Focal Adhesion Kinase during Development

Focal adhesion kinase (FAK) regulates numerous cellular functions and is critical for processes ranging from embryo development to cancer progression. Although autophosphorylation on Tyr-397 appears required for FAK functions in vitro, its role in vivo has not been established. We addressed this question using a mutant mouse (fakΔ) deleted of exon 15, which encodes Tyr-397. The resulting mutant protein FAKΔ is an active kinase expressed at normal levels. Our results demonstrate that the requirement for FAK autophosphorylation varies during development. FAK^Δ/Δ embryos developed normally up to embryonic day (E) 12.5, contrasting with the lethality at E8.5 of FAK-null embryos. Thus, autophosphorylation on Tyr-397 is not required for FAK to achieve its functions until late mid-gestation. However, FAK^Δ/Δ embryos displayed hemorrhages, edema, delayed artery formation, vascular remodeling defects, multiple organ abnormalities, and overall developmental retardation at E13.5–14.5, and died thereafter demonstrating that FAK autophosphorylation is also necessary for normal development. Fibroblasts derived from mutant embryos had a normal stellate morphology and expression of focal adhesion proteins, Src family members, p53, and Pyk2. In contrast, in FAK^Δ/Δ fibroblasts and endothelial cells, spreading and lamellipodia formation were altered with an increased size and number of focal adhesions, enriched in FAKΔ. FAK mutation also decreased fibroblast proliferation. These results show that the physiological functions of FAK in vivo are achieved through both autophosphorylation-independent and autophosphorylation-dependent mechanisms.     

骨桥蛋白趋化和趋触作用对FAK和ILK磷酸化修饰的影响

为探讨可溶性(趋化作用)或锚定(趋触作用)形式的骨桥蛋白(OPN)与整合素相互作用对下游信号分子黏着斑激酶(FAK)和整合素偶联激酶(ILK)磷酸化修饰的影响,分别用包被于培养瓶上锚定型或加在培养液中的可溶性OPN刺激血管平滑肌细胞(VSMC)后,观察FAK和ILK的磷酸化及FAK与ILK相互作用的变化。结果显示,包被于培养瓶上的OPN通过趋触作用促进VSMC黏附和伸展,接种45min时,黏附细胞数达对照组的2.4倍(P<0.05);OPN的趋触及趋化作用均可诱导FAK磷酸化、ILK去磷酸化并抑制FAK与ILK结合;转染可表达整合素β3亚单位胞内区的表达质粒pEGFP-C3-β3CD能阻断OPN与整合素相互作用所引发的FAK磷酸化及ILK去磷酸化。研究结果表明,OPN的趋触和趋化作用对整合素下游信号分子FAK和ILK的影响是一致的,且这些作用是由整合素β3亚单位胞内区所介导的。     

Visualizing and Manipulating Focal Adhesion Kinase Regulation in Live Cells

Focal Adhesion Kinase (FAK) is essential for cell migration and plays an important role in tumor metastasis. However, the complex intermolecular and intramolecular interactions that regulate FAK activity at the focal adhesion remain unresolved. We have engineered a toolbox of FRET sensors that retain all of the individual FAK domains but modulate a key intramolecular regulatory interaction between the band 4.1/ezrin/radixin/moesin (FERM) and kinase domains of FAK. We demonstrate systematic control and quantitative measurement of the FERM-kinase interaction at focal adhesions, which in turn allows us to control cell migration. Using these sensors, we find that Tyr-397 phosphorylation, rather than kinase activity of FAK, is the key determinant of cell migration. Our sensors directly demonstrate, for the first time, a pH-dependent change in a protein-protein interaction at a macromolecular structure in live cells. The FERM-kinase interaction at focal adhesions is enhanced at acidic pH, with a concomitant decrease in Tyr-397 phosphorylation, providing a potential mechanism for enhanced migration of cancer cells.