Focal adhesion kinase (FAK) activates and stabilizes IGF-1 receptor

Recent studies have shown a direct association between IGF-1R and FAK, two important mediators of cell growth, survival and migration. However, the mechanism by which FAK affects IGF-1R function remains unknown. This study investigates the potential role of FAK in mediating activation and stability of IGF-1R. Autophosphorylation and phosphorylation capacities of wild type and mutant IGF-1R were studied. Surprisingly, we found that the mutant IGF-1R lacking the three core tyrosine residues in the activation-loop can be phosphorylated although it is unable to undergo autophosphorylation, suggesting that another kinase possesses the ability to phosphorylate IGF-1R. By using wild type MEFs and FAK−/− MEFs we could demonstrate that FAK mediates activation-loop independent phosphorylation, as well as Akt and ERK activation. Furthermore, the stability of IGF-1R was decreased upon FAK siRNA or inactivation. Taken together, our data suggest a role for FAK in phosphorylation, signaling and stability of the IGF-1R.     

Focal adhesion kinase (FAK), a multifunctional protein

Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase localized to regions called focal adhesions. Many stimuli can induce tyrosine phosphorylation and activation of FAK, including integrins and growth factors. The major site of autophosphorylation, tyrosine 397, is a docking site for the SH2 domains of Src family proteins. The other sites of phosphorylation are phosphorylated by Src kinases. Phosphorylated FAK binds proteins of focal adhesion and can activate them directly or indirectly by phosphorylation. These activated proteins forming the FAK complex facilitate the generation of downstream signals necessary to regulate cell functions, like motility, survival and proliferation. Dysregulation of FAK could participate in the development of cancer. This review will focus upon the mechanisms by which FAK transmits biochemical signals and elicits biological effects.     

Focal adhesion kinase (FAK) regulates insulin-stimulated glycogen synthesis in hepatocytes.

Experimental data support a role for FAK, an important component of the integrin signaling pathway, in insulin action. To test the hypothesis that FAK plays a regulatory role in hepatic insulin action, we overexpressed wild type (WT), a kinase inactive (KR), or a COOH-terminal focal adhesion targeting (FAT) sequence-truncated mutant of FAK in HepG2 hepatoma cells. In control untransfected (NON) and vector (CMV2)- and WT-transfected cells, insulin stimulated an expected 54 +/- 13, 37 +/- 4, and 47 +/- 12 increase in [U-(14)C]glucose incorporation into glycogen, respectively. This was entirely abolished in the presence of either KR (-1 +/- 7%) or FAT mutants (0 +/- 8%, n = 5, p < 0.05 for KR or FAT versus other groups), and this was associated with a significant attenuation of incremental insulin-stimulated glycogen synthase (GS) activity. Insulin-stimulated serine phosphorylation of Akt/protein kinase B was significantly impaired in mutant-transfected cells. Moreover, the ability of insulin to inactivate GS kinase-3beta (GSK-3beta), the regulatory enzyme immediately upstream of GS, by serine phosphorylation (308 +/- 16, 321 +/- 41, and 458 +/- 34 optical densitometric units (odu) in NON, CMV2, and WT, respectively, p < 0.02 for WT versus CMV2) was attenuated in the presence of either FAT (205 +/- 14, p < 0.01) or KR (189 +/- 4, p < 0.005) mutants. FAK co-immunoprecipitated with GSK-3beta, but only in cells overexpressing the KR (374 +/- 254 odu) and FAT (555 +/- 308) mutants was this association stimulated by insulin compared with NON (-209 +/- 92), CMV2 (-47 +/- 70), and WT (-39 +/- 31 odu). This suggests that FAK and GSK-3beta form both a constitutive association and a transient complex upon insulin stimulation, the dissociation of which requires normal function and localization of FAK. We conclude that FAK regulates the activity of Akt/protein kinase B and GSK-3beta and the association of GSK-3beta with FAK to influence insulin-stimulated glycogen synthesis in hepatocytes. Insulin action may be subject to regulation by the integrin signaling pathway, ensuring that these growth and differentiation-promoting pathways act in a coordinated and/or complementary manner.     

Roles for focal adhesion kinase (FAK) in blastomere abscission and vesicle trafficking during cleavage in the sea urchin embryo

Is focal adhesion kinase (FAK) needed for embryonic cleavage? We find that FAK is expressed during early cleavage divisions of sea urchin embryos as determined by polyclonal antibodies to the Lytechinus variegatus protein. FAK is absent in eggs and zygotes and then cycles in abundance during the first cleavages after fertilization. It is maximal at anaphase, similar to the destruction and synthesis of cyclin proteins. To investigate whether FAK is needed during early cleavage, we interfered with its function by microinjecting eggs with anti-FAK antibodies or with FAK antisense morpholino oligonucleotides. Both treatments led to regression of the cleavage furrow. FAK knockdown with antibodies or morpholino oligonucleotides also resulted in an over-accumulation of endocytic vesicles. Thus, FAK could be restricting endocytosis or increasing exocytosis in localized areas important for abscission. FAK appears to be necessary for successful cleavage. These results are the first to document a functional role for FAK during embryonic cleavage.     

cAMP-dependent protein kinase in sea urchin embryos

cAMP-dependent protein kinase in the supernatant fraction of the homogenate of sea urchin eggs and embryos obtained by centrifugation at 105,000g was investigated in the present study. In the previous report, the dissociation constant between cAMP-binding proteins and cAMP changed during the development. This suggests that the nature of cAMP-dependent protein kinase, which has been well established to be the major cAMP receptor, changes during the development. In the present study, four protein kinases were separated through DEAE-cellulose column from the supernatant of unfertilized egg homogenate. One of them was cAMP-dependent protein kinase. The others were cAMP-independent ones. One among them was phosvitin kinase, and the others were not identified at present. The activity of cAMP-dependent protein kinase gradually increased during a period from fertilization to the swimming blastula stage. During this period, cleavages occurred at a high rate, and the rate decreased after hatching out. Thus, it is supposed that cAMP-dependent protein kinase in the supernatant may take a part in the mechanism of cleavage. The activity, however, became very low at the mesenchyme blastula, the gastrula, and the pluteus stages. cAMP-binding capacity was observed in the sedimentable fraction and the supernatant fraction, respectively, obtained by 105,000g centrifugation at all stages examined. If the structure-bound cAMP-binding protein is also cAMP-dependent protein kinase, it may play different roles in the mechanism of development.     

Focal adhesion kinase (FAK) perspectives in mechanobiology: implications for cell behaviour

Mechanobiology is a scientific interface discipline emerging from engineering and biology. With regard to tissue-regenerative cell-based strategies, mechanobiological concepts, including biomechanics as a target for cell and human mesenchymal stem cell behaviour, are on the march. Based on the periodontium as a paradigm, this mini-review discusses the key role of focal-adhesion kinase (FAK) in mechanobiology, since it is involved in mediating the transformation of environmental biomechanical signals into cell behavioural responses via mechanotransducing signalling cascades. These processes enable cells to adjust quickly to environmental cues, whereas adjustment itself relies on the specific intramolecular phosphorylation of FAK tyrosine residues and the multiple interactions of FAK with distinct partners. Furthermore, interaction-triggered mechanotransducing pathways govern the dynamics of focal adhesion sites and cell behaviour. Facets of behaviour not only include cell spreading and motility, but also proliferation, differentiation and apoptosis. In translational terms, identified and characterized biomechanical parameters can be incorporated into innovative concepts of cell- and tissue-tailored clinically applied biomaterials controlling cell behaviour as desired.     

Achievement of thermotolerance trough hsps phosphorylation in sea urchin embryos.

TPA treatment of sea urchin embryos is able to induce thermotolerance. Evidence is provided that TPA treatment induces phosphorylation of a constitutive stress protein of 38 KDa.     

Uniaxial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation

We investigated the role of tyrosine phosphorylation of FAK in the stretch-induced MAPKs (extracellular signal-regulated kinase (ERK), p38MAPK) activation in mutant FAK-transfected fibroblasts. In response to uniaxial cyclic stretch (1 Hz, 120% in length), the levels of tyrosine phosphorylation of the Tyr-397 and Tyr-925 of FAK in control cells increased and peaked at 5 min (2.75 +/- 0.51, n = 3), and 20 min (2.98 +/- 0.58, n = 3), respectively, and the activities of MAPKs increased and peaked at approximately 10 min. On the other hand, in the mutant FAK-transfected cells, the stretch-induced MAPKs activation was significantly inhibited. The stretch-induced activation of MAPKs was also significantly abolished by either treatment with Gd(3+) or extracellular Ca(2+) removal which may inhibit intracellular Ca(2+) increase caused by the activation of cation selective (Ca(2+)-permeable) stretch activated (SACatC) channels. These results suggest that the stretch-induced tyrosine-phosphorylation of FAK via SACatC activation is critical for the stretch-induced MAPKs activation.     

Cholinergic receptors in early (pre-nervous) sea urchin embryos

Agonists of nicotinic acetylcholine receptors (nAChR) nicotine and 1-acetyl-4-methylpiperazine do not act on the early sea urchin embryogenesis but evoke calcium shock in both oocytes and early embryos under certain conditions. Many nAChR ligands protect both oocytes and embryos against this shock. There seem to exist putative nAChR on the cell surface of the early sea urchin oocytes and early embryos. Pre-nervous acetylcholine seems to be functionally coupled via these receptors with the second messengers, endogenous activators of the protein kinase C.     

Actin gene expression in developing sea urchin embryos.

We show that the synthesis of actin is regulated developmentally during early sea urchin embryogenesis and that the level of synthesis of this protein parallels the steady-state amounts of the actin messenger ribonucleic acids (RNA). An in vitro translation and RNA blotting analysis of embryo RNA from several stages of early development indicated that during the first 8 h after fertilization there was a low and relatively constant level of actin messenger RNA in the embryo. Between 8 and 13 h of development, the amount of actin messenger RNA began to increase both in the cytoplasm and on polysomes, and by 18 h the amounts of actin message per embryo had risen between approximately 10- and 25-fold in the cytoplasm and between 15- and 40-fold on polysomes. Two size classes of actin messenger RNA (2.2 and 1.8 kilobases) were identified in unfertilized eggs and in all of the developmental stages examined. The amount of each actin message class increased over a similar time interval during early development. However, the amounts of these size classes in the cytoplasm relative to each other shifted between the earliest stages examined (2 to 5 h) and the hatching blastula stage (18 h), with the ratio of the 1.8-kilobase actin messenger RNA to the 2.2-kilobase actin messenger RNA increasing almost threefold during this period.     

Focal adhesion kinase (FAK)-related non-kinase inhibits myofibroblast differentiation through differential MAPK activation in a FAK-dependent manner

Transforming growth factor (TGF)-beta1 induces fibroblast transdifferentiation to myofibroblasts, a process that requires the involvement of integrin-mediated signaling and focal adhesion kinase (FAK). FAK-related non-kinase (FRNK) is known for its role in inhibiting integrin-mediated cell migration; however, its role in myofibroblast differentiation has not been defined. Here, we report that FRNK abrogates TGF-beta1-induced myofibroblast differentiation in vitro and in vivo. TGF-beta1 can induce alpha-smooth muscle actin (alpha-SMA) expression in the presence or absence of FAK; however, TGF-beta1-induced alpha-SMA expression is reduced (approximately 73%) in FAK-deficient fibroblasts. Although both ERK and p38 MAPK activation is required for maximal TGF-beta1-induced alpha-SMA expression, ERK is the major signaling intermediate in cells that express FAK. In contrast, p38 MAPK is the dominant mediator of TGF-beta1-induced alpha-SMA expression in FAK-deficient cells. FRNK overexpression blocks TGF-beta1-induced ERK or p38 MAPK activation in the presence, and surprisingly, in the absence of FAK. The loss of FRNK was tested in vivo during experimentally induced pulmonary fibrosis in mice. FRNK knock-out mice have a greater increase in alpha-SMA-expressing cells in response to a pulmonary fibrotic stimulus in vivo, as compared with congenic wild type mice. This is the first time that FRNK loss has been shown to modify the pathobiology in any animal disease model. Together, the data demonstrate that FRNK negatively regulates myofibroblast differentiation in vitro and in vivo. These data further suggest that modulation FRNK expression may be a novel avenue for therapeutic intervention in tissue fibrosis.     

Focal adhesion kinase (FAK) binds RET kinase via its FERM domain, priming a direct and reciprocal RET-FAK transactivation mechanism

Whether RET is able to directly phosphorylate and activate downstream targets independently of the binding of proteins that contain Src homology 2 or phosphotyrosine binding domains and whether mechanisms in trans by cytoplasmic kinases can modulate RET function and signaling remain largely unexplored. In this study, oligopeptide arrays were used to screen substrates directly phosphorylated by purified recombinant wild-type and oncogenic RET kinase domain in the presence or absence of small molecule inhibitors. The results of the peptide array were validated by enzyme kinetics, in vitro kinase, and cell-based experiments. The identification of focal adhesion kinase (FAK) as a direct substrate for RET kinase revealed (i) a RET-FAK transactivation mechanism consisting of direct phosphorylation of FAK Tyr-576/577 by RET and a reciprocal phosphorylation of RET by FAK, which crucially is able to rescue the kinase-impaired RET K758M mutant and (ii) that FAK binds RET via its FERM domain. Interestingly, this interaction is abolished upon RET phosphorylation, indicating that RET binding to the FERM domain of FAK is a priming step for RET-FAK transactivation. Finally, our data indicate that FAK inhibitors could be used as potential therapeutic agents for patients with multiple endocrine neoplasia type 2 tumors because both, treatment with the FAK kinase inhibitor NVP-TAE226 and FAK down-regulation by siRNA reduced RET phosphorylation and signaling as well as the proliferation and survival of tumor and transfected cell lines expressing oncogenic RET.     

Nanog increases focal adhesion kinase (FAK) promoter activity and expression and directly binds to FAK protein to be phosphorylated

Nanog and FAK were shown to be overexpressed in cancer cells. In this report, the Nanog overexpression increased FAK expression in 293, SW480, and SW620 cancer cells. Nanog binds the FAK promoter and up-regulates its activity, whereas Nanog siRNA decreases FAK promoter activity and FAK mRNA. The FAK promoter contains four Nanog-binding sites. The site-directed mutagenesis of these sites significantly decreased up-regulation of FAK promoter activity by Nanog. EMSA showed the specific binding of Nanog to each of the four sites, and binding was confirmed by ChIP assay. Nanog directly binds the FAK protein by pulldown and immunoprecipitation assays, and proteins co-localize by confocal microscopy. Nanog binds the N-terminal domain of FAK. In addition, FAK directly phosphorylates Nanog in a dose-dependent manner by in vitro kinase assay and in cancer cells in vivo. The site-directed mutagenesis of Nanog tyrosines, Y35F and Y174F, blocked phosphorylation and binding by FAK. Moreover, overexpression of wild type Nanog increased filopodia/lamellipodia formation, whereas mutant Y35F and Y174F Nanog did not. The wild type Nanog increased cell invasion that was inhibited by the FAK inhibitor and increased by FAK more significantly than with the mutants Y35F and Y174F Nanog. Down-regulation of Nanog with siRNA decreased cell growth reversed by FAK overexpression. Thus, these data demonstrate the regulation of the FAK promoter by Nanog, the direct binding of the proteins, the phosphorylation of Nanog by FAK, and the effect of FAK and Nanog cross-regulation on cancer cell morphology, invasion, and growth that plays a significant role in carcinogenesis.     

Focal adhesion kinase pp125FAK is associated with both intercellular junctions and matrix adhesion sites in vivo

Previous studies have characterized pp125^FAK as a focal adhesion (FA)-associated non-receptor tyrosine kinase. However, there are few data available on the expression and localization of this kinase in tissues. In this study we show that in human tissues the highest expression of pp125^FAK is found in some developing epithelia, where pp125^FAK is associated with either intercellular junctions or with sites of adhesion to the basement membrane, whereas the same adult tissues show only a faint reactivity. Connective tissue cells do not show any reactivity for pp125^FAK in vivo, but developing arterial smooth muscle expresses pp125^FAK at high levels. The expression pattern in malignant tissues is variable, but most carcinomas do not express this kinase. In primary cultures of human amnion epithelial cells pp125^FAK first becomes associated with the polarized adhesion lamellae, but is subsequently translocated to the forming adherens junctions (AJs). Later upon culturing pp125^FAK becomes associated with prominent FAs, as in cultured cell lines. Taken together, our results suggest that the association of pp125^FAK with FAs in cultured cells is principally due to a process of adaptation, whereas in vivo pp125^FAK mainly functions as a regulatory component of intercellular AJs and cell-matrix adhesions of developing epithelia and also in developing arterial smooth muscle.     

Rho-kinase in sea urchin eggs and embryos

The activation of sea urchin eggs at fertilization provides an ideal system for studying the molecular events involved in cellular activation. Rho GTPases, which are key signaling enzymes in eukaryotes, are involved in sustaining the activation of sea urchin eggs; however, their downstream effectors have not yet been characterized. In somatic cells, RhoA regulates a serine/threonine kinase known as Rho-kinase (ROCK). The activity of ROCK in early sea urchin development has been inferred, but not tested directly. A ROCK gene was identified in the sea urchin (Strongylocentrotus purpuratus) genome and the sequence of its cDNA determined. The sea urchin ROCK (SpROCK) sequence predicts a protein of 158 kDa with >72% and 45% identities with different protein orthologues of the kinase catalytic domain and the complete protein sequence, respectively. SpROCK mRNA levels are high in unfertilized eggs and decrease to 35% after 15 min postfertilization and remain low up to the 4 cell stage. Antibodies to the human ROCK-I kinase domain revealed SpROCK to be concentrated in the cortex of eggs and early embryos. Co-immunoprecipitation assays indicate that RhoA and SpROCK are physically associated. This association is destroyed by treatment with the C3 exoenzyme and with the ROCK antagonist H-1152. H-1152 also inhibited DNA synthesis in embryos. We conclude that the Rho-dependent signaling pathway, via SpROCK, is essential for early embryonic development.