Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.10.2 (focal adhesion kinase)
 44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Src is activated in response to a variety of growth factors and hormones that bind G protein-coupled receptors (GPCRs), and its activity is regulated by phosphorylation at key sites, including the autophosphorylation site Tyr-418 and the inhibitory site Tyr-529. To better understand the mechanisms controlling Src activation, we examined Src phosphorylation in Swiss 3T3 fibroblasts stimulated with bombesin and in IEC-18 intestinal epithelial cells stimulated with angiotensin II (Ang II). Phosphorylation at Src Tyr-418, the activation loop site, was rapidly and markedly increased after GPCR agonist addition in both cell types. However, treatment of intact cells with the selective Src family kinase inhibitor PP2, at concentrations which abolished Src-mediated phosphorylation of focal adhesion kinase (FAK) at Tyr-577, unexpectedly led to increased phosphorylation at Src Tyr-418 and diminished phosphorylation at Tyr-529. In Swiss 3T3 cells, PP2 enhanced Tyr-418 phosphorylation after 1 min of bombesin stimulation, while in IEC-18 cells, PP2 increased Ang II-stimulated Tyr-418 phosphorylation at all times tested. These results imply that a distinct, non-Src family kinase may be responsible for phosphorylating Src at Tyr-418 in intact fibroblasts and epithelial cells stimulated by GPCR agonists.
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PMID:Bombesin and angiotensin II rapidly stimulate Src phosphorylation at Tyr-418 in fibroblasts and intestinal epithelial cells through a PP2-insensitive pathway. 1545 Oct 29

Malignant glioma is the major brain tumor in adults and has a poor prognosis. The failure to control invasive cell subpopulations may be the key reason for local glioma recurrence after radical tumor resection and may contribute substantially to the failure of the other treatment modalities such as radiation therapy and chemotherapy. As a model for this invasion, we have implanted spheroids from a human glioma cell line (U251) in three-dimensional collagen type I matrices, which these cells readily invade. We first observed that the Src family kinase-specific pharmacologic inhibitors PP2 and SU6656 significantly inhibited the invasion of the cells in this assay. We confirmed this result by showing that expression of two inhibitors of Src family function, dominant-negative-Src and CSK, also suppressed glioma cell invasion. To characterize this effect at the level of the cytoskeleton, we used fluorescent time-lapse microscopy on U251 cells stably expressing a YFP-actin construct and observed a rapid change in actin dynamics following addition of PP2 in both two-dimensional and three-dimensional cultures. In monolayer cultures, PP2 caused the disappearance of peripheral membrane ruffles within minutes. In three-dimensional cultures, PP2 induced the loss of actin bursting at the leading tip of the invadopodium. The inhibition of Src family activity is thus a potential therapeutic approach to treat highly invasive malignant glioma.
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PMID:SRC regulates actin dynamics and invasion of malignant glial cells in three dimensions. 1556 76

We have identified the Yes kinase in zebrafish eggs and investigated its role in development of the zebrafish embryo. In situ hybridization as well as immunofluorescence techniques demonstrated that Yes kinase is maternally expressed and is localized to the cortical region of the unfertilized egg. Fertilization resulted in concentration of Yes kinase to the blastodisc where it continued to be localized to the blastoderm cells through cleavage, gastrulation, and later development. Yes kinase activity was found to decrease abruptly at fertilization, then increase progressively during epiboly, and was maintained at high levels throughout gastrulation. The role of Yes kinase in development was tested by treating embryos with chemical protein tyrosine kinase (PTK) inhibitors such as 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) and by injection of antisense morpholinos. Both treatments resulted in the arrest of development at the beginning of the epiboly. Co-immunoprecipitation studies demonstrated that Yes kinase participates in a stable complex with focal adhesion kinase (FAK), which is phosphorylated in vitro. These results demonstrate that Yes kinase plays an important role in epiboly and indicate that Yes kinase participates in signaling by focal adhesion kinase during early development.
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PMID:Role of Yes kinase during early zebrafish development. 1557 45

We have previously described regulation of focal adhesion kinase (FAK) by its amino-terminal FERM-like domain through an autoinhibitory interaction with its kinase domain (Cooper, L. A., Shen, T. L., and Guan, J. L. (2003) Mol. Cell. Biol. 23, 8030-8041). Here we show that the first two subdomains of the FERM-like domain are independently capable of inhibiting phosphorylation of FAK in trans. We characterized several point mutations within the first subdomain of the FERM-like domain and find that mutation of Lys-38 to alanine results in a FAK mutant that is strongly hyperphosphorylated when expressed in mammalian cells, and promotes increased phosphorylation of the FAK substrate paxillin. A second mutation of Lys-78 to alanine results in a FAK mutant that is underphosphorylated, but can be activated by extracellular matrix stimuli. Like deletion of the amino terminus itself the K38A mutation is phosphorylated in suspension. The Delta375 truncation mutant of FAK is strongly phosphorylated both when Tyr-397 is mutated to phenylalanine, and in the presence of the Src inhibitor, PP2, suggesting that removal of the amino terminus can render FAK Src independent. This is in contrast to the K38A mutant that is not phosphorylated in the Y397F background, and which shows decreased phosphorylation in the presence of the Src inhibitor PP2, suggesting that regulation of FAK by Src is a secondary step in its activation. The K38A mutation weakens the interaction between the amino terminus of FAK and its own kinase domain, and disrupts the ability of the amino terminus to inhibit the phosphorylation of FAK in trans. The K38A mutation of FAK also increases the ability of FAK to promote cell cycle progression and cell migration, suggesting that hyperphosphorylation of this mutant can positively affect FAK function in cells. Together, these data strongly suggest a role for the first FAK subdomain of the FERM domain in its normal regulation and function in the cell.
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PMID:Residues within the first subdomain of the FERM-like domain in focal adhesion kinase are important in its regulation. 1561 Nov 37

In the present study, we demonstrated that Ang II provokes a transitory enhancement of focal adhesion kinase (FAK) and paxillin phosphorylation in human umbilical endothelial cells (HUVEC). Moreover, Ang II induces a time- and dose-dependent augmentation in cell migration, but does not affect HUVEC proliferation. The effect of Ang II on FAK and paxillin phosphorylation was markedly attenuated in cells pretreated with wortmannin and LY294002, indicating that phosphoinositide 3-kinase (PI3K) plays an important role in regulating FAK activation. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific inhibitor PP2 for Src family kinases, demonstrating the involvement of protein tyrosine kinases, and particularly Src family of tyrosine kinases, in the downstream signalling pathway of Ang II receptors. Furthermore, FAK and paxillin phosphorylation was markedly blocked after treatment of HUVEC with AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) phosphorylation. Pretreatment of cells with inhibitors of PI3K, Src family tyrosine kinases, and EGFR also decreased HUVEC migration. In conclusion, these results suggest that Ang II mediates an increase in FAK and paxillin phosphorylation and induces HUVEC migration through signal transduction pathways dependent on PI3K and Src tyrosine kinase activation and EGFR transactivation.
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PMID:Angiotensin II induces focal adhesion kinase/paxillin phosphorylation and cell migration in human umbilical vein endothelial cells. 1565 90

The expression of the receptor protein tyrosine phosphatase r-PTPeta is drastically reduced in rat and human malignant thyroid cells, whereas its restoration reverts the neoplastic phenotype of retrovirally transformed rat thyroid cells. Moreover, reduced levels and loss of heterozygosity of DEP-1, the human homolog of r-PTPeta, have been found in many human neoplasias. Here, we report that the r-PTPeta protein binds to c-Src in living cells and dephosphorylates the c-Src inhibitory tyrosine phosphorylation site (Tyr 529), thereby increasing c-Src tyrosine kinase activity in malignant rat thyroid cells stably transfected with r-PTPeta. Tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin was enhanced in r-PTPeta-expressing cells. This was associated with increased adhesion of malignant r-PTPeta-transfected thyroid cells vs both untransfected cells and cells stably transfected with an inactive r-PTPeta mutant. Treatment of rat thyroid cells with the c-Src inhibitor PP2 decreased cell adhesion to a higher extent in r-PTPeta-transfected cells than in mock-transfected or stably transfected cells with the inactive r-PTPeta mutant, indicating that r-PTPeta regulates cell-substratum adhesion by activating c-Src. Interestingly, the extent of both c-Src dephosphorylation at Tyr 529, FAK and paxillin phosphorylation, and the increased cell adhesion were associated with the degree of r-PTPeta expression.
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PMID:The rat tyrosine phosphatase eta increases cell adhesion by activating c-Src through dephosphorylation of its inhibitory phosphotyrosine residue. 2742 94

In several previous studies, we performed sensitivity analysis to gauge the relative importance of different atomic partial charges in determining protein-ligand binding. In this work, we gain further insights by decomposing these results into three contributions: desolvation, intramolecular interactions, and intermolecular interactions, again based on a Poisson continuum electrostatics model. Three protein kinase-inhibitor systems have been analyzed: CDK2-deschloroflavopiridol, PKA-PKI, and LCK-PP2. Although our results point out the importance of specific intermolecular interactions to the binding affinity, they also reveal the remarkable contributions from the solvent-mediated intramolecular interactions in some cases. Thus, it is necessary to look beyond analyzing protein-ligand interactions to understand protein-ligand recognition or to gain insights into designing ligands and proteins. In analyzing the contributions of the three components to the overall binding free energy, the PKA-PKI system with a much larger ligand was found to behave differently from the other two systems with smaller ligands. In the former case, the intermolecular interactions are very favorable, and together with the favorable solvent-mediated intramolecular interactions, they overcome the large desolvation penalties to give a favorable electrostatics contribution to the overall binding affinity. On the other hand, the other two systems with smaller ligands only present modest intermolecular interactions and they are not or are only barely sufficient to overcome the desolvation penalty even with the aid of the favorable intramolecular contributions. As a result, the binding affinity of these two systems do not or only barely benefit from electrostatics contributions.
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PMID:Relative contributions of desolvation, inter- and intramolecular interactions to binding affinity in protein kinase systems. 1575 3

Neurite growth is influenced by many factors, including the availability of trophic support as well as the extracellular environment. In this study, we have investigated whether attachment to a permissive culture substrate such as laminin is sufficient to promote neurite outgrowth from dorsal root ganglion neurons in the absence of added nerve growth factor (NGF) and whether this attachment can enhance the response of these neurons to NGF. Adult dorsal root ganglia neurons plated on surfaces coated with a thin film of laminin exhibited increased neurite outgrowth. This effect was integrin-dependent as it was attenuated by treatment with RGD (arginine-glycine-aspartate) peptides and by a beta1-integrin blocking antibody. The addition of NGF resulted in a significant increase in the integrin-dependent outgrowth. We have correlated this increase in growth with increased expression of integrin subunits and activation of known downstream signaling intermediates such as focal adhesion kinase, Src, and Akt. We have also examined pathway cooperation through the use of an Src-specific inhibitor, PP2, and a beta1-integrin blocking antibody, beta1i, by observing downstream signaling intermediates in both integrin and growth factor signaling pathways. These results are among the first to detail the importance of interactions between neurotrophin- and integrin-activated signaling in adult primary neurons.
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PMID:Integrin activation and neurotrophin signaling cooperate to enhance neurite outgrowth in sensory neurons. 1584 70

Aldosterone is currently recognized as a risk hormone for cardiovascular disease. However, the cellular mechanism by which aldosterone acts on vasculature has not been well understood. In the present study, we investigated whether aldosterone affects angiotensin-converting enzyme (ACE) gene expression in rat endothelial cells. Cultured rat aortic endothelial cells (RAECs) from Sprague-Dawley rats were used in the study. ACE mRNA levels and its enzyme activities in RAECs were examined by real-time RT-PCR and enzyme assay using hippuryl-His-Leu as substrates, respectively. Aldosterone significantly increased steady-state ACE mRNA levels and its enzymatic activities. This effect was dose dependent and time dependent and abolished by mineralocorticoid receptor antagonist spironolactone or transcription inhibitor actinomycin D. Dexamethasone also increased steady-state ACE mRNA levels, whose effect was completely blocked by glucocorticoid receptor antagonist RU486, but not by spironolactone. By contrast, the aldosterone-induced ACE mRNA expression was only partially blocked by RU486. The stimulatory effect of aldosterone on ACE mRNA expression was completely blocked by a protein tyrosine kinase inhibitor (genistein) and JAK2 inhibitor (AG490), partially by Src kinase inhibitor (PP2) and epidermal growth factor receptor kinase inhibitor (AG1478), but not by platelet-derived growth factor receptor kinase inhibitor (AG1296). Transfection of dominant-negative JAK2 construct, but not wild-type construct, significantly blocked the aldosterone-induced ACE mRNA up-regulation. Furthermore, aldosterone induced phosphorylation of JAK2, whose effect was blocked by spironolactone and actinomycin D. In conclusion, the present study demonstrates for the first time that aldosterone induces ACE gene expression and its enzyme activity mainly via a mineralocorticoid receptor-mediated and JAK2-dependent pathway in rat endothelial cells. This may constitute a positive feedback loop for a local renin-angiotensin system, possibly involved in the development of aldosterone-induced endothelial dysfunction and vascular injury.
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PMID:Aldosterone induces angiotensin converting enzyme gene expression via a JAK2-dependent pathway in rat endothelial cells. 1593 31

Exposure of cultured cerebellar granule neurons (24 h serum-starved) during 3 min to 30% hyposmotic medium activated the tyrosine kinase receptor ErbB4 in the absence of its ligand. Hyposmolarity also activated the non-receptor tyrosine kinases, Src, focal adhesion kinase (FAK), extracellular signal-regulated protein kinase (ERK)1/2, and the tyrosine kinase target phosphatidyl-inositol-3-kinase (PI3K). The hyposmotic-induced activation of these kinases required the prior phosphorylation of ErbB4 as shown by the effect of ErbB4 blockade with AG213 reducing by 85-95% the phosphorylation of FAK and ERK1/2, by 74% and 36% that of PI3K and Src, respectively. These results suggest a key role of ErbB4 as a signal integrator of events associated with hyposmolarity. PI3K seems to be an important connecting element in the signaling network evoked by the hyposmolarity/ErbB4 activation as: (i) the p85 regulatory subunit of PI3K co-immunoprecipitates with ErbB4 and with FAK; (ii) PI3K blockade with wortmannin reduced the hyposmotic activation of FAK (90%) and ERK1/2 (84-91%). Inhibition of Src with PP2 reduced ErbB4 phosphorylation and inhibited the subsequent cytosolic kinase activation with the same potency as ErbB4 blockade. These results point to Src and ErbB4 and as early targets of the hyposmotic stimulus and osmosignaling. The functional significance for cell volume regulation of the ErbB4-Src-PI3K signaling cascade is indicated by the 48-66% decrease of the hyposmotic taurine efflux observed by inhibition of these kinases.
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PMID:Hyposmolarity-induced ErbB4 phosphorylation and its influence on the non-receptor tyrosine kinase network response in cultured cerebellar granule neurons. 1593 39


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