Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Intestinal epithelial cells are subject to repetitive deformation during peristalsis and villous motility, whereas the mucosa atrophies during sepsis or ileus when such stimuli are abnormal. Such repetitive deformation stimulates intestinal epithelial proliferation via focal adhesion kinase (FAK) and extracellular signal-regulated kinases (ERK). However, the upstream mediators of these effects are unknown. We investigated whether Src and Rac1 mediate deformation-induced FAK and ERK phosphorylation and proliferation in human Caco-2 and rat IEC-6 intestinal epithelial cells. Cells cultured on collagen-I were subjected to an average 10% cyclic strain at 10 cycles/min. Cyclic strain activated Rac1 and induced Rac1 translocation to cell membranes. Mechanical strain also induced rapid sustained phosphorylation of c-Src at Tyr(418), Rac1 at Ser(71), FAK at Tyr(397) and Tyr(576), and ERK1/2 at Thr(202)/Tyr(204). The mitogenic effect of cyclic strain was blocked by inhibition of Src (PP2 or short interfering RNA) or Rac1 (NSC23766). Src or Rac1 inhibition also prevented strain-induced FAK phosphorylation at Tyr(576) and ERK phosphorylation but not FAK phosphorylation at Tyr(397). Reducing FAK using short interfering RNA blocked strain-induced mitogenicity and attenuated ERK phosphorylation but not Src or Rac1 phosphorylation. Src inhibition blocked strain-induced Rac1 phosphorylation, but Rac inhibition did not alter Src phosphorylation. Transfection of a two-tyrosine phosphorylation-deficient FAK mutant Y576F/Y577F prevented activation of cotransfected myc-ERK2 by cyclic strain. Repetitive deformation induced by peristalsis or villus motility may support the gut mucosa by a pathway involving Src, Rac1, FAK, and ERK. This pathway may present important targets for interventions to prevent mucosal atrophy during prolonged ileus or fasting.
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PMID:Repetitive deformation activates focal adhesion kinase and ERK mitogenic signals in human Caco-2 intestinal epithelial cells through Src and Rac1. 1708 51

Severe neurologic sequelae have been reported with the use of lidocaine after spinal anesthesia. This is considered a consequence of the high concentrations reached in the cerebrospinal fluid. We have previously shown that lidocaine increases the phosphorylation of focal adhesion kinase (FAK, a nonreceptor tyrosine kinase playing a role in neuronal plasticity and cell death). Here, we compared the effects of lidocaine and bupivacaine on FAK phosphorylation and cleaved caspase 3 expression in rat hippocampal slices. Slices were treated with increasing concentrations of lidocaine (4.3 nM to 4.3 mM) or bupivacaine (3.4 nM to 3.4 mM) in the presence or absence of the specific inhibitor of the FAK tyrosine kinase PP2 (10 microM). Caspase 3 expression and FAK phosphorylation were examined by immunoblotting. Lidocaine induced a concentration-related increase in FAK phosphorylation while the bupivacaine effect was biphasic. The maximal effect observed with millimolar lidocaine concentrations was significantly more than with clinically equipotent bupivacaine concentrations (4.3 x 10(-3) M lidocaine: 168% +/- 20%, mean value +/- sd; 10(-3) M bupivacaine: 145% +/- 19% P < 0.001). The expression of cleaved caspase 3 was increased by lidocaine, but not bupivacaine, at millimolar concentrations and was blocked by PP2. Our results indicate that millimolar concentrations of lidocaine, but not bupivacaine, increase cleaved caspase 3 expression. The role of FAK phosphorylation in this effect remains to be clarified.
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PMID:The effects of lidocaine and bupivacaine on protein expression of cleaved caspase 3 and tyrosine phosphorylation in the rat hippocampal slice. 1717 55

Idiopathic pulmonary arterial hypertension (iPAH) is associated with human herpesvirus 8 (HHV8) infection and demonstrates pathological angiogenesis similar to that observed with another HHV8-linked disease, namely Kaposi Sarcoma (KS). Importantly, the HHV8 encoded viral G-protein-coupled receptor (vGPCR) induces KS lesions in a murine model. Investigating the impact of vGPCR expression on the angiogenic activity of human pulmonary arterial endothelial cells (HPAEC) can yield insight into the pathobiology of HHV8-associated vascular disorders, particularly PAH. Cultured HPAECs were transduced with retroviral vectors carrying either control or vGPCR coding regions. vGPCR expression selectively activated matrix metalloproteinase (MMP)-2, a pivotal matrix modulating enzyme during angiogenesis. A membrane type 1 MMP (MT1-MMP) neutralizing antibody and the tissue inhibitor of metalloproteinases-2 (TIMP-2) independently blocked vGPCR-induced MMP-2 activation. vGPCR expression concordantly promoted MMP-2 activation by increasing MT1-MMP expression while decreasing TIMP-2 expression. vGPCR activated Src kinase as demonstrated by phosphorylation of Src and its substrate focal adhesion kinase (FAK). vGPCR promoted angiogenesis of HPAECs as demonstrated by a substantial increase in tubulogenesis in vitro. The Src inhibitors PP2 and SU6656 significantly diminished vGPCR-induced MMP-2 activation and tubulogenesis. Our findings indicate that vGPCR induces MMP-2 activation in HPAECs through regulation of MT1-MMP and TIMP-2 expression. vGPCR activates Src and inhibition of such activation abrogates proMMP-2 activation and in vitro angiogenesis induced by vGCPR. The current study implicates vGPCR as an etiological agent in iPAH and identifies Src and MMP-2 as potential therapeutic targets in HHV8 associated KS and iPAH.
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PMID:Activation of proMMP-2 and Src by HHV8 vGPCR in human pulmonary arterial endothelial cells. 1722 63

Substratum surface topography is a powerful modulator of cell behaviour, but how it influences intracellular signaling is largely unknown. We investigated the influence of microfabricated topographies on the activation of nonreceptor tyrosine kinases Src, FAK and ERK 1/2, as well as the transcription factor, Runx2, in rat osteoblasts, cultured on substrata that varied in their ability to promote bone-like tissue formation. Total tyrosine phosphorylation increased on grooves, tapered pits, and gap cornered boxes, relative to the levels found on smooth surfaces, with the greatest activity at 1 week. Src levels was higher on smooth than on any other surface, but FAK and ERK 1/2 phosphorylation were highest on groove and gap-cornered boxes up to 6 weeks. Inhibition of Src phosphorylation with PP2 inhibited FAK and ERK 1/2 phosphorylation on grooves, but had no detectable effect on either FAK or ERK 1/2 on smooth substratum. We suggest that osteoblast response to substrata with specific topographical features requires FAK-Y397-Src-Y416 complexes for ERK 1/2 phosphorylation, but on smooth surfaces, Src independent methods of ERK 1/2 activation are present.
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PMID:The effect of substratum topography on osteoblast adhesion mediated signal transduction and phosphorylation. 1721 38

Pulmonary epithelial cells are exposed to repetitive deformation during physiological breathing and mechanical ventilation. Such deformation may influence pulmonary growth, development, and barotrauma. Although deformation stimulates proliferation and activates extracellular signal-regulated kinases (ERK1/2) in human pulmonary epithelial H441 cells, the upstream mechanosensors that induce ERK activation are poorly understood. We investigated whether c-Src or focal adhesion kinase (FAK) mediates cyclic mechanical strain-induced ERK1/2 activation and proliferation in human pulmonary epithelial (NCI-H441) cells. The H441 and A549 cells were grown on collagen I-precoated membranes and were subjected to an average 10% cyclic mechanical strain at 20 cycles/min. Cyclic strain activated Src within 2 min by increasing phosphorylation at Tyr(418), followed by rapid phosphorylation of FAK at Tyr(397) and Tyr(576) and ERK1/2 at Thr(202)/Tyr(204) (n = 5, P < 0.05). Twenty-four (A549 cells) and 24-72 h (H441 cells) of cyclic mechanical strain increased cell numbers compared with static culture. Twenty-four hours of cyclic strain also increased H441 FAK, Src, and ERK phosphorylation without affecting total FAK, Src, or ERK protein. The mitogenic effect was blocked by Src (10 micromol/l PP2 or short interfering RNA targeted to Src) or MEK (50 micromol/l PD-98059) inhibition. PP2 also blocked strain-induced phosphorylation of FAK-Tyr(576) and ERK-Thr(202)/Tyr(204) but not FAK-Tyr(397). Reducing FAK by FAK-targeted short interfering RNA blocked mechanical strain-induced mitogenicity and significantly attenuated strain-induced ERK activation but not strain-induced Src phosphorylation. Together, these results suggest that repetitive mechanical deformation induced by ventilation supports pulmonary epithelial proliferation by a pathway involving Src, FAK, and then ERK signaling.
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PMID:Src and focal adhesion kinase mediate mechanical strain-induced proliferation and ERK1/2 phosphorylation in human H441 pulmonary epithelial cells. 1721 24

The results presented here demonstrate that focal adhesion kinase (FAK) Tyr-861 is the predominant tyrosine phosphorylation site stimulated by hyperosmotic stress in a variety of cell types, including epithelial cell lines (ileum-derived IEC-18, colon-derived Caco2, and stomach-derived NCI-N87), FAK null fibroblasts re-expressing FAK, and Src family kinase triple null fibroblasts (SYF cells) in which c-Src has been restored (YF cells). We show that hyperosmotic stress-stimulated FAK phosphorylation in epithelial cells is inhibited by Src family kinase inhibitors PP2 and SU6656 and that it does not occur in SYF cells. Unexpectedly, hyperosmotic stress-induced phosphorylation of FAK at Tyr-397, Tyr-576, and most dramatically at Tyr-861 was completely insensitive to the F-actin-disrupting agents, latrunculin A and cytochalasin D. Finally, we show that in FAK null cells exposed to hyperosmotic stress or growth factor withdrawal, re-expression of wild type FAK restored cell survival, whereas re-expression of FAK mutated from tyrosine to phenylalanine at position 861 (FAKY861F) did not. Our results indicate that FAK Tyr-861 phosphorylation is required for mammalian cell survival of hyperosmotic stress. Furthermore, the results suggest that FAK is an upstream regulator (rather than downstream effector) of F-actin reorganization in response to hyperosmotic stress. We propose that FAK/c-Src bipartite enzyme is a sensor of cytoplasmic shrinkage, and that the phosphorylation on FAK Tyr-861 by Src and subsequent reorganization of F-actin can initiate an anti-apoptotic signaling pathway that protects cells from hyperosmotic stress.
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PMID:Preferential phosphorylation of focal adhesion kinase tyrosine 861 is critical for mediating an anti-apoptotic response to hyperosmotic stress. 1728 81

Cancer cell adhesion is traditionally viewed as random, occurring if the cell's receptors match the substrate. Cancer cells are subjected to pressure and shear during growth against a constraining stroma, surgical manipulation, and passage through the venous and lymphatic system. Cells shed into a cavity such as the abdomen postoperatively also experience increased pressure from postoperative edema. Increased extracellular pressure stimulates integrin-mediated cancer cell adhesion via FAK and Src. PI 3-kinase (PI3K) inhibitors (LY294002 or wortmannin), Akt inhibitors, or Akt1 siRNA blocked adhesion stimulated by 15 mmHg pressure in SW620 or primary human malignant colonocytes. Pressure activated PI3K, tyrosine-phosphorylated and membrane-translocated the p85 subunit, and phosphorylated Akt. PI3K inhibitor (LY294002) prevented pressure-stimulated Akt Ser473 and FAK Tyr397, but not FAK576 or Src416 phosphorylation. PP2 inhibited PI3K activity and Akt phosphorylation. FAK siRNA did not affect pressure-induced PI3K activation but blocked Akt phosphorylation. Pressure also stimulated FAK or FAKY397F mutant translocation to the membrane. Akt inhibitor IV blocked pressure-induced Akt and FAK translocation. Pressure activated Src- and PI3K-dependently induced p85 interaction with FAK, and FAK with beta1 integrin. These results delineate a novel force-activated inside-out Src/PI3K/FAK/Akt pathway by which cancer cells regulate their own adhesion. These signals may be potential targets for inhibition of metastatic adhesion.
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PMID:FAK association with multiple signal proteins mediates pressure-induced colon cancer cell adhesion via a Src-dependent PI3K/Akt pathway. 1731 26

The chemotactic peptide formyl-methionyl-leucyl-phenilalanine (fMLP) triggers intracellular protein tyrosine phosphorylation leading to neutrophil activation. Deficiency of the Src family kinases Hck and Fgr have previously been found to regulate fMLP-induced degranulation. In this study, we further investigate fMLP signaling in hck-/-fgr-/- neutrophils and find that they fail to activate a respiratory burst and display reduced F-actin polymerization in response to fMLP. Additionally, albeit migration of both hck-/-fgr-/-mouse neutrophils and human neutrophils incubated with the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) through 3-microm pore size Transwells was normal, deficiency, or inhibition, of Src kinases resulted in a failure of neutrophils to migrate through 1-microm pore size Transwells. Among MAPKs, phosphorylation of ERK1/2 was not different, phosphorylation of p38 was only partially affected, and phosphorylation of JNK was markedly decreased in fMLP-stimulated hck-/-fgr-/- neutrophils and in human neutrophils incubated with PP2. An increase in intracellular Ca(2+) concentration and phosphorylation of Akt/PKB occurred normally in fMLP-stimulated hck-/-fgr-/- neutrophils, indicating that activation of both phosphoinositide-specific phospholipase C and PI3K is independent of Hck and Fgr. In contrast, phosphorylation of the Rho/Rac guanine nucleotide exchange factor Vav1 and the Rac target p21-activated kinases were markedly reduced in both hck-/-fgr-/- neutrophils and human neutrophils incubated with a PP2. Consistent with these findings, PP2 inhibited Rac2 activation in human neutrophils. We suggest that Hck and Fgr act within a signaling pathway triggered by fMLP receptors that involves Vav1 and p21-activated kinases, leading to respiratory burst and F-actin polymerization.
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PMID:The Src family kinases Hck and Fgr regulate neutrophil responses to N-formyl-methionyl-leucyl-phenylalanine. 1733 87

In chronic myeloid leukemia (CML), resistance to imatinib is diverse. In addition to BCR-ABL-dependent mechanisms, BCR-ABL-independent mechanisms have been proposed. Here we established and characterized novel CML cell lines, an imatinib-sensitive cell line, MYL, and an imatinib-resistant subline, MYL-R. Treatment with imatinib inhibited phosphorylation of BCR-ABL and CrkL in both MYL and MYL-R, even though imatinib-induced apoptosis was preferentially observed in MYL than MYL-R, indicating that the resistance is based on a BCR-ABL-independent mechanism. MYL-R showed elevated expressions of Lyn mRNA, Lyn protein, phosphorylated Lyn, and phosphorylated STAT5. Silencing of Lyn by short-interfering RNA (siRNA) in MYL-R, but not in MYL, induced significant growth-inhibition, increased caspase-3 activity, and induced partial recovery from imatinib-resistance. Expression of Bcl-2, previously reported to be associated with Lyn-mediated resistance, was not elevated in MYL-R. Expression of Bim, which plays an important role in imatinib-induced cell-killing, was not suppressed in MYL-R. These results imply that diverse mechanisms of resistance exist among cell types. Treatment of MYL-R cells with various reagents known to have anti-leukemic activity revealed that zoledronic acid and the farnesyl transferase inhibitor (SCH 66336) showed strong synergism with imatinib; interferon alpha, PP2, CGP76030, and FK228 (depsipeptide) showed synergism; whereas soluble TRAIL and As2O3 showed additivity or antagonism, and 17-AAG and radicicol showed antagonism. Treatment with either PP2 or zoledronic acid induced greater growth-reduction in MYL-R than MYL. Taken together, Lyn may play an important role in imatinib-resistance in MYL-R. Some novel reagents, including siRNA targeting Lyn, may have good potential to overcome this resistance.
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PMID:Establishment and characterization of a novel imatinib-sensitive chronic myeloid leukemia cell line MYL, and an imatinib-resistant subline MYL-R showing overexpression of Lyn. 1743 77

The specificities of 65 compounds reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70-80 protein kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)-raptor (regulatory associated protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule inhibitors of protein kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes.
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PMID:The selectivity of protein kinase inhibitors: a further update. 1785 Feb 14


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