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

Recent studies suggest that ischemia activates Src and members of the mitogen-activated protein (MAP) kinase superfamily and their downstream effectors, including big MAP kinase 1 (BMK1) and p90 ribosomal S6 kinase (p90RSK). It has also been reported that adenosine is released during ischemia and involved in triggering the protective mechanism of ischemic preconditioning. To assess the roles of Src and adenosine in ischemia-induced MAP kinases activation, we utilized the Src inhibitor PP2 (4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and the adenosine receptor antagonist 8-(p-sulfophenyl) theophylline (SPT) in perfused guinea pig hearts. PP2 (1 microm) inhibited ischemia-induced Src, BMK1 and JNK activation but not JAK2 and p38 activation. SPT inhibited ischemia-mediated p38 and JNK activation. These results demonstrate that Src family kinase and adenosine regulate MAP kinases by parallel pathways. Preconditioning significantly improved both recovery of developed pressure and dp/dt in isolated guinea pig hearts. Since the protective effect of preconditioning was blocked by PP2 (1 microm) and SPT (50 microm), we next investigated the regulation of Src, MAP kinases and p90RSK during preconditioning. The activity and time course of ERK1/2 was not changed, but p90RSK activation by reperfusion was completely inhibited by preconditioning. In contrast, the activation by ischemia of Src, BMK1, p38 and JNK was significantly faster in preconditioned hearts. Maximal BMK1 activation by ischemia was also significantly enhanced by preconditioning. These data suggest important roles for Src family kinases and adenosine in mediating preconditioning, and suggest specific roles for individual MAP kinases in preconditioning.
J Mol Cell Cardiol 2001 Nov
PMID:Src family kinase and adenosine differentially regulate multiple MAP kinases in ischemic myocardium: modulation of MAP kinases activation by ischemic preconditioning. 1170 43

Quantification of mRNAs from extremely small human samples remains a challenge. Requiring minimal amounts of tissue and no post-reaction manipulation, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is an attractive method to quantitatively assess the expression of rare mRNAs. We evaluated the applicability of the technique on RNA extracted from human endomyocardial biopsies and isolated cardiomyocytes, and compared the technique to the RT-competitive PCR approach. Primers and probes were designed to amplify the three subtypes of human beta -adrenoceptors (beta1-, beta2- and beta3 AR), as well as reference genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Hypoxanthine-guanine phosphoribosyltransferase (HPRT), and the oncogene ABL by real-time RT-PCR. Specific primers and a deleted competitor were synthetized to compare the quantitation of the beta 3 AR mRNA expression by RT-competitive PCR. We validated the technique on human cardiomyocytes either freshly isolated or selectively excised from fixed sections of human myocardium by Laser Capture Microdissection. The standard curves obtained for the cDNA's analysed showed mean slopes comprised between -3.3 and -3.7. Inter- and intra-assay variability of gene quantitation was reflected by mean values of the variance coefficients of Ct of 4.84+/-1.13% and 2.73+/-0.39% or 3.32+/-1.03% and 2.21+/-0.24% (corresponding to percent variances of copy numbers of 83.07+/-12.72% and 34.45+/-9.03% or 47.40+/-8.59% and 23.83+/-3.16%) for human beta3 AR and GAPDH genes, respectively. The expression of GAPDH, HPRT and ABL mRNA was characterized by a very low dispersion of individual values across cardiac pathologies, suggesting that these genes may be used as reference genes in quantitative PCR studies. Finally, we applied the technique to detect rare mRNAs, such as beta -AR mRNAs, from small human endomyocardial biopsies and even isolated cardiomyocytes. Real-time RT-PCR is appropriate to quantitate rare messenger RNAs, including in extremely small human tissue samples. This method appears very promising for futures studies of gene expression in several pathophysiological conditions, including heart failure.
J Mol Cell Cardiol 2001 Dec
PMID:Real-time RT-PCR for the detection of beta-adrenoceptor messenger RNAs in small human endomyocardial biopsies. 1173 59

Urocortin (Ucn), is a peptide related to hypothalamic corticotrophin-releasing factor (CRF) and binds with a high affinity to the CRF-R2 beta receptor which is expressed in the heart. Ucn promotes cardiac myocyte survival against hypoxia reoxygenation (HR) injury and this involves activation of the mitogen activated protein kinase pathway (MEK1/2 p42/44 MAPK). In this study we report that Ucn stimulates the phosphorylation of protein kinase B (PKB/Akt) via phosphatidylinositol (PI) 3-OH kinase (PI-3 kinase). To investigate the signalling pathways that mediate the anti-apoptotic and cell survival effect of Ucn in hypoxia reoxygenation (HR), gene based inhibitors of MEK1/2, PI-3 kinase and Akt were over-expressed in rat neonatal cardiac myocytes and cell survival effects against HR were assessed. The dominant negative mutants of the MEK1/2, PI-3 kinase and Akt inhibited Ucn mediated cardioprotection in HR and active PI-3 kinase was itself cardioprotective. In addition, chemical inhibitors of the PI-3 kinase pathway, wortmannin and LY294002 inhibit Ucn mediated cardioprotection in HR in both neonatal and adult cardiac myocytes. Hence the PI-3 kinase/Akt pathway is required in addition to MEK1/2 to mediate Ucn cardioprotection in HR. To our knowledge this is the first report of the activation of the PI-3 kinase/Akt pathway by a member of the CRF family of peptides.
J Mol Cell Cardiol 2002 Apr
PMID:Activation of protein kinase B/Akt by urocortin is essential for its ability to protect cardiac cells against hypoxia/reoxygenation-induced cell death. 1199 36

Activation of the local and systemic renin-angiotensin system is directly and indirectly involved in mechanisms of vascular remodeling during chronic hypertension. This study investigated the effect of angiotensin II (AII) on rat vascular smooth muscle cell (VSMC) migration towards platelet-derived growth factor-BB (PDGF-BB) in vitro. Pre-treatment with AII (1 microM) for 48 or 72 h induced a significant increase in PDGF-BB-directed migration by 77 +/- 21 % and 58 +/- 24 %, respectively (both p < 0.01). This effect was concentration dependent and inhibited by the selective angiotensin receptor type I (AT(1)) blocker DUP 753. PDGF-directed migration of VSMCs was significantly inhibited by antibodies against beta(3)-and beta(5)-integrins, indicating an important role of these integrins in VSMC migration. However, AII augmented migration was not accompanied by an increased expression of beta(3)- and beta(5)-integrin mRNA and protein levels in VSMCs. Inhibition of the mitogen-activated protein kinase ERK 1/2 with PD 98059 (30 microM) completely abolished the effect of AII on PDGF-BB-directed VSMC migration (p < 0.01). The proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK) are cytoskeleton-associated protein kinases participating in integrin-dependent signaling. Therefore, expression and phosphorylation of these kinases was determined 48 h after AII treatment, revealing a significant increase in Pyk2 and FAK protein levels (up to 2-fold, both p < 0.05) and increased phosphorylation of Pyk2 (2-fold, p < 0.05) and ERK 1/2 (4-fold, p < 0.05) as compared to controls. Furthermore, immunofluorescence and Western blot analysis demonstrated a translocation of Pyk2 from the plasma membrane to the cytosol, as well as a perinuclear enrichment of ERK 1/2 protein 48 h after AII treatment. In conclusion, our data suggest that changes in the levels of Pyk2 and ERK 1/2 phosphorylation, responsible for integrin-dependent signaling, as well as their subcellular translocation are important for the enhanced chemotactic response of VSMCs after AII pre-treatment.
Basic Res Cardiol 2002 Jul
PMID:Angiotensin II-augmented migration of VSMCs towards PDGF-BB involves Pyk2 and ERK 1/2 activation. 1211 Oct 44

Although the cardioprotection of late preconditioning (PC) is known to be mediated by both inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), the signaling mechanism responsible for COX-2 upregulation and the interaction between iNOS and COX-2 remain unknown. A total of 122 mice were used to address this issue. In wild-type mice preconditioned with six cycles of 4-min coronary occlusion-4-min reperfusion, ischemic PC resulted in rapid activation of nuclear STAT1/3 through tyrosine phosphorylation (STAT1: 339 +/- 48% of control; STAT3: 389 +/- 46% of control) and increased STAT1/3-DNA binding activity (687 +/- 58% of control) at 30 min after PC, with subsequent upregulation of COX-2 protein (373 +/- 60% of control) and activity(increased myocardial levels of PGE2, PGF(2alpha), and 6-keto-PGF(1alpha)) at 24 h. However, COX-1 protein was not changed 24 h after ischemic PC. Pretreatment with the Janus tyrosine kinase (JAK) inhibitor AG-490 before the six occlusion-reperfusion cycles blocked both the tyrosine phosphorylation of STAT1/3 and the subsequent upregulation of COX-2 protein, demonstrating a necessary role of the JAK-STAT pathway in the induction of COX-2. Targeted disruption of the iNOS gene (iNOS-/-) did not block the increased expression of COX-2 protein 24 h after ischemic PC but completely blocked the increase in COX-2 activity, whereas targeted disruption of the COX-2 gene (COX-2-/-) did not alter ischemic PC-induced iNOS induction. Immunoprecipitation of preconditioned heart tissues with anti-COX-2 antibodies followed by immunoblotting with anti-iNOS antibodies revealed that the increased iNOS protein co-precipitated with COX-2. We conclude that (i) the upregulation of COX-2 protein expression after ischemic PC is mediated by a JAK1/2-STAT1/3-signaling cascade; (ii) COX-2 activity requires upregulated iNOS and iNOS-derived NO; and (iii) COX-2 forms complexes with iNOS, supporting a direct interaction between these two proteins. To our knowledge, this is the first evidence that myocardial COX-2 is upregulated via a JAK1/2-STAT1/3 pathway.
J Mol Cell Cardiol 2003 May
PMID:Mechanism of cyclooxygenase-2 upregulation in late preconditioning. 1273 34

In pressure-overloaded myocardium, our recent study demonstrated cytoskeletal assembly of c-Src and other signaling proteins which was partially mimicked in vitro using adult feline cardiomyocytes embedded in three-dimensional (3D) collagen matrix and stimulated with an integrin-binding Arg-Gly-Asp (RGD) peptide. In the present study, we improved this model further to activate c-Src and obtain a full assembly of the focal adhesion complex (FAC), and characterized c-Src localization and integrin subtype(s) involved. RGD dose response experiments revealed that c-Src activation occurs subsequent to its cytoskeletal recruitment and is accompanied by p130Cas cytoskeletal binding and focal adhesion kinase (FAK) Tyr925 phosphorylation. When cardiomyocytes expressing hexahistidine-tagged c-Src via adenoviral gene delivery were used for RGD stimulation, the expressed c-Src exhibited relocation: (i) biochemical analysis revealed c-Src movement from the detergent-soluble to the -insoluble cytoskeletal fraction and (ii) confocal microscopic analysis showed c-Src movement from a nuclear/perinuclear to a sarcolemmal region. RGD treatment also caused sarcolemmal co-localization of FAK and vinculin. Characterization of integrin subtypes revealed that beta3, but not beta1, integrin plays a predominant role: (i) expression of cytoplasmic domain of beta1A integrin did not affect the RGD-stimulated FAC formation and (ii) both pressure-overloaded myocardium and RGD-stimulated cardiomyocytes exhibited phosphorylation of beta3 integrin at Tyr773/785 sites but not beta1 integrin at Thr788/789 sites. Together these data indicate that RGD treatment in cardiomyocytes causes beta3 integrin activation and c-Src sarcolemmal localization, that subsequent c-Src activation is accompanied by p130Cas binding and FAK Tyr925 phosphorylation, and that these events might be crucial for growth and remodeling of hypertrophying adult cardiomyocytes.
J Mol Cell Cardiol 2003 Jun
PMID:Focal complex formation in adult cardiomyocytes is accompanied by the activation of beta3 integrin and c-Src. 1278 85

We have found that neuregulin-1beta (NRG-1beta) is expressed in the cardiac microvascular endothelium, and promotes the growth and survival of cardiac myocytes in culture through the activation of erbB2 and erbB4 receptor tyrosine kinases. In this study, we examined the role of NRG-1/erbB signaling in protection of cardiac myocytes from anthracycline-induced apoptosis in vitro to determine the coupling between erbB receptor subtypes and cytoprotective signaling. Treatment of neonatal rat ventricular myocytes with NRG-1beta inhibited daunorubicin-induced apoptosis as shown by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining for DNA fragmentation as well as flow cytometric quantification of apoptotic myocytes. Daunorubicin-induced activation of caspase-3 in cardiomyocytes was similarly inhibited by NRG-1beta. The phosphoinositol-3-kinase (PI3-kinase) inhibitor wortmannin prevented the effects of NRG-1beta on daunorubicin-induced apoptosis and activation of caspase-3. NRG-1beta treatment induced rapid activation of Akt/PKB that was inhibited by wortmannin, and adenoviral-mediated overexpression of a dominant-negative Akt prevented the protective effect of NRG-1beta. Akt activation by NRG-1beta was prevented by the tyrphostin AG1478, which we show inhibits erbB4 activation by NRG-1beta. In contrast, the erbB2-specific tyrphostin AG879 had no effect on NRG-1beta activation of Akt. Myocyte treatment with an activating antibody to erbB2 caused phosphorylation of erbB2, and led to activation of Erk but not Akt. Treatment with the erbB2 antibody had no effect on anthracycline-induced apoptosis. Thus, NRG-1beta protects against anthracycline-induced apoptosis via erbB4-dependent activation of the PI3-kinase/Akt pathway.
J Mol Cell Cardiol 2003 Dec
PMID:Neuregulin-1 protects ventricular myocytes from anthracycline-induced apoptosis via erbB4-dependent activation of PI3-kinase/Akt. 1465 73

Rat neonatal ventricular myocytes exposed to simulated ischaemia and reperfusion (SI/R) were used as an in vitro model to delineate the role(s) of extracellular signal-regulated kinase (ERK), p38 and c-Jun NH(2)-terminal protein kinase (JNK), as well as PKB in apoptosis. Exposure of the myocytes to SI (simulated ischaemia - energy depletion induced by KCN and 2-deoxy- D-glucose) reduced cell viability, as measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, and stimulated apoptosis as evidenced by caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage. However, morphological evidence of increased apoptosis, detected by staining with Hoechst 33342, was only seen in response to reperfusion. This suggests that although ischaemic conditions are sufficient to induce cellular markers of apoptosis (PARP cleavage and caspase-3 activation), reperfusion is required to complete the apoptotic pathway in these cells. Furthermore, SI resulted in a rapid, strong, biphasic activation of p38 concomitant with a weak and transient activation of the two ERK isoenzymes, p42/p44-MAPK. Reperfusion for 5 minutes resulted in a strong phosphorylation of p42/p44-MAPK, while no additional p38 activation was seen at this stage. On the other hand, p46/p54-MAPK (JNK) was phosphorylated in response to 5 minutes of reperfusion only and not during SI alone. A peak of PKB/Akt (Ser(473)) activity was seen within 5 minutes of exposure to SI, whereas PKB/Akt (Thr(308)) phosphorylation remained at the baseline level. Both PKB/Akt phosphorylation sites (Ser(473) and Thr(308)) were phosphorylated after 5 minutes of reperfusion. Inhibition of PI-3-kinase activity, using wortmannin, decreased phosphorylation on both sites during SI. However, only SI/R-induced PKB/Akt phosphorylation on Thr(308) was reduced by wortmannin. Myocytes pre-treated with SB203580, a p38-inhibitor, displayed a significant increase in cell viability [63.67 +/- 1.85 to 84.33 +/- 4.8% (p < 0.05)] and attenuation of the apoptotic index during SI/R [22.6 +/- 2.94% to 9 +/- 0.43% (p < 0.001)], while SP600125, a specific JNK inhibitor, caused a significant increase in caspase-3 activation [1.66 +/- 0.03 fold to 2.56 +/- 0.27 fold (p < 0.001)] and apoptotic index [22.6 +/- 2.94% to 32.75 +/- 6.13% (p < 0.05)]. However, PD98059, an ERK inhibitor, failed to affect apoptosis during SI/R. Inhibition of PI-3-kinase prevented the increase in mitochondrial viability usually observed during reperfusion. Interestingly, wortmannin caused a significant increase in PARP cleavage during reperfusion, but had no effect on caspase-3 activation or the apoptotic index. Our results suggest that p38 has a pro-apoptotic role while JNK phosphorylation is protective in our cell model and that these kinases act via caspase-3 to prevent or promote cell survival in response to SI/R-induced injury.
Basic Res Cardiol 2004 Sep
PMID:p38 and JNK have distinct regulatory functions on the development of apoptosis during simulated ischaemia and reperfusion in neonatal cardiomyocytes. 1530 13

The immediate protective effect of erythropoietin (EPO) against ischemia in heart suggests a role beyond hematopoiesis and the treatment of anemia. We determined the role of JAK/STAT and Ras/Rac/MAPK in the protective effect of EPO against ischemia-reperfusion injury in infant rabbit heart. EPO (1.0 U/ml) administered 15 minutes prior to 30-minutes global ischemia and 35 minutes reperfusion resulted in increased recovery of postischemic ventricular developed pressure in rabbit hearts. EPO exerted its immediate cardioprotective effect via activation of multiple signaling pathways by: 1) phosphorylation and activation of JAK1/2, STAT3 and STAT5A but not of STAT1alpha and STAT5B, 2) phosphorylation and activation of PI(3) kinase and its downstream kinases Akt and Rac, 3) activation of PKCepsilon, Raf, MEK1/2, p42/44 MAPK and p38 MAPK. Pretreatment with Wortmannin abolished EPO-induced Akt activation and phosphorylation. Pretreatment with Chelerythrine followed by EPO treatment resulted in partial inhibition of Raf activation, and abolished PKCepsilon and p38 MAPK activation without any effect on Akt, MEK1/2 and p42/44 MAPK. PD98059 abolished MEK1/2 and p42/44 MAPK activation with no effect on Akt, Raf and p38 MAPK activation. SB203580 inhibited only p38 MAPK activation by EPO. We can conclude EPO increases immediate cardioprotection through the activation of multiple signal transduction pathways.
Basic Res Cardiol 2005 May
PMID:Erythropoietin protects the infant heart against ischemia-reperfusion injury by triggering multiple signaling pathways. 1561 43

Thyroid hormone is known to cause hypertrophy, tachycardia, vasorelaxation, and enhanced contractile function. The exact mechanisms responsible for these effects are unknown but classical regulation of gene expression through binding to nuclear receptors has been widely implicated. Data have also accumulated suggesting that TH can exert effects through non-classical mechanisms involving activation of signal transduction pathways. Whether thyroid hormone can activate signal transduction pathways in the heart is unknown. In this study, we treated neonatal rat cardiomyocytes with T3 and determined the expression and phosphorylation of signaling molecules. T3 caused specific activation of Akt/PKB signaling after 24 h of treatment. Since Akt is known to protect against cell death, cells were serum-starved in the presence or absence of T3 to determine whether T3 could protect against serum starvation-induced cell death. Indeed, myocytes treated with T3 displayed enhanced sarcomeric structure after 4 days of serum starvation. T3 increased cell viability as measured by MTT assays, prevented DNA laddering, and reduced TUNEL positive cells, which was associated with increased phosphorylated Akt and glycogen synthase kinase 3beta (GSK-3beta). The protective effect of T3 on cell viability, DNA laddering and TUNEL positive cells were blocked by LY294002, a phosphoinositide-3 kinase (PI3K) inhibitor that blocks Akt signaling. Overall these data suggest that T3 can activate Akt in cardiomyocytes which protects myocytes against cell death.
J Mol Cell Cardiol 2005 Nov
PMID:Thyroid hormone activates Akt and prevents serum starvation-induced cell death in neonatal rat cardiomyocytes. 1617 8


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