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)

Recently, we have demonstrated that in PC12 cells activation of the Ras/extracellular signal-regulated kinase pathway in response to membrane depolarization or bradykinin is mediated by calcium-dependent transactivation of the epidermal growth factor receptor (EGFR). Here we address the question whether Ca(2+)-calmodulin-dependent protein kinase (CaM kinase) has a role in the EGFR transactivation signal. Using compounds that selectively interfere with either CaM kinase activity or calmodulin function, we show that KCl-mediated membrane depolarization-triggered, but not bradykinin-mediated signals involve CaM kinase function upstream of the EGFR. Although both depolarization-induced calcium influx and bradykinin stimulation of PC12 cells were found to induce c-fos transcription through EGFR activation, the former signal is CaM kinase-dependent and the latter was shown to be independent. As PYK2 is also activated upon elevation of intracellular calcium, we investigated the potential involvement of this cytoplasmic tyrosine kinase in EGFR transactivation. Interestingly, we observed that inhibition of CaM kinase activity in PC12 cells abrogated tyrosine phosphorylation of PYK2 upon KCl but not bradykinin treatment. Nevertheless, PYK2 activation in response to both stimuli appeared to be mediated by pathways parallel to EGFR transactivation. Our data demonstrate the existence of two distinct calcium-dependent mechanisms leading either to EGFR-mediated extracellular signal-regulated activation or to PYK2 tyrosine phosphorylation. Both pathways either in concert or independently might contribute to the definition of biological responses in neuronal cell types.
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PMID:Distinct calcium-dependent pathways of epidermal growth factor receptor transactivation and PYK2 tyrosine phosphorylation in PC12 cells. 1040 47

Vascular smooth muscle cell (VSMC) proliferation is a prominent feature of the atherosclerotic process occurring after endothelial injury. A vascular wall kallikrein-kinin system has been described. The contribution of this system to vascular disease is undefined. In the present study we characterized the signal transduction pathway leading to mitogen-activated protein kinase (MAPK) activation in response to bradykinin (BK) in VSMC. Addition of 10(-10)-10(-7) M BK to VSMC resulted in a rapid and concentration-dependent increase in tyrosine phosphorylation of several 144- to 40-kDa proteins. This effect of BK was abolished by the B(2)-kinin receptor antagonist HOE-140, but not by the B(1)-kinin receptor antagonist des-Arg(9)-Leu(8)-BK. Immunoprecipitation with anti-phosphotyrosine antibodies followed by immunoblot revealed that 10(-9) M BK induced tyrosine phosphorylation of focal adhesion kinase (p125(FAK)). BK (10(-8) M) promoted the association of p60(src) with the adapter protein growth factor receptor binding protein-2 and also induced a significant increase in MAPK activity. Pertussis and cholera toxins did not inhibit BK-induced MAPK tyrosine phosphorylation. Protein kinase C downregulation by phorbol 12-myristate 13-acetate and/or inhibitors to protein kinase C, p60(src) kinase, and MAPK kinase inhibited BK-induced MAPK tyrosine phosphorylation. These findings provide evidence that activation of the B(2)-kinin receptor in VSMC leads to generation of multiple second messengers that converge to activate MAPK. The activation of this crucial kinase by BK provides a strong rationale to investigate the mitogenic actions of BK on VSMC proliferation in disease states of vascular injury.
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PMID:Mechanisms of MAPK activation by bradykinin in vascular smooth muscle cells. 1044 1

Stimulation of quiescent Swiss 3T3 cells with bombesin induces a rapid increase in the formation of complexes between focal adhesion kinase (FAK) and Src family members, which can be extracted with a buffer containing Triton, deoxycholate, and SDS but not with a buffer containing Triton alone. An increase in complex formation between FAK and Src in response to bombesin could be detected within 1 min, reached a maximum after 10 min, and declined toward base-line levels after 60 min of bombesin treatment. Bradykinin, endothelin, and lysophosphatidic acid also stimulated FAK-Src complex formation. Bombesin stimulated FAK/Src association through a Ca(2+) and phosphatidylinositol 3'-kinase-independent pathway that requires the integrity of the actin filament network and is partly dependent on functional protein kinase C. Treatment with the selective Src kinase inhibitor PP-2 inhibited both FAK activation and phosphorylation of FAK at Tyr(577) induced by bombesin in intact cells. Platelet-derived growth factor at low concentrations (1-10 ng/ml) also induced FAK-Src complex formation via a pathway that depended on the integrity of the actin cytoskeleton and phosphatidylinositol 3'-kinase. Thus, G protein-coupled receptor agonists and platelet-derived growth factor promote complex formation between endogenous FAK and Src in attached cells through different signal transduction pathways.
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PMID:Bombesin and platelet-derived growth factor induce association of endogenous focal adhesion kinase with Src in intact Swiss 3T3 cells. 1049 97

Related adhesion focal tyrosine kinase (RAFTK) (also known as PYK2) is a cytoplasmic tyrosine kinase related to the focal adhesion kinase (FAK) p125(FAK). RAFTK is rapidly phosphorylated on tyrosine residues in response to various stimuli, such as tumor necrosis factor-alpha, changes in osmolarity, elevation in intracellular calcium concentration, lysophosphatidic acid, and bradykinin. Overexpression of RAFTK induces activation of c-Jun amino-terminal kinase (also known as stress-activated protein kinase), mitogen-activated protein kinase (MAPK), and p38 MAPK. The present studies demonstrate that RAFTK binds constitutively to the protein tyrosine phosphatase SHPTP1. In contrast to PTP1B, overexpression of wild-type SHPTP1 blocks tyrosine phosphorylation of RAFTK. The results further demonstrate that RAFTK is a direct substrate of SHPTP1 in vitro. Moreover, treatment of PC12 cells with bradykinin is associated with inhibition in tyrosine phosphorylation of RAFTK in the presence of SHPTP1. Furthermore, in contrast to the phosphatase-dead SHPTP1 C453S mutant, overexpression of wild-type SHPTP1 blocks interaction of RAFTK with the SH2-domain of c-Src and inhibits RAFTK-mediated MAPK activation. Significantly, cotransfection of RAFTK with SHPTP1 did not inhibit RAFTK-mediated c-Jun amino-terminal kinase activation. Taken together, these findings suggest that SHPTP1 plays a negative role in PYK2/RAFTK signaling by dephosphorylating RAFTK.
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PMID:Negative regulation of PYK2/related adhesion focal tyrosine kinase signal transduction by hematopoietic tyrosine phosphatase SHPTP1. 1052 52

The effect of phosphoinositide depletion on focal adhesion kinase (FAK) signaling was investigated in two neuronal cell lines. Treatment of either SH-SY5Y neuroblastoma cells or PC12 cells with wortmannin, at a concentration that inhibits phosphatidylinositol 4-kinase activity, led to a selective depletion of phosphatidylinositol 4-phosphate without significantly altering phosphatidylinositol 4,5-bisphosphate (PIP2) content. An enhanced tyrosine phosphorylation of FAK elicited by agonist occupancy of phospholipase C-coupled receptors (muscarinic cholinergic in SH-SY5Y neuroblastoma or bradykinin in PC12 cells) was blocked completely by wortmannin. Under the above conditions, phosphoinositide resynthesis was prevented, and as a consequence, receptor stimulation led to a marked depletion of PIP2. In contrast, the increased tyrosine phosphorylation of FAK elicited by agents that do not activate phospholipase C (phenylarsine oxide, lysophosphatidic acid, or phorbol ester) persisted in the presence of wortmannin. However, the ability of these agents to elicit an increase in FAK phosphorylation was also prevented if PIP2 was depleted by activation of a phospholipase C-coupled receptor in the presence of wortmannin. The results suggest that agonist-sensitive pools of PIP2 must be maintained for FAK signaling to occur in response to a mechanistically diverse range of stimuli.
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PMID:Attenuation of focal adhesion kinase signaling following depletion of agonist-sensitive pools of phosphatidylinositol 4,5-bisphosphate. 1053 51

The relationship between cell proliferation, protein tyrosine phosphorylation, phosphotyrosine kinase activity and bradykinin receptor activation in rat mesangial cells was investigated. We demonstrated that bradykinin (BK), through the B2 receptor, induced a dose-dependent inhibition of mesangial cell proliferation stimulated by fetal calf serum. We next found that BK induced a dose-dependent inhibition of phospho-tyrosine kinase activity. Treatments with pertussis-toxin, inhibition of phospholipase C and protein kinase C inhibitors and chelation of free cytosolic calcium did not change the bradykinin-induced inhibition of phosphotyrosine kinase. Western blot analysis of phosphotyrosinated proteins demonstrated that BK reduced tyrosine phosphorylation of several proteins among which we identified the 125-focal adhesion kinase. Taken together, these data suggest for the first time that, in proliferating rat mesangial cells, B2 receptor stimulation is able to induce, via a pertussis insensitive pathway, the inhibition of tyrosine kinase activity and mesangial cell proliferation.
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PMID:Bradykinin-induced inhibition of cell proliferation and tyrosine kinase activity in rat mesangial cells. 1060 80

We investigated the relationship between protein tyrosine phosphorylation and bradykinin (BK) receptor activation in rat mesangial cells (MC). Stimulation of the B2 receptor resulted in a dual effect consisting of an independent activation and inhibition of tyrosine kinase activity (TKA). The activation was rapid and transient, reaching a peak value at 30 s whereas the inhibition was observed at 5 min and persisted up to 10 min. Treatments with pertussis-toxin and U73122 showed that only the BK-induced stimulation of TKA is dependent on phospholipase C activation via a pertussis-toxin sensitive G-protein. In addition, BK induced an increase in tyrosine phosphatase activity. Western-blot analysis demonstrated that the dual effect of BK on TKA was associated with both an increase and a decrease in tyrosine phosphorylation of the p125-focal adhesion kinase (FAK). Moreover, BK was able to reduce the maximal stimulated MC cell proliferation induced by fetal calf serum. These data show that in rat MC, B2 receptor stimulation activates and inhibits two independent tyrosine kinase signaling pathways associated with tyrosine phosphorylation of p125-FAK that might be implicated in MC proliferation.
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PMID:Effect of bradykinin on tyrosine kinase and phosphatase activities and cell proliferation in mesangial cells. 1061 90

Substance P (SP) analogues including [d-Arg(1),d-Trp(5,7,9), Leu(11)]SP are broad spectrum neuropeptide antagonists and potential anticancer agents, but their mechanism of action is not fully understood. Here, we examined the mechanism of action of [d-Arg(1), d-Trp(5,7,9),Leu(11)]SP as an inhibitor of G protein-coupled receptor (GPCR)-mediated signal transduction and cellular DNA synthesis in Swiss 3T3 cells. Addition of [d-Arg(1),d-Trp(5,7,9), Leu(11)]SP, at 10 micrometer, caused a striking rightward shift in the dose-response curves of DNA synthesis induced by bombesin, bradykinin, or vasopressin and markedly inhibited the activation of p42(mapk) (ERK-2) and p44(mapk) (ERK-1) induced by these GPCR agonists. In addition, this SP analogue also prevented the protein kinase C-dependent activation of protein kinase D induced by these agonists. [d-Arg(1),d-Trp(5,7,9),Leu(11)]SP, at a concentration (10 micrometer) that inhibited these G(q)-mediated events, also prevented GPCR agonist-induced responses mediated through the G proteins of the G(12) subfamily. These include bombesin-induced assembly of focal adhesions, formation of parallel arrays of actin stress fibers, increase in the tyrosine phosphorylation of focal adhesion kinase (FAK), p130(Cas), and paxillin, and formation of a complex between FAK and Src. We conclude that [d-Arg(1),d-Trp(5,7,9),Leu(11)]SP acts as a mitogenic antagonist of neuropeptide GPCRs blocking signal transduction via both G(q) and G(12).
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PMID:[D-Arg(1),D-Trp(5,7,9),Leu(11)]Substance P inhibits bombesin-induced mitogenic signal transduction mediated by both G(q) and G(12) in Swiss 3T3cells. 1088 May 15

Quenched fluorescence peptides were used to investigate the substrate specificity requirements for recombinant wild-type angiotensin I-converting enzyme (ACE) and two full-length mutants bearing a single functional active site (N- or C-domain). We assayed two series of bradykinin-related peptides flanked by o-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp), namely, Abz-GFSPFXQ-EDDnp and Abz-GFSPFRX-EDDnp (X = natural amino acids), in which the fluorescence appeared when Abz/EDDnp are separated by substrate hydrolysis. Abz-GFSPFFQ-EDDnp was preferentially hydrolyzed by the C-domain while Abz-GFSPFQQ-EDDnp exhibits higher N-domain specificity. Internally quenched fluorescent analogues of N-acetyl-SDKP-OH were also synthesized and assayed. Abz-SDK(Dnp)P-OH, in which Abz and Dnp (2,4-dinitrophenyl) are the fluorescent donor-acceptor pair, was cleaved at the D-K(Dnp) bond with high specificity by the ACE N-domain (k(cat)/K(m) = 1.1 microM(-)(1) s(-)(1)) being practically resistant to hydrolysis by the C-domain. The importance of hydroxyl-containing amino acids at the P(2) position for N-domain specificity was shown by performing the kinetics of hydrolysis of Abz-TDK(Dnp)P-OH and Abz-YDK(Dnp)P-OH. The peptides Abz-YRK(Dnp)P-OH and Abz-FRK(Dnp)P-OH which were hydrolyzed by wild-type ACE with K(m) values of 5.1 and 4.0 microM and k(cat) values of 246 and 210 s(-)(1), respectively, have been shown to be excellent substrates for ACE. The differentiation of the catalytic specificity of the C- and N-domains of ACE seems to depend on very subtle variations on substrate-specific amino acids. The presence of a free C-terminal carboxyl group or an aromatic moiety at the same substrate position determines specific interactions with the ACE active site which is regulated by chloride and seems to distinguish the activities of both domains.
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PMID:Peptidase specificity characterization of C- and N-terminal catalytic sites of angiotensin I-converting enzyme. 1091 58

Protein kinase A (PKA) plays an essential role in the depolarization-induced c-fos expression in PC12 cells although the exact mechanism is unknown. Here we demonstrate that PKA is required for depolarization-induced activation of both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase in PC12 cells. In addition, we have found that the depolarization-induced tyrosine phosphorylation of proline-rich tyrosine kinase (PYK) 2, a key calcium-sensitive upstream mediator of MAP kinase activation, is profoundly blocked by PKA inhibition. In contrast to the depolarization-induced signaling, the ERK and PYK2 activation by bradykinin (1 microM), a G-protein coupled receptor agonist, was not blocked by PKA inhibition. These findings suggest that PKA inhibition prevents depolarization-induced PYK2/MAP kinase pathway activation, thereby inhibiting the early gene expression.
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PMID:Protein kinase A activity is required for depolarization-induced proline-rich tyrosine kinase 2 and mitogen-activated protein kinase activation in PC12 cells. 1092 66


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