Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thrombin-induced endothelial monolayer hyperpermeability is thought to result from increased F-actin stress fiber-related contractile tension, a process regulated by the small GTP-binding protein Rho. We tested whether this process was dependent on the Rho-associated protein kinase, ROCK, using a specific ROCK inhibitor, Y-27632. The effects of Y-27632 on thrombin-induced myosin light chain phosphorylation (MLCP) and tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)) and paxillin were measured by Western blotting. F-actin organization and content were analyzed by digital imaging, and endothelial monolayer permeability was measured in bovine pulmonary artery endothelial cell (EC) monolayers using a size-selective permeability assay. Y-27632 enhanced EC monolayer barrier function due to a decline in small-pore number that was associated with increased EC surface area, reduced F-actin content, and reorganization of F-actin to beta-catenin-containing cell-cell adherens junctions. Although Y-27632 prevented thrombin-induced MLCP, stress fiber formation, and the increased phosphotyrosine content of paxillin and p125(FAK), it attenuated but did not prevent the thrombin-induced formation of large paracellular holes. These data indicate that thrombin-induced stress fiber formation is ROCK dependent. In contrast, thrombin-induced paracellular hole formation occurs in a ROCK-independent manner, whereas thrombin-induced monolayer hyperpermeability appears to be partially ROCK dependent.
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PMID:ROCK mediates thrombin's endothelial barrier dysfunction. 1089 31

Freshly isolated peripheral blood monocytes lack focal adhesion kinase (p125(FAK)) but activate a second member of this kinase family, calcium-dependent tyrosine kinase (CADTK; also known as Pyk2/CAKbeta/RAFTK/FAK2), upon adhesion or stimulation with chemokines. To study the role of CADTK in monocyte adherence and motility, we performed immunocytochemical localization that showed CADTK at the leading edge and ruffling lamellipodial structures in freshly isolated, adhered human monocytes. We next introduced CADTK/CAKbeta-related non-kinase (CRNK), the C-terminal noncatalytic domain of CADTK, into monocytes by electroporation and showed that it inhibited CADTK autophosphorylation. Introduction of the fusion protein glutathione S-transferase (GST)-CRNK also reduced (i) cell spreading, as reflected in a reduced cell area 30 min after adhesion, (ii) adhesion-induced phosphotyrosine increases and redistribution into lamellipodia, and (iii) adhesion-induced extracellular signal-regulated protein kinase (ERK) activation. In control experiments, introduction of GST or GST-C3 transferase (an inhibitor of RhoA GTPase activity) by electroporation did not affect these parameters. Monocytes adhered in the presence of autologous serum were highly motile even after introduction of GST (83% motile cells). However, only 26% of monocytes with introduced GST-CRNK were motile. In contrast, GST-CRNK-treated monocytes were fully capable of phagocytosis and adhesion-induced cytokine gene induction, suggesting that CADTK is not involved in these cellular activities and that GST-CRNK introduction does not inhibit global monocyte functions. These results suggest that CADTK is crucial for the in vitro monocyte cytoskeletal reorganization necessary for cell motility and is likely to be required in vivo for recruitment to sites of inflammation.
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PMID:Inhibition of the calcium-dependent tyrosine kinase (CADTK) blocks monocyte spreading and motility. 1106 41

G-protein-coupled receptors are a large group of integral membranal receptors, which in response to ligand binding initiate diverse downstream signaling. Here we studied the gonadotropin-releasing hormone (GnRH) receptor, which uses Gq for its downstream signaling. We show that extracellular signal-regulated kinase (ERK) activation is fully dependent on protein kinase C (PKC), but only partially dependent on Src, dynamin, and Ras. Receptor tyrosine kinases, FAK, Gbetagamma, and beta-arrestin, which were implicated in some G-protein-coupled receptor signaling to MAPK cascades, do not play a role in the GnRH to ERK pathway. Our results suggest that the activation of ERK by GnRH involves two distinct signaling pathways, which converge at the level of Raf-1. The main pathway involves a direct activation of Raf-1 by PKC, and this step is partially dependent on a second pathway consisting of Ras activation, which occurs in a dynamin-dependent manner, downstream of Src.
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PMID:Role of dynamin, Src, and Ras in the protein kinase C-mediated activation of ERK by gonadotropin-releasing hormone. 2855 Jan 41

Megakaryocytic genes such as alphaIIbbeta3 can be expressed by malignant cells as part of the disturbances in their gene regulation. However, the function of the gene product greatly depends on the interaction of the ectopic protein with the new environment. The outside-in signaling of the ectopically expressed alphaIIbbeta3 integrin was studied in B16a murine melanoma cells using a monoclonal antibody, specifically directed to the activated conformation of alphaIIbbeta3, PAC-1 and the physiological ligand, fibrinogen. Ligation of alphaIIbbeta3 induced down-regulation of FAK but serine phosphorylation of three protein bands, 20/21, 85 and 140 kDa within 1-15 min. Flow cytometry indicated that the ligation of the receptor in B16a cells induces approximately 50% increase in phosphoserine positive cells within 5-15 min. 12-lipoxygenase is placed downstream in the signaling pathway, since ligation of alphaIIbbeta3 induces 12-HETE production within 5 min and pretreatment of tumor cells with select lipoxygenase inhibitior, Baicalein, prevents the increase in serine phosphorylation. Confocal microscopy of adherent tumor cells demonstrated rearrangement of actin filaments upon alphaIIbbeta3 ligation paralleled by downregulation of p125FAK and phoshotyrosine+ adhesion plaques and translocation of PKCalpha to stress fibers and cortical actin. PKC appears to be the major effector serine kinase of the alphaIIbbeta3-coupled signaling pathway, since pretreatment of tumor cells with a select PKC inhibitor, Calphostin C, prevents the ligation-induced serine phosphorylation. Previous studies have indicated a role for the 12-lipoxygenase-PKC signaling pathway in platelet aggregation as well as tumor invasion, therefore the involvement of this cascade in the signaling of the ectopic alphaIIbbeta3 integrin may partially explain its role in tumor progression.
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PMID:Ectopic alphaIIbbeta3 integrin signaling involves 12-lipoxygenase- and PKC-mediated serine phosphorylation events in melanoma cells. 1143 81

We have investigated signaling pathways leading to angiotensin II (Ang II) activation of mitogen-activated protein kinase (MAPK) in hepatocytes. MAPK activation by Ang II was abolished by the Ang II type 1 (AT1) receptor antagonist losartan, but not by the Ang II type 2 (AT2) receptor antagonist PD123319. Ang II (100 nM) induced a rapid phosphorylation of Src (peak approximately 2 min) and focal adhesion kinase (FAK, peak approximately 5 min) followed by a decrease to basal levels in 30 min. An increased association between FAK and Src in response to Ang II was detected after 1 min, which declined to basal levels after 30 min. Treatment with the Src kinase inhibitor PP-1 inhibited FAK phosphorylation. Downregulation of PKC, intracellular Ca2+ chelator BAPTA or inhibitors of PKC, Src kinase, MAPK kinase (MEK), Ca2+/calmodulin dependent protein kinase, phosphatidylinositol 3-kinase all blocked Ang II-induced MAPK phosphorylation. In contrast to other cells, there was no evidence for the role of EGF receptor transactivation in the activation of MAPK by Ang II. However, PDGF receptor phosphorylation is involved in the Ang II stimulated MAPK activation. Furthermore, Src/FAK and Ca/CaM kinase activation serve as potential links between the Ang II receptor and MAPK activation. These studies offer insight into the signaling network upstream of MAPK activation by AT1 receptor in hepatocytes.
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PMID:Angiotensin II activation of focal adhesion kinase and pp60c-Src in relation to mitogen-activated protein kinases in hepatocytes. 1203 95

MAP kinase can be activated by integrin-dependent adhesion in a FAK-dependent manner. Cell-cell contact inhibition is continuously active in controlling cell growth and the loss of cell-cell contact inhibition is correlated with the malignant characteristics of cancer cells. In this study we showed that cell adhesion to fibronectin for 1 h activated MAP kinase phosphorylation. However, when non-tumorigenic HSG cells, MCF-10A cells, or 293 cells were plated on fibronectin-coated substrates for 1 h at high cell density (which favors cell-cell contact), MAP kinase phosphorylation was not enhanced. Tumorigenic breast cancer cells, BT474, Cama, MCF-7, MDA-MB-231 and SKBR3, did not show inhibition of MAP kinase phosphorylation but rather enhanced MAP kinase phosphorylation when cultured at high density on fibronectin-coated substrates. Adhesion of HSG cells to fibronectin also increased FAK phosphorylation and this FAK phosphorylation was partially inhibited when cells were cultured at high density. Expression of Raf-1 catalytic domain-GFP in HSG cells could overcome the cell density-dependent inhibition of MAP kinase phosphorylation and FAK phosphorylation. The expression of Raf-1-catalytic domain-GFP also upregulated the expression of alphav integrin and promoted cell-cell adhesion in HSG cells. These results suggest that the active form of Raf-1 may interrupt cell-cell contact inhibition by promoting alphav integrin expression, which has been implicated in cell aggregation.
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PMID:Role of Raf-1 and FAK in cell density-dependent regulation of integrin-dependent activation of MAP kinase. 1211 85

Death-associated protein kinase (DAP-kinase) is a calcium/calmodulin-dependent serine/threonine kinase, and participates in various apoptosis systems. However, its apoptosis-promoting mechanism is poorly understood. Here, we demonstrate that DAP-kinase suppresses integrin-mediated cell adhesion and signal transduction, whereas dominant-negative interference of this kinase promotes adhesion. This effect of DAP-kinase is neither a consequence of apoptosis nor a result of decreased expression of integrins. Rather, DAP-kinase downregulates integrin activity through an inside-out mechanism. We present evidence indicating that this adhesion-inhibitory effect accounts for a major mechanism of the apoptosis induced by DAP-kinase. First, in growth-arrested fibroblasts, DAP-kinase triggers apoptosis in cells plated on fibronectin, but does not affect the death of cells on poly-l-lysine. Second, in epithelial cells, DAP-kinase induces apoptosis in the anoikis-sensitive MCF10A cells, but not in the anoikis-resistant BT474 cells. Most importantly, the apoptosis-promoting effect of DAP-kinase is completely abolished by enforced activation of integrin-mediated signaling pathways from either integrin itself or its downstream effector, FAK. Finally, we show that integrin or FAK activation blocks the ability of DAP-kinase to upregulate p53. Our results indicate that DAP-kinase exerts apoptotic effects by suppressing integrin functions and integrin-mediated survival signals, thereby activating a p53-dependent apoptotic pathway.
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PMID:DAP-kinase induces apoptosis by suppressing integrin activity and disrupting matrix survival signals. 1237 Feb 43

Ochratoxin A (OTA), a metabolite produced by strains of Aspergillus and Penicillium, has nephritogenic, carcinogenic, and teratogenic activity in animals and humans. Nanomolar concentrations of OTA promote apoptosis in a cell-type specific fashion. In this study, we have analyzed the molecular mechanism by which OTA affects COS cell adhesion and signaling resulting in an apoptotic response. OTA, at noncytotoxic doses, was able to detach collagen- and fibronectin-adherent cells from immobilized substratum. However, prior to inducing detachment of adherent cells, OTA caused apoptosis as measured by caspase-3 activation. The treatment of adherent cells by OTA caused a reduction of tyrosine phosphorylation levels of FAK and of the adapter protein paxillin. The down-regulation of FAK preceded apoptosis and cell detachment induced by OTA. The mycotoxin was also able to cause a decrease of the phosphorylation levels of the two Shc isoforms, P66 and P52, in adherent cells. Since these Shc isoforms have been implicated in the activation of protein kinase c-Src, which is required for FAK tyrosine phosphorylation, the observed dephosphorylation of FAK and of the FAK substrate paxillin by OTA could be ascribed to the early down-regulation of Shc isoforms. However, whether FAK and Shc phosphorylation contribute both to the same pathway leading to the induction of apoptosis by OTA or are involved in two parallel signaling pathways remains to be investigated.
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PMID:Ochratoxin A affects COS cell adhesion and signaling. 1457 39

Platelet-activating factor (PAF), a phospholipid second messenger, has diverse physiological functions, including responses in differentiated endothelial cells to external stimuli. We used human umbilical vein endothelial cells (HUVECs) as a model system. We show that PAF activated pertussis toxin-insensitive G alpha(q) protein upon binding to its seven transmembrane receptor. Elevated cAMP levels were observed via activation of adenylate cyclase, which activated protein kinase A (PKA) and was attenuated by a PAF receptor antagonist, blocking downstream activity. Phosphorylation of Src by PAF required G alpha(q) protein and adenylate cyclase activation; there was an absolute requirement of PKA for PAF-induced Src phosphorylation. Immediate (1 min) PAF-induced STAT-3 phosphorylation required the activation of G alpha(q) protein, adenylate cyclase, and PKA, and was independent of these intermediates at delayed (30 min) and prolonged (60 min) PAF exposure. PAF activated PLC beta 3 through its G alpha(q) protein-coupled receptor, whereas activation of phospholipase C gamma 1 (PLC gamma 1) by PAF was independent of G proteins but required the involvement of Src at prolonged PAF exposure (60 min). We demonstrate for the first time in vascular endothelial cells: (i) the involvement of signaling intermediates in the PAF-PAF receptor system in the induction of TIMP2 and MT1-MMP expression, resulting in the coordinated proteolytic activation of MMP2, and (ii) a receptor-mediated signal transduction cascade for the tyrosine phosphorylation of FAK by PAF. PAF exposure induced binding of p130(Cas), Src, SHC, and paxillin to FAK. Clearly, PAF-mediated signaling in differentiated endothelial cells is critical to endothelial cell functions, including cell migration and proteolytic activation of MMP2.
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PMID:Activation of platelet-activating factor receptor-coupled G alpha q leads to stimulation of Src and focal adhesion kinase via two separate pathways in human umbilical vein endothelial cells. 1461 36

The bioactive component of mildly oxidized low-density lipoproteins, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), activates tissue factor expression and monocyte adhesion to endothelial cells (EC) from systemic circulation, but blocks expression of inflammatory adhesion molecules (VCAM, E-selectin) and neutrophil adhesion associated with EC acute inflammatory response to bacterial lypopolysacharide (LPS). Due to constant exposure to oxygen free radicals, lipids in the injured lung are especially prone to oxidative modification and increased OxPAPC generation. In this study, we focused on OxPAPC-mediated intracellular signaling mechanisms that lead to physiological responses in pulmonary endothelial cells. Our results demonstrate that OxPAPC treatment activated in a time-dependent fashion protein kinase C (PKC), protein kinase A (PKA), Raf/MEK1,2/Erk-1,2 MAP kinase cascade, JNK MAP kinase and transient protein tyrosine phosphorylation in human pulmonary artery endothelial cells (HPAEC), whereas nonoxidized PAPC was without effect. Pharmacological inhibition of PKC and tyrosine kinases blocked activation of Erk-1,2 kinase cascade upstream of Raf. OxPAPC did not affect myosin light chain (MLC) phosphorylation, but increased phosphorylation of cofillin, a molecular regulator of actin polymerization. Finally, OxPAPC induced p60Src-dependent tyrosine phosphorylation of focal adhesion proteins paxillin and FAK. Our results suggest a critical involvement of PKC and tyrosine phosphorylation in OxPAPC-induced activation of Erk-1,2 MAP kinase cascade associated with regulation of specific gene expression, and demonstrate rapid phosphorylation of cytoskeletal proteins, which indicates OxPAPC-induced EC remodeling.
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PMID:Signal transduction pathways activated in human pulmonary endothelial cells by OxPAPC, a bioactive component of oxidized lipoproteins. 1470 99


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