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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The platelet receptor for von Willebrand factor (VWF), glycoprotein (GP) Ib-IX, mediates initial platelet adhesion and activation. It is known that the cytoplasmic domain of GPIbbeta is phosphorylated at Ser(166) by cAMP-dependent protein kinase (PKA). To understand the physiological role of GPIbbeta phosphorylation, a GPIb-IX mutant replacing Ser(166) of GPIbbeta with alanine (S166A) and a deletion mutant lacking residues 166-181 of GPIbbeta (Delta165) were constructed. These mutants, expressed in Chinese hamster ovary (CHO) cells, showed an enhanced VWF-binding function compared with wild type GPIb-IX. Treatment of CHO cells expressing wild type GPIb-IX with a PKA inhibitor, PKI, reduced Ser(166) phosphorylation and also enhanced VWF binding to GPIb-IX. Furthermore, cells expressing S166A or Delta165 mutants showed a significantly enhanced adhesion to immobilized VWF under flow conditions. Consistent with the studies in CHO cells, treatment of platelets with PKI enhanced VWF binding to platelets. In contrast, a PKA stimulator, forskolin, reduced VWF binding and VWF-induced platelet agglutination, which was reversed by PKI. Thus, PKA-mediated phosphorylation of GPIbbeta at Ser(166) negatively regulates VWF binding to GPIb-IX and is one of the mechanisms by which PKA mediates platelet inhibition.
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PMID:Regulation of glycoprotein Ib-IX-von Willebrand factor interaction by cAMP-dependent protein kinase-mediated phosphorylation at Ser 166 of glycoprotein Ib(beta). 1236 48

It is currently accepted that cGMP-dependent protein kinase (PKG) inhibits platelet activation. Here, we show that PKG plays an important stimulatory role in platelet activation. Expression of recombinant PKG in a reconstituted cell model enhanced von Willebrand factor (vWF)-induced activation of the platelet integrin alpha(IIb)beta(3). PKG knockout mice showed impaired platelet responses to vWF or low doses of thrombin and prolonged bleeding time. Human platelet aggregation induced by vWF or low-dose thrombin was inhibited by PKG inhibitors but enhanced by cGMP. Furthermore, a cGMP-enhancing agent, sildenafil, promoted vWF- or thrombin-induced platelet aggregation. The cGMP-stimulated platelet responses are biphasic, consisting of an initial transient stimulatory response that promotes platelet aggregation and a subsequent inhibitory response that limits the size of thrombi.
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PMID:A stimulatory role for cGMP-dependent protein kinase in platelet activation. 1252 95

Platelets play a key role in hemostasis through their ability to rapidly adhere to activated or injured endothelium, subendothelial matrix proteins, and other activated platelets. A strong equilibrium between activating and inhibiting processes is essential for normal platelet and vascular function, impairment of this equilibrium being associated with either thrombophilic or bleeding disorders. Both cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) have been established as crucial and synergistic intracellular messengers that mediate the effects of platelet inhibitors such as nitric oxide (NO) and prostacyclin (PG-I2). However, it was recently suggested that a rapid cGMP/cGMP-dependent protein kinase (cGK)-mediated extracellular signal-related kinase (ERK) phosphorylation promotes platelet activation. This hypothesis was examined here by evaluating established and proposed cGK activators/inhibitors with respect to their capacity to promote either platelet activation or inhibition. In particular, the regulatory role of cGK for ERK phosphorylation and thrombin-, thromboxane-, and VWF-induced platelet activation was investigated. The data obtained do not support the concept that cGK-mediated ERK phosphorylation promotes platelet activation but confirm the inhibitory role of cGK in platelet function. One explanation for these discrepancies is the novel finding that extracellular cGMP analogs potently and rapidly inhibit thrombin-, thromboxane-, and VWF-induced human platelet signaling and activation by a cGK-independent mechanism.
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PMID:Potent inhibition of human platelets by cGMP analogs independent of cGMP-dependent protein kinase. 1464 96

Glycoprotein Ib-IX-V (GPIb-IX-V) mediates platelet tethering to von Willebrand factor (VWF), recruiting platelets into the thrombus, and activates integrin alphaIIbbeta3 through a pathway that is dependent on Src kinases. In addition, recent reports indicate that activation of alphaIIbbeta3 by VWF is dependent on protein kinase G (PKG) and mitogen-activated protein (MAP) kinases. The present study compares the importance of these signaling pathways in the activation of alphaIIbbeta3 by GPIb-IX-V. In contrast to a recent report, VWF did not promote an increase in cyclic guanosine monophosphate (cGMP), while agents that elevate cGMP, such as the nitrous oxide (NO) donor glyco-SNAP-1 (N-(beta-D-glucopyranosyl)-N2-acetyl-S-nitroso-D,L-penicillaminamide) or the type 5 phosphosdiesterase inhibitor, sildenafil, inhibited rather than promoted activation of alphaIIbbeta3 by GPIb-IX-V and blocked aggregate formation on collagen at an intermediate rate of shear (800 s(-1)). Additionally, sildenafil increased blood flow in a rabbit model of thrombus formation in vivo. A novel inhibitor of the MAP kinase pathway, which is active in plasma, PD184161, had no effect on aggregate formation on collagen under flow conditions, whereas a novel inhibitor of Src kinases, which is also active in plasma, PD173952, blocked this response. These results demonstrate a critical role for Src kinases but not MAP kinases in VWF-dependent platelet activation and demonstrate an inhibitory role for cGMP-elevating agents in regulating this process.
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PMID:GPIb-dependent platelet activation is dependent on Src kinases but not MAP kinase or cGMP-dependent kinase. 1515 73

Endothelial cells exhibit regulated exocytosis in response to inflammatory mediators such as thrombin and histamine. The exocytosis of Weibel-Palade bodies (WPBs) containing von Willebrand factor, P-selectin, and interleukin-8 within minutes after stimulation is important for vascular homeostasis. SNARE proteins are key components of the exocytic machinery in neurons and some secretory cells, but their role in regulating exocytosis in endothelial cells is not well understood. We examined the function of SNARE proteins in mediating exocytosis of WPBs in endothelial cells. We identified the presence of syntaxin 4, syntaxin 3, and the high affinity syntaxin 4-regulatory protein Munc18c in human lung microvascular endothelial cells. Small interfering RNA-induced knockdown of syntaxin 4 (but not of syntaxin 3) inhibited exocytosis of WPBs as detected by the reduction in thrombin-induced cell surface P-selectin expression. Thrombin ligation of protease-activated receptor-1 activated the phosphorylation of syntaxin 4 and Munc18c, which, in turn, disrupted the interaction between syntaxin 4 and Munc18. Protein kinase Calpha activation was required for the phosphorylation of syntaxin 4 and Munc18c as well as the cell surface expression of P-selectin. We also observed that syntaxin 4 knockdown inhibited the adhesion of neutrophils to thrombin-activated endothelial cells, demonstrating the functional role of syntaxin 4 in promoting endothelial adhesivity. Thus, protease-activated receptor-1-induced protein kinase Calpha activation and phosphorylation of syntaxin 4 and Munc18c are required for the cell surface expression of P-selectin and the consequent binding of neutrophils to endothelial cells.
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PMID:Protease-activated receptor-1 activation of endothelial cells induces protein kinase Calpha-dependent phosphorylation of syntaxin 4 and Munc18c: role in signaling p-selectin expression. 1557 73

We have identified a new protein kinase in Dictyostelium discoideum that carries the same conserved class of "alpha-kinase" catalytic domain as reported previously in myosin heavy chain kinases (MHCKs) in this amoeba but that has a completely novel domain organization. The protein contains an N-terminal von Willebrand factor A (vWFA)-like motif and is therefore named VwkA. Manipulation of VwkA expression level via high copy number plasmids (VwkA++ cells) or gene disruption (vwkA null cells) results in an array of cellular defects, including impaired growth and multinucleation in suspension culture, impaired development, and alterations in myosin II abundance and assembly. Despite sequence similarity to MHCKs, the purified protein failed to phosphorylate myosin II in vitro. Autophosphorylation activity, however, was enhanced by calcium/calmodulin, and the enzyme can be precipitated from cellular lysates with calmodulin-agarose, suggesting that VwkA may directly bind calmodulin. VwkA is cytosolic in distribution but enriched on the membranes of the contractile vacuole and Golgi-like structures in the cell. We propose that VwkA likely acts indirectly to influence myosin II abundance and assembly behavior and possibly has broader roles than previously characterized alpha kinases in this organism, which all seem to be MHCKs.
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PMID:Identification and characterization of a novel alpha-kinase with a von Willebrand factor A-like motif localized to the contractile vacuole and Golgi complex in Dictyostelium discoideum. 1572 26

Integrin activation (inside-out signaling) in platelets can be initiated by agonists such as von Willebrand factor (VWF) and thrombin. Here we show that a mitogen-activated protein kinase (MAPK), p38, plays an important role in the activation of integrin alphaIIb beta3 induced by VWF and thrombin. A dominant-negative mutant of p38, p38AF, inhibits alphaIIb beta3 activation induced by VWF binding to its receptor, the platelet glycoprotein Ib-IX (GPIb-IX), and p38 inhibitors diminish platelet aggregation induced by VWF or low-dose thrombin. The inhibitory effect of p38 inhibitor is unlikely to be caused by the previous suggested effect on cyclo-oxygenase, as inhibition also was observed in the presence of high concentrations of cyclo-oxygenase inhibitor, aspirin. VWF or thrombin induces p38 activation, which is inhibited in cGMP-dependent protein kinase (PKG)-knockout mouse platelets and PKG inhibitor-treated human platelets, indicating that activation of p38 is downstream from PKG in the signaling pathway. p38AF or p38 inhibitors diminish PKG-induced phosphorylation of extracellular stimuli-responsive kinase (ERK), which also is important in integrin activation. Thus, p38 plays an important role in mediating PKG-dependent activation of ERK. These data delineate a novel signaling pathway in which platelet agonists sequentially activate PKG, p38, and ERK pathways leading to integrin activation.
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PMID:Sequential activation of p38 and ERK pathways by cGMP-dependent protein kinase leading to activation of the platelet integrin alphaIIb beta3. 1621 Mar 41

Thrombin-mediated endothelial-cell release of von Willebrand factor (VWF) and P-selectin functionally links protease-activated receptors (PARs) to thrombosis and inflammation. VWF release can be stimulated by both Ca2+ and cAMP, and, although both VWF and P-selectin are found in Weibel-Palade bodies (WPBs), we found that their release could be differentially regulated. In these studies, human umbilical vein endothelial cells stimulated with cAMP or PAR2-AP led to a delayed release of VWF and significantly less P-selectin release compared with histamine, thrombin, or PAR1-AP. Dose-response studies revealed that PAR2-AP was significantly less efficacious in promoting the release of P-selectin compared with VWF. PAR2-AP-induced robust stimulation of intracellular Ca2+ coupled with a significantly greater inhibitory effect of calcium chelation on release of VWF compared with cell-surface expression of P-selectin, suggests an additional Ca2+-independent pathway involved in release of P-selectin. PAR2-AP failed to increase global cAMP levels; however, inhibition of protein kinase A led to a significant attenuation of PAR2-AP-mediated release of VWF. Confocal microscopy studies revealed that PAR2 and forskolin caused preferential release of a population of Weibel-Palade bodies (WPBs) consisting of only VWF. Thus, WPBs are pharmacologically and morphologically heterogeneous, and distinct granule populations are susceptible to differential regulation.
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PMID:Differential regulation of endothelial exocytosis of P-selectin and von Willebrand factor by protease-activated receptors and cAMP. 1633 77

Isolation of endothelial progenitors from human umbilical cord blood generated great hope in vascular tissue engineering. However, before clinical use, progenitor derived endothelial cells (PDECs) have to be compared with mature endothelial cells (ECs). The aim of this study was to explore the behavior of PDECs exposed to a proinflammatory cytokine (interleukin-1alpha; IL-1alpha) according to the mitogen-activated protein (MAP) kinase and nuclear factor (NF)-kappaB signal transduction pathways as well as procoagulant activity (PCA). CD34(+) mononuclear cells were isolated using magnetic beads, cultured, and compared with human saphenous vein ECs (HSVECs). PDECs express endothelial markers: CD31, VE-cadherin, von Willebrand factor, KDR, and incorporate acetylated low-density lipoprotein (Dil-Ac-LDL). IL-1alpha similarly activates c-Jun N-terminal protein kinase (JNK) and p38 pathways in HSVECs and PDECs, whereas extracellular signal-related kinase (ERK)1/2 phosphorylation is lower in PDECs than in HSVECs. Low ERK1/2 phosphorylation in PDECs was specific to IL-1alpha as vascular endothelial growth factor (VEGF) similarly stimulated ERK1/2 pathway. With respect to inhibitor of NF-kappa B (Ikappa B) degradation, NF-kappa B translocation and phosphorylation, the NF-kappa B pathway is comparable in HSVECs and PDECs after stimulation. PCA and tissue factor level induced by IL-1alpha are lower in PDECs than in HSVECs. Thus, our data show that PDECs display the characteristics of functional mature ECs under IL-1alpha stimulation. However, we observed significant differences between PDECs and HSVECs related to both ERK1/2 pathway activation and tissue factor production.
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PMID:Signal transduction and procoagulant state of human cord blood--progenitor-derived endothelial cells after interleukin-1alpha stimulation. 1757 11

Engagement of the adhesion receptor glycoprotein (GP) Ib-IX-V by von Willebrand factor (VWF) mediates platelet adhesion to damaged vessels and triggers platelet activation and thrombus formation in heart attack and stroke. GPIb-IX-V contains distinct 14-3-3zeta-binding sites at the GPIb alpha C-terminus involving phosphorylation of Ser609, an upstream site involving phosphorylated Ser587/Ser590, and a protein kinase A (PKA)-dependent site on GPIb beta involving Ser166. 14-3-3zeta regulates the VWF-binding affinity of GPIb-IX-V and inhibiting 14-3-3zeta association blocks receptor signaling, suggesting a key functional role for 14-3-3zeta. We used deletion mutants of GPIb alpha expressed in Chinese hamster ovary (CHO) cells to define the relationship of 14-3-3zeta binding to another GPIb-IX-V-associated signaling protein, phosphoinositide 3-kinase (PI3-kinase). Pull-down experiments involving glutathione S-transferase (GST)-PI3-kinase/p85-subunit and GST-14-3-3zeta indicated that both proteins interacted with contiguous GPIb alpha sequences 580 to 590/591 to 610. Deleting these, but not upstream sequences of GPIb alpha expressed in CHO cells, inhibited VWF/ristocetin-dependent Akt phosphorylation, relative to wild-type receptor, confirming this region encompassed a functional PI3-kinase-binding site. Pull-down experiments with GST-p85 truncates indicated the GPIb alpha-binding region involved the p85 breakpoint cluster region (BCR) domain, containing RSXSXP. However, pull-down of GPIb-IX was unaltered by mutation/deletion/phosphorylation of this potential 14-3-3zeta-binding sequence in mutant constructs of GST-p85, suggesting PI3-kinase bound GPIb alpha independently of 14-3-3zeta; 14-3-3zeta inhibitor peptide R18 also blocked pull-down of receptor by GST-14-3-3zeta but not GST-p85, and GST-p85 pull-downs were unaffected by excess 14-3-3zeta. Together, these data suggest the GPIb alpha C-terminus regulates signaling through independent association of 14-3-3zeta and PI3-kinase.
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PMID:A functional 14-3-3zeta-independent association of PI3-kinase with glycoprotein Ib alpha, the major ligand-binding subunit of the platelet glycoprotein Ib-IX-V complex. 1829 48


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