Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The triazolopyrimidine trapidil has been found in controlled clinical trials to prevent restenosis after vascular injury. Although trapidil is widely regarded as a platelet-derived growth factor receptor (PDGF) antagonist, its precise mode of action is still unknown. This study was designed to investigate the inhibition of mitogenesis by trapidil in cultured bovine coronary artery smooth muscle cells (SMC) and to identify major signal transduction pathways involved. Trapidil inhibited PDGF-BB-induced mitogenesis in SMC in a concentration-dependent manner. Comparable inhibitory effects were obtained after stimulation of smooth muscle cells by phorbol ester, which suggests that the action of trapidil was not restricted to PDGF receptor-mediated mechanisms. Trapidil also inhibited PDGF- and phorbol ester-induced mitogen-activated protein kinase as well as Raf-1 kinase activity. As a possible target of trapidil, stimulation of cellular protein kinase A (PKA) activity was detected. Trapidil also induced the phosphorylation of vasodilator-stimulated phosphoprotein in SMC. Antimitogenic effects of trapidil were completely abolished by PKA inhibitors. Neither a direct stimulation of cAMP formation nor a phosphodiesterase inhibition was observed at antimitogenic concentrations of trapidil. However, trapidil directly activated purified PKA holoenzyme in a cAMP-independent manner. In conclusion, trapidil exerts its antimitogenic effects on SMC by direct activation of PKA. Thus, PKA-mediated inhibition of the Raf-1/MAP kinase pathway may be involved in the antimitogenic actions of the compound.
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PMID:Antimitogenic effects of trapidil in coronary artery smooth muscle cells by direct activation of protein kinase A. 968 64

Proliferation of vascular smooth muscle cells (SMC) in response to platelet-derived growth factor (PDGF) and other mitogens plays an important role in restenosis following coronary angioplasty. Elevation of adenosine 3',5'-cyclic monophosphate (cAMP) concentration in SMC has been shown to inhibit SMC mitogenesis and could be obtained either directly by stimulation of adenylyl cyclase-coupled receptors or indirectly by inhibition of cAMP-specific phosphodiesterase (PDE4) or the cyclic guanosine 3', 5'-monophosphate-inhibitable phosphodiesterase (PDE3). This study compared the effects of the selective PDE3 inhibitors trequinsin and quazinone with the selective PDE4 inhibitors Ro 20-1724 and rolipram on PDGF-induced DNA synthesis, mitogen-activated protein (MAP) kinase activation, cAMP levels, and protein kinase A (PKA) activation in SMC. Both PDE3 and PDE4 inhibitors stimulated intracellular PKA activation as seen from phosphorylation of vasodilator-stimulated phosphoprotein (VASP). However, only PDE3 inhibitors, and not inhibitors of PDE4, reduced PDGF-induced DNA synthesis and inhibited p42/p44 MAP kinase phosphorylation. At antimitogenic concentrations, the PDE3 inhibitors had only minor effects on cAMP levels. In contrast, PDE4 inhibitors increased the forskolin-induced cellular cAMP concentration 13- to 17-fold above control. These data demonstrate that inhibitors of PDE3 are potent antimitogenic agents and that a general increase in cellular cAMP levels and PKA activation per se are not sufficient to inhibit PDGF-induced SMC mitogenesis.
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PMID:Inhibition of platelet-derived growth factor-induced mitogenesis by phosphodiesterase 3 inhibitors: role of protein kinase A in vascular smooth muscle cell mitogenesis. 1085 33

Several major functions of type I cGMP-dependent protein kinase (cGK I) have been established in smooth muscle cells, platelets, endothelial cells, and cardiac myocytes. Here we demonstrate that cGK Ibeta is endogenously expressed in freshly purified human peripheral blood T lymphocytes and inhibits their proliferation and interleukin 2 release. Incubation of human T cells with the NO donor, sodium nitroprusside, or the membrane-permeant cGMP analogs PET-cGMP and 8-pCPT-cGMP, activated cGK I and produced (i) a distinct pattern of phosphorylation of vasodilator-stimulated phosphoprotein, (ii) stimulation of the mitogen-activated protein kinases ERK1/2 and p38 kinase, and, upon anti-CD3 stimulation, (iii) inhibition of interleukin 2 release and (iv) inhibition of cell proliferation. cGK I was lost during in vitro culturing of primary T cells and was not detectable in transformed T cell lines. The proliferation of these cGK I-deficient cells was not inhibited by even high cGMP concentrations indicating that cGK I, but not cGMP-regulated phosphodiesterases or channels, cAMP-dependent protein kinase, or other potential cGMP mediators, was responsible for inhibition of T cell proliferation. Consistent with this, overexpression of cGK Ibeta, but not an inactive cGK Ibeta mutant, restored cGMP-dependent inhibition of cell proliferation of Jurkat cells. Thus, the NO/cGMP/cGK signaling system is a negative regulator of T cell activation and proliferation and of potential significance for counteracting inflammatory or lymphoproliferative processes.
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PMID:Activation of cGMP-dependent protein kinase Ibeta inhibits interleukin 2 release and proliferation of T cell receptor-stimulated human peripheral T cells. 1107 64

Phosphorylation of heat shock protein 27 (Hsp27) in human platelets by mitogen-activated protein kinase-activated protein kinase (MAPKAP) 2 is associated with signaling events involved in platelet aggregation and regulation of microfilament organization. We now show that Hsp27 is also phosphorylated by cGMP-dependent protein kinase (cGK), a signaling system important for the inhibition of platelet aggregation. Stimulation of washed platelets with 8-para-chlorophenylthio-cGMP, a cGK specific activator, resulted in a time-dependent phosphorylation of Hsp27. This is supported by the ability of cGK to phosphorylate Hsp27 in vitro to an extent comparable with the cGK-mediated phosphorylation of its established substrate vasodilator-stimulated phosphoprotein. Studies with Hsp27 mutants identified threonine 143 as a yet uncharacterized phosphorylation site in Hsp27 specifically targeted by cGK. To test the hypothesis that cGK could inhibit platelet aggregation by phosphorylating Hsp27 and interfering with the MAPKAP kinase phosphorylation of Hsp27, the known MAPKAP kinase 2-phosphorylation sites (Ser15, Ser78, and Ser82) as well as Thr143 were replaced by negatively charged amino acids, which are considered to mimic phosphate groups, and tested in actin polymerization experiments. Mimicry at the MAPKAP kinase 2 phosphorylation sites led to mutants with a stimulating effect on actin polymerization. Mutation of the cGK-specific site Thr143 alone had no effect on actin polymerization, but in the MAPKAP kinase 2 phosphorylation-mimicking mutant, this mutation reduced the stimulation of actin polymerization significantly. These data suggest that phosphorylation of Hsp27 and Hsp27-dependent regulation of actin microfilaments contribute to the inhibitory effects of cGK on platelet function.
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PMID:Heat shock protein 27 is a substrate of cGMP-dependent protein kinase in intact human platelets: phosphorylation-induced actin polymerization caused by HSP27 mutants. 1138 10

Sprouty and the Sprouty-related protein, Spred (Sprouty-related Ena/vasodilator-stimulated phosphoprotein homology-1 (EVH1) domain-containing protein), inhibit Ras-dependent extracellular signal-regulated kinase (ERK) signaling induced by a variety of growth factors. Since Sprouty proteins have been shown to inhibit not only ERK activation but also cell migration, we postulated that Spreds also inhibit cellular migration. Using stably highly metastatic LM8 cells infected with the Spred1-Sendai virus vector, we demonstrated that Spred1 inhibits the metastasis of LM8 cells in nude mice. Spred1 overexpression also inhibited migration of cells in vitro in response to chemokines, CCL19 and CCL21. We also found that Spred1 overexpression dissolved actin-stress fibers. Both EVH1 domain and C-terminal Sprouty-related domain were required for actin reassembly. Spred1 and Spred2 suppressed constitutively activated RhoA (V14RhoA)-induced stress fiber formation and serum response factor activation. Spred1 bound to activated RhoA, but not cdc42 and Rac. Spred1 also inhibited chemokine-induced RhoA activation and active RhoA-induced Rho-kinase activation. These data suggest that Spreds are key regulators of RhoA-mediated cell motility and signal transduction. Furthermore, our study suggests that the induction of Spreds could be a novel strategy for preventing cancer cell metastasis.
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PMID:The Sprouty-related protein, Spred, inhibits cell motility, metastasis, and Rho-mediated actin reorganization. 1518 77

Sprouty and Spred {Sprouty-related EVH1 [Ena/VASP (vasodilator-stimulated phosphoprotein) homology 1] domain} proteins have been identified as antagonists of growth factor signalling pathways. We show here that Spred-1 and Spred-2 appear to have distinct mechanisms whereby they induce their effects, as the Sprouty domain of Spred-1 is not required to block MAPK (mitogen-activated protein kinase) activation, while that of Spred-2 is required. Similarly, deletion of the C-terminal Sprouty domain of Spred-1 does not affect cell-cycle progression of G(0)-synchronized cells through to S-phase following growth factor stimulation, while the Sprouty domain is required for Spred-2 function. We also demonstrate that the inhibitory function of Spred proteins is restricted to the Ras/MAPK pathway, that tyrosine phosphorylation is not required for this function, and that the Sprouty domain mediates heterodimer formation of Spred proteins. Growth-factor-mediated activation of the small GTPases, Ras and Rap1, was able to be regulated by Spred-1 and Spred-2, without affecting receptor activation. Taken together, these results highlight the potential for different functional roles of the Sprouty domain within the Spred family of proteins, suggesting that Spred proteins may use different mechanisms to induce inhibition of the MAPK pathway.
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PMID:Distinct requirements for the Sprouty domain for functional activity of Spred proteins. 1568 64

Spreds (Sprouty-related proteins with an Ena/Vasodilator-stimulated phosphoprotein homology-1 domain) are a new protein family inhibiting the mitogen-activated protein kinase (MAPK) signaling pathway. Different RNA and protein studies already revealed an almost ubiquitous Spred-2 expression pattern. But until now, only few data were available on the in situ Spred-2 promoter activity. Here, we show a detailed in situ analysis of a mouse strain with a trapped Spred-2 gene, bringing a beta-galactosidase and neomycin fusion gene (beta-geo) under the control of the endogenous Spred-2 promoter. This allowed us to monitor Spred-2 promoter activity in practically every organ and their corresponding sub-compartments. X-Gal staining of newborn and adult mice revealed a nearly congruent Spred-2 promoter activity pattern. Our detailed data provide information for further studies of the still enigmatic physiological functions of Spred-2 in various organs by identifying the tissues with strong Spred-2 promoter activity.
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PMID:Tissue-specific Spred-2 promoter activity characterized by a gene trap approach. 1637 60

Compartmentation of cAMP signaling been demonstrated to be attributable to the structural association of protein kinase A (PKA) (via association with A-kinase anchoring proteins [AKAPs]) with phosphodiesterase and AKAP-dependent effector molecules. However, other mechanisms contributing to compartmentalization have not been rigorously explored, including the possibility that different isoforms of adenylyl cyclase (AC) may be functionally "compartmentalized" because of differential association with tethering or signaling molecules. To this end, we examined the effect of adenoviral transduction of representative AC isoforms (AC1, AC2, AC5, and AC6) on cellular cAMP production, PKA activation, extracellular signal-regulated kinase (ERK) activation, cell doubling and proliferation, as well as arborization responses (an index of cAMP-mediated cytoskeletal re-organization) in vascular smooth muscle cells. When isoforms were expressed at levels to achieve comparable forskolin-stimulated AC activity, only gene transfer of AC6 significantly enhanced PKA-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation and arborization responses. Treatment of control cells, which express AC6 endogenously, as well as vascular smooth overexpressing the AC6 isoform with small interfering RNA directed against AC6, significantly suppressed both isoproterenol-stimulated cAMP accumulation and arborization. Notably, the selective effects of AC6 expression were abrogated in the presence of phosphodiesterase suppression. In contrast, only the expression of AC1 enhanced forskolin-stimulated association of ERK with AC, demonstrated by coimmuno-isolation of ERK with Flag-tagged AC1, but not with Flag-tagged AC6. To determine whether these isoform-selective effects of AC were unique to differentiated and morphologically compartmentalized vascular smooth muscle cells or were a general property of these isoforms, we examined the consequence of expression of these various isoforms in human embryonic kidney (HEK) cells. Indeed, we observed similar isoform-dependent association of AC1 with ERK, activation of ERK by stimulation of AC1 with forskolin, and AC1-dependent lengthening of doubling time, indicating that these properties of AC1 are cell autologous and likely result from AC1-dependent protein-protein interactions. In aggregate, these findings suggest that isoform-selective signaling complexes likely contribute to various functional consequences of cAMP elevation in vascular smooth muscle cells.
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PMID:Adenylyl cyclase isoform-selective regulation of vascular smooth muscle proliferation and cytoskeletal reorganization. 1703 46

Sprouty-related proteins with an EVH1 domain (Spreds) belong to a new protein family harboring a conserved N-terminal EVH1 domain, which is related to the VASP (vasodilator-stimulated phosphoprotein) EVH1 domain (Enabled/VASP homology 1 domain) and a C-terminal Sprouty-related domain, typical for Sprouty proteins. Spreds were, like Sproutys, initially discovered as inhibitors of the Ras/MAPK pathway, and the SPR (Sprouty-related) domains of both protein families seem to be very important for many protein interactions and cellular processes. VASP was initially characterized as a proline-rich substrate of protein kinases A and G in human platelets and later shown to be a scaffold protein, regulating both signal transduction pathways and the actin filament system. The VASP-EVH1 domain is known to bind specifically to a FP(4) binding motif, which is, for example, present in the focal adhesion proteins vinculin and zyxin. In this review we give a structural and functional overview on these three protein families and ask whether nature plays a modular protein domain puzzle with stable exchangeable elements or if these closely related domains have various functions when pasted in a different protein context.
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PMID:The VASP-Spred-Sprouty domain puzzle. 1698 6

Resveratrol has been reported to have antiplatelet activity; however, the detailed mechanisms have not yet been resolved. This study aimed to systematically examine the detailed mechanisms of resveratrol in the prevention of platelet activation in vitro and in vivo. Resveratrol (0.05-0.25 micromol/l) showed stronger inhibition of platelet aggregation stimulated by collagen (1 microg/ml) than other agonists. Resveratrol (0.15 and 0.25 micromol/l) inhibited collagen-induced platelet activation accompanied by [Ca(+2)]i mobilization, thromboxane A(2) (TxA(2)) formation, phosphoinositide breakdown, and protein kinase C (PKC) activation. Resveratrol markedly increased levels of NO/cyclic guanosine monophosphate (GMP), and cyclic GMP-induced vasodilator-stimulated phosphoprotein phosphorylation. Resveratrol markedly inhibited p38 mitogen-activated protein kinase (MAPK) but not Jun N-terminal kinase or extracellular signal-regulated kinase-2 phosphorylation in washed platelets. Resveratrol-reduced hydroxyl radical (OH(-)) formation in the electron spin resonance study. In an in vivo study, resveratrol (5 mg/kg) significantly prolonged platelet plug formation of mice. In conclusion, the main findings of this study suggest that the inhibitory effects of resveratrol possibly involve (i) inhibition of the p38 MAPK-cytosolic phospholipase A(2)-arachidonic acid-TxA(2)-[Ca(+2)]i cascade and (ii) activation of NO/cyclic GMP, resulting in inhibition of phospholipase C and/or PKC activation. Resveratrol is likely to exert significant protective effects in thromboembolic-related disorders by inhibiting platelet aggregation.
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PMID:Inhibitory mechanisms of resveratrol in platelet activation: pivotal roles of p38 MAPK and NO/cyclic GMP. 1786 48


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