Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The small GTPase Rho; functions as a molecular switch that regulates various cellular processes such as cell adhesion, motility, gene expression and cytokinesis. We previously isolated several putative Rho; targets including rhophilin which bound selectively to the GTP-bound form of Rho;. Rhophilin is expressed highly in testis and is localized specifically in sperm flagella. The presence of a PDZ domain at the carboxy terminus of rhophilin suggested that rhophilin works as an adaptor molecule. To test this hypothesis, we employed a yeast two hybrid system using the rhophilin PDZ domain as a bait, and screened a mouse testis cDNA library. We isolated several positive clones containing the same insert. The open reading frame of the cDNA encoded a novel protein of 212 amino acids designated as ropporin from a Japanese word 'oppo' (the tail). The amino-terminal 40 amino acid sequence of ropporin showed high homology to that of the regulatory subunit of type II cAMP-dependent protein kinase, which is involved in dimerization and binding to A-kinase anchoring proteins. Consistently, a yeast two hybrid assay and gel filtration of recombinant ropporin indicated that ropporin dimerizes through this domain. Deletion analysis indicated that the carboxy-terminal four amino acids are essential for binding of ropporin to rhophilin, and ropporin and RhoV14 coprecipitated in the presence of rhophilin in vitro. Northern blot analysis showed that ropporin is exclusively expressed in testis, and induced at the late stage of spermatogenesis. This induction paralleled that of rhophilin. Immunocytochemistry using anti-ropporin antibody showed that ropporin is localized in the principal piece and the end piece of sperm flagella. Electronmicroscopy revealed that ropporin is mostly localized in the inner surface of the fibrous sheath while rhophilin is present in the outer surface of the outer dense fiber. These results suggest that rhophilin and ropporin may form a complex in sperm flagella.
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PMID:Ropporin, a sperm-specific binding protein of rhophilin, that is localized in the fibrous sheath of sperm flagella. 1059 29

Epac belongs to a new family of proteins that can directly mediate the action of the intracellular second messenger cAMP by activating a downstream small GTPase Rap1. The Epac/Rap1 pathway represents a novel cAMP-signaling cascade that is independent of the cAMP-dependent protein kinase (PKA). In this study, we have used fluorescence microscopy to probe the intracellular targeting of Epac during different stages of the cell division cycle and the structural features that are important for Epac localization. Our results suggest Epac, endogenous or expressed as a green fluorescent protein fusion protein, is mainly localized to the nuclear membrane and mitochondria during interphase in COS-7 cells. Deletion mutagenesis analysis reveals that whereas the DEP domain is responsible for membrane association, the mitochondrial-targeting sequence is located at the N terminus. Although Epac predominantly exhibits perinuclear localization in interphase, the subcellular localization of Epac is cell cycle-dependent. Epac disassociates from the nuclear membrane and localizes to the mitotic spindle and centrosomes in metaphase. At the end of the cell cycle, Epac is observed to reassociate with the nuclear envelope and concentrate around the contractile ring. Furthermore, overexpression of Epac in COS-7 cells leads to an increase in multinuclear cell populations. These results suggest that Epac may play an important role in mitosis.
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PMID:Cell cycle-dependent subcellular localization of exchange factor directly activated by cAMP. 1200 Jul 63

Phosphorylation of myofilament proteins by kinases such as cAMP-dependent protein kinase and protein kinase C has been shown to lead to altered thin-filament protein-protein interactions and modulation of cardiac function in vitro. In the present study, we report that a small GTPase-dependent kinase, p21-activated kinase (PAK), increases the calcium sensitivity of Triton-skinned cardiac muscle fiber bundles. Constitutively active PAK3 caused an average 1.25-fold (25.0+/-6.0%, n=6) increase in force at pCa 5.75, 1.44-fold (44.0+/-7.78%, n=6) at pCa 6.25, and 2.41-fold (141.2+/-23.7%, n=4) at pCa 6.5, representing a change in pCa50 value of approximately 0.25. Constitutively active PAK3 produced no change in force under conditions of relaxation (pCa 8.0) or maximal contraction (pCa 4.5). Furthermore, an inactive, kinase-dead form of PAK3 failed to produce any change in force development at any pCa value. The myofilament proteins phosphorylated by PAK3, at pCa 6.5, are desmin, troponin T, troponin I, and an unidentified 70-kDa protein. Importantly, cardiac troponin I was found to be phosphorylated at serine 149 of human cardiac troponin I, representing a novel phosphorylation site. These findings suggest a novel mechanism of modulating the calcium sensitivity of cardiac muscle contraction.
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PMID:p21-activated kinase increases the calcium sensitivity of rat triton-skinned cardiac muscle fiber bundles via a mechanism potentially involving novel phosphorylation of troponin I. 1224 69

The fibrous sheath is a unique cytoskeletal structure surrounding the axoneme and outer dense fibers and defines the extent of the principal piece region of the sperm flagellum. It consists of two longitudinal columns connected by closely arrayed semicircular ribs that assemble from distal to proximal throughout spermiogenesis. The fibrous sheath is believed to influence the degree of flexibility, plane of flagellar motion, and the shape of the flagellar beat. Nearly half of the protein in fibrous sheaths isolated from mouse sperm is AKAP4. This protein and two others, AKAP3 and TAKAP-80, have anchoring sites for cAMP-dependent protein kinase. AKAP3 also anchors ropporin, a spermatogenic cell-specific protein that is linked through rhophilin to the small GTPase Rho. Other proteins associated with the fibrous sheath include two enzymes in the glycolytic pathway. Glyceraldehyde 3-phosphate dehydrogenase-s (GAPDS) is the product of a gene expressed only in spermatogenic cells, while hexokinase type 1-s (HK1-S) is derived from alternative transcripts present only in spermatogenic cells. Most of the other glycolytic enzymes in sperm have unique structural or functional properties. The fibrous sheath also contains a spermatogenic cell-specific member of the mu-class glutathione S-transferase family (GSTM5) and an intermediate filament-like protein (FS39). These and other observations indicate that the fibrous sheath functions as a scaffold for proteins in signaling pathways that might be involved in regulating sperm maturation, motility, capacitation, hyperactivation, and/or acrosome reaction and for enzymes in the glycolytic pathway that provide energy for the hyperactivated motility of sperm that allows them to penetrate the zona pellucida.
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PMID:Fibrous sheath of mammalian spermatozoa. 1267 26

Adenosine A(2B) receptors have been suggested to influence cell differentiation and proliferation. Human adenosine A(2B) receptors expressed in Chinese hamster ovary cells mediate phosphorylation and activation of the extracellular signal-regulated kinase (ERK1/2). Already low concentrations of agonists such as 5'-N-ethylcarboxamidoadenosine (NECA) are effective. Phosphorylation of the stress-activated protein kinase p38 was also potently induced by NECA (EC(50) 18.5 nM). These NECA-induced effects were mimicked by forskolin and 8-Br-cAMP. Inhibition of cAMP-dependent protein kinase (PKA) using H89 (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide)) blocked phosphorylation of the cAMP response element-binding protein (CREB) and p38, but did not decrease NECA-induced ERK1/2 phosphorylation. NECA activated the small GTPase Rap1, and this was also not blocked by H89. Inhibition of phosphatidylinositol-3'-kinase (PI3K) by wortmannin inhibited adenosine A(2B) receptor-mediated ERK1/2 phosphorylation and activation of Rap1, without affecting CREB and p38 phosphorylation. A(2B) receptor-stimulated protein kinase B phosphorylation was sensitive to wortmannin, but not to H89. Thus, stimulation of adenosine A(2B) receptors activates both ERK1/2 and p38 via cAMP, but the downstream pathways are markedly different. ERK1/2 activation was dependent on PI3K but not on PKA. p38 activation by NECA was instead independent of PI3K but required cAMP and PKA. The potent activation of both MAPKs suggests a physiological role.
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PMID:The G(s)-coupled adenosine A(2B) receptor recruits divergent pathways to regulate ERK1/2 and p38. 1451 97

Cell surface glycoconjugates are thought to mediate cell-cell recognition and to play roles in neuronal development and functions. We demonstrated here that exposure of neuronal cells to nanomolar levels of glyco-chains with an N-acetylgalactosamine (GalNAc) residue at the non-reducing termini (GalNAc-S) such as GalNAcbeta4(Neu5Acalpha3)Galbeta4GlcCer (GM2) ganglioside, its oligosaccharide portion, GalNAcbeta4Galbeta4GlcCer (Gg(3)) Cer, GalNAcalpha3GalNAcbeta3Galalpha4Galbeta4GlcCer (Gb(5)) Cer (Forssman hapten) and alpha1-4 linked oligomers of GalNAc, induced a rapid and transient activation of cAMP-dependent protein kinase (PKA) in subplasmalemma. The treatment was accompanied by peripheral actin polymerization and filopodia formation in NG108-15 cells and primary cultured hippocampal neurons, but not in glial cells. A cAMP-dependent protein kinase (PKA) selective inhibitor and an adenylate cyclase inhibitor blocked both PKA activation and the subsequent filopodia formation. A small GTPase cdc42 was a potential downstream target of GalNAc-S-activated PKA. These results suggest that extracellular GalNAc-S serve as potential regulators of the filopodia formation in neuronal cells by triggering the activation of PKA followed by cdc42 up-regulation via a cell surface receptor-like component. Filopodia formation induced by GalNAc-S may have a physiological relevance because long-term exposure to GalNAc-S enhanced F-actin-rich dendrite generation of primary cultured hippocampal neurons, and PKA-dependent dendritic outgrowth and branch formation of primary cultured cerebellar Purkinje neurons, in which actin isoforms were localized to motile structures in dendrites. These findings provide evidence for a novel GalNAc/PKA-signaling cascade in regulating some neuronal maturation.
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PMID:Extracellular carbohydrate-signal triggering cAMP-dependent protein kinase-dependent neuronal actin-reorganization. 1464 65

We demonstrate here that growth hormone (GH) stimulates the activation of RhoA and its substrate Rho kinase (ROCK) in NIH-3T3 cells. GH-stimulated formation of GTP-bound RhoA requires JAK2-dependent dissociation of RhoA from its negative regulator p190 RhoGAP. Inactivation of RhoA does not affect GH-stimulated JAK2 tyrosine phosphorylation nor p44/42 MAPK activity. However, RhoA and ROCK activities are required for GH-stimulated, Stat5-mediated transcription. RhoA-dependent enhancement of GH-stimulated, Stat5-mediated transcription is due to repression of histone deacetylase 6 activity recruited by transcription cofactor p300 that negatively regulates GH-stimulated, Stat5-mediated transcription. We also demonstrate that RhoA is the pivot for cAMP-dependent protein kinase inhibition of GH-stimulated, Stat5-mediated transcription as a consequence of cAMP-dependent protein kinase inactivation of RhoA through serine residue 188 of RhoA. We have therefore provided a novel mechanism by which a Ras-like small GTPase, RhoA, can regulate Stat5-mediated transcription.
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PMID:RhoA/ROCK activation by growth hormone abrogates p300/histone deacetylase 6 repression of Stat5-mediated transcription. 1510 57

cAMP is a second messenger controlling various cellular processes through cAMP-dependent protein kinase (cAPK, PKA) and cyclic nucleotide-gated ion channels. Recently, the PKA-independent-cAMP-mediated signaling pathway by means of exchange protein directly activated by cAMP (Epac) has been demonstrated. Epac is a guanine nucleotide-exchange factor (GEF) for Rap, a Ras-like small GTPase. To investigate this new target for cAMP in development, we have isolated Xepac, the Xenopus laevis homologue of Epac by cDNA library screening. Xepac (Xepac1) encodes 890 amino acids, which have 57% identity with human Epac1 and 59% with that of rat Epac1 in amino acids. Whole-mount in situ hybridization and reverse transcriptase-polymerase chain reaction analysis show that XEpac is expressed both maternally and zygotically and is restricted within the developing hatching gland. Intriguingly, overexpression of XEpac induces the anterior markers XAG-1 and XOtx2 and can convert ectoderm into cement- and hatching gland-expressing cells. These results suggest that XEpac contains anterior positional information.
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PMID:XEpac, a guanine nucleotide-exchange factor for Rap GTPase, is a novel hatching gland specific marker during the Xenopus embryogenesis. 1575 76

Epac, a guanine nucleotide exchange factor for the small GTPase Rap, binds to and is activated by the second messenger cAMP. In sperm, there are a number of signaling pathways required to achieve egg-fertilizing ability that depend upon an intracellular rise of cAMP. Most of these processes were thought to be mediated by cAMP-dependent protein kinases. Here we report a new dependence for the cAMP-induced acrosome reaction involving Epac. The acrosome reaction is a specialized type of regulated exocytosis leading to a massive fusion between the outer acrosomal and the plasma membranes of sperm cells. Ca2+ is the archetypical trigger of regulated exocytosis, and we show here that its effects on acrosomal release are fully mediated by cAMP. Ca2+ failed to trigger acrosomal exocytosis when intracellular cAMP was depleted by an exogenously added phosphodiesterase or when Epac was sequestered by specific blocking antibodies. The nondiscriminating dibutyryl-cAMP and the Epac-selective 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate analogues triggered the acrosome reaction in the effective absence of extracellular Ca2+. This indicates that cAMP, via Epac activation, has the ability to drive the whole cascade of events necessary to bring exocytosis to completion, including tethering and docking of the acrosome to the plasma membrane, priming of the fusion machinery, mobilization of intravesicular Ca2+, and ultimately, bilayer mixing and fusion. cAMP-elicited exocytosis was sensitive to anti-alpha-SNAP, anti-NSF, and anti-Rab3A antibodies, to intra-acrosomal Ca2+ chelators, and to botulinum toxins but was resistant to cAMP-dependent protein kinase blockers. These experiments thus identify Epac in human sperm and evince its indispensable role downstream of Ca2+ in exocytosis.
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PMID:Calcium-induced acrosomal exocytosis requires cAMP acting through a protein kinase A-independent, Epac-mediated pathway. 1640 49

Cyclic AMP regulates multiple neuronal functions, including neurite outgrowth and axonal regeneration. GPR3, GPR6, and GPR12 make up a family of constitutively active G protein-coupled receptors (GPCRs) that share greater than 50% identity and 65% similarity at the amino acid level. They are highly expressed in the central nervous system, and their expression in various cell lines results in constitutive stimulation of cAMP production. When the constitutively active GPCRs were overexpressed in rat cerebellar granule neurons in culture, the transfected neurons exhibited significantly enhanced neurite outgrowth and overcame growth inhibition caused by myelin-associated glycoprotein. GPR12-mediated neurite outgrowth was the most prominent and was shown to depend on G(s) and cAMP-dependent protein kinase. Moreover, the GPR12-mediated rescue from myelin-associated glycoprotein inhibition was attributable to cAMP-dependent protein kinase-mediated inhibition of the small GTPase, RhoA. Among the three receptors, GPR3 was revealed to be enriched in the developing rat cerebellar granule neurons. When the endogenous GPR3 was knocked down, significant reduction of neurite growth was observed, which was reversed by expression of either GPR3 or GPR12. Taken together, our results indicate that expression of the constitutively active GPCRs up-regulates cAMP production in neurons, stimulates neurite outgrowth, and counteracts myelin inhibition. Further characterization of the GPCRs in developing and injured mammalian neurons should provide insights into how basal cAMP levels are regulated in neurons and could establish a firm scientific foundation for applying receptor biology to treatment of various neurological disorders.
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PMID:Neural expression of G protein-coupled receptors GPR3, GPR6, and GPR12 up-regulates cyclic AMP levels and promotes neurite outgrowth. 1728 43


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