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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

Clone A colon carcinoma cells develop fan-shaped lamellae and exhibit random migration when plated on laminin, processes that depend on the ligation of the alpha6beta4 integrin. Here, we report that expression of a dominant negative RhoA (N19RhoA) in clone A cells inhibited alpha6beta4-dependent membrane ruffling, lamellae formation, and migration. In contrast, expression of a dominant negative Rac (N17Rac1) had no effect on these processes. Using the Rhotekin binding assay to assess RhoA activation, we observed that engagement of alpha6beta4 by either antibody-mediated clustering or laminin attachment resulted in a two- to threefold increase in RhoA activation, compared with cells maintained in suspension or plated on collagen. Antibody-mediated clustering of beta1 integrins, however, actually suppressed Rho A activation. The alpha6beta4-mediated interaction of clone A cells with laminin promoted the translocation of RhoA from the cytosol to membrane ruffles at the edges of lamellae and promoted its colocalization with beta1 integrins, as assessed by immunofluorescence microscopy. In addition, RhoA translocation was blocked by inhibiting phosphodiesterase activity and enhanced by inhibiting the activity of cAMP-dependent protein kinase. Together, these results establish a specific integrin-mediated pathway of RhoA activation that is regulated by cAMP and that functions in lamellae formation and migration.
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PMID:RhoA function in lamellae formation and migration is regulated by the alpha6beta4 integrin and cAMP metabolism. 1064 58

WAVE proteins are members of the Wiskott-Aldrich syndrome protein (WASP) family of scaffolding proteins that coordinate actin reorganization by coupling Rho-related small molecular weight GTPases to the mobilization of the Arp2/3 complex. We identified WAVE-1 in a screen for rat brain A kinase-anchoring proteins (AKAPs), which bind to the SH3 domain of the Abelson tyrosine kinase (Abl). Recombinant WAVE-1 interacts with cAMP-dependent protein kinase (PKA) and Abl kinases when expressed in HEK-293 cells, and both enzymes co-purify with endogenous WAVE from brain extracts. Mapping studies have defined binding sites for each kinase. Competition experiments suggest that the PKA-WAVE-1 interaction may be regulated by actin as the kinase binds to a site overlapping a verprolin homology region, which has been shown to interact with actin. Immunocytochemical analyses in Swiss 3T3 fibroblasts suggest that the WAVE-1 kinase scaffold is assembled dynamically as WAVE, PKA and Abl translocate to sites of actin reorganization in response to platelet-derived growth factor treatment. Thus, we propose a previously unrecognized function for WAVE-1 as an actin-associated scaffolding protein that recruits PKA and Abl.
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PMID:Scar/WAVE-1, a Wiskott-Aldrich syndrome protein, assembles an actin-associated multi-kinase scaffold. 1097 Aug 52

Small GTPase Rho and cGMP/cGMP-dependent protein kinase (cGK) pathways exert opposing effects in specific systems such as vascular contraction and growth. However, the direct interaction between these pathways has remained elusive. We demonstrate that cGK phosphorylates RhoA in vitro at Ser188, the same residue phosphorylated by cAMP-dependent protein kinase. In HeLa cells transfected with constitutively active cGK (C-cGK), stress fiber formation induced by lysophosphatidic acid or V14RhoA was blocked. By contrast, C-cGK failed to inhibit stress fiber formation in cells transfected with mutant RhoA with substitution of Ser188 to Ala. C-cGK did not affect actin reorganization induced by Rac1 or Rho-associated kinase, one of the effectors for RhoA. Furthermore, C-cGK expression inhibited the membrane translocation of RhoA. Collectively, our findings suggest that cGK phosphorylates RhoA at Ser188 and inactivates RhoA signaling. The physiological relevance of the direct interaction between RhoA and cGK awaits further investigation.
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PMID:cGMP-dependent protein kinase phosphorylates and inactivates RhoA. 1116 91

The cAMP-dependent protein kinase (PKA) is targeted to specific subcellular compartments through its interaction with A-kinase anchoring proteins (AKAPs). AKAPs contain an amphipathic helix domain that binds to the type II regulatory subunit of PKA (RII). Synthetic peptides containing this amphipathic helix domain bind to RII with high affinity and competitively inhibit the binding of PKA with AKAPs. Addition of these anchoring inhibitor peptides to spermatozoa inhibits motility (Vijayaraghavan, S., Goueli, S. A., Davey, M. P., and Carr, D. W. (1997) J. Biol. Chem. 272, 4747-4752). However, inhibition of the PKA catalytic activity does not mimic these peptides, suggesting that the peptides are disrupting the interaction of AKAP(s) with proteins other than PKA. Using the yeast two-hybrid system, we have now identified two sperm-specific human proteins that interact with the amphipathic helix region of AKAP110. These proteins, ropporin (a protein previously shown to interact with the Rho signaling pathway) and AKAP-associated sperm protein, are 39% identical to each other and share a strong sequence similarity with the conserved domain on the N terminus of RII that is involved in dimerization and AKAP binding. Mutation of conserved residues in ropporin or RII prevents binding to AKAP110. These data suggest that sperm contains several proteins that bind to AKAPs in a manner similar to RII and imply that AKAPs may have additional and perhaps unique functions in spermatozoa.
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PMID:Identification of sperm-specific proteins that interact with A-kinase anchoring proteins in a manner similar to the type II regulatory subunit of PKA. 1127 69

Members of the Rho family of small GTPases, such as Rho and Rac, are required for actin cytoskeletal reorganization during the migration of carcinoma cells. Phosphodiesterases are necessary for this migration because they alleviate cAMP-dependent protein kinase (PKA)-mediated inhibition of RhoA (O'Connor, K. L., Shaw, L. M., and Mercurio, A. M. (1998) J. Cell Biol. 143, 1749-1760; O'Connor K. L., Nguyen, B.-K., and Mercurio, A. M. (2000), J. Cell Biol. 148, 253-258). In this study, we report that the migration of breast and squamous carcinoma cells toward either lysophosphatidic acid or epidermal growth factor involves not only phosphodiesterase activity but also cooperative signaling from PKA. Furthermore, we demonstrate that Rac1 activation in response to chemoattractant or beta(1) integrin clustering is regulated by PKA and that Rac1 is required for this migration. Also, we find that beta(1) integrin signaling stimulates the rapid and transient activation of PKA. A novel implication of these findings is that carcinoma cell migration is controlled by cAMP-dependent as well as cAMP inhibitory signaling mechanisms.
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PMID:Protein kinase A regulates Rac and is required for the growth factor-stimulated migration of carcinoma cells. 1160 81

Cell surface integrin receptors and Rho family GTPases function together to mediate adhesion-dependent events in cells. We have shown that the attachment of Swiss 3T3 cells to a cellulose substratum (Cuprophan, CU) activates adenylyl cyclase, which catalyses cyclic AMP (cAMP) production. CU adsorbs vitronectin poorly, prevents cell spreading and causes cells to aggregate. By contrast, spread cells on polystyrene (PS) contain low cAMP concentrations. We have now investigated the shift between integrin signalling-Rho A and the cAMP pathway. CU did not support the formation of focal contacts and stress fibres. The plasma membranes of cells on CU had less Rho A than those of cells on PS. Also, blocking vitronectin (VN) or fibronectin (FN)-integrin receptors with echistatin, which activates cAMP production, decreased Rho A in the plasma membrane of cells attached to PS. But adsorption of VN or FN onto CU, which limits the production of the cAMP, increased the cell membrane Rho A. Adding an inhibitor of cAMP-dependent protein kinase PKA to the medium also increased the plasma membrane Rho A in aggregated cells attached to CU. These results highlight the importance of cAMP, generated by cell attachment to substratum, as a gating element in integrin-Rho A signalling.
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PMID:Cyclic AMP--dependent aggregation of Swiss 3T3 cells on a cellulose substratum (Cuprophan) and decreased cell membrane Rho A. 1201 76

Much evidence indicates that cAMP-dependent protein kinase (PKA) prevents increased endothelial permeability induced by inflammatory mediators. We investigated the hypothesis that PKA inhibits Rho GTPases, which are regulator proteins believed to mediate endothelial barrier dysfunction. Stimulation of human microvascular endothelial cells (HMEC) with thrombin (10 nM) increased activated RhoA (RhoA-GTP) within 1 min, which remained elevated approximately fourfold over control for 15 min. The activation was accompanied by RhoA translocation to the cell membrane. However, thrombin did not activate Cdc42 or Rac1 within similar time points, indicating selectivity of activation responses by Rho GTPases. Pretreatment of HMEC with 10 micro M forskolin plus 1 micro M IBMX (FI) to elevate intracellular cAMP levels inhibited both thrombin-induced RhoA activation and translocation responses. FI additionally inhibited thrombin-mediated dissociation of RhoA from guanine nucleotide dissociation inhibitor (GDI) and enhanced in vivo incorporation of (32)P by GDI. HMEC pretreated in parallel with FI showed >50% reduction in time for the thrombin-mediated resistance drop to return to near baseline and inhibition of approximately 23% of the extent of resistance drop. Infection of HMEC with replication-deficient adenovirus containing the protein kinase A inhibitor gene (PKA inhibitor) blocked both the FI-mediated protective effects on RhoA activation and resistance changes. In conclusion, the results provide evidence that PKA inhibited RhoA activation in endothelial cells, supporting a signaling mechanism of protection against vascular endothelial barrier dysfunction.
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PMID:PKA inhibits RhoA activation: a protection mechanism against endothelial barrier dysfunction. 1258 8

LTD4 induces a calcium signal that consists of a mobilization from internal stores regulated by PTX-insensitive G protein, Rho, and, an influx across the plasma membrane regulated by PTX-sensitive Gi protein in human intestinal epithelial cells. The LTD4 induced mobilization of Ca2+ is mediated by a PH domain dependent association between PLC-gamma 1 and G beta gamma subunits. This interaction requires Src kinase activity as well as the association of this kinase with PLC-gamma 1, suggesting a G beta gamma mediated recruitment of proteins to the plasma membrane and formation of a signaling complex which is essential for the downstream Ca2+ signal. We found a cAMP-dependent protein kinase activity upstream of tyrosine kinase(s) and downstream of Gi protein, that is essential for LTD4-induced Ca2+ influx. This model of the LTD4-induced Ca2+ signaling pathways in intestinal epithelial cells is outlined schematically in Fig. 1.
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PMID:Leukotriene D4-induced calcium signaling in human intestinal epithelial cells. 1266 84

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


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