Anchoring Proteins (anchoring + protein)

Distribution by Scientific Domains
Distribution within Life Sciences

Kinds of Anchoring Proteins

  • kinase anchoring protein


  • Selected Abstracts


    Poster Sessions AP01: Gene Expression and Regulation

    JOURNAL OF NEUROCHEMISTRY, Issue 2001
    J. M. Calandria
    The formation of Cortico-Thalamic projections requires the precise spatial and temporal expression of proteins that are involved in the different stages of synaptogenesis. We reasoned that the underlying molecular mechanism of this process is the differential expression of genes that code for stage specific proteins. Our research objective was to identify the differential expressed mRNAs during the main stages of synapses formation, which starts at embryonic day 12 (E12) and finishes on the first postnatal days in the rat. We approach this problem using Differential Display technique on three distinct ages of rat cerebral cortex that were: E13, E18 and postnatal day 0 (P0). We found 80 differential bands using 54 random primers and 18 of them were cloned and sequenced. The sequence analysis showed among others, a cDNA fragment highly homologous with the human A Kinase Anchoring Protein 450/350 also called CG-NAP. We found that this cDNA fragment homologous to AKAP was up regulated at E15 when cortical cells are undergoing active axogenesis. The expression pattern of this cDNA was confirmed by Real Time PCR. Our findings suggest a possible function for AKAP 450 in the regulation of the state of phosphorylation of centrosomal components during the initial stages of synapses formation during the establishment of Cortico-Thalamic connection. [source]


    The Effects of Disruption of A Kinase Anchoring Protein,Protein Kinase A Association on Protein Kinase A Signalling in Neuroendocrine Melanotroph Cells of Xenopus laevis

    JOURNAL OF NEUROENDOCRINOLOGY, Issue 7 2006
    G. J. H. Corstens
    Abstract The secretory activity of melanotroph cells from Xenopus laevis is regulated by multiple neurotransmitters that act through adenylyl cyclase. Cyclic adenosine monophosphate (cAMP), acting on protein kinase A (PKA), stimulates the frequency of intracellular Ca2+ oscillations and the secretory activity of the melanotroph cell. Anchoring of PKA near target proteins is essential for many PKA-regulated processes, and the family of A kinase anchoring proteins (AKAPs) is involved in the compartmentalisation of PKA type II (PKA II) regulatory subunits. In the present study, we determined to what degree cAMP signalling in Xenopus melanotrophs depends on compartmentalised PKA II. For this purpose, a membrane-permeable stearated form of Ht31 (St-Ht31), which dislodges PKA II from AKAP (thus disrupting PKA II signalling), was used. The effect of St-Ht31 on both secretion of radiolabelled peptides and intracellular Ca2+ signalling by superfused Xenopus melanotrophs was assessed. St-Ht31 stimulated secretion but had no effect on Ca2+ signalling. We conclude Xenopus melanotrophs possess a St-Ht31-sensitive PKA II that is associated with the exocytosis machinery and, furthermore, that Ca2+ signalling is regulated by an AKAP-independent signalling system. Moreover, our results support a recent proposal that AKAP participates in regulating PKA activity independently from cAMP. [source]


    Localization of the A kinase anchoring protein AKAP79 in the human hippocampus

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2000
    Attila Sík
    Abstract The phosphorylation state of the proteins, regulated by phosphatases and kinases, plays an important role in signal transduction and long-term changes in neuronal excitability. In neurons, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and calcineurin (CN) are attached to a scaffold protein, A kinase anchoring protein (AKAP), thought to anchor these three enzymes to specific sites of action. However, the localization of AKAP, and the predicted sites of linked phosphatase and kinase activities, are still unknown at the fine structural level. In the present study, we investigated the distribution of AKAP79 in the hippocampus from postmortem human brains and lobectomy samples from patients with intractable epilepsy, using preembedding immunoperoxidase and immunogold histochemical methods. AKAP79 was found in the CA1, presubicular and subicular regions, mostly in pyramidal cell dendrites, whereas pyramidal cells in the CA3, CA2 regions and dentate granule cells were negative both in postmortem and in surgical samples. In some epileptic cases, the dentate molecular layer and hilar interneurons also became immunoreactive. At the subcellular level, AKAP79 immunoreactivity was present in postsynaptic profiles near, but not attached to, the postsynaptic density of asymmetrical (presumed excitatory) synapses. We conclude that the spatial selectivity for the action of certain kinases and phosphatases regulating various ligand- and voltage-gated channels may be ensured by the selective presence of their anchoring protein, AKAP79, at the majority of glutamatergic synapses in the CA1, but not in the CA2/CA3 regions, suggesting profound differences in signal transduction and long-term synaptic plasticity between these regions of the human hippocampus. [source]


    A yeast two-hybrid system using Sp17 identified Ropporin as a novel cancer,testis antigen in hematologic malignancies

    INTERNATIONAL JOURNAL OF CANCER, Issue 7 2007
    Zhanfei Li
    Abstract Since most intracellular proteins are expressed with their ligands, ligands of cancer,testis (CT) antigens may also be CT in their distribution. Applying Sperm protein 17 (Sp17) as the bait in a yeast 2-hybrid system of a testicular cDNA library, 17 interacting clones were isolated and all encoded Ropporin, a spermatogenic cell-specific protein that serves as an anchoring protein for the A-kinase anchoring protein, AKAP110. Ropporin showed a very restricted normal tissue gene expression, detected only in testis and fetal liver. Ropporin mRNA could also be detected in tumor cells from patients with multiple myeloma, chronic lymphocytic leukemia and acute myeloid leukemia. Interestingly, expression of Sp17 did not necessarily predict for the expression of Ropporin suggesting that their coexpression in these tumor cells was random rather than coordinated. Ropporin gene expression in tumor cells is associated with the presence of high titer IgG antibodies against Ropporin, suggesting the in vivo translation of the mRNA into protein and the immunogenicity of the protein to the autologous hosts. Using a CT antigen as the bait in a yeast 2-hybrid system may, therefore, identify novel tumor antigen. Our results also suggest that Ropporin is a novel CT antigen in hematologic malignancies. © 2007 Wiley-Liss, Inc. [source]


    Bicarbonate-Induced phosphorylation of p270 protein in mouse sperm by cAMP-Dependent protein kinase

    MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2008
    Masako Kaneto
    Abstract Signaling by cAMP-dependent protein kinase (PKA) plays an important role in the regulation of mammalian sperm motility. However, it has not been determined how PKA signaling leads to changes in motility, and specific proteins responsible for these changes have not yet been identified as PKA substrates. Anti-phospho-(Ser/Thr) PKA substrate antibodies detected a sperm protein with a relative molecular weight of 270,000 (p270), which was phosphorylated within 1 min after incubation in a medium supporting capacitation. Phosphorylation of p270 was induced by bicarbonate or a cAMP analog, but was blocked by the PKA inhibitor H-89, indicating that p270 is likely a PKA substrate in sperm. In addition, phosphorylation of p270 was inhibited by stearated peptide st-Ht31, suggesting that p270 is phosphorylated by PKA associated with an A-kinase anchoring protein (AKAP). AKAP4 is the major fibrous sheath protein of mammalian sperm and tethers regulatory subunits of PKA to localize phosphorylation events. Phosphorylation of p270 occurred in sperm lacking AKAP4, suggesting that AKAP4 is not involved directly in the phosphorylation event. Phosphorylated p270 was enriched in fractionated sperm tails and appeared to be present in multiple compartments including a detergent-resistant membrane fraction. PKA phosphorylation of p270 within 1 min of incubation under capacitation conditions suggests that this protein may have an important role in the initial signaling events that lead to the activation and subsequent hyperactivation of sperm motility. Mol. Reprod. Dev. 75: 1045,1053, 2007. © 2007 Wiley-Liss, Inc. [source]


    TEM, FISH and molecular studies in infertile men with pericentric inversion of chromosome 9

    ANDROLOGIA, Issue 4 2006
    G. Collodel
    Summary Pericentric inversions involving the secondary constriction (qh) region of chromosome 9 are considered to be normal variants of human karyotype. A number of investigators have suggested that chromosomal anomalies can contribute to human infertility causing spermatogenetic derangement. The present study was aimed at verifying the influence of chromosome 9 inversion on human spermatogenesis. Semen samples of 18 male carriers of chromosome 9 inversion, analysed by light microscopy, revealed that five patients were azoospermic. PCR analysis demonstrated that two of them also had Y microdeletions. The other 13 showed generally normal sperm concentrations and reduced motility. The morphological characteristics of sperm were studied by TEM and the data were elaborated by a mathematical formula. Sperm pathologies resulted more frequently in the studied group compared to controls, particularly apoptosis. Partial sequences of the A-kinase anchoring protein (Akap) 4 and 3 genes were performed in all patients, as a previous study by our group highlighted Dysplasia of Fibrous Sheath (DFS) defect in two men with inv 9 investigations. The possible effect of chromosome 9 inversion on meiotic chromosome segregation was investigated by FISH, which showed an increased incidence of diploidy. We hypothesized that this inversion could have variable effects on spermatogenesis, from azoospermia to severely altered sperm morphology, motility and meiotic segregation. [source]


    The Risk of Cardiac Events and Genotype-Based Management of LQTS Patients

    ANNALS OF NONINVASIVE ELECTROCARDIOLOGY, Issue 1 2009
    Ph.D., oskot M.D., yna Markiewicz-
    This review discusses the risk of cardiac events and genotype-based management of LQTS. We describe here the genetic background of long QT syndrome and the eleven different genes for ion-channels and a structural anchoring protein associated with that disorder. Clinical Background section discusses the risk of cardiac events associated with different LQTS types. Management and Prevention section describes in turn gene-specific therapy, which was based on the identification of the gene defect and the dysfunction of the associated transmembrane ion channel. In patients affected by LQTS, genetic analysis is useful for risk stratification and for making therapeutic decisions. A recent study reported a quite novel pathogenic mechanism for LQTS and suggested that treatments aimed at scaffolding proteins rather than specific ion channels may be an alternative to antiarrhythmic strategy in the future. [source]


    AKAP-independent localization of type-II protein kinase A to dynamic actin microspikes

    CYTOSKELETON, Issue 9 2009
    Robert L. Rivard
    Abstract Regulation of the cyclic AMP-dependent protein kinase (PKA) in subcellular space is required for cytoskeletal dynamics and chemotaxis. Currently, spatial regulation of PKA is thought to require the association of PKA regulatory (R) subunits with A-kinase anchoring proteins (AKAPs). Here, we show that the regulatory RII, subunit of PKA associates with dynamic actin microspikes in an AKAP-independent manner. Both endogenous RII, and a GFP-RII, fusion protein co-localize with F-actin in microspikes within hippocampal neuron growth cones and the leading edge lamellae of NG108-15 cells. Live-cell imaging demonstrates that RII,-associated microspikes are highly dynamic and that the coupling of RII, to actin is tight, as the movement of both actin and RII, are immediately and coincidently stopped by low-dose cytochalasin D. Importantly, co-localization of RII, and actin in these structures is resistant to displacement by a cell-permeable disrupter of PKA-AKAP interactions. Biochemical fractionation confirms that a substantial pool of PKA RII, is associated with the detergent-insoluble cytoskeleton and is resistant to extraction by a peptide inhibitor of AKAP interactions. Finally, mutation of the AKAP-binding domain of RII, fails to disrupt its association with actin microspikes. These data provide the first demonstration of the physical association of a kinase with such dynamic actin structures, as well as the first demonstration of the ability of type-II PKA to localize to discrete subcellular structures independently of canonical AKAP function. This association is likely to be important for microfilament dynamics and cell migration and may prime the investigation of novel mechanisms for localizing PKA activity. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source]


    Protein kinase A RII-like (R2D2) proteins exhibit differential localization and AKAP interaction,

    CYTOSKELETON, Issue 7 2008
    Amy E. Hanlon Newell
    Abstract A-kinase anchoring proteins (AKAPs) bind to protein kinase A (PKA) via an amphipathic helix domain that interacts with a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins (ROPN1, ASP, SP17, and CABYR) also contain a highly conserved RII dimerization/docking (R2D2) domain, suggesting all four proteins may interact with all AKAPs in a manner similar to RII. All four of these proteins were originally detected in the flagellum of mammalian sperm. In this report, we demonstrate that all four R2D2 proteins are expressed in a wide variety of tissues and three of the proteins SP17, CABYR, and ASP are located in motile cilia of human bronchus and fallopian tubes. In addition, we detect SP17 in primary cilia. We also provide evidence that ROPN1 and ASP bind to a variety of AKAPs and this interaction can be disrupted with anchoring inhibitor peptides. The interaction of SP17 and CABYR with AKAPs appears to be much more limited. None of the R2D2 proteins appears to bind cAMP, a fundamental characteristic of the regulatory subunits of PKA. These observations suggest that R2D2 proteins utilize docking interactions with AKAPs to accomplish their function of regulating cilia and flagella. Based on location, affinity for AKAPs and lack of affinity for cAMP, it appears that each R2D2 protein has a unique role in this process. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source]


    Elements of the C-terminal t peptide of acetylcholinesterase that determine amphiphilicity, homomeric and heteromeric associations, secretion and degradation

    FEBS JOURNAL, Issue 8 2004
    Stéphanie Belbeoc'h
    The C-terminal t peptide (40 residues) of vertebrate acetylcholinesterase (AChE) T subunits possesses a series of seven conserved aromatic residues and forms an amphiphilic ,-helix; it allows the formation of homo-oligomers (monomers, dimers and tetramers) and heteromeric associations with the anchoring proteins, ColQ and PRiMA, which contain a proline-rich motif (PRAD). We analyzed the influence of mutations in the t peptide of Torpedo AChET on oligomerization and secretion. Charged residues influenced the distribution of homo-oligomers but had little effect on the heteromeric association with QN, a PRAD-containing N-terminal fragment of ColQ. The formation of homo-tetramers and QN -linked tetramers required a central core of four aromatic residues and a peptide segment extending to residue 31; the last nine residues (32,40) were not necessary, although the formation of disulfide bonds by cysteine C37 stabilized T4 and T4,QN tetramers. The last two residues of the t peptide (EL) induced a partial intracellular retention; replacement of the C-terminal CAEL tetrapeptide by KDEL did not prevent tetramerization and heteromeric association with QN, indicating that these associations take place in the endoplasmic reticulum. Mutations that disorganize the ,-helical structure of the t peptide were found to enhance degradation. Co-expression with QN generally increased secretion, mostly as T4,QN complexes, but reduced it for some mutants. Thus, mutations in this small, autonomous interaction domain bring information on the features that determine oligomeric associations of AChET subunits and the choice between secretion and degradation. [source]


    Role of protein kinase C-dependent A-kinase anchoring proteins in lysophosphatidic acid-induced cAMP signaling in human diploid fibroblasts

    AGING CELL, Issue 6 2006
    Ji-Heon Rhim
    Summary Previously, we reported that lysophosphatidic acid (LPA)-induced adenosine 3,,5,-cyclic monophosphate (cAMP) production by human diploid fibroblasts depends on the age of the fibroblasts. In this study, we examined the role of A-kinase anchoring proteins (AKAP) in the regulation of LPA-stimulated cAMP production in senescent fibroblasts. We found that levels of protein kinase C (PKC)-dependent AKAPs, such as Gravin and AKAP79, were elevated in senescent cells. Co-immunoprecipitation experiments revealed that Gravin and AKAP79 do not associate with adenylyl cyclase type 2 (AC2) but bind to AC4/6, which interacts with calcium-dependent PKCs ,/, both in young and senescent fibroblasts. When the expression of Gravin and AKAP79 was blocked by small interference RNA transfection, the basal level of cAMP was greatly reduced and the cAMP status after LPA treatment was also reversed. Protein kinase A showed a similar pattern in terms of its basal activity and LPA-dependent modulation. These data suggest that Gravin and to a lesser extent, AKAP79, may play important roles in maintaining the basal AC activity and in coupling the AC systems to inhibitory signals such as Gi, in young cells, and to stimulatory signals such as PKCs in senescent cells. This study also demonstrates that Gravin is especially important for the long-term activation of PKC by LPA in senescent cells. We conclude that LPA-dependent increased level of cAMP in senescent human diploid fibroblasts is associated with increases in Gravin levels resulting in its increased binding with and activation of calcium-dependent PKC ,/, and AC4/6. [source]


    The Effects of Disruption of A Kinase Anchoring Protein,Protein Kinase A Association on Protein Kinase A Signalling in Neuroendocrine Melanotroph Cells of Xenopus laevis

    JOURNAL OF NEUROENDOCRINOLOGY, Issue 7 2006
    G. J. H. Corstens
    Abstract The secretory activity of melanotroph cells from Xenopus laevis is regulated by multiple neurotransmitters that act through adenylyl cyclase. Cyclic adenosine monophosphate (cAMP), acting on protein kinase A (PKA), stimulates the frequency of intracellular Ca2+ oscillations and the secretory activity of the melanotroph cell. Anchoring of PKA near target proteins is essential for many PKA-regulated processes, and the family of A kinase anchoring proteins (AKAPs) is involved in the compartmentalisation of PKA type II (PKA II) regulatory subunits. In the present study, we determined to what degree cAMP signalling in Xenopus melanotrophs depends on compartmentalised PKA II. For this purpose, a membrane-permeable stearated form of Ht31 (St-Ht31), which dislodges PKA II from AKAP (thus disrupting PKA II signalling), was used. The effect of St-Ht31 on both secretion of radiolabelled peptides and intracellular Ca2+ signalling by superfused Xenopus melanotrophs was assessed. St-Ht31 stimulated secretion but had no effect on Ca2+ signalling. We conclude Xenopus melanotrophs possess a St-Ht31-sensitive PKA II that is associated with the exocytosis machinery and, furthermore, that Ca2+ signalling is regulated by an AKAP-independent signalling system. Moreover, our results support a recent proposal that AKAP participates in regulating PKA activity independently from cAMP. [source]


    Novel alternatively spliced mRNA (1c) of the protein kinase A RIα subunit is implicated in haploid germ cell specific expression

    MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2001
    Maria K. Dahle
    Abstract By using 5′ RACE on rat testis cDNA we identified three alternatively spliced mRNAs of the RIα subunit of cAMP‐dependent protein kinase that differed in their 5′ untranslated regions. Two of these 5′‐regions showed similarity with the human RIα exons 1a and 1b, while the third (1c) constituted a novel mRNA splice variant. Northern blot analysis showed that the 1c mRNA was specifically expressed in testis and only in postmeiotic germ cells. In contrast, the RIα 1b and RIα 1a mRNAs were present both in premeiotic germ cells and somatic cells of the testis, and the expression of both RIα 1a and 1b mRNAs were stimulated by cAMP in Sertoli cells. In sperm, the RIα protein was expressed after meiosis, and targeted to various subcellular structures via anchoring proteins. The RIα 1c haploid‐specific mRNA, therefore, may be important for the regulation of RIα expression in sperm. Mol. Reprod. Dev. 59:11–16, 2001. © 2001 Wiley‐Liss, Inc. [source]


    cAMP microdomains and L-type Ca2+ channel regulation in guinea-pig ventricular myocytes

    THE JOURNAL OF PHYSIOLOGY, Issue 3 2007
    Sunita Warrier
    Many different receptors can stimulate cAMP synthesis in the heart, but not all elicit the same functional responses. For example, it has been recognized for some time that prostaglandins such as PGE1 increase cAMP production and activate PKA, but they do not elicit responses like those produced by ,-adrenergic receptor (,AR) agonists such as isoproterenol (isoprenaline), even though both stimulate the same signalling pathway. In the present study, we confirm that isoproterenol, but not PGE1, is able to produce cAMP-dependent stimulation of the L-type Ca2+ current in guinea pig ventricular myocytes. This is despite finding evidence that these cells express EP4 prostaglandin receptors, which are known to activate Gs -dependent signalling pathways. Using fluorescence resonance energy transfer-based biosensors that are either freely diffusible or bound to A kinase anchoring proteins, we demonstrate that the difference is due to the ability of isoproterenol to stimulate cAMP production in cytosolic and caveolar compartments of intact cardiac myocytes, while PGE1 only stimulates cAMP production in the cytosolic compartment. Unlike other receptor-mediated responses, compartmentation of PGE1 responses was not due to concurrent activation of a Gi -dependent signalling pathway or phosphodiesterase activity. Instead, compartmentation of the PGE1 response in cardiac myocytes appears to be due to transient stimulation of cAMP in a microdomain that can communicate directly with the bulk cytosolic compartment but not the caveolar compartment associated with ,AR regulation of L-type Ca2+ channel function. [source]