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John D. Scott - One of the best experts on this subject based on the ideXlab platform.
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drugs that regulate local cell signaling akap targeting as a therapeutic option
Annual Review of Pharmacology and Toxicology, 2021Co-Authors: Paula J Bucko, John D. ScottAbstract:Cells respond to environmental cues by mobilizing signal transduction cascades that engage protein kinases and phosphoprotein phosphatases. Correct organization of these enzymes in space and time enables the efficient and precise transmission of chemical signals. The cyclic AMP-dependent protein kinase A is compartmentalized through its association with A-kinase anchoring proteins (AKAPs). AKAPs are a family of multivalent scaffolds that constrain signaling enzymes and effectors at subcellular locations to drive essential physiological events. More recently, it has been recognized that defective signaling in certain endocrine disorders and cancers proceeds through pathological AKAP complexes. Consequently, pharmacologically targeting these macromolecular complexes unlocks new therapeutic opportunities for a growing number of clinical indications. This review highlights recent findings on AKAP signaling in disease, particularly in certain cancers, and offers an overview of peptides and small molecules that locally regulate AKAP-binding partners.
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AKAP
2015Co-Authors: Jessica L. Esseltine, John D. ScottAbstract:signaling com towards the next ge, U Reviewtenets of AKAP signaling: specificity, sensitivity, localiza-tion, and temporal control. Recently, investigators are shifting their attention from cells in culture towards in vivo models in order to understand how anchored signaling pathways are per-turbed during disease. This comes at a time when approx-imately 50 % of marketed pharmaceuticals target G protein-coupled receptors (GPCRs) and increasing numbers of kinase inhibitor drugs are entering the quired for binding to PP2B was originally mapped more than 10 years ago, recent work has identified a specific sequence within this region, which bears striking resem-blance to the PIxIxIT motif in NFAT [11,12]. The authors showed that this region was responsible for the AKAP79/ 150-mediated activation of NFAT by PP2B [11,12]. In rat hippocampal neurons NFAT signaling is initiated via AKAP79/150-anchored Cav1.2-mediated increases in in-tracellular Ca2+, PP2B activation, and NFAT dephosphor-ylation, allowing NFAT translocation to the nucleus where it regulates gene transcription. Additionally, AKAP150-dependent nuclear translocation of NFAT regulates gene expression of KCNQ2 and KCNQ3 potassium channels and is abolished in AKAP150 null mice [13]. Activation of AKAP-anchored PP2B involves local Ca2+ influx through the L-type voltage-gated Ca2+ channel, Cav1.2. However, negative feedback regulation of the chan-nel also appears to be modulated by PP2B as deletion of the 0165-6147/ $ – see front matte
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akap anchored pka maintains neuronal l type calcium channel activity and nfat transcriptional signaling
Cell Reports, 2014Co-Authors: Jonathan G Murphy, Jennifer L Sanderson, John D. Scott, William A Sather, William A Catterall, Jessica A Gorski, Mark L DellacquaAbstract:L-type voltage-gated Ca2+ channels (LTCC) couple neuronal excitation to gene transcription. LTCC activity is elevated by the cyclic AMP (cAMP)-dependent protein kinase (PKA) and depressed by the Ca2+-dependent phosphatase calcineurin (CaN), and both enzymes are localized to the channel by A-kinase anchoring protein 79/150 (AKAP79/150). AKAP79/150 anchoring of CaN also promotes LTCC activation of transcription through dephosphorylation of the nuclear factor of activated T cells (NFAT). We report here that the basal activity of AKAP79/150-anchored PKA maintains neuronal LTCC coupling to CaN-NFAT signaling by preserving LTCC phosphorylation in opposition to anchored CaN. Genetic disruption of AKAP-PKA anchoring promoted redistribution of the kinase out of postsynaptic dendritic spines, profound decreases in LTCC phosphorylation and Ca2+ influx, and impaired NFAT movement to the nucleus and activation of transcription. Thus, LTCC-NFAT transcriptional signaling in neurons requires precise organization and balancing of PKA and CaN activities in the channel nanoenvironment, which is only made possible by AKAP79/150 scaffolding.
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intrinsic disorder within an akap protein kinase a complex guides local substrate phosphorylation
eLife, 2013Co-Authors: Donelson F Smith, Lorene K Langeberg, John D. Scott, Jessica L. Esseltine, Steve L Reichow, Dan Shi, Tamir GonenAbstract:Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an ∼16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates. Cell-based analyses suggest that the catalytic subunit remains within type-II PKA-AKAP18γ complexes upon cAMP elevation. We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.01319.001.
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engineering a kinase anchoring protein akap selective regulatory subunits of protein kinase a pka through structure based phage selection
Journal of Biological Chemistry, 2013Co-Authors: Matthew G Gold, Christopher K Means, Catherine T Pawson, Jason J Stephany, Lorene K Langeberg, Douglas M. Fowler, Stanley Fields, John D. ScottAbstract:PKA is retained within distinct subcellular environments by the association of its regulatory type II (RII) subunits with A-kinase anchoring proteins (AKAPs). Conventional reagents that universally disrupt PKA anchoring are patterned after a conserved AKAP motif. We introduce a phage selection procedure that exploits high-resolution structural information to engineer RII mutants that are selective for a particular AKAP. Selective RII (RSelect) sequences were obtained for eight AKAPs following competitive selection screening. Biochemical and cell-based experiments validated the efficacy of RSelect proteins for AKAP2 and AKAP18. These engineered proteins represent a new class of reagents that can be used to dissect the contributions of different AKAP-targeted pools of PKA. Molecular modeling and high-throughput sequencing analyses revealed the molecular basis of AKAP-selective interactions and shed new light on native RII-AKAP interactions. We propose that this structure-directed evolution strategy might be generally applicable for the investigation of other protein interaction surfaces.
Mark L Dellacqua - One of the best experts on this subject based on the ideXlab platform.
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akap79 150 recruits the transcription factor nfat to regulate signaling to the nucleus by neuronal l type ca2 channels
Molecular Biology of the Cell, 2019Co-Authors: Kevin C Crosby, Jonathan G Murphy, Philip J Dittmer, William A Sather, Mark L DellacquaAbstract:In neurons, regulation of activity-dependent transcription by the nuclear factor of activated T-cells (NFAT) depends upon Ca2+ influx through voltage-gated L-type calcium channels (LTCC) and NFAT translocation to the nucleus following its dephosphorylation by the Ca2+-dependent phosphatase calcineurin (CaN). CaN is recruited to the channel by A-kinase anchoring protein (AKAP) 79/150, which binds to the LTCC C-terminus via a modified leucine-zipper (LZ) interaction. Here we sought to gain new insights into how LTCCs and signaling to NFAT are regulated by this LZ interaction. RNA interference-mediated knockdown of endogenous AKAP150 and replacement with human AKAP79 lacking its C-terminal LZ domain resulted in loss of depolarization-stimulated NFAT signaling in rat hippocampal neurons. However, the LZ mutation had little impact on the AKAP-LTCC interaction or LTCC function, as measured by Forster resonance energy transfer, Ca2+ imaging, and electrophysiological recordings. AKAP79 and NFAT coimmunoprecipitated when coexpressed in heterologous cells, and the LZ mutation disrupted this association. Critically, measurements of NFAT mobility in neurons employing fluorescence recovery after photobleaching and fluorescence correlation spectroscopy provided further evidence for an AKAP79 LZ interaction with NFAT. These findings suggest that the AKAP79/150 LZ motif functions to recruit NFAT to the LTCC signaling complex to promote its activation by AKAP-anchored calcineurin.
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camkii regulates the depalmitoylation and synaptic removal of the scaffold protein akap79 150 to mediate structural long term depression
Journal of Biological Chemistry, 2017Co-Authors: Kevin M Woolfrey, Mark L Dellacqua, Holly R Robertson, Heather Oleary, Dayton J Goodell, Eric A Horne, Steven J Coultrap, Ulrich K BayerAbstract:Both long-term potentiation (LTP) and depression (LTD) of excitatory synapse strength require the Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) and its autonomous activity generated by Thr-286 autophosphorylation. Additionally, LTP and LTD are correlated with dendritic spine enlargement and shrinkage that are accompanied by the synaptic accumulation or removal, respectively, of the AMPA-receptor regulatory scaffold protein A-kinase anchoring protein (AKAP) 79/150. We show here that the spine shrinkage associated with LTD indeed requires synaptic AKAP79/150 removal, which in turn requires CaMKII activity. In contrast to normal CaMKII substrates, the substrate sites within the AKAP79/150 N-terminal polybasic membrane-cytoskeletal targeting domain were phosphorylated more efficiently by autonomous compared with Ca2+/CaM-stimulated CaMKII activity. This unusual regulation was mediated by Ca2+/CaM binding to the substrate sites resulting in protection from phosphorylation in the presence of Ca2+/CaM, a mechanism that favors phosphorylation by prolonged, weak LTD stimuli versus brief, strong LTP stimuli. Phosphorylation by CaMKII inhibited AKAP79/150 association with F-actin; it also facilitated AKAP79/150 removal from spines but was not required for it. By contrast, LTD-induced spine removal of AKAP79/150 required its depalmitoylation on two Cys residues within the N-terminal targeting domain. Notably, such LTD-induced depalmitoylation was also blocked by CaMKII inhibition. These results provide a mechanism how CaMKII can indeed mediate not only LTP but also LTD through regulated substrate selection; however, in the case of AKAP79/150, indirect CaMKII effects on palmitoylation are more important than the effects of direct phosphorylation. Additionally, our results provide the first direct evidence for a function of the well-described AKAP79/150 trafficking in regulating LTD-induced spine shrinkage.
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the palmitoyl acyltransferase dhhc2 regulates recycling endosome exocytosis and synaptic potentiation through palmitoylation of akap79 150
The Journal of Neuroscience, 2015Co-Authors: Kevin M Woolfrey, Jennifer L Sanderson, Mark L DellacquaAbstract:Phosphorylation and dephosphorylation of AMPA-type ionotropic glutamate receptors (AMPARs) by kinases and phosphatases and interactions with scaffold proteins play essential roles in regulating channel biophysical properties and trafficking events that control synaptic strength during NMDA receptor-dependent synaptic plasticity, such as LTP and LTD. We previously demonstrated that palmitoylation of the AMPAR-linked scaffold protein A-kinase anchoring protein (AKAP) 79/150 is required for its targeting to recycling endosomes in dendrites, where it regulates exocytosis from these compartments that is required for LTP-stimulated enlargement of postsynaptic dendritic spines, delivery of AMPARs to the plasma membrane, and maintenance of synaptic potentiation. Here, we report that the recycling endosome-resident palmitoyl acyltransferase DHHC2 interacts with and palmitoylates AKAP79/150 to regulate these plasticity signaling mechanisms. In particular, RNAi-mediated knockdown of DHHC2 expression in rat hippocampal neurons disrupted stimulation of exocytosis from recycling endosomes, enlargement of dendritic spines, AKAP recruitment to spines, and potentiation of AMPAR-mediated synaptic currents that occur during LTP. Importantly, expression of a palmitoylation-independent lipidated AKAP mutant in DHHC2-deficient neurons largely restored normal plasticity regulation. Thus, we conclude that DHHC2-AKAP79/150 signaling is an essential regulator of dendritic recycling endosome exocytosis that controls both structural and functional plasticity at excitatory synapses.
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akap anchored pka maintains neuronal l type calcium channel activity and nfat transcriptional signaling
Cell Reports, 2014Co-Authors: Jonathan G Murphy, Jennifer L Sanderson, John D. Scott, William A Sather, William A Catterall, Jessica A Gorski, Mark L DellacquaAbstract:L-type voltage-gated Ca2+ channels (LTCC) couple neuronal excitation to gene transcription. LTCC activity is elevated by the cyclic AMP (cAMP)-dependent protein kinase (PKA) and depressed by the Ca2+-dependent phosphatase calcineurin (CaN), and both enzymes are localized to the channel by A-kinase anchoring protein 79/150 (AKAP79/150). AKAP79/150 anchoring of CaN also promotes LTCC activation of transcription through dephosphorylation of the nuclear factor of activated T cells (NFAT). We report here that the basal activity of AKAP79/150-anchored PKA maintains neuronal LTCC coupling to CaN-NFAT signaling by preserving LTCC phosphorylation in opposition to anchored CaN. Genetic disruption of AKAP-PKA anchoring promoted redistribution of the kinase out of postsynaptic dendritic spines, profound decreases in LTCC phosphorylation and Ca2+ influx, and impaired NFAT movement to the nucleus and activation of transcription. Thus, LTCC-NFAT transcriptional signaling in neurons requires precise organization and balancing of PKA and CaN activities in the channel nanoenvironment, which is only made possible by AKAP79/150 scaffolding.
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balanced interactions of calcineurin with akap79 regulate ca2 calcineurin nfat signaling
Nature Structural & Molecular Biology, 2012Co-Authors: Matthew D Pink, Mark L Dellacqua, Patrick G. Hogan, Jonathan G Murphy, Alexander SteinAbstract:In hippocampal neurons, the scaffold protein AKAP79 recruits the phosphatase calcineurin to L-type Ca(2+) channels and couples Ca(2+) influx to activation of calcineurin and of its substrate, the transcription factor NFAT. Here we show that an IAIIIT anchoring site in human AKAP79 binds the same surface of calcineurin as the PxIxIT recognition peptide of NFAT, albeit more strongly. A modest decrease in calcineurin-AKAP affinity due to an altered anchoring sequence is compatible with NFAT activation, whereas a further decrease impairs activation. Counterintuitively, increasing calcineurin-AKAP affinity increases recruitment of calcineurin to the scaffold but impairs NFAT activation; this is probably due to both slower release of active calcineurin from the scaffold and sequestration of active calcineurin by 'decoy' AKAP sites. We propose that calcineurin-AKAP79 scaffolding promotes NFAT signaling by balancing strong recruitment of calcineurin with its efficient release to communicate with NFAT.
Kjetil Taskén - One of the best experts on this subject based on the ideXlab platform.
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molecular mechanisms for camp mediated immunoregulation in t cells role of anchored protein kinase a signaling units
Frontiers in Immunology, 2016Co-Authors: Vanessa L Wehbi, Kjetil TaskénAbstract:The cyclic AMP/protein kinase A (cAMP/PKA) pathway is one of the most common and versatile signal pathways in eukaryotic cells. A kinase anchoring proteins (AKAPs) target PKA to specific substrates and distinct subcellular compartments providing spatial and temporal specificity for mediation of biological effects channeled through the cAMP/PKA pathway. In the immune system cAMP is a potent negative regulator of T cell receptor (TCR)-mediated activation of effector T cells (Teff) acting through a proximal PKA/Csk/Lck pathway anchored via a scaffold consisting of the AKAP Ezrin holding PKA, the linker protein EBP50 and the anchoring protein PAG holding Csk. As PKA activates Csk and Csk inhibits Lck this pathway in response to cAMP shuts down proximal T cell activation. This immunomodulating pathway in Teff mediates clinically important responses to regulatory T cell (Treg) suppression and inflammatory mediators such as prostaglandins, adrenergic stimuli, adenosine and a number of other ligands. A major inducer of T cell cAMP levels is prostaglandin E2 (PGE2) acting through EP2 and EP4 prostanoid receptors. PGE2 plays a crucial role in the normal physiological control of immune homeostasis as well as in inflammation and cancer immune evasion. Peripherally induced regulatory T cells express COX-2, secrete prostaglandin E2 (PGE2) and elicit the immunosuppressive cAMP pathway in effector T cells as one tumor immune evasion mechanism. Moreover, a cAMP increase can also be induced by indirect mechanisms such as intercellular transfer between T cells. Indeed, Treg, known to have elevated levels of intracellular cAMP, may mediate their suppressive function by transferring cAMP to Teff through gap junctions, which we speculate could also be regulated by PKA/AKAP complexes. In this review we present an updated overview on the influence of cAMP-mediated immunoregulatory mechanisms acting through localized cAMP signaling and the therapeutical increasing prospects of AKAPs disruptors in T cell immune function.
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molecular mechanisms for camp mediated immunoregulation in t cells role of anchored protein kinase a signaling units
Frontiers in Immunology, 2016Co-Authors: Vanessa L Wehbi, Kjetil TaskénAbstract:The cyclic AMP/protein kinase A (cAMP/PKA) pathway is one of the most common and versatile signal pathways in eukaryotic cells. A-kinase anchoring proteins (AKAPs) target PKA to specific substrates and distinct subcellular compartments providing spatial and temporal specificity for mediation of biological effects channeled through the cAMP/PKA pathway. In the immune system, cAMP is a potent negative regulator of T cell receptor-mediated activation of effector T cells (Teff) acting through a proximal PKA/Csk/Lck pathway anchored via a scaffold consisting of the AKAP Ezrin holding PKA, the linker protein EBP50, and the anchoring protein phosphoprotein associated with glycosphingolipid-enriched microdomains holding Csk. As PKA activates Csk and Csk inhibits Lck, this pathway in response to cAMP shuts down proximal T cell activation. This immunomodulating pathway in Teff mediates clinically important responses to regulatory T cell (Treg) suppression and inflammatory mediators, such as prostaglandins (PGs), adrenergic stimuli, adenosine, and a number of other ligands. A major inducer of T cell cAMP levels is PG E2 (PGE2) acting through EP2 and EP4 prostanoid receptors. PGE2 plays a crucial role in the normal physiological control of immune homeostasis as well as in inflammation and cancer immune evasion. Peripherally induced Tregs express cyclooxygenase-2, secrete PGE2, and elicit the immunosuppressive cAMP pathway in Teff as one tumor immune evasion mechanism. Moreover, a cAMP increase can also be induced by indirect mechanisms, such as intercellular transfer between T cells. Indeed, Treg, known to have elevated levels of intracellular cAMP, may mediate their suppressive function by transferring cAMP to Teff through gap junctions, which we speculate could also be regulated by PKA/AKAP complexes. In this review, we present an updated overview on the influence of cAMP-mediated immunoregulatory mechanisms acting through localized cAMP signaling and the therapeutical increasing prospects of AKAPs disruptors in T-cell immune function.
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characterization of a kinase anchoring disruptors using a solution based assay
Biochemical Journal, 2006Co-Authors: Anne Jorunn Stokka, John D. Scott, Friedrich W. Herberg, Cathrine R Carlson, Frank Gesellchen, Kjetil TaskénAbstract:Subcellular localization of PKA (cAMP-dependent protein kinase or protein kinase A) is determined by protein–protein interactions between its R (regulatory) subunits and AKAPs (A-kinase-anchoring proteins). In the present paper, we report the development of the Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen™) as a means to characterize AKAP-based peptide competitors of PKA anchoring. In this assay, the prototypic anchoring disruptor Ht31 efficiently competed in RIIα isoform binding with RII-specific and dual-specificity AKAPs (IC50 values of 1.4±0.2 nM and 6±1 nM respectively). In contrast, RIα isoform binding to a dual-specific AKAP was less efficiently competed (IC50 of 156±10 nM). Characterization of two RI-selective anchoring disruptors, RIAD (RI-anchoring disruptor) and PV-38 revealed that RIAD (IC50 of 13±1 nM) was 20-fold more potent than PV-38 (IC50 of 304±17 nM) and did not compete in the RIIα–AKAP interaction. We also observed that the kinetics of RII displacement from pre-formed PKA–AKAP complexes and competition of RII–AKAP complex formation by Ht31 differed by an order of magnitude when the component parts were mixed in vitro. No such difference in potency was seen for RIα–AKAP complexes. Thus the AlphaScreen assay may prove to be a valuable tool for detailed characterization of a variety of PKA–AKAP complexes.
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a kinase anchoring protein akap interaction and dimerization of the riα and riβ regulatory subunits of protein kinase a in vivo by the yeast two hybrid system
Journal of Molecular Biology, 2003Co-Authors: Cathrine R Carlson, Anja Ruppelt, Kjetil TaskénAbstract:Abstract Protein kinase A (PKA) regulatory (R) subunits dimerize through an N-terminal motif. Such dimerization is necessary for binding to PKA anchoring proteins (AKAPs) and targeting of PKA to its site of action. In the present study, we used the yeast two-hybrid system as an in vivo bio-reporter assay and analyzed the formation of homo- and heterodimeric complexes of RIα and RIβ as well as AKAP binding of RI dimers. Native polyacrylamide gel electrophoresis (PAGE) of yeast extracts confirmed the two-hybrid data. Both RIα- and RIβ homodimers as well as an RIα:RIβ heterodimer were observed. Single, double and one triple mutation were introduced into the RIα and RIβ subunits and dimerization properties of the mutants were analyzed. Consistent with previous reports, RIα(C37H) dimerized, although the disulfide bridges were disrupted, whereas the additional mutation of F47 or F52 abolished the dimerization. Corresponding mutations (C38H, F48A, F53A) in RIβ were not sufficient to abolish the RIβ dimerization, indicating that additional or other amino acids are important. RIα:RIβ heterodimers of the mutants were formed at intermediate stringency. Analysis of ternary complexes by the yeast two-hybrid system revealed that RIα and RIβ homodimers as well as an RIα:RIβ heterodimer and several of the mutants were able to bind to the R-binding domain of AKAP149/D-AKAP1. Furthermore, an RIβ:AKAP149 complex was identified following introduction of RIβ into HEK293 cells. Importantly, RIβ revealed AKAP binding properties similar to those of RIα, indicating that RIβ holoenzymes may be anchored.
Elona Gusho - One of the best experts on this subject based on the ideXlab platform.
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murine akap7 has a 2 5 phosphodiesterase domain that can complement an inactive murine coronavirus ns2 gene
Mbio, 2014Co-Authors: Susan R. Weiss, Elona Gusho, Rong Zhang, Babal K Jha, Joshua M Thornbrough, Beihua Dong, Christina Gaughan, Ruth Elliott, Robert H SilvermanAbstract:Viral 2',5'-phosphodiesterases (2',5'-PDEs) help disparate RNA viruses evade the antiviral activity of interferon (IFN) by degrading 2',5'-oligoadenylate (2-5A) activators of RNase L. A kinase anchoring proteins (AKAPs) bind the regulatory subunits of protein kinase A (PKA) to localize and organize cyclic AMP (cAMP) signaling during diverse physiological processes. Among more than 43 AKAP isoforms, AKAP7 appears to be unique in its homology to viral 2',5'-PDEs. Here we show that mouse AKAP7 rapidly degrades 2-5A with kinetics similar to that of murine coronavirus (mouse hepatitis virus [MHV]) strain A59 ns2 and human rotavirus strain WA VP3 proteins. To determine whether AKAP7 could substitute for a viral 2',5'-PDE, we inserted AKAP7 cDNA into an MHV genome with an inactivated ns2 gene. The AKAP7 PDE domain or N-terminally truncated AKAP7 (both lacking a nuclear localization motif), but not full-length AKAP7 or a mutant, AKAP7(H185R), PDE domain restored the infectivity of ns2 mutant MHV in bone marrow macrophages and in livers of infected mice. Interestingly, the AKAP7 PDE domain and N-terminally deleted AKAP7 were present in the cytoplasm (the site of MHV replication), whereas full-length AKAP7 was observed only in nuclei. We suggest the possibility that viral acquisition of the host AKAP7 PDE domain might have occurred during evolution, allowing diverse RNA viruses to antagonize the RNase L pathway. Importance: Early virus-host interactions determine whether an infection is established, highlighting the need to understand fundamental mechanisms regulating viral pathogenesis. Recently, our laboratories reported a novel mode of regulation of the IFN antiviral response. We showed that the coronavirus MHV accessory protein ns2 antagonizes the type I IFN response, promoting viral replication and hepatitis. ns2 confers virulence by cleaving 2',5'-oligoadenylate (2-5A) activators of RNase L in macrophages. We also reported that the rotavirus VP3 C-terminal domain (VP3-CTD) cleaves 2-5A and that it may rescue ns2 mutant MHV. Here we report that a cellular protein, AKAP7, has an analogous 2',5'-phosphodiesterase (2',5'-PDE) domain that is able to restore the growth of chimeric MHV expressing inactive ns2. The proviral effect requires cytoplasmic localization of the AKAP7 PDE domain. We speculate that AKAP7 is the ancestral precursor of viral proteins, such as ns2 and VP3, that degrade 2-5A to evade the antiviral activity of RNase L.
William A Sather - One of the best experts on this subject based on the ideXlab platform.
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akap79 150 recruits the transcription factor nfat to regulate signaling to the nucleus by neuronal l type ca2 channels
Molecular Biology of the Cell, 2019Co-Authors: Kevin C Crosby, Jonathan G Murphy, Philip J Dittmer, William A Sather, Mark L DellacquaAbstract:In neurons, regulation of activity-dependent transcription by the nuclear factor of activated T-cells (NFAT) depends upon Ca2+ influx through voltage-gated L-type calcium channels (LTCC) and NFAT translocation to the nucleus following its dephosphorylation by the Ca2+-dependent phosphatase calcineurin (CaN). CaN is recruited to the channel by A-kinase anchoring protein (AKAP) 79/150, which binds to the LTCC C-terminus via a modified leucine-zipper (LZ) interaction. Here we sought to gain new insights into how LTCCs and signaling to NFAT are regulated by this LZ interaction. RNA interference-mediated knockdown of endogenous AKAP150 and replacement with human AKAP79 lacking its C-terminal LZ domain resulted in loss of depolarization-stimulated NFAT signaling in rat hippocampal neurons. However, the LZ mutation had little impact on the AKAP-LTCC interaction or LTCC function, as measured by Forster resonance energy transfer, Ca2+ imaging, and electrophysiological recordings. AKAP79 and NFAT coimmunoprecipitated when coexpressed in heterologous cells, and the LZ mutation disrupted this association. Critically, measurements of NFAT mobility in neurons employing fluorescence recovery after photobleaching and fluorescence correlation spectroscopy provided further evidence for an AKAP79 LZ interaction with NFAT. These findings suggest that the AKAP79/150 LZ motif functions to recruit NFAT to the LTCC signaling complex to promote its activation by AKAP-anchored calcineurin.
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akap anchored pka maintains neuronal l type calcium channel activity and nfat transcriptional signaling
Cell Reports, 2014Co-Authors: Jonathan G Murphy, Jennifer L Sanderson, John D. Scott, William A Sather, William A Catterall, Jessica A Gorski, Mark L DellacquaAbstract:L-type voltage-gated Ca2+ channels (LTCC) couple neuronal excitation to gene transcription. LTCC activity is elevated by the cyclic AMP (cAMP)-dependent protein kinase (PKA) and depressed by the Ca2+-dependent phosphatase calcineurin (CaN), and both enzymes are localized to the channel by A-kinase anchoring protein 79/150 (AKAP79/150). AKAP79/150 anchoring of CaN also promotes LTCC activation of transcription through dephosphorylation of the nuclear factor of activated T cells (NFAT). We report here that the basal activity of AKAP79/150-anchored PKA maintains neuronal LTCC coupling to CaN-NFAT signaling by preserving LTCC phosphorylation in opposition to anchored CaN. Genetic disruption of AKAP-PKA anchoring promoted redistribution of the kinase out of postsynaptic dendritic spines, profound decreases in LTCC phosphorylation and Ca2+ influx, and impaired NFAT movement to the nucleus and activation of transcription. Thus, LTCC-NFAT transcriptional signaling in neurons requires precise organization and balancing of PKA and CaN activities in the channel nanoenvironment, which is only made possible by AKAP79/150 scaffolding.
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akap79 150 anchoring of calcineurin controls neuronal l type ca2 channel activity and nuclear signaling
Neuron, 2007Co-Authors: Seth F Oliveria, Mark L Dellacqua, William A SatherAbstract:Neuronal L-type calcium channels contribute to dendritic excitability and activity-dependent changes in gene expression that influence synaptic strength. Phosphorylation-mediated enhancement of L-type channels containing the CaV1.2 pore-forming subunit is promoted by A-kinase anchoring proteins (AKAPs) that target cAMP-dependent protein kinase (PKA) to the channel. Although PKA increases L-type channel activity in dendrites and dendritic spines, the mechanism of enhancement in neurons remains poorly understood. Here, we show that CaV1.2 interacts directly with AKAP79/150, which binds both PKA and the Ca2+/calmodulin-activated phosphatase calcineurin (CaN). Cotargeting of PKA and CaN by AKAP79/150 confers bidirectional regulation of L-type current amplitude in transfected HEK293 cells and hippocampal neurons. However, anchored CaN dominantly suppresses PKA enhancement of the channel. Additionally, activation of the transcription factor NFATc4 via local Ca2+ influx through L-type channels requires AKAP79/150, suggesting that this signaling complex promotes neuronal L channel signaling to the nucleus through NFATc4.