title: Receptor-mediated Ca2+ and PKC signaling triggers the loss of cortical PKA compartmentalization through the redistribution of gravin.
abstract: A-Kinase Anchoring Proteins (AKAPs) direct the flow of cellular information by positioning multiprotein signaling complexes into proximity with effector proteins. However, certain AKAPs are not stationary but can undergo spatiotemporal redistribution in response to stimuli. Gravin, a 300kD AKAP that intersects with a diverse signaling array, is localized to the plasma membrane but has been shown to translocate to the cytosol following the elevation of intracellular calcium ([Ca(2+)]i). Despite the potential for gravin redistribution to impact multiple signaling pathways, the dynamics of this event remain poorly understood. In this study, quantitative microscopy of cells expressing gravin-EGFP revealed that Ca(2+) elevation caused the complete translocation of gravin from the cell cortex to the cytosol in as little as 60s of treatment with ionomycin or thapsigargin. In addition, receptor mediated signaling was also shown to cause gravin redistribution following ATP treatment, and this event required both [Ca(2+)]i elevation and PKC activation. To understand the mechanism for Ca(2+) mediated gravin dynamics, deletion of calmodulin-binding domains revealed that a fourth putative calmodulin binding domain called CB4 (a.a. 670-694) is critical for targeting gravin to the cell cortex despite its location downstream of gravin's membrane-targeting domains, which include an N-terminal myristoylation site and three polybasic domains. Finally, confocal microscopy of cells co-transfected with gravin-EYFP and PKA RII-ECFP revealed that gravin redistribution mediated by ionomycin, thapsigargin, and ATP each triggered the gravin-dependent loss of PKA localized at the cell cortex. Our results support the hypothesis that gravin redistribution regulates cross-talk between PKA-dependent signaling and receptor-mediated events involving Ca(2+) and PKC.
authors.affiliation: Department of Basic Sciences, UND School of Medicine and Health Sciences, 501 N Columbia Rd., Grand Forks, ND 58202-9037, USA.
authors.forename: Micah B
authors.initials: MB
authors.lastname: Schott
authors.forename: Bryon
authors.initials: B
authors.lastname: Grove
mesh_terms: D054758:A Kinase Anchor Proteins / Q000235:genetics* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D061207:Calcium Ionophores / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D015398:Signal Transduction; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D054758:A Kinase Anchor Proteins; C491162:AKAP12 protein, human; D001426:Bacterial Proteins; D061207:Calcium Ionophores; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; C485184:enhanced green fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D015759:Ionomycin; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium
keywords: 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester); A-Kinase Anchoring Protein; AKAP; AKAP12; ATP; BAPTA-AM; BIM; CB4; CaM; Calcium; ECFP; EGFP; EYFP; G protein coupled receptor; GPCR; Gravin; IM; InsP3; PB1–3; PDE4; PKA; PKC; PLC; Purinergic receptor; RII; SERCA; SES; SOCE; SSeCKS; Src-Suppressed C-Kinase Substrate; Tg; WT; [Ca(2+)](i); adenosine triphosphate; bisindolylmaleimide; calcium–calmodulin; calmodulin binding domain 4; enhanced cyan fluorescent protein; enhanced green fluorescent protein; enhanced yellow fluorescent protein; inositol triphosphate; intracellular calcium concentration; ionomycin; phosphodiesterase type 4; phospholipase C; polybasic domains 1 through 3; protein kinase A; protein kinase C; regulatory subunit of type II PKA; sarco/endoplasmic reticulum calcium ATPase; standard extracellular solution; store-operated calcium entry; thapsigargin; wild-type/full-length; β2-adrenergic receptor; β2AR