{"gene":"AKAP6","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":1995,"finding":"AKAP6 (AKAP100) was identified as an A-kinase anchoring protein that binds the RIIα regulatory subunit of PKA and targets type II PKA to the sarcoplasmic reticulum in cardiac and skeletal muscle cells.","method":"Interaction cloning with RIIα probe, solid-phase overlay binding assay, immunohistochemistry, cAMP-agarose affinity chromatography","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (in vitro binding, affinity purification, immunolocalization) in a foundational paper with >90 citations","pmids":["7721854"],"is_preprint":false},{"year":1998,"finding":"AKAP100 (AKAP6) localizes to multiple subcellular compartments in adult cardiac myocytes—nucleus, sarcolemma, intercalated disc, Z-line, and transverse tubule/junctional sarcoplasmic reticulum—and co-localizes specifically with RII (not RI) subunits of PKA.","method":"Immunofluorescence, confocal microscopy, double immunostaining with α-actinin and ryanodine receptor antibodies, immunoblotting","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal localization methods with functional specificity (RII vs RI discrimination), >90 citations","pmids":["9679148"],"is_preprint":false},{"year":1999,"finding":"mAKAP (AKAP6) is targeted to the nuclear membrane of differentiated myocytes through two regions containing spectrin-like repeat sequences (residues 772–915 and 915–1065); overexpression of these targeting domains displaces endogenous mAKAP from the nuclear membrane, demonstrating saturable targeting.","method":"mAKAP-GFP fusion construct imaging, dominant-negative displacement experiments, nuclear membrane fractionation","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — live-cell imaging with GFP fusions plus functional displacement experiments, >150 citations","pmids":["10413680"],"is_preprint":false},{"year":2001,"finding":"mAKAP assembles a cAMP signaling module comprising PKA and PDE4D3 at the nuclear envelope in heart tissue; anchored PKA phosphorylates and activates PDE4D3 to reduce local cAMP levels (negative feedback loop), and disruption of PKA–mAKAP interaction prevents enhancement of PDE4D3 activity.","method":"Co-immunoprecipitation from cardiac tissue, functional kinase and phosphodiesterase activity assays, dominant-negative PKA-anchoring disruption","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical reconstitution in cardiac tissue plus functional activity assays, >380 citations","pmids":["11296225"],"is_preprint":false},{"year":2001,"finding":"The mAKAP complex at the cardiac nuclear envelope also contains ryanodine receptors and protein phosphatase 2A; mAKAP-bound ryanodine receptor can be regulated by PKA-mediated phosphorylation within the complex.","method":"Co-immunoprecipitation, immunohistochemistry, tissue fractionation","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP and fractionation with multiple antibodies, >125 citations","pmids":["11590243"],"is_preprint":false},{"year":2003,"finding":"mAKAP-anchored PKA directly increases PKA-dependent phosphorylation of the skeletal muscle ryanodine receptor (RyR1) and augments Ca2+ efflux through RyR1; a PKA-anchoring-deficient mutant mAKAP-P fails to enhance RyR1 phosphorylation or Ca2+ release.","method":"CHO cell expression of mAKAP vs. mAKAP-P (PKA-anchoring mutant), immunoelectron microscopy, PKA phosphorylation assay, cytosolic Ca2+ transient measurements with caffeine/forskolin","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis of anchoring domain combined with functional phosphorylation and Ca2+ assays","pmids":["12709444"],"is_preprint":false},{"year":2004,"finding":"PKA phosphorylation of PDE4D3 on Ser-13 increases the affinity of PDE4D3 for mAKAP, thereby enhancing recruitment of PDE4D3 to the mAKAP complex and facilitating faster cAMP signal termination.","method":"In vitro binding assays, cellular co-immunoprecipitation, site-directed mutagenesis of PDE4D3 Ser-13","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — in vitro assay plus mutagenesis plus cellular validation, >85 citations","pmids":["15182229"],"is_preprint":false},{"year":2005,"finding":"The mAKAP complex coordinates two integrated cAMP effector feedback loops: anchored PKA activates PDE4D3 (negative feedback), while an mAKAP-associated ERK5 module suppresses PDE4D3; PDE4D3 also acts as adaptor to recruit Epac1, enabling cAMP-dependent attenuation of ERK5. Anchored ERK5 can induce cardiomyocyte hypertrophy.","method":"Co-immunoprecipitation, pharmacological and dominant-negative disruption, ERK5 kinase assay, cardiomyocyte hypertrophy assay (cell size measurement)","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods, reconstituted feedback loops, functional hypertrophy readout, >450 citations","pmids":["16177794"],"is_preprint":false},{"year":2005,"finding":"mAKAP facilitates PKA-catalyzed phosphorylation of the ryanodine receptor Ca2+ channel; calcineurin Aβ associates with mAKAP; and the mAKAP complex is required for full activation of the pro-hypertrophic transcription factor NFATc in response to adrenergic signaling.","method":"Co-immunoprecipitation of calcineurin with mAKAP, siRNA knockdown of mAKAP, expression of PKA-anchoring-deficient mAKAP mutant, NFATc transcriptional reporter assay, cardiomyocyte hypertrophy assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — RNAi + dominant-negative + co-IP + functional transcription/hypertrophy assays, >100 citations","pmids":["16306226"],"is_preprint":false},{"year":2005,"finding":"Nesprin-1α serves as the receptor for mAKAP at the nuclear envelope: nesprin-1α is inserted into the nuclear envelope via a C-terminal klarsicht transmembrane domain and its N-terminal spectrin repeat dimerization domain directly binds the third spectrin repeat of mAKAP, targeting mAKAP to the nuclear envelope.","method":"Direct binding assay between nesprin-1α and mAKAP spectrin repeat domains, overexpression-mediated displacement, co-immunoprecipitation","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 2 — direct binding plus domain mapping plus functional displacement, >100 citations","pmids":["15652351"],"is_preprint":false},{"year":2008,"finding":"mAKAP organizes ubiquitin E3 ligases at the perinuclear region that control HIF-1α stability; depletion of mAKAP or disruption of its perinuclear targeting alters HIF-1α stability and transcriptional activation of hypoxia-responsive genes in cardiomyocytes.","method":"Co-immunoprecipitation of E3 ligases with mAKAP, siRNA knockdown, HIF-1α stability assay (immunoblot under normoxia/hypoxia), transcriptional reporter assay","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 2 — co-IP of E3 ligases plus RNAi plus functional HIF-1α stability and transcription assays","pmids":["19109240"],"is_preprint":false},{"year":2010,"finding":"PP2A associated with mAKAP complexes dephosphorylates PDE4D3 at Ser-54; the PP2A holoenzyme in this complex contains the B56δ targeting subunit, which is itself a PKA substrate whose phosphorylation by anchored PKA enhances phosphatase activity 2-fold, constituting a cAMP-induced positive feedback loop. The C-terminal mAKAP domain (residues 2085–2319) mediates PP2A binding.","method":"Domain mapping by deletion constructs, co-immunoprecipitation from heart tissue, phosphatase activity assay, site-directed mutagenesis of B56δ PKA phosphorylation site, PDE4D3 activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — domain mapping, mutagenesis, in vitro phosphatase activity, and cardiac tissue co-IP with multiple orthogonal methods","pmids":["20106966"],"is_preprint":false},{"year":2012,"finding":"mAKAP organizes a calcineurin/MEF2 signaling complex in myocytes; calcineurin–MEF2 association is dependent on mAKAP expression, and disruption of calcineurin binding to mAKAP (via a competing calcineurin-binding domain peptide) blunts MEF2 transcriptional activity during myoblast differentiation and inhibits adrenergic-induced cardiomyocyte hypertrophy.","method":"Co-immunoprecipitation from C2C12 cells and cardiac myocytes, dominant-negative calcineurin-binding domain peptide, MEF2 transcriptional reporter assay, myoblast differentiation assay","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 2 — co-IP plus dominant-negative disruption plus functional transcription and differentiation assays","pmids":["23261540"],"is_preprint":false},{"year":2012,"finding":"mAKAP directly binds MEF2 transcription factors through discrete domains; disruption of MEF2/mAKAP binding blocks MEF2 activation in early myoblast differentiation and inhibits myotube formation and expression of differentiation markers.","method":"Co-immunoprecipitation, direct domain binding assay, dominant-interference with MEF2/mAKAP binding peptide, myogenic differentiation assay (myotube formation, differentiation marker expression)","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 2 — direct domain binding plus functional differentiation readout with dominant-negative approach","pmids":["22484155"],"is_preprint":false},{"year":2013,"finding":"The naturally occurring human mAKAP mutation P1400S (in the PDE4D3-binding site) significantly reduces binding to PDE4D3 without affecting PKA binding; S2195F (in the PP2A-binding site) increases PKA binding and PKA activity; L717V (flanking the spectrin repeat domain) increases PKA binding without changing activity. These confirm specific binding domains for each partner.","method":"Immunoprecipitation, surface plasmon resonance (Biacore-2000), PKA activity assay, Ca2+ measurement","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis validated by SPR plus co-IP plus functional activity assays","pmids":["23806656"],"is_preprint":false},{"year":2015,"finding":"AKAP6 knockdown in skeletal myoblasts halts myotube formation and decreases myogenin and myosin heavy chain expression; AKAP6 promotes myogenin expression through MEF2A, and myogenin in turn binds an E-box site on the AKAP6 promoter to upregulate AKAP6 expression (positive feedback loop). In vivo shAKAP6 delivery impairs muscle regeneration after cardiotoxin injury.","method":"siRNA knockdown of AKAP6, shRNA-lentivirus delivery in vivo, myoblast differentiation assay, chromatin immunoprecipitation (ChIP), luciferase promoter assay, motor function testing","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 — RNAi plus ChIP plus promoter reporter plus in vivo rescue experiment","pmids":["26563778"],"is_preprint":false},{"year":2019,"finding":"AKAP6 co-localizes and physically interacts with phospholamban (PLN) at the perinuclear/sarcoplasmic reticulum region in cardiomyocytes; AKAP6 co-expression promotes Ca2+ uptake activity of SERCA1 in the presence of PLN.","method":"Co-immunoprecipitation (HEK-293T and adult rat cardiomyocytes), immunofluorescence colocalization, Ca2+ uptake functional assay","journal":"Physiological reports","confidence":"Medium","confidence_rationale":"Tier 3 — co-IP plus colocalization plus one functional assay, single lab","pmids":["31325238"],"is_preprint":false},{"year":2020,"finding":"AKAP6 is a key organizer of the nuclear envelope MTOC in cardiomyocytes and osteoclasts: its spectrin repeats anchor centrosomal proteins (Pcnt, AKAP9) to the nuclear envelope via nesprin-1α. AKAP6 and AKAP9 together form a protein platform tethering the Golgi to the nucleus, enabling two pools of microtubules. Ectopic AKAP6 expression in epithelial cells is sufficient to recruit endogenous centrosomal proteins to the nuclear envelope. AKAP6 is required for cardiomyocyte hypertrophy and osteoclast bone resorption.","method":"Immunofluorescence, co-immunoprecipitation, gain-of-function (ectopic AKAP6 expression), loss-of-function (AKAP6 KD/KO), MTOC activity assays, cardiomyocyte hypertrophy assay, osteoclast bone resorption assay","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including gain- and loss-of-function with defined cellular phenotype, replicated in two cell types","pmids":["33295871"],"is_preprint":false},{"year":2021,"finding":"The transcription factor myogenin is required and sufficient to drive NE-MTOC formation by inducing transcription of muscle/NE-MTOC-specific isoforms of AKAP6 and nesprin-1α; overexpression of AKAP6β and nesprin-1α together is sufficient to recruit endogenous MTOC proteins to the nuclear envelope in myoblasts even without myogenin.","method":"Loss- and gain-of-function experiments (siRNA, overexpression), promoter/transcriptional reporter assays, bioinformatics, immunofluorescence for MTOC protein recruitment","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — promoter studies plus gain/loss-of-function with clear molecular readout, orthogonal to prior AKAP6 MTOC paper","pmids":["34605406"],"is_preprint":false},{"year":2024,"finding":"AKAP6 anchors calcineurin (CaN) and NFATc4 in neurons; BDNF-mediated neuroprotection requires AKAP6-anchored CaN to activate NFATc4 transcription, and NFATc4 acts downstream of BDNF neuroprotection in vivo.","method":"Co-immunoprecipitation of CaN/NFATc4 with AKAP6, dominant-negative disruption of CaN anchoring, NFATc4 transcriptional reporter, NFATc4-/- mouse neuroprotection assay","journal":"Molecular brain","confidence":"Medium","confidence_rationale":"Tier 2-3 — co-IP plus dominant-negative plus in vivo KO, single lab with limited citations","pmids":["39578909"],"is_preprint":false},{"year":2025,"finding":"Activation of the Wnt/β-catenin pathway upregulates AKAP6 expression (AKAP6 is a target gene of canonical Wnt signaling), which in turn enhances PKA-mediated phosphorylation of RyR2, causing sarcoplasmic reticulum calcium leakage and cardiac dysfunction.","method":"Transcriptome analysis, Wnt pathway activation/inhibition (cycloheximide block), RyR2 phosphorylation assay, AKAP6 overexpression/knockdown, Ca2+ imaging in cardiomyocytes","journal":"Journal of molecular cell biology","confidence":"Medium","confidence_rationale":"Tier 2-3 — transcriptomics plus functional RyR2 phosphorylation and Ca2+ assays, single recent paper","pmids":["40097291"],"is_preprint":false}],"current_model":"AKAP6 (mAKAP) functions as a perinuclear scaffold protein—anchored to the nuclear envelope via direct binding of its spectrin repeats to nesprin-1α—that assembles dynamic signalosomes in striated muscle and neurons, coordinating PKA, PDE4D3, PP2A, Epac1, ERK5, ryanodine receptors, calcineurin, MEF2, HIF-1α-regulatory E3 ligases, and phospholamban into integrated cAMP/Ca2+/MAPK feedback circuits that control cardiac hypertrophy, myogenic differentiation, hypoxic gene expression, and, additionally, serves as the key organizer of the nuclear envelope microtubule-organizing center by recruiting centrosomal proteins (Pcnt, AKAP9) through its spectrin repeats downstream of myogenin-driven transcription."},"narrative":{"teleology":[{"year":1995,"claim":"Establishing that AKAP6 is an A-kinase anchoring protein resolved how type II PKA is targeted to specific subcellular compartments in cardiac and skeletal muscle.","evidence":"Interaction cloning with RIIα, overlay binding assay, and cAMP-agarose affinity chromatography in cardiac tissue","pmids":["7721854"],"confidence":"High","gaps":["Exact subcellular targeting mechanism unknown","No identified substrates of the anchored PKA pool","Functional consequence of PKA anchoring not tested"]},{"year":1999,"claim":"Mapping nuclear envelope targeting to two spectrin-like repeat regions explained how AKAP6 achieves perinuclear localization in differentiated myocytes, distinguishing it from other AKAPs.","evidence":"GFP-fusion construct imaging and dominant-negative displacement of endogenous mAKAP from the nuclear membrane","pmids":["10413680","9679148"],"confidence":"High","gaps":["Identity of the nuclear envelope receptor for AKAP6 unknown","Whether spectrin repeats mediate protein-protein interactions beyond targeting unclear"]},{"year":2001,"claim":"Discovery that AKAP6 co-assembles PKA, PDE4D3, ryanodine receptors, and PP2A into one complex established the concept of an integrated cAMP/Ca²⁺ signaling module with built-in negative feedback at the nuclear envelope.","evidence":"Co-immunoprecipitation from cardiac tissue, kinase and phosphodiesterase activity assays, dominant-negative PKA-anchoring disruption","pmids":["11296225","11590243"],"confidence":"High","gaps":["Stoichiometry and dynamics of the complex not determined","Whether ryanodine receptor regulation by this complex operates in vivo unclear"]},{"year":2005,"claim":"Identification of dual opposing feedback loops (PKA→PDE4D3 activation vs. ERK5→PDE4D3 inhibition, with Epac1 attenuating ERK5) and calcineurin/NFATc recruitment revealed how AKAP6 integrates cAMP and MAPK signaling to control cardiomyocyte hypertrophy.","evidence":"Co-IP, pharmacological and dominant-negative disruption, ERK5 kinase assay, NFATc reporter, cardiomyocyte hypertrophy cell-size measurement","pmids":["16177794","16306226"],"confidence":"High","gaps":["In vivo cardiac phenotype of AKAP6 disruption not yet shown","Whether ERK5 module operates independently of PKA feedback in pathological hypertrophy unclear"]},{"year":2005,"claim":"Identification of nesprin-1α as the nuclear envelope receptor that directly binds the third spectrin repeat of AKAP6 solved how AKAP6 is anchored at the nuclear envelope.","evidence":"Direct binding assay between nesprin-1α and mAKAP spectrin repeat domains, overexpression displacement, co-immunoprecipitation","pmids":["15652351"],"confidence":"High","gaps":["Structural basis of spectrin repeat–nesprin interaction not resolved","Whether other nuclear envelope proteins contribute to anchoring unknown"]},{"year":2008,"claim":"Showing that AKAP6 organizes E3 ubiquitin ligases controlling HIF-1α stability expanded the scaffold's role beyond kinase/phosphatase signaling to include regulation of hypoxic gene expression.","evidence":"Co-IP of E3 ligases with mAKAP, siRNA knockdown, HIF-1α stability immunoblot under normoxia/hypoxia, transcriptional reporter","pmids":["19109240"],"confidence":"High","gaps":["Identity and specificity of the E3 ligases in the complex not fully defined","Whether HIF-1α regulation is relevant outside cardiomyocytes unknown"]},{"year":2010,"claim":"Characterization of PP2A (B56δ subunit) as a PKA-activated phosphatase within the AKAP6 complex that dephosphorylates PDE4D3 uncovered a cAMP-induced positive feedback loop layered onto the existing negative feedback circuit.","evidence":"Domain mapping, cardiac tissue co-IP, phosphatase activity assay, B56δ PKA-site mutagenesis","pmids":["20106966"],"confidence":"High","gaps":["Net outcome of simultaneous positive and negative feedback on local cAMP dynamics not modeled quantitatively","Whether other PP2A substrates in the complex exist unknown"]},{"year":2012,"claim":"Demonstrating that AKAP6 directly binds MEF2 and bridges calcineurin to MEF2 established the scaffold as essential for myogenic transcription and differentiation beyond its kinase-anchoring role.","evidence":"Co-IP, direct domain binding, dominant-negative peptide interference, MEF2 reporter, myotube formation assay","pmids":["23261540","22484155"],"confidence":"High","gaps":["Which MEF2 isoform is preferentially bound in vivo unknown","Structural basis of MEF2–AKAP6 interface not determined"]},{"year":2015,"claim":"Discovery of a positive feedback loop in which AKAP6 promotes myogenin expression via MEF2A, and myogenin in turn transactivates the AKAP6 promoter, explained how AKAP6 levels are amplified during myogenesis and linked AKAP6 to in vivo muscle regeneration.","evidence":"siRNA/shRNA knockdown, ChIP showing myogenin at AKAP6 E-box, luciferase promoter assay, in vivo cardiotoxin injury model","pmids":["26563778"],"confidence":"High","gaps":["Whether the feedback loop is sufficient for full differentiation or requires additional cofactors unknown","Temporal dynamics of the feedback loop during regeneration not resolved"]},{"year":2020,"claim":"Establishing AKAP6 as the key organizer of the nuclear envelope MTOC — recruiting Pcnt and AKAP9 via its spectrin repeats and nesprin-1α — redefined AKAP6 function beyond signaling scaffold to include cytoskeletal architecture essential for cardiomyocyte hypertrophy and osteoclast bone resorption.","evidence":"Gain-of-function (ectopic AKAP6 in epithelial cells recruits centrosomal proteins), loss-of-function (KD/KO), MTOC activity assays, hypertrophy and resorption assays in two cell types","pmids":["33295871"],"confidence":"High","gaps":["How AKAP6-mediated MTOC function is coordinated with its signaling scaffold role is unclear","Whether AKAP6 recruits additional MTOC components beyond Pcnt and AKAP9 unknown"]},{"year":2021,"claim":"Showing that myogenin is both necessary and sufficient to induce the muscle-specific AKAP6β and nesprin-1α isoforms that together reconstitute NE-MTOC formation placed AKAP6 MTOC assembly under direct transcriptional control of the myogenic program.","evidence":"siRNA and overexpression of myogenin, promoter/transcriptional reporter, immunofluorescence for MTOC protein recruitment in myoblasts","pmids":["34605406"],"confidence":"High","gaps":["Whether other transcription factors cooperate with myogenin for AKAP6 induction unknown","Mechanism by which NE-MTOC formation feeds back on differentiation not explored"]},{"year":2024,"claim":"Extension of the AKAP6–calcineurin–NFATc axis to neurons demonstrated that AKAP6-anchored calcineurin activates NFATc4 downstream of BDNF for neuroprotection, broadening AKAP6 function beyond striated muscle.","evidence":"Co-IP of CaN/NFATc4 with AKAP6, dominant-negative disruption, NFATc4 reporter, NFATc4−/− mouse neuroprotection assay","pmids":["39578909"],"confidence":"Medium","gaps":["Single-lab finding; independent replication in neuronal systems needed","Identity of neuroprotective NFATc4 target genes downstream of AKAP6 unknown"]},{"year":2025,"claim":"Identification of AKAP6 as a Wnt/β-catenin target gene whose upregulation enhances PKA-mediated RyR2 phosphorylation and SR calcium leak linked an upstream developmental pathway to AKAP6-dependent calcium dysregulation in cardiac dysfunction.","evidence":"Transcriptomics, Wnt pathway activation/inhibition, RyR2 phosphorylation assay, AKAP6 overexpression/knockdown, Ca²⁺ imaging in cardiomyocytes","pmids":["40097291"],"confidence":"Medium","gaps":["Whether Wnt-driven AKAP6 upregulation is relevant in human heart failure not established","Mechanism of β-catenin regulation of AKAP6 promoter not mapped"]},{"year":null,"claim":"A full structural model of the AKAP6 signalosome — including how simultaneous binding of PKA, PDE4D3, PP2A, ERK5, calcineurin, MEF2, and MTOC components is coordinated on one scaffold — and in vivo phenotyping of AKAP6 knockout in adult heart remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of any AKAP6 multi-protein complex","No conditional cardiac-specific AKAP6 knockout phenotype published","Quantitative modeling of the integrated feedback circuits has not been performed"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,3,7,8,11,12,17]}],"localization":[{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[2,9,17]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,2]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[17,18]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,7,8,11,20]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[13,15,18]},{"term_id":"R-HSA-397014","term_label":"Muscle contraction","supporting_discovery_ids":[1,5,16]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[17,18]}],"complexes":["mAKAP-PKA-PDE4D3-Epac1-ERK5 signalosome","mAKAP-calcineurin-MEF2 complex","mAKAP-nesprin-1α-Pcnt-AKAP9 NE-MTOC platform","mAKAP-PP2A(B56δ) complex"],"partners":["PRKAR2A","PDE4D3","SYNE1","PPP3CA","MEF2A","PCNT","AKAP9","PLN"],"other_free_text":[]},"mechanistic_narrative":"AKAP6 (mAKAP) is a perinuclear scaffold protein in striated muscle and neurons that integrates cAMP, calcium, and MAPK signaling at the nuclear envelope to control cardiac hypertrophy, myogenic differentiation, hypoxic gene expression, and microtubule organization. Anchored to the nuclear envelope via direct binding of its spectrin repeats to nesprin-1α, AKAP6 assembles a signalosome containing PKA, PDE4D3, PP2A (B56δ), Epac1, ERK5, calcineurin, ryanodine receptors, and MEF2 transcription factors, enabling coordinated feedback loops in which PKA activates PDE4D3 to dampen local cAMP while ERK5 opposes this effect, and calcineurin activates NFATc and MEF2 to drive hypertrophic and myogenic transcription [PMID:11296225, PMID:16177794, PMID:23261540]. AKAP6 also serves as the key organizer of the nuclear envelope microtubule-organizing center (NE-MTOC) by recruiting centrosomal proteins Pcnt and AKAP9 through its spectrin repeats, a process driven by myogenin-dependent transcriptional induction of AKAP6 and nesprin-1α [PMID:33295871, PMID:34605406]. In neurons, AKAP6-anchored calcineurin activates NFATc4 downstream of BDNF to mediate neuroprotection, and in cardiomyocytes Wnt/β-catenin signaling upregulates AKAP6 to enhance PKA-dependent RyR2 phosphorylation and SR calcium release [PMID:39578909, PMID:40097291]."},"prefetch_data":{"uniprot":{"accession":"Q13023","full_name":"A-kinase anchor protein 6","aliases":["A-kinase anchor protein 100 kDa","AKAP 100","Protein kinase A-anchoring protein 6","PRKA6","mAKAP"],"length_aa":2319,"mass_kda":256.7,"function":"Binds to type II regulatory subunits of protein kinase A and anchors/targets them to the nuclear membrane or sarcoplasmic reticulum. May act as an adapter for assembling multiprotein complexes","subcellular_location":"Sarcoplasmic reticulum; Nucleus membrane","url":"https://www.uniprot.org/uniprotkb/Q13023/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AKAP6","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/AKAP6","total_profiled":1310},"omim":[{"mim_id":"606057","title":"RAP GUANINE NUCLEOTIDE EXCHANGE FACTOR 3; RAPGEF3","url":"https://www.omim.org/entry/606057"},{"mim_id":"604691","title":"A-KINASE ANCHOR PROTEIN 6; AKAP6","url":"https://www.omim.org/entry/604691"},{"mim_id":"600620","title":"FK506-BINDING PROTEIN 1B; FKBP1B","url":"https://www.omim.org/entry/600620"},{"mim_id":"600129","title":"PHOSPHODIESTERASE 4D; PDE4D","url":"https://www.omim.org/entry/600129"},{"mim_id":"188830","title":"PROTEIN KINASE, cAMP-DEPENDENT, REGULATORY, TYPE I, ALPHA; PRKAR1A","url":"https://www.omim.org/entry/188830"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"heart muscle","ntpm":51.1},{"tissue":"skeletal muscle","ntpm":44.4},{"tissue":"tongue","ntpm":47.4}],"url":"https://www.proteinatlas.org/search/AKAP6"},"hgnc":{"alias_symbol":["KIAA0311","mAKAP","AKAP100","PRKA6","ADAP6"],"prev_symbol":[]},"alphafold":{"accession":"Q13023","domains":[{"cath_id":"1.20.58.60","chopping":"766-938","consensus_level":"medium","plddt":79.971,"start":766,"end":938},{"cath_id":"1.20.58.60","chopping":"963-1068","consensus_level":"medium","plddt":79.1276,"start":963,"end":1068},{"cath_id":"1.20.58.60","chopping":"1075-1226","consensus_level":"medium","plddt":69.8801,"start":1075,"end":1226},{"cath_id":"1.20.58","chopping":"61-170","consensus_level":"medium","plddt":70.6499,"start":61,"end":170}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13023","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13023-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13023-F1-predicted_aligned_error_v6.png","plddt_mean":42.16},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AKAP6","jax_strain_url":"https://www.jax.org/strain/search?query=AKAP6"},"sequence":{"accession":"Q13023","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13023.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13023/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13023"}},"corpus_meta":[{"pmid":"16177794","id":"PMC_16177794","title":"The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways.","date":"2005","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/16177794","citation_count":450,"is_preprint":false},{"pmid":"11296225","id":"PMC_11296225","title":"mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module.","date":"2001","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/11296225","citation_count":389,"is_preprint":false},{"pmid":"10413680","id":"PMC_10413680","title":"mAKAP: an A-kinase anchoring protein targeted to the nuclear membrane of differentiated myocytes.","date":"1999","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/10413680","citation_count":154,"is_preprint":false},{"pmid":"11590243","id":"PMC_11590243","title":"mAKAP and the ryanodine receptor are part of a multi-component signaling complex on the cardiomyocyte nuclear envelope.","date":"2001","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/11590243","citation_count":128,"is_preprint":false},{"pmid":"16306226","id":"PMC_16306226","title":"The mAKAP complex participates in the induction of cardiac myocyte hypertrophy by adrenergic receptor signaling.","date":"2005","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/16306226","citation_count":109,"is_preprint":false},{"pmid":"15652351","id":"PMC_15652351","title":"Nesprin-1alpha contributes to the targeting of mAKAP to the cardiac myocyte nuclear envelope.","date":"2005","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/15652351","citation_count":100,"is_preprint":false},{"pmid":"7721854","id":"PMC_7721854","title":"Cloning and characterization of A-kinase anchor protein 100 (AKAP100). A protein that targets A-kinase to the sarcoplasmic reticulum.","date":"1995","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7721854","citation_count":94,"is_preprint":false},{"pmid":"9679148","id":"PMC_9679148","title":"A-kinase anchoring protein 100 (AKAP100) is localized in multiple subcellular compartments in the adult rat heart.","date":"1998","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/9679148","citation_count":90,"is_preprint":false},{"pmid":"15182229","id":"PMC_15182229","title":"PKA-phosphorylation of PDE4D3 facilitates recruitment of the mAKAP signalling complex.","date":"2004","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/15182229","citation_count":89,"is_preprint":false},{"pmid":"20106966","id":"PMC_20106966","title":"cAMP-stimulated protein phosphatase 2A activity associated with muscle A kinase-anchoring protein (mAKAP) signaling complexes inhibits the phosphorylation and activity of the cAMP-specific phosphodiesterase PDE4D3.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20106966","citation_count":81,"is_preprint":false},{"pmid":"16460834","id":"PMC_16460834","title":"The mAKAP signaling complex: integration of cAMP, calcium, and MAP kinase signaling pathways.","date":"2006","source":"European journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/16460834","citation_count":56,"is_preprint":false},{"pmid":"12709444","id":"PMC_12709444","title":"Targeting of protein kinase A by muscle A kinase-anchoring protein (mAKAP) regulates phosphorylation and function of the skeletal muscle ryanodine receptor.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12709444","citation_count":56,"is_preprint":false},{"pmid":"19109240","id":"PMC_19109240","title":"mAKAP compartmentalizes oxygen-dependent control of HIF-1alpha.","date":"2008","source":"Science signaling","url":"https://pubmed.ncbi.nlm.nih.gov/19109240","citation_count":54,"is_preprint":false},{"pmid":"25551320","id":"PMC_25551320","title":"mAKAP-a master scaffold for cardiac remodeling.","date":"2015","source":"Journal of cardiovascular pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/25551320","citation_count":44,"is_preprint":false},{"pmid":"33295871","id":"PMC_33295871","title":"AKAP6 orchestrates the nuclear envelope microtubule-organizing center by linking golgi and nucleus via AKAP9.","date":"2020","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/33295871","citation_count":43,"is_preprint":false},{"pmid":"23261540","id":"PMC_23261540","title":"Regulation of MEF2 transcriptional activity by calcineurin/mAKAP complexes.","date":"2012","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/23261540","citation_count":35,"is_preprint":false},{"pmid":"17487687","id":"PMC_17487687","title":"The mAKAP signalosome and cardiac myocyte hypertrophy.","date":"2007","source":"IUBMB life","url":"https://pubmed.ncbi.nlm.nih.gov/17487687","citation_count":30,"is_preprint":false},{"pmid":"22484155","id":"PMC_22484155","title":"Myocyte enhancer factor 2 (MEF2) tethering to muscle selective A-kinase anchoring protein (mAKAP) is necessary for myogenic differentiation.","date":"2012","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/22484155","citation_count":23,"is_preprint":false},{"pmid":"26563778","id":"PMC_26563778","title":"AKAP6 inhibition impairs myoblast differentiation and muscle regeneration: Positive loop between AKAP6 and myogenin.","date":"2015","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/26563778","citation_count":20,"is_preprint":false},{"pmid":"34605406","id":"PMC_34605406","title":"Myogenin controls via AKAP6 non-centrosomal microtubule-organizing center formation at the nuclear envelope.","date":"2021","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/34605406","citation_count":16,"is_preprint":false},{"pmid":"23806656","id":"PMC_23806656","title":"Protein kinase A and phosphodiesterase-4D3 binding to coding polymorphisms of cardiac muscle anchoring protein (mAKAP).","date":"2013","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/23806656","citation_count":15,"is_preprint":false},{"pmid":"31325238","id":"PMC_31325238","title":"AKAP6 and phospholamban colocalize and interact in HEK-293T cells and primary murine cardiomyocytes.","date":"2019","source":"Physiological reports","url":"https://pubmed.ncbi.nlm.nih.gov/31325238","citation_count":7,"is_preprint":false},{"pmid":"40097291","id":"PMC_40097291","title":"Wnt/β-catenin pathway induces cardiac dysfunction via AKAP6-mediated RyR2 phosphorylation and sarcoplasmic reticulum calcium leakage.","date":"2025","source":"Journal of molecular cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/40097291","citation_count":2,"is_preprint":false},{"pmid":"39578909","id":"PMC_39578909","title":"AKAP6 controls NFATc4 activity for BDNF-mediated neuroprotection.","date":"2024","source":"Molecular brain","url":"https://pubmed.ncbi.nlm.nih.gov/39578909","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11694,"output_tokens":5236,"usd":0.056811},"stage2":{"model":"claude-opus-4-6","input_tokens":8854,"output_tokens":3642,"usd":0.20298},"total_usd":0.259791,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"AKAP6 (AKAP100) was identified as an A-kinase anchoring protein that binds the RIIα regulatory subunit of PKA and targets type II PKA to the sarcoplasmic reticulum in cardiac and skeletal muscle cells.\",\n      \"method\": \"Interaction cloning with RIIα probe, solid-phase overlay binding assay, immunohistochemistry, cAMP-agarose affinity chromatography\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (in vitro binding, affinity purification, immunolocalization) in a foundational paper with >90 citations\",\n      \"pmids\": [\"7721854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"AKAP100 (AKAP6) localizes to multiple subcellular compartments in adult cardiac myocytes—nucleus, sarcolemma, intercalated disc, Z-line, and transverse tubule/junctional sarcoplasmic reticulum—and co-localizes specifically with RII (not RI) subunits of PKA.\",\n      \"method\": \"Immunofluorescence, confocal microscopy, double immunostaining with α-actinin and ryanodine receptor antibodies, immunoblotting\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal localization methods with functional specificity (RII vs RI discrimination), >90 citations\",\n      \"pmids\": [\"9679148\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"mAKAP (AKAP6) is targeted to the nuclear membrane of differentiated myocytes through two regions containing spectrin-like repeat sequences (residues 772–915 and 915–1065); overexpression of these targeting domains displaces endogenous mAKAP from the nuclear membrane, demonstrating saturable targeting.\",\n      \"method\": \"mAKAP-GFP fusion construct imaging, dominant-negative displacement experiments, nuclear membrane fractionation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — live-cell imaging with GFP fusions plus functional displacement experiments, >150 citations\",\n      \"pmids\": [\"10413680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"mAKAP assembles a cAMP signaling module comprising PKA and PDE4D3 at the nuclear envelope in heart tissue; anchored PKA phosphorylates and activates PDE4D3 to reduce local cAMP levels (negative feedback loop), and disruption of PKA–mAKAP interaction prevents enhancement of PDE4D3 activity.\",\n      \"method\": \"Co-immunoprecipitation from cardiac tissue, functional kinase and phosphodiesterase activity assays, dominant-negative PKA-anchoring disruption\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical reconstitution in cardiac tissue plus functional activity assays, >380 citations\",\n      \"pmids\": [\"11296225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The mAKAP complex at the cardiac nuclear envelope also contains ryanodine receptors and protein phosphatase 2A; mAKAP-bound ryanodine receptor can be regulated by PKA-mediated phosphorylation within the complex.\",\n      \"method\": \"Co-immunoprecipitation, immunohistochemistry, tissue fractionation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP and fractionation with multiple antibodies, >125 citations\",\n      \"pmids\": [\"11590243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"mAKAP-anchored PKA directly increases PKA-dependent phosphorylation of the skeletal muscle ryanodine receptor (RyR1) and augments Ca2+ efflux through RyR1; a PKA-anchoring-deficient mutant mAKAP-P fails to enhance RyR1 phosphorylation or Ca2+ release.\",\n      \"method\": \"CHO cell expression of mAKAP vs. mAKAP-P (PKA-anchoring mutant), immunoelectron microscopy, PKA phosphorylation assay, cytosolic Ca2+ transient measurements with caffeine/forskolin\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis of anchoring domain combined with functional phosphorylation and Ca2+ assays\",\n      \"pmids\": [\"12709444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PKA phosphorylation of PDE4D3 on Ser-13 increases the affinity of PDE4D3 for mAKAP, thereby enhancing recruitment of PDE4D3 to the mAKAP complex and facilitating faster cAMP signal termination.\",\n      \"method\": \"In vitro binding assays, cellular co-immunoprecipitation, site-directed mutagenesis of PDE4D3 Ser-13\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro assay plus mutagenesis plus cellular validation, >85 citations\",\n      \"pmids\": [\"15182229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The mAKAP complex coordinates two integrated cAMP effector feedback loops: anchored PKA activates PDE4D3 (negative feedback), while an mAKAP-associated ERK5 module suppresses PDE4D3; PDE4D3 also acts as adaptor to recruit Epac1, enabling cAMP-dependent attenuation of ERK5. Anchored ERK5 can induce cardiomyocyte hypertrophy.\",\n      \"method\": \"Co-immunoprecipitation, pharmacological and dominant-negative disruption, ERK5 kinase assay, cardiomyocyte hypertrophy assay (cell size measurement)\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods, reconstituted feedback loops, functional hypertrophy readout, >450 citations\",\n      \"pmids\": [\"16177794\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"mAKAP facilitates PKA-catalyzed phosphorylation of the ryanodine receptor Ca2+ channel; calcineurin Aβ associates with mAKAP; and the mAKAP complex is required for full activation of the pro-hypertrophic transcription factor NFATc in response to adrenergic signaling.\",\n      \"method\": \"Co-immunoprecipitation of calcineurin with mAKAP, siRNA knockdown of mAKAP, expression of PKA-anchoring-deficient mAKAP mutant, NFATc transcriptional reporter assay, cardiomyocyte hypertrophy assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — RNAi + dominant-negative + co-IP + functional transcription/hypertrophy assays, >100 citations\",\n      \"pmids\": [\"16306226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Nesprin-1α serves as the receptor for mAKAP at the nuclear envelope: nesprin-1α is inserted into the nuclear envelope via a C-terminal klarsicht transmembrane domain and its N-terminal spectrin repeat dimerization domain directly binds the third spectrin repeat of mAKAP, targeting mAKAP to the nuclear envelope.\",\n      \"method\": \"Direct binding assay between nesprin-1α and mAKAP spectrin repeat domains, overexpression-mediated displacement, co-immunoprecipitation\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct binding plus domain mapping plus functional displacement, >100 citations\",\n      \"pmids\": [\"15652351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"mAKAP organizes ubiquitin E3 ligases at the perinuclear region that control HIF-1α stability; depletion of mAKAP or disruption of its perinuclear targeting alters HIF-1α stability and transcriptional activation of hypoxia-responsive genes in cardiomyocytes.\",\n      \"method\": \"Co-immunoprecipitation of E3 ligases with mAKAP, siRNA knockdown, HIF-1α stability assay (immunoblot under normoxia/hypoxia), transcriptional reporter assay\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP of E3 ligases plus RNAi plus functional HIF-1α stability and transcription assays\",\n      \"pmids\": [\"19109240\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PP2A associated with mAKAP complexes dephosphorylates PDE4D3 at Ser-54; the PP2A holoenzyme in this complex contains the B56δ targeting subunit, which is itself a PKA substrate whose phosphorylation by anchored PKA enhances phosphatase activity 2-fold, constituting a cAMP-induced positive feedback loop. The C-terminal mAKAP domain (residues 2085–2319) mediates PP2A binding.\",\n      \"method\": \"Domain mapping by deletion constructs, co-immunoprecipitation from heart tissue, phosphatase activity assay, site-directed mutagenesis of B56δ PKA phosphorylation site, PDE4D3 activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — domain mapping, mutagenesis, in vitro phosphatase activity, and cardiac tissue co-IP with multiple orthogonal methods\",\n      \"pmids\": [\"20106966\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"mAKAP organizes a calcineurin/MEF2 signaling complex in myocytes; calcineurin–MEF2 association is dependent on mAKAP expression, and disruption of calcineurin binding to mAKAP (via a competing calcineurin-binding domain peptide) blunts MEF2 transcriptional activity during myoblast differentiation and inhibits adrenergic-induced cardiomyocyte hypertrophy.\",\n      \"method\": \"Co-immunoprecipitation from C2C12 cells and cardiac myocytes, dominant-negative calcineurin-binding domain peptide, MEF2 transcriptional reporter assay, myoblast differentiation assay\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus dominant-negative disruption plus functional transcription and differentiation assays\",\n      \"pmids\": [\"23261540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"mAKAP directly binds MEF2 transcription factors through discrete domains; disruption of MEF2/mAKAP binding blocks MEF2 activation in early myoblast differentiation and inhibits myotube formation and expression of differentiation markers.\",\n      \"method\": \"Co-immunoprecipitation, direct domain binding assay, dominant-interference with MEF2/mAKAP binding peptide, myogenic differentiation assay (myotube formation, differentiation marker expression)\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct domain binding plus functional differentiation readout with dominant-negative approach\",\n      \"pmids\": [\"22484155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The naturally occurring human mAKAP mutation P1400S (in the PDE4D3-binding site) significantly reduces binding to PDE4D3 without affecting PKA binding; S2195F (in the PP2A-binding site) increases PKA binding and PKA activity; L717V (flanking the spectrin repeat domain) increases PKA binding without changing activity. These confirm specific binding domains for each partner.\",\n      \"method\": \"Immunoprecipitation, surface plasmon resonance (Biacore-2000), PKA activity assay, Ca2+ measurement\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis validated by SPR plus co-IP plus functional activity assays\",\n      \"pmids\": [\"23806656\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"AKAP6 knockdown in skeletal myoblasts halts myotube formation and decreases myogenin and myosin heavy chain expression; AKAP6 promotes myogenin expression through MEF2A, and myogenin in turn binds an E-box site on the AKAP6 promoter to upregulate AKAP6 expression (positive feedback loop). In vivo shAKAP6 delivery impairs muscle regeneration after cardiotoxin injury.\",\n      \"method\": \"siRNA knockdown of AKAP6, shRNA-lentivirus delivery in vivo, myoblast differentiation assay, chromatin immunoprecipitation (ChIP), luciferase promoter assay, motor function testing\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — RNAi plus ChIP plus promoter reporter plus in vivo rescue experiment\",\n      \"pmids\": [\"26563778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"AKAP6 co-localizes and physically interacts with phospholamban (PLN) at the perinuclear/sarcoplasmic reticulum region in cardiomyocytes; AKAP6 co-expression promotes Ca2+ uptake activity of SERCA1 in the presence of PLN.\",\n      \"method\": \"Co-immunoprecipitation (HEK-293T and adult rat cardiomyocytes), immunofluorescence colocalization, Ca2+ uptake functional assay\",\n      \"journal\": \"Physiological reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP plus colocalization plus one functional assay, single lab\",\n      \"pmids\": [\"31325238\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"AKAP6 is a key organizer of the nuclear envelope MTOC in cardiomyocytes and osteoclasts: its spectrin repeats anchor centrosomal proteins (Pcnt, AKAP9) to the nuclear envelope via nesprin-1α. AKAP6 and AKAP9 together form a protein platform tethering the Golgi to the nucleus, enabling two pools of microtubules. Ectopic AKAP6 expression in epithelial cells is sufficient to recruit endogenous centrosomal proteins to the nuclear envelope. AKAP6 is required for cardiomyocyte hypertrophy and osteoclast bone resorption.\",\n      \"method\": \"Immunofluorescence, co-immunoprecipitation, gain-of-function (ectopic AKAP6 expression), loss-of-function (AKAP6 KD/KO), MTOC activity assays, cardiomyocyte hypertrophy assay, osteoclast bone resorption assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including gain- and loss-of-function with defined cellular phenotype, replicated in two cell types\",\n      \"pmids\": [\"33295871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The transcription factor myogenin is required and sufficient to drive NE-MTOC formation by inducing transcription of muscle/NE-MTOC-specific isoforms of AKAP6 and nesprin-1α; overexpression of AKAP6β and nesprin-1α together is sufficient to recruit endogenous MTOC proteins to the nuclear envelope in myoblasts even without myogenin.\",\n      \"method\": \"Loss- and gain-of-function experiments (siRNA, overexpression), promoter/transcriptional reporter assays, bioinformatics, immunofluorescence for MTOC protein recruitment\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — promoter studies plus gain/loss-of-function with clear molecular readout, orthogonal to prior AKAP6 MTOC paper\",\n      \"pmids\": [\"34605406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AKAP6 anchors calcineurin (CaN) and NFATc4 in neurons; BDNF-mediated neuroprotection requires AKAP6-anchored CaN to activate NFATc4 transcription, and NFATc4 acts downstream of BDNF neuroprotection in vivo.\",\n      \"method\": \"Co-immunoprecipitation of CaN/NFATc4 with AKAP6, dominant-negative disruption of CaN anchoring, NFATc4 transcriptional reporter, NFATc4-/- mouse neuroprotection assay\",\n      \"journal\": \"Molecular brain\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-IP plus dominant-negative plus in vivo KO, single lab with limited citations\",\n      \"pmids\": [\"39578909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Activation of the Wnt/β-catenin pathway upregulates AKAP6 expression (AKAP6 is a target gene of canonical Wnt signaling), which in turn enhances PKA-mediated phosphorylation of RyR2, causing sarcoplasmic reticulum calcium leakage and cardiac dysfunction.\",\n      \"method\": \"Transcriptome analysis, Wnt pathway activation/inhibition (cycloheximide block), RyR2 phosphorylation assay, AKAP6 overexpression/knockdown, Ca2+ imaging in cardiomyocytes\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — transcriptomics plus functional RyR2 phosphorylation and Ca2+ assays, single recent paper\",\n      \"pmids\": [\"40097291\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AKAP6 (mAKAP) functions as a perinuclear scaffold protein—anchored to the nuclear envelope via direct binding of its spectrin repeats to nesprin-1α—that assembles dynamic signalosomes in striated muscle and neurons, coordinating PKA, PDE4D3, PP2A, Epac1, ERK5, ryanodine receptors, calcineurin, MEF2, HIF-1α-regulatory E3 ligases, and phospholamban into integrated cAMP/Ca2+/MAPK feedback circuits that control cardiac hypertrophy, myogenic differentiation, hypoxic gene expression, and, additionally, serves as the key organizer of the nuclear envelope microtubule-organizing center by recruiting centrosomal proteins (Pcnt, AKAP9) through its spectrin repeats downstream of myogenin-driven transcription.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"AKAP6 (mAKAP) is a perinuclear scaffold protein in striated muscle and neurons that integrates cAMP, calcium, and MAPK signaling at the nuclear envelope to control cardiac hypertrophy, myogenic differentiation, hypoxic gene expression, and microtubule organization. Anchored to the nuclear envelope via direct binding of its spectrin repeats to nesprin-1α, AKAP6 assembles a signalosome containing PKA, PDE4D3, PP2A (B56δ), Epac1, ERK5, calcineurin, ryanodine receptors, and MEF2 transcription factors, enabling coordinated feedback loops in which PKA activates PDE4D3 to dampen local cAMP while ERK5 opposes this effect, and calcineurin activates NFATc and MEF2 to drive hypertrophic and myogenic transcription [PMID:11296225, PMID:16177794, PMID:23261540]. AKAP6 also serves as the key organizer of the nuclear envelope microtubule-organizing center (NE-MTOC) by recruiting centrosomal proteins Pcnt and AKAP9 through its spectrin repeats, a process driven by myogenin-dependent transcriptional induction of AKAP6 and nesprin-1α [PMID:33295871, PMID:34605406]. In neurons, AKAP6-anchored calcineurin activates NFATc4 downstream of BDNF to mediate neuroprotection, and in cardiomyocytes Wnt/β-catenin signaling upregulates AKAP6 to enhance PKA-dependent RyR2 phosphorylation and SR calcium release [PMID:39578909, PMID:40097291].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Establishing that AKAP6 is an A-kinase anchoring protein resolved how type II PKA is targeted to specific subcellular compartments in cardiac and skeletal muscle.\",\n      \"evidence\": \"Interaction cloning with RIIα, overlay binding assay, and cAMP-agarose affinity chromatography in cardiac tissue\",\n      \"pmids\": [\"7721854\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Exact subcellular targeting mechanism unknown\", \"No identified substrates of the anchored PKA pool\", \"Functional consequence of PKA anchoring not tested\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Mapping nuclear envelope targeting to two spectrin-like repeat regions explained how AKAP6 achieves perinuclear localization in differentiated myocytes, distinguishing it from other AKAPs.\",\n      \"evidence\": \"GFP-fusion construct imaging and dominant-negative displacement of endogenous mAKAP from the nuclear membrane\",\n      \"pmids\": [\"10413680\", \"9679148\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the nuclear envelope receptor for AKAP6 unknown\", \"Whether spectrin repeats mediate protein-protein interactions beyond targeting unclear\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Discovery that AKAP6 co-assembles PKA, PDE4D3, ryanodine receptors, and PP2A into one complex established the concept of an integrated cAMP/Ca²⁺ signaling module with built-in negative feedback at the nuclear envelope.\",\n      \"evidence\": \"Co-immunoprecipitation from cardiac tissue, kinase and phosphodiesterase activity assays, dominant-negative PKA-anchoring disruption\",\n      \"pmids\": [\"11296225\", \"11590243\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and dynamics of the complex not determined\", \"Whether ryanodine receptor regulation by this complex operates in vivo unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identification of dual opposing feedback loops (PKA→PDE4D3 activation vs. ERK5→PDE4D3 inhibition, with Epac1 attenuating ERK5) and calcineurin/NFATc recruitment revealed how AKAP6 integrates cAMP and MAPK signaling to control cardiomyocyte hypertrophy.\",\n      \"evidence\": \"Co-IP, pharmacological and dominant-negative disruption, ERK5 kinase assay, NFATc reporter, cardiomyocyte hypertrophy cell-size measurement\",\n      \"pmids\": [\"16177794\", \"16306226\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo cardiac phenotype of AKAP6 disruption not yet shown\", \"Whether ERK5 module operates independently of PKA feedback in pathological hypertrophy unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identification of nesprin-1α as the nuclear envelope receptor that directly binds the third spectrin repeat of AKAP6 solved how AKAP6 is anchored at the nuclear envelope.\",\n      \"evidence\": \"Direct binding assay between nesprin-1α and mAKAP spectrin repeat domains, overexpression displacement, co-immunoprecipitation\",\n      \"pmids\": [\"15652351\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of spectrin repeat–nesprin interaction not resolved\", \"Whether other nuclear envelope proteins contribute to anchoring unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showing that AKAP6 organizes E3 ubiquitin ligases controlling HIF-1α stability expanded the scaffold's role beyond kinase/phosphatase signaling to include regulation of hypoxic gene expression.\",\n      \"evidence\": \"Co-IP of E3 ligases with mAKAP, siRNA knockdown, HIF-1α stability immunoblot under normoxia/hypoxia, transcriptional reporter\",\n      \"pmids\": [\"19109240\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity and specificity of the E3 ligases in the complex not fully defined\", \"Whether HIF-1α regulation is relevant outside cardiomyocytes unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Characterization of PP2A (B56δ subunit) as a PKA-activated phosphatase within the AKAP6 complex that dephosphorylates PDE4D3 uncovered a cAMP-induced positive feedback loop layered onto the existing negative feedback circuit.\",\n      \"evidence\": \"Domain mapping, cardiac tissue co-IP, phosphatase activity assay, B56δ PKA-site mutagenesis\",\n      \"pmids\": [\"20106966\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Net outcome of simultaneous positive and negative feedback on local cAMP dynamics not modeled quantitatively\", \"Whether other PP2A substrates in the complex exist unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrating that AKAP6 directly binds MEF2 and bridges calcineurin to MEF2 established the scaffold as essential for myogenic transcription and differentiation beyond its kinase-anchoring role.\",\n      \"evidence\": \"Co-IP, direct domain binding, dominant-negative peptide interference, MEF2 reporter, myotube formation assay\",\n      \"pmids\": [\"23261540\", \"22484155\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which MEF2 isoform is preferentially bound in vivo unknown\", \"Structural basis of MEF2–AKAP6 interface not determined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Discovery of a positive feedback loop in which AKAP6 promotes myogenin expression via MEF2A, and myogenin in turn transactivates the AKAP6 promoter, explained how AKAP6 levels are amplified during myogenesis and linked AKAP6 to in vivo muscle regeneration.\",\n      \"evidence\": \"siRNA/shRNA knockdown, ChIP showing myogenin at AKAP6 E-box, luciferase promoter assay, in vivo cardiotoxin injury model\",\n      \"pmids\": [\"26563778\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the feedback loop is sufficient for full differentiation or requires additional cofactors unknown\", \"Temporal dynamics of the feedback loop during regeneration not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Establishing AKAP6 as the key organizer of the nuclear envelope MTOC — recruiting Pcnt and AKAP9 via its spectrin repeats and nesprin-1α — redefined AKAP6 function beyond signaling scaffold to include cytoskeletal architecture essential for cardiomyocyte hypertrophy and osteoclast bone resorption.\",\n      \"evidence\": \"Gain-of-function (ectopic AKAP6 in epithelial cells recruits centrosomal proteins), loss-of-function (KD/KO), MTOC activity assays, hypertrophy and resorption assays in two cell types\",\n      \"pmids\": [\"33295871\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How AKAP6-mediated MTOC function is coordinated with its signaling scaffold role is unclear\", \"Whether AKAP6 recruits additional MTOC components beyond Pcnt and AKAP9 unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showing that myogenin is both necessary and sufficient to induce the muscle-specific AKAP6β and nesprin-1α isoforms that together reconstitute NE-MTOC formation placed AKAP6 MTOC assembly under direct transcriptional control of the myogenic program.\",\n      \"evidence\": \"siRNA and overexpression of myogenin, promoter/transcriptional reporter, immunofluorescence for MTOC protein recruitment in myoblasts\",\n      \"pmids\": [\"34605406\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other transcription factors cooperate with myogenin for AKAP6 induction unknown\", \"Mechanism by which NE-MTOC formation feeds back on differentiation not explored\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extension of the AKAP6–calcineurin–NFATc axis to neurons demonstrated that AKAP6-anchored calcineurin activates NFATc4 downstream of BDNF for neuroprotection, broadening AKAP6 function beyond striated muscle.\",\n      \"evidence\": \"Co-IP of CaN/NFATc4 with AKAP6, dominant-negative disruption, NFATc4 reporter, NFATc4−/− mouse neuroprotection assay\",\n      \"pmids\": [\"39578909\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding; independent replication in neuronal systems needed\", \"Identity of neuroprotective NFATc4 target genes downstream of AKAP6 unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identification of AKAP6 as a Wnt/β-catenin target gene whose upregulation enhances PKA-mediated RyR2 phosphorylation and SR calcium leak linked an upstream developmental pathway to AKAP6-dependent calcium dysregulation in cardiac dysfunction.\",\n      \"evidence\": \"Transcriptomics, Wnt pathway activation/inhibition, RyR2 phosphorylation assay, AKAP6 overexpression/knockdown, Ca²⁺ imaging in cardiomyocytes\",\n      \"pmids\": [\"40097291\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Wnt-driven AKAP6 upregulation is relevant in human heart failure not established\", \"Mechanism of β-catenin regulation of AKAP6 promoter not mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A full structural model of the AKAP6 signalosome — including how simultaneous binding of PKA, PDE4D3, PP2A, ERK5, calcineurin, MEF2, and MTOC components is coordinated on one scaffold — and in vivo phenotyping of AKAP6 knockout in adult heart remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No high-resolution structure of any AKAP6 multi-protein complex\", \"No conditional cardiac-specific AKAP6 knockout phenotype published\", \"Quantitative modeling of the integrated feedback circuits has not been performed\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 3, 7, 8, 11, 12, 17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [2, 9, 17]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [17, 18]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 7, 8, 11, 20]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [13, 15, 18]},\n      {\"term_id\": \"R-HSA-397014\", \"supporting_discovery_ids\": [1, 5, 16]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [17, 18]}\n    ],\n    \"complexes\": [\n      \"mAKAP-PKA-PDE4D3-Epac1-ERK5 signalosome\",\n      \"mAKAP-calcineurin-MEF2 complex\",\n      \"mAKAP-nesprin-1α-Pcnt-AKAP9 NE-MTOC platform\",\n      \"mAKAP-PP2A(B56δ) complex\"\n    ],\n    \"partners\": [\n      \"PRKAR2A\",\n      \"PDE4D3\",\n      \"SYNE1\",\n      \"PPP3CA\",\n      \"MEF2A\",\n      \"PCNT\",\n      \"AKAP9\",\n      \"PLN\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}