{"gene":"AKAP7","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2001,"finding":"AKAP15 (AKAP7α) directly interacts with the C-terminal domain of the CaV1.1 alpha1 subunit via a leucine zipper (LZ) motif, anchoring PKA to skeletal muscle L-type Ca2+ channels; disruption of the LZ interaction inhibits voltage-dependent potentiation of L-type Ca2+ channels.","method":"Direct binding assays, leucine zipper disruption mutagenesis, electrophysiology in skeletal muscle cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutagenesis with functional readout (electrophysiology), direct binding established, clear mechanistic pathway","pmids":["11733497"],"is_preprint":false},{"year":1998,"finding":"AKAP15 (AKAP7) co-purifies with rat brain sodium channels and anchors PKA to the Nav1.2 alpha subunit; AKAP15 was identified by mass spectrometry in purified sodium channel preparations and co-immunoprecipitates with the sodium channel alpha subunit, enabling PKA phosphorylation of four serine residues on the channel.","method":"Co-purification, immunoprecipitation, immunoblot, mass spectrometry, gel overlay assay, in vitro PKA phosphorylation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, mass spectrometry identification, in vitro phosphorylation assay with defined sites, multiple orthogonal methods","pmids":["9748250"],"is_preprint":false},{"year":2002,"finding":"AKAP-15 (AKAP7) binds specifically to intracellular loop I-II (L(I-II)) of Nav1.2a sodium channels, targeting PKA directly to its phosphorylation sites (S554, S573, S576, S687); PKC phosphorylation of S576 enhances subsequent PKA modulation requiring additional phosphorylation at S687, revealing convergent multi-site regulation.","method":"Protein-protein interaction analysis of AKAP-15 with intracellular loops, site-directed mutagenesis of serine residues, expression and electrophysiology in heterologous system","journal":"Molecular and cellular neurosciences","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutagenesis at multiple defined sites combined with functional electrophysiology and binding assays","pmids":["12359152"],"is_preprint":false},{"year":2007,"finding":"The central domain of AKAP18δ (AKAP7δ) is a member of the 2H phosphoesterase family, featuring two conserved His-x-Thr motifs; X-ray crystallography reveals this domain specifically binds AMP and CMP in a groove between two pseudo-2-fold-related lobes, with AMP affinity in the physiological concentration range.","method":"Bioinformatics, X-ray crystallography, nucleotide co-crystallization screening","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with ligand binding defined, single lab but rigorous structural method","pmids":["18082768"],"is_preprint":false},{"year":2014,"finding":"Mouse AKAP7 contains a functional 2',5'-phosphodiesterase (2',5'-PDE) domain that rapidly degrades 2',5'-oligoadenylate (2-5A) activators of RNase L; the PDE domain requires cytoplasmic localization for antiviral activity (as shown by complementation of ns2-mutant coronavirus), while full-length AKAP7 localizes to the nucleus and cannot complement. A single point mutation AKAP7(H185R) abolishes PDE activity.","method":"Biochemical 2-5A degradation assay, viral complementation in bone marrow macrophages and mice, site-directed mutagenesis (H185R), subcellular localization by immunofluorescence","journal":"mBio","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro enzymatic assay, mutagenesis, in vivo viral complementation, and localization experiments all converging","pmids":["24987090"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of AKAP18β PKA-binding domain bound to the D/D domain of PKA RIIα reveals three hydrophilic anchor points outside the core PKA-binding helix that mediate contacts with the D/D domain; in vitro and cell-based experiments confirm these anchor points are required for RII subunit interaction with AKAP18.","method":"X-ray crystallography, in vitro binding assays, cell-based interaction experiments, sequence analysis of anchor points","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional validation in vitro and in cells, single lab with multiple orthogonal methods","pmids":["27102985"],"is_preprint":false},{"year":2016,"finding":"Genetic ablation of AKAP7 specifically from dentate granule cells disrupts mossy fiber–CA3 LTP initiated by cAMP and impairs pattern separation behavior, establishing that the AKAP7/PKA complex in mossy fiber projections is essential for presynaptic PKA-dependent plasticity and spatial discrimination.","method":"Conditional knockout mouse (dentate granule cell-specific AKAP7 deletion), electrophysiology (LTP assay), behavioral testing (pattern separation)","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific KO with defined electrophysiological and behavioral phenotypes, clear pathway placement","pmids":["27911261"],"is_preprint":false},{"year":2012,"finding":"AKAP7 knockout mice (all isoforms deleted) show normal cardiomyocyte responses to β-adrenergic stimulation: Ca2+ current, intracellular Ca2+ transients, Ca2+ reuptake, and phosphorylation of CaV1.2 and phospholamban are unaffected, indicating AKAP7 is not required for regulation of Ca2+ handling in mouse ventricular cardiomyocytes.","method":"AKAP7 global knockout mouse, whole-cell patch clamp, fluorescent Ca2+ indicator, immunoblot for substrate phosphorylation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with multiple orthogonal physiological readouts; rigorous negative result","pmids":["23035250"],"is_preprint":false},{"year":2012,"finding":"AKAP7γ and AKAP7α both interact with multiple PKC isoenzymes via multi-site binding on both proteins; AKAP7 scaffolding enhances PKC substrate phosphorylation (shown by FRET-based activity reporter) and restricts PKC mobility within cells (shown by FRAP and virtual modeling).","method":"Surface plasmon resonance, protein biochemistry pulldowns, FRET-based PKC activity reporter, FRAP imaging","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple orthogonal binding and functional methods in single lab; FRET and FRAP provide functional and localization evidence","pmids":["22670899"],"is_preprint":false},{"year":2006,"finding":"AKAP18 isoforms and PDE4 family phosphodiesterases are differentially localized in renal collecting duct principal cells, where AKAP-anchored PKA participates in AVP-stimulated aquaporin-2 (AQP2) phosphorylation and redistribution to the plasma membrane.","method":"Immunofluorescence localization in renal principal cells, functional context of AVP/AQP2 signaling","journal":"European journal of cell biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — localization by immunofluorescence in single study, limited mechanistic follow-up specific to AKAP7","pmids":["16500722"],"is_preprint":false},{"year":2022,"finding":"AKAP7γ (long isoform) is highly mobile within cardiomyocytes as demonstrated by FRAP of GFP-tagged AKAP7γ; PKA activation accelerates AKAP7γ-GFP wash-out upon saponin permeabilization, indicating PKA signaling increases AKAP7γ mobility, which may contribute to spatial propagation of β-adrenergic signaling to SR Ca2+ uptake.","method":"FRAP of GFP-tagged AKAP7γ in rabbit ventricular cardiomyocytes, saponin permeabilization wash-out assay with PKA activation","journal":"Function (Oxford, England)","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct live-cell FRAP imaging with functional perturbation (PKA activation), single lab","pmids":["35620477"],"is_preprint":false},{"year":2025,"finding":"Long AKAP18 isoforms (AKAP7γ/δ) scaffold PKA together with ubiquitin-specific proteinase USP4 at cardiac sarcomere Z bands via the AKAP18 2'-phosphoesterase domain; AKAP18-anchored PKA phosphorylates USP4 at Ser829 near its active site, stimulating USP4 deubiquitinase activity. Pharmacological PKA inhibition or AKAP7 gene deletion decreases calcium flux through SERCA2, establishing the AKAP18/PKA/USP4 complex as a regulator of SR Ca2+ reuptake.","method":"Proximity-proteomics (BioID) in cardiomyocytes, co-immunoprecipitation, in vitro PKA phosphorylation assay, phospho-specific antibody (pSer829), AKAP7 knockout mouse, pharmacological PKA inhibition, calcium flux measurement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — proximity proteomics, Co-IP, in vitro phosphorylation, mutagenesis-implied active site proximity, KO mouse with functional readout, multiple orthogonal methods in single study","pmids":["40449590"],"is_preprint":false}],"current_model":"AKAP7 (AKAP15/AKAP18) is a scaffolding protein that anchors PKA (and PKC) to defined subcellular locations—including skeletal muscle L-type Ca2+ channels (via leucine zipper), brain Na+ channels (via loop I-II binding), and the cardiac sarcoplasmic reticulum (via long isoforms γ/δ)—to enable spatially precise phosphorylation of ion channels and Ca2+-handling proteins; additionally, its central 2H phosphoesterase domain can bind AMP and degrade 2',5'-oligoadenylates, and its long isoforms recruit USP4 at sarcomere Z bands where AKAP18-anchored PKA phosphorylates USP4-Ser829 to enhance SR Ca2+ reuptake, while its mossy fiber-localized AKAP7/PKA complex is essential for cAMP-dependent LTP and hippocampal pattern separation."},"narrative":{"mechanistic_narrative":"AKAP7 (AKAP15/AKAP18) is an A-kinase anchoring protein that tethers PKA—and PKC—to defined subcellular sites to enable spatially restricted phosphorylation of ion channels and Ca2+-handling proteins [PMID:11733497, PMID:9748250, PMID:22670899]. It anchors PKA to skeletal muscle L-type Ca2+ channels through a leucine-zipper interaction with the CaV1.1 alpha1 C-terminus, where disrupting this interaction blocks voltage-dependent channel potentiation [PMID:11733497], and to brain Nav1.2 sodium channels by binding the intracellular I-II loop, positioning PKA at defined serine sites whose phosphorylation is gated by prior PKC modification [PMID:9748250, PMID:12359152]. PKA recruitment is mediated by a PKA-binding domain that contacts the RIIα D/D domain through anchor points flanking the core binding helix [PMID:27102985]. Beyond scaffolding, the central 2H phosphoesterase domain of long isoforms binds AMP and functions as a 2',5'-phosphodiesterase that degrades the 2-5A activators of RNase L, an activity abolished by the H185R mutation and dependent on cytoplasmic localization for antiviral function [PMID:18082768, PMID:24987090]. In the heart, long isoforms (AKAP7γ/δ) scaffold PKA together with the deubiquitinase USP4 at sarcomere Z bands via the phosphoesterase domain; anchored PKA phosphorylates USP4 at Ser829 to stimulate its deubiquitinase activity and promote SERCA2-mediated SR Ca2+ reuptake [PMID:40449590]. In the brain, the AKAP7/PKA complex in dentate granule cell mossy fibers is required for cAMP-dependent presynaptic LTP and pattern-separation behavior [PMID:27911261]. Notably, global AKAP7 deletion leaves β-adrenergic Ca2+ handling in mouse ventricular cardiomyocytes intact, indicating its cardiac role is context- and isoform-specific rather than essential for canonical β-adrenergic Ca2+ regulation [PMID:23035250].","teleology":[{"year":1998,"claim":"Established that AKAP7 physically links PKA to voltage-gated sodium channels, defining its role as a channel-targeting anchor.","evidence":"Co-purification, reciprocal Co-IP, mass spectrometry, and in vitro PKA phosphorylation of Nav1.2 from rat brain","pmids":["9748250"],"confidence":"High","gaps":["Did not map the channel-binding interface","Functional consequence for channel gating not yet defined"]},{"year":2001,"claim":"Showed that AKAP7 anchoring of PKA to L-type Ca2+ channels is functionally required for channel modulation, linking the scaffold directly to channel physiology.","evidence":"Leucine-zipper disruption mutagenesis with electrophysiology in skeletal muscle cells","pmids":["11733497"],"confidence":"High","gaps":["Whether the same LZ motif targets other channel types not addressed","Structural basis of LZ recognition not resolved"]},{"year":2002,"claim":"Defined the sodium-channel binding site and revealed convergent PKC/PKA multi-site regulation gated by AKAP7-anchored kinase positioning.","evidence":"Loop I-II interaction mapping, serine site-directed mutagenesis, heterologous electrophysiology","pmids":["12359152"],"confidence":"High","gaps":["In vivo relevance of multi-site gating not established","Stoichiometry of PKC/PKA co-anchoring unknown"]},{"year":2007,"claim":"Discovered an unanticipated enzymatic identity for the AKAP18δ central domain as a nucleotide-binding 2H phosphoesterase, beyond pure scaffolding.","evidence":"Bioinformatics and X-ray crystallography with AMP/CMP co-crystallization","pmids":["18082768"],"confidence":"High","gaps":["Catalytic substrate in cells not yet identified","Physiological role of nucleotide binding undefined at this stage"]},{"year":2012,"claim":"Tested whether AKAP7 is required for cardiac Ca2+ handling and found it dispensable for β-adrenergic regulation, bounding its cardiac function.","evidence":"Global AKAP7 knockout mouse with patch clamp, Ca2+ imaging, and substrate phospho-immunoblots","pmids":["23035250"],"confidence":"High","gaps":["Possible redundancy with other AKAPs not excluded","Isoform- or microdomain-specific roles not resolved by global deletion"]},{"year":2012,"claim":"Extended AKAP7 scaffolding to PKC, showing it constrains kinase mobility and enhances substrate phosphorylation.","evidence":"SPR, pulldowns, FRET activity reporter, and FRAP imaging","pmids":["22670899"],"confidence":"Medium","gaps":["Single-lab functional readouts","In vivo PKC substrates of the AKAP7/PKC complex not identified"]},{"year":2014,"claim":"Assigned a catalytic antiviral function to the phosphoesterase domain as a 2',5'-PDE that destroys RNase L activators, with localization gating activity.","evidence":"In vitro 2-5A degradation, H185R mutagenesis, viral complementation in macrophages and mice, immunofluorescence","pmids":["24987090"],"confidence":"High","gaps":["How nuclear full-length AKAP7 is excluded from cytoplasmic antiviral function unclear","Endogenous regulation of this activity in human cells not defined"]},{"year":2016,"claim":"Resolved how AKAP7 engages PKA, defining anchor points outside the canonical helix required for RIIα binding.","evidence":"Crystal structure of AKAP18β PKA-binding domain with RIIα D/D, with in vitro and cell-based validation","pmids":["27102985"],"confidence":"High","gaps":["RI subunit selectivity not addressed","Whether anchor points are regulated dynamically unknown"]},{"year":2016,"claim":"Demonstrated a required physiological role for AKAP7 in presynaptic plasticity and behavior, placing the scaffold in cAMP-dependent LTP.","evidence":"Dentate granule cell-specific conditional knockout with LTP electrophysiology and pattern separation behavior","pmids":["27911261"],"confidence":"High","gaps":["Presynaptic PKA substrates mediating the LTP defect not identified","Molecular targeting of AKAP7 at mossy fiber terminals unresolved"]},{"year":2022,"claim":"Showed AKAP7γ is dynamically mobile and that PKA activation increases its mobility, offering a mechanism for spatial spread of β-adrenergic signaling.","evidence":"FRAP and saponin permeabilization wash-out of GFP-AKAP7γ in rabbit cardiomyocytes","pmids":["35620477"],"confidence":"Medium","gaps":["Functional consequence of mobility for Ca2+ handling inferred, not demonstrated","Single-lab live-cell assay"]},{"year":2025,"claim":"Linked AKAP7 long isoforms to SR Ca2+ reuptake via a Z-band complex in which anchored PKA activates USP4 deubiquitinase, integrating scaffolding with ubiquitin signaling.","evidence":"BioID proximity proteomics, Co-IP, in vitro PKA phosphorylation, pSer829 antibody, AKAP7 KO mouse, PKA inhibition, calcium flux","pmids":["40449590"],"confidence":"High","gaps":["USP4 deubiquitination substrates controlling SERCA2 not identified","Reconciliation with the dispensable Ca2+-handling phenotype of global KO not fully addressed"]},{"year":null,"claim":"How AKAP7 isoform diversity, subcellular localization, and dual scaffolding/enzymatic activities are coordinated to select specific substrates in each tissue remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No unified model linking phosphoesterase catalysis to anchoring function","Tissue-specific isoform targeting determinants not mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,2,5,8,11]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,11]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[3,4]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[4]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[11]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,5,11]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[6]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[4]},{"term_id":"R-HSA-397014","term_label":"Muscle contraction","supporting_discovery_ids":[11]}],"complexes":["AKAP18/PKA/USP4 Z-band complex"],"partners":["PRKAR2A","CACNA1S","SCN2A","USP4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O43687","full_name":"A-kinase anchor protein 7 isoforms alpha and beta","aliases":["A-kinase anchor protein 18 kDa","AKAP 18","Protein kinase A-anchoring protein 7 isoforms alpha/beta","PRKA7 isoforms alpha/beta"],"length_aa":104,"mass_kda":11.5,"function":"Targets the cAMP-dependent protein kinase (PKA) to the plasma membrane, and permits functional coupling to the L-type calcium channel. The membrane-associated form reduces epithelial sodium channel (ENaC) activity, whereas the free cytoplasmic form may negatively regulate ENaC channel feedback inhibition by intracellular sodium","subcellular_location":"Apical cell membrane","url":"https://www.uniprot.org/uniprotkb/O43687/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AKAP7","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"PRKACA","stoichiometry":0.2},{"gene":"SNRPD2","stoichiometry":0.2},{"gene":"SNRPF","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/AKAP7","total_profiled":1310},"omim":[{"mim_id":"604693","title":"A-KINASE ANCHOR PROTEIN 7; AKAP7","url":"https://www.omim.org/entry/604693"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"adrenal gland","ntpm":54.4}],"url":"https://www.proteinatlas.org/search/AKAP7"},"hgnc":{"alias_symbol":["AKAP18","AKAP15"],"prev_symbol":[]},"alphafold":{"accession":"O43687","domains":[{"cath_id":"1.20.5","chopping":"48-76","consensus_level":"medium","plddt":93.4414,"start":48,"end":76}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43687","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43687-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43687-F1-predicted_aligned_error_v6.png","plddt_mean":68.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AKAP7","jax_strain_url":"https://www.jax.org/strain/search?query=AKAP7"},"sequence":{"accession":"O43687","fasta_url":"https://rest.uniprot.org/uniprotkb/O43687.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43687/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43687"}},"corpus_meta":[{"pmid":"11733497","id":"PMC_11733497","title":"A novel leucine zipper targets AKAP15 and cyclic AMP-dependent protein kinase to the C terminus of the skeletal muscle Ca2+ channel and modulates its function.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11733497","citation_count":103,"is_preprint":false},{"pmid":"12359152","id":"PMC_12359152","title":"Molecular mechanism of convergent regulation of brain Na(+) channels by protein kinase C and protein kinase A anchored to AKAP-15.","date":"2002","source":"Molecular and cellular neurosciences","url":"https://pubmed.ncbi.nlm.nih.gov/12359152","citation_count":77,"is_preprint":false},{"pmid":"9748250","id":"PMC_9748250","title":"AKAP15 anchors cAMP-dependent protein kinase to brain sodium channels.","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9748250","citation_count":73,"is_preprint":false},{"pmid":"23035250","id":"PMC_23035250","title":"Cardiomyocytes from AKAP7 knockout mice respond normally to adrenergic stimulation.","date":"2012","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/23035250","citation_count":54,"is_preprint":false},{"pmid":"18082768","id":"PMC_18082768","title":"AKAP18 contains a phosphoesterase domain that binds AMP.","date":"2007","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/18082768","citation_count":54,"is_preprint":false},{"pmid":"16500722","id":"PMC_16500722","title":"Spatial organisation of AKAP18 and PDE4 isoforms in renal collecting duct principal cells.","date":"2006","source":"European journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/16500722","citation_count":46,"is_preprint":false},{"pmid":"24987090","id":"PMC_24987090","title":"Murine AKAP7 has a 2',5'-phosphodiesterase domain that can complement an inactive murine coronavirus ns2 gene.","date":"2014","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/24987090","citation_count":38,"is_preprint":false},{"pmid":"28446746","id":"PMC_28446746","title":"Peripheral blood AKAP7 expression as an early marker for lymphocyte-mediated post-stroke blood brain barrier disruption.","date":"2017","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/28446746","citation_count":36,"is_preprint":false},{"pmid":"27911261","id":"PMC_27911261","title":"Targeted deletion of AKAP7 in dentate granule cells impairs spatial discrimination.","date":"2016","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/27911261","citation_count":32,"is_preprint":false},{"pmid":"27102985","id":"PMC_27102985","title":"AKAP18:PKA-RIIα structure reveals crucial anchor points for recognition of regulatory subunits of PKA.","date":"2016","source":"The Biochemical 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chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40449590","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9795,"output_tokens":3181,"usd":0.03855,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10752,"output_tokens":3746,"usd":0.073705,"stage2_stop_reason":"end_turn"},"total_usd":0.112255,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"AKAP15 (AKAP7α) directly interacts with the C-terminal domain of the CaV1.1 alpha1 subunit via a leucine zipper (LZ) motif, anchoring PKA to skeletal muscle L-type Ca2+ channels; disruption of the LZ interaction inhibits voltage-dependent potentiation of L-type Ca2+ channels.\",\n      \"method\": \"Direct binding assays, leucine zipper disruption mutagenesis, electrophysiology in skeletal muscle cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutagenesis with functional readout (electrophysiology), direct binding established, clear mechanistic pathway\",\n      \"pmids\": [\"11733497\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"AKAP15 (AKAP7) co-purifies with rat brain sodium channels and anchors PKA to the Nav1.2 alpha subunit; AKAP15 was identified by mass spectrometry in purified sodium channel preparations and co-immunoprecipitates with the sodium channel alpha subunit, enabling PKA phosphorylation of four serine residues on the channel.\",\n      \"method\": \"Co-purification, immunoprecipitation, immunoblot, mass spectrometry, gel overlay assay, in vitro PKA phosphorylation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, mass spectrometry identification, in vitro phosphorylation assay with defined sites, multiple orthogonal methods\",\n      \"pmids\": [\"9748250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"AKAP-15 (AKAP7) binds specifically to intracellular loop I-II (L(I-II)) of Nav1.2a sodium channels, targeting PKA directly to its phosphorylation sites (S554, S573, S576, S687); PKC phosphorylation of S576 enhances subsequent PKA modulation requiring additional phosphorylation at S687, revealing convergent multi-site regulation.\",\n      \"method\": \"Protein-protein interaction analysis of AKAP-15 with intracellular loops, site-directed mutagenesis of serine residues, expression and electrophysiology in heterologous system\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutagenesis at multiple defined sites combined with functional electrophysiology and binding assays\",\n      \"pmids\": [\"12359152\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The central domain of AKAP18δ (AKAP7δ) is a member of the 2H phosphoesterase family, featuring two conserved His-x-Thr motifs; X-ray crystallography reveals this domain specifically binds AMP and CMP in a groove between two pseudo-2-fold-related lobes, with AMP affinity in the physiological concentration range.\",\n      \"method\": \"Bioinformatics, X-ray crystallography, nucleotide co-crystallization screening\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with ligand binding defined, single lab but rigorous structural method\",\n      \"pmids\": [\"18082768\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Mouse AKAP7 contains a functional 2',5'-phosphodiesterase (2',5'-PDE) domain that rapidly degrades 2',5'-oligoadenylate (2-5A) activators of RNase L; the PDE domain requires cytoplasmic localization for antiviral activity (as shown by complementation of ns2-mutant coronavirus), while full-length AKAP7 localizes to the nucleus and cannot complement. A single point mutation AKAP7(H185R) abolishes PDE activity.\",\n      \"method\": \"Biochemical 2-5A degradation assay, viral complementation in bone marrow macrophages and mice, site-directed mutagenesis (H185R), subcellular localization by immunofluorescence\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro enzymatic assay, mutagenesis, in vivo viral complementation, and localization experiments all converging\",\n      \"pmids\": [\"24987090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structure of AKAP18β PKA-binding domain bound to the D/D domain of PKA RIIα reveals three hydrophilic anchor points outside the core PKA-binding helix that mediate contacts with the D/D domain; in vitro and cell-based experiments confirm these anchor points are required for RII subunit interaction with AKAP18.\",\n      \"method\": \"X-ray crystallography, in vitro binding assays, cell-based interaction experiments, sequence analysis of anchor points\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional validation in vitro and in cells, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"27102985\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Genetic ablation of AKAP7 specifically from dentate granule cells disrupts mossy fiber–CA3 LTP initiated by cAMP and impairs pattern separation behavior, establishing that the AKAP7/PKA complex in mossy fiber projections is essential for presynaptic PKA-dependent plasticity and spatial discrimination.\",\n      \"method\": \"Conditional knockout mouse (dentate granule cell-specific AKAP7 deletion), electrophysiology (LTP assay), behavioral testing (pattern separation)\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific KO with defined electrophysiological and behavioral phenotypes, clear pathway placement\",\n      \"pmids\": [\"27911261\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"AKAP7 knockout mice (all isoforms deleted) show normal cardiomyocyte responses to β-adrenergic stimulation: Ca2+ current, intracellular Ca2+ transients, Ca2+ reuptake, and phosphorylation of CaV1.2 and phospholamban are unaffected, indicating AKAP7 is not required for regulation of Ca2+ handling in mouse ventricular cardiomyocytes.\",\n      \"method\": \"AKAP7 global knockout mouse, whole-cell patch clamp, fluorescent Ca2+ indicator, immunoblot for substrate phosphorylation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with multiple orthogonal physiological readouts; rigorous negative result\",\n      \"pmids\": [\"23035250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"AKAP7γ and AKAP7α both interact with multiple PKC isoenzymes via multi-site binding on both proteins; AKAP7 scaffolding enhances PKC substrate phosphorylation (shown by FRET-based activity reporter) and restricts PKC mobility within cells (shown by FRAP and virtual modeling).\",\n      \"method\": \"Surface plasmon resonance, protein biochemistry pulldowns, FRET-based PKC activity reporter, FRAP imaging\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple orthogonal binding and functional methods in single lab; FRET and FRAP provide functional and localization evidence\",\n      \"pmids\": [\"22670899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"AKAP18 isoforms and PDE4 family phosphodiesterases are differentially localized in renal collecting duct principal cells, where AKAP-anchored PKA participates in AVP-stimulated aquaporin-2 (AQP2) phosphorylation and redistribution to the plasma membrane.\",\n      \"method\": \"Immunofluorescence localization in renal principal cells, functional context of AVP/AQP2 signaling\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — localization by immunofluorescence in single study, limited mechanistic follow-up specific to AKAP7\",\n      \"pmids\": [\"16500722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"AKAP7γ (long isoform) is highly mobile within cardiomyocytes as demonstrated by FRAP of GFP-tagged AKAP7γ; PKA activation accelerates AKAP7γ-GFP wash-out upon saponin permeabilization, indicating PKA signaling increases AKAP7γ mobility, which may contribute to spatial propagation of β-adrenergic signaling to SR Ca2+ uptake.\",\n      \"method\": \"FRAP of GFP-tagged AKAP7γ in rabbit ventricular cardiomyocytes, saponin permeabilization wash-out assay with PKA activation\",\n      \"journal\": \"Function (Oxford, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct live-cell FRAP imaging with functional perturbation (PKA activation), single lab\",\n      \"pmids\": [\"35620477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Long AKAP18 isoforms (AKAP7γ/δ) scaffold PKA together with ubiquitin-specific proteinase USP4 at cardiac sarcomere Z bands via the AKAP18 2'-phosphoesterase domain; AKAP18-anchored PKA phosphorylates USP4 at Ser829 near its active site, stimulating USP4 deubiquitinase activity. Pharmacological PKA inhibition or AKAP7 gene deletion decreases calcium flux through SERCA2, establishing the AKAP18/PKA/USP4 complex as a regulator of SR Ca2+ reuptake.\",\n      \"method\": \"Proximity-proteomics (BioID) in cardiomyocytes, co-immunoprecipitation, in vitro PKA phosphorylation assay, phospho-specific antibody (pSer829), AKAP7 knockout mouse, pharmacological PKA inhibition, calcium flux measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — proximity proteomics, Co-IP, in vitro phosphorylation, mutagenesis-implied active site proximity, KO mouse with functional readout, multiple orthogonal methods in single study\",\n      \"pmids\": [\"40449590\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AKAP7 (AKAP15/AKAP18) is a scaffolding protein that anchors PKA (and PKC) to defined subcellular locations—including skeletal muscle L-type Ca2+ channels (via leucine zipper), brain Na+ channels (via loop I-II binding), and the cardiac sarcoplasmic reticulum (via long isoforms γ/δ)—to enable spatially precise phosphorylation of ion channels and Ca2+-handling proteins; additionally, its central 2H phosphoesterase domain can bind AMP and degrade 2',5'-oligoadenylates, and its long isoforms recruit USP4 at sarcomere Z bands where AKAP18-anchored PKA phosphorylates USP4-Ser829 to enhance SR Ca2+ reuptake, while its mossy fiber-localized AKAP7/PKA complex is essential for cAMP-dependent LTP and hippocampal pattern separation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AKAP7 (AKAP15/AKAP18) is an A-kinase anchoring protein that tethers PKA—and PKC—to defined subcellular sites to enable spatially restricted phosphorylation of ion channels and Ca2+-handling proteins [#0, #1, #8]. It anchors PKA to skeletal muscle L-type Ca2+ channels through a leucine-zipper interaction with the CaV1.1 alpha1 C-terminus, where disrupting this interaction blocks voltage-dependent channel potentiation [#0], and to brain Nav1.2 sodium channels by binding the intracellular I-II loop, positioning PKA at defined serine sites whose phosphorylation is gated by prior PKC modification [#1, #2]. PKA recruitment is mediated by a PKA-binding domain that contacts the RIIα D/D domain through anchor points flanking the core binding helix [#5]. Beyond scaffolding, the central 2H phosphoesterase domain of long isoforms binds AMP and functions as a 2',5'-phosphodiesterase that degrades the 2-5A activators of RNase L, an activity abolished by the H185R mutation and dependent on cytoplasmic localization for antiviral function [#3, #4]. In the heart, long isoforms (AKAP7γ/δ) scaffold PKA together with the deubiquitinase USP4 at sarcomere Z bands via the phosphoesterase domain; anchored PKA phosphorylates USP4 at Ser829 to stimulate its deubiquitinase activity and promote SERCA2-mediated SR Ca2+ reuptake [#11]. In the brain, the AKAP7/PKA complex in dentate granule cell mossy fibers is required for cAMP-dependent presynaptic LTP and pattern-separation behavior [#6]. Notably, global AKAP7 deletion leaves β-adrenergic Ca2+ handling in mouse ventricular cardiomyocytes intact, indicating its cardiac role is context- and isoform-specific rather than essential for canonical β-adrenergic Ca2+ regulation [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established that AKAP7 physically links PKA to voltage-gated sodium channels, defining its role as a channel-targeting anchor.\",\n      \"evidence\": \"Co-purification, reciprocal Co-IP, mass spectrometry, and in vitro PKA phosphorylation of Nav1.2 from rat brain\",\n      \"pmids\": [\"9748250\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map the channel-binding interface\", \"Functional consequence for channel gating not yet defined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Showed that AKAP7 anchoring of PKA to L-type Ca2+ channels is functionally required for channel modulation, linking the scaffold directly to channel physiology.\",\n      \"evidence\": \"Leucine-zipper disruption mutagenesis with electrophysiology in skeletal muscle cells\",\n      \"pmids\": [\"11733497\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the same LZ motif targets other channel types not addressed\", \"Structural basis of LZ recognition not resolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined the sodium-channel binding site and revealed convergent PKC/PKA multi-site regulation gated by AKAP7-anchored kinase positioning.\",\n      \"evidence\": \"Loop I-II interaction mapping, serine site-directed mutagenesis, heterologous electrophysiology\",\n      \"pmids\": [\"12359152\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of multi-site gating not established\", \"Stoichiometry of PKC/PKA co-anchoring unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Discovered an unanticipated enzymatic identity for the AKAP18δ central domain as a nucleotide-binding 2H phosphoesterase, beyond pure scaffolding.\",\n      \"evidence\": \"Bioinformatics and X-ray crystallography with AMP/CMP co-crystallization\",\n      \"pmids\": [\"18082768\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic substrate in cells not yet identified\", \"Physiological role of nucleotide binding undefined at this stage\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Tested whether AKAP7 is required for cardiac Ca2+ handling and found it dispensable for β-adrenergic regulation, bounding its cardiac function.\",\n      \"evidence\": \"Global AKAP7 knockout mouse with patch clamp, Ca2+ imaging, and substrate phospho-immunoblots\",\n      \"pmids\": [\"23035250\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Possible redundancy with other AKAPs not excluded\", \"Isoform- or microdomain-specific roles not resolved by global deletion\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended AKAP7 scaffolding to PKC, showing it constrains kinase mobility and enhances substrate phosphorylation.\",\n      \"evidence\": \"SPR, pulldowns, FRET activity reporter, and FRAP imaging\",\n      \"pmids\": [\"22670899\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab functional readouts\", \"In vivo PKC substrates of the AKAP7/PKC complex not identified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Assigned a catalytic antiviral function to the phosphoesterase domain as a 2',5'-PDE that destroys RNase L activators, with localization gating activity.\",\n      \"evidence\": \"In vitro 2-5A degradation, H185R mutagenesis, viral complementation in macrophages and mice, immunofluorescence\",\n      \"pmids\": [\"24987090\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How nuclear full-length AKAP7 is excluded from cytoplasmic antiviral function unclear\", \"Endogenous regulation of this activity in human cells not defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Resolved how AKAP7 engages PKA, defining anchor points outside the canonical helix required for RIIα binding.\",\n      \"evidence\": \"Crystal structure of AKAP18β PKA-binding domain with RIIα D/D, with in vitro and cell-based validation\",\n      \"pmids\": [\"27102985\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RI subunit selectivity not addressed\", \"Whether anchor points are regulated dynamically unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated a required physiological role for AKAP7 in presynaptic plasticity and behavior, placing the scaffold in cAMP-dependent LTP.\",\n      \"evidence\": \"Dentate granule cell-specific conditional knockout with LTP electrophysiology and pattern separation behavior\",\n      \"pmids\": [\"27911261\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Presynaptic PKA substrates mediating the LTP defect not identified\", \"Molecular targeting of AKAP7 at mossy fiber terminals unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed AKAP7γ is dynamically mobile and that PKA activation increases its mobility, offering a mechanism for spatial spread of β-adrenergic signaling.\",\n      \"evidence\": \"FRAP and saponin permeabilization wash-out of GFP-AKAP7γ in rabbit cardiomyocytes\",\n      \"pmids\": [\"35620477\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of mobility for Ca2+ handling inferred, not demonstrated\", \"Single-lab live-cell assay\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked AKAP7 long isoforms to SR Ca2+ reuptake via a Z-band complex in which anchored PKA activates USP4 deubiquitinase, integrating scaffolding with ubiquitin signaling.\",\n      \"evidence\": \"BioID proximity proteomics, Co-IP, in vitro PKA phosphorylation, pSer829 antibody, AKAP7 KO mouse, PKA inhibition, calcium flux\",\n      \"pmids\": [\"40449590\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"USP4 deubiquitination substrates controlling SERCA2 not identified\", \"Reconciliation with the dispensable Ca2+-handling phenotype of global KO not fully addressed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How AKAP7 isoform diversity, subcellular localization, and dual scaffolding/enzymatic activities are coordinated to select specific substrates in each tissue remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No unified model linking phosphoesterase catalysis to anchoring function\", \"Tissue-specific isoform targeting determinants not mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 2, 5, 8, 11]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 11]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 5, 11]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"R-HSA-397014\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"complexes\": [\"AKAP18/PKA/USP4 Z-band complex\"],\n    \"partners\": [\"PRKAR2A\", \"CACNA1S\", \"SCN2A\", \"USP4\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}