{"gene":"AKAP9","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":1999,"finding":"AKAP450/CG-NAP was identified as a centrosomal scaffolding protein that binds RIIα (regulatory subunit of PKA type II) via a putative RII-binding amphipathic helix (around amino acid 2556), co-precipitates with PP2A catalytic subunit (when PR130 B-subunit is expressed), and co-precipitates with PP1 catalytic subunit in HeLa cells. The protein localizes to centrosome throughout the cell cycle, the midbody at telophase, and the Golgi apparatus at interphase.","method":"RII overlay screening, immunoprecipitation, immunofluorescence, mutation analysis of RII-binding site","journal":"The EMBO journal / The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP and direct binding assays replicated across multiple labs (PMID:10202149 and PMID:10358086), with mutation analysis identifying the RII-binding site","pmids":["10202149","10358086","9915845"],"is_preprint":false},{"year":2000,"finding":"The C-terminal ~90 amino acid PACT domain of AKAP450 is necessary and sufficient for centrosomal targeting; fusion of this domain to a reporter confers centrosomal localization, overexpression displaces endogenous pericentrin, and the isolated C-terminal domain associates with calmodulin when isolated from transfected cells.","method":"GFP fusion reporter localization, overexpression displacement assay, calmodulin association from transfected cells","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct functional mapping of targeting domain with reporter, dominant-negative displacement, and binding assay; replicated conceptually across AKAP450 and pericentrin","pmids":["11263498"],"is_preprint":false},{"year":1998,"finding":"Yotiao (a splice variant/isoform encoded by AKAP9) was identified as a binding partner of the NR1 subunit of the NMDA receptor in a C1 exon-dependent manner; yotiao co-immunoprecipitates with NR1 from heterologous cells and from rat brain, fractionates with postsynaptic density and cytoskeletal proteins, and colocalizes with NR1 at neuromuscular junctions.","method":"Yeast two-hybrid screen, co-immunoprecipitation from brain and heterologous cells, immunofluorescence co-localization, subcellular fractionation","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid confirmed by reciprocal Co-IP from brain tissue and heterologous cells, multiple orthogonal methods","pmids":["9482789"],"is_preprint":false},{"year":1999,"finding":"Yotiao interacts with PKA regulatory subunit RII via an RII-binding site constituted by amino acids 1452–1469, with a Kd of 50–90 nM in vitro; a stable complex of Yotiao, RIIβ, and NR1 was immunoprecipitated from whole rat brain.","method":"Yeast two-hybrid, in vitro binding assay with purified C-terminal Yotiao fragment, co-immunoprecipitation from rat brain","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro binding with Kd measurement plus reciprocal Co-IP from brain; single lab but multiple orthogonal methods","pmids":["10618500"],"is_preprint":false},{"year":2002,"finding":"CG-NAP/AKAP450 anchors the gamma-tubulin ring complex (γ-TuRC) at the centrosome: its N-terminal region associates with γ-TuRC indirectly by binding GCP2 and/or GCP3, while its C-terminal region interacts with calmodulin. Antibody inhibition of CG-NAP (or kendrin, or both combined) moderately to strongly inhibits microtubule nucleation from isolated centrosomes.","method":"Co-immunoprecipitation of endogenous proteins, yeast two-hybrid for calmodulin interaction, antibody inhibition of microtubule nucleation from isolated centrosomes","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro nucleation assay with antibody inhibition plus Co-IP; replicated with two scaffold proteins and multiple γ-TuRC components","pmids":["12221128"],"is_preprint":false},{"year":2003,"finding":"A fraction of the small GTPase Ran is tightly associated with the centrosome via AKAP450; when AKAP450 is delocalized from the centrosome, Ran is also delocalized, and microtubule regrowth and anchoring are impaired despite persistent γ-tubulin association with the centrosome.","method":"Immunofluorescence, immunoelectron microscopy, biochemical fractionation, dominant-negative AKAP450 displacement","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (IF, IEM, biochemistry) and functional consequence (MT regrowth defect); single lab but rigorous","pmids":["14517334"],"is_preprint":false},{"year":2003,"finding":"Displacement of endogenous AKAP450 from centrosomes (by overexpression of its C-terminal centrosome-targeting domain) delocalizes centrosomal PKA type IIα, impairs cytokinesis, increases ploidy in HeLa cells, arrests diploid RPE1 cells in G1, and interrupts centriole duplication.","method":"Overexpression of dominant-negative C-terminal domain, immunofluorescence, flow cytometry cell cycle analysis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean dominant-negative perturbation with defined phenotypes in two cell lines, multiple readouts","pmids":["12808041"],"is_preprint":false},{"year":2002,"finding":"CG-NAP/AKAP450 interacts with CK1δ and CK1ε (but not other CK1 isoforms) via a 182 amino acid fragment; this fragment co-immunoprecipitates with CK1δ/ε from mammalian cells, co-localizes with endogenous CK1δ at the centrosome, and when targeted to the plasma membrane is sufficient to re-localize CK1δ to the membrane, establishing CG-NAP as the centrosomal anchor for CK1δ/ε.","method":"Yeast two-hybrid, co-immunoprecipitation, immunofluorescence co-localization, membrane-targeting re-localization experiment","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by Co-IP from mammalian cells plus functional re-localization experiment; multiple orthogonal methods","pmids":["12270714"],"is_preprint":false},{"year":2009,"finding":"AKAP450 is required for microtubule nucleation at the Golgi apparatus: depletion of AKAP450 abolishes Golgi MT nucleation, and depletion of the cis-Golgi protein GM130 disorganizes the AKAP450 network and impairs MT nucleation. AKAP450 binds the cis-side of the Golgi in an MT-independent, GM130-dependent manner.","method":"siRNA depletion, live-cell MT regrowth assay, brefeldin A treatment, immunofluorescence","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean siRNA loss-of-function with specific MT nucleation readout, identification of GM130-dependent recruitment mechanism, replicated with pharmacological perturbation","pmids":["19242490"],"is_preprint":false},{"year":2004,"finding":"Yotiao interacts with the type 1 InsP3 receptor (InsP3R1) via the leucine/isoleucine zipper (LIZ) motif in the InsP3R1 coupling domain and the fourth LIZ motif in AKAP9/Yotiao; this interaction mediates PKA-InsP3R1 association in brain, is isoform-specific (type 1 only), and promotes association of InsP3R1 with the NR1 NMDA receptor as well as indirect association with PP1.","method":"Biochemical co-immunoprecipitation from brain, in vitro binding, domain mapping with LIZ mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple biochemical experiments in one study including domain mapping and isoform specificity; Co-IP from brain tissue","pmids":["14982933"],"is_preprint":false},{"year":2004,"finding":"Yotiao (AKAP9) directly associates with the IKs potassium channel complex (KCNQ1/KCNE1), recruits PKA and PP1 to the channel, and exerts direct allosteric effects on channel gating that are distinct from its role in coordinating PKA phosphorylation — demonstrated by studying channels mutated to simulate phosphorylation.","method":"Electrophysiology (patch-clamp), co-immunoprecipitation, channel mutagenesis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — electrophysiological functional assay combined with biochemical Co-IP; replicated and extended in subsequent publications","pmids":["15528278"],"is_preprint":false},{"year":2005,"finding":"Yotiao itself is a PKA substrate: Ser-43 in the N-terminus of Yotiao is phosphorylated by PKA in response to β-adrenergic receptor stimulation. Alanine substitution of Ser-43 abolishes PKA phosphorylation of Yotiao and markedly diminishes the functional (voltage-dependent activation and kinetics) response of the IKs channel to cAMP, without preventing PKA phosphorylation of KCNQ1 or KCNQ1 binding to Yotiao.","method":"Phospho-specific antibody, mutagenesis (S43A), β-adrenergic stimulation, electrophysiology","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — site-directed mutagenesis identifying specific PKA phosphorylation site combined with functional electrophysiology; clean separation of effects","pmids":["16002409"],"is_preprint":false},{"year":2008,"finding":"Yotiao (AKAP9) directly interacts with adenylyl cyclase (AC) isoforms 1, 2, 3, and 9 but not AC5 or AC6; it inhibits AC2 and AC3 enzymatic activity but has no effect on AC1 or AC9. The N-terminus of AC2 binds directly to amino acids 808–957 of Yotiao. Disruption of Yotiao-AC interactions increases brain AC activity by ~40%, establishing Yotiao as a direct regulator of cAMP production.","method":"Co-immunoprecipitation, enzymological AC activity assay, domain mapping with truncations, peptide competition","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro enzymatic assay combined with Co-IP domain mapping and peptide competition; multiple orthogonal methods in one study","pmids":["18772391"],"is_preprint":false},{"year":2000,"finding":"CG-NAP anchors hypophosphorylated/immature PKCε at the Golgi/centrosome area via direct binding to PKCε's catalytic domain; sufficiently phosphorylated (mature) PKCε does not bind CG-NAP. Phosphorylation-site mutants (T566A or S729A) of PKCε co-localize with CG-NAP at Golgi/centrosome, while wild-type PKCε distributes in cytosol upon maturation.","method":"Co-immunoprecipitation, in vitro binding assay, pulse-chase, mutagenesis, immunofluorescence","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro binding with mutagenesis plus pulse-chase and immunofluorescence; multiple orthogonal methods establishing phosphorylation-dependent binding","pmids":["10945988"],"is_preprint":false},{"year":2005,"finding":"AKAP9-BRAF fusion protein results from paracentric inversion of chromosome 7q, creating an in-frame fusion of AKAP9 exons 1–8 with BRAF exons 9–18. The fusion protein retains the BRAF kinase domain but lacks the autoinhibitory N-terminal portion, displays elevated kinase activity, and transforms NIH3T3 cells; it was preferentially found in radiation-induced papillary thyroid carcinomas.","method":"Molecular cloning, kinase activity assay, NIH3T3 transformation assay, PCR/sequencing of tumor samples","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — kinase activity measurement plus functional transformation assay; multiple methods in single rigorous study","pmids":["15630448"],"is_preprint":false},{"year":2002,"finding":"AKAP350A contains a distinct Golgi apparatus targeting motif between amino acids 3259 and 3307 that is functionally distinguishable from the adjacent centrosomal PACT domain (amino acids 3308–3324); GFP chimeras of the carboxyl-terminal regions defined these two non-overlapping targeting domains.","method":"GFP chimeric construct localization, brefeldin A treatment, immunofluorescence","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct domain mapping with GFP fusions and pharmacological confirmation; single lab, multiple constructs","pmids":["12163481"],"is_preprint":false},{"year":2002,"finding":"AKAP350 associates with all CLIC family members via a 133 amino acid domain; specifically, CLIC5B (a novel CLIC isoform) co-localizes and co-immunoprecipitates with AKAP350 at the Golgi apparatus, and this association is disrupted by brefeldin A treatment.","method":"Yeast two-hybrid, co-immunoprecipitation, GFP targeting constructs, immunofluorescence, brefeldin A treatment","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — yeast two-hybrid confirmed by Co-IP from cells and co-localization; single lab","pmids":["12163479"],"is_preprint":false},{"year":2004,"finding":"AKAP350 interacts with CIP4 (and structurally related proteins FBP17, FBP17b) via yeast two-hybrid and pull-down. CIP4 is phosphorylated by PKA in vitro, and forskolin stimulates CIP4 phosphorylation in situ. Disruption of the CIP4-AKAP350 interaction or AKAP350 knockdown by RNAi leads to changes in Golgi structure.","method":"Yeast two-hybrid, GST pull-down, in vitro PKA phosphorylation assay, RNAi knockdown, immunofluorescence of Golgi morphology","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay plus pull-down plus RNAi functional readout; single lab, multiple orthogonal methods","pmids":["15047863"],"is_preprint":false},{"year":2007,"finding":"CG-NAP is recruited to the Golgi apparatus via interaction with the dynein-dynactin complex: CG-NAP possesses two microtubule-binding domains, co-immunoprecipitates with dynactin subunit p150(Glued), and the p150(Glued)-binding region of CG-NAP when targeted to mitochondria recruits mitochondria to the pericentriolar area. Overexpression of this region causes Golgi fragmentation similar to dynamitin overexpression.","method":"Co-immunoprecipitation, microtubule co-sedimentation, mitochondria-targeting re-localization, overexpression/dominant-negative, immunofluorescence","journal":"Genes to cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional re-localization assay and dominant-negative; single lab, multiple orthogonal methods","pmids":["17352745"],"is_preprint":false},{"year":2005,"finding":"The centrosome-targeting region of CG-NAP (CG-NAP/D) causes centrosome amplification by recruiting cyclin E-cdk2 to centrosomes; CG-NAP/D co-immunoprecipitates active cyclin-cdk complexes (histone H1 kinase activity), centrosome fractions from CG-NAP/D cells have increased cdk2, and amplification is suppressed by a mutant cyclin E unable to bind cdk2.","method":"Overexpression of targeting domain, centrosome counting, co-immunoprecipitation of kinase activity, dominant-negative cyclin E, immunofluorescence","journal":"Genes to cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase activity measurement combined with domain-dependent localization and genetic epistasis with cyclin E mutant; single lab","pmids":["15670215"],"is_preprint":false},{"year":2010,"finding":"AKAP9 interacts with Epac1 and facilitates microtubule polymerization in endothelial cells; AKAP9 silencing abolishes Epac1-stimulated microtubule growth and the ability of Epac1 activation to enhance barrier function via integrin adhesion at cell-cell contacts, despite intact Rap1 activation, cortical actin, and VE-cadherin adhesion.","method":"siRNA knockdown, live-cell microtubule dynamics imaging, co-immunoprecipitation (Epac1-AKAP9), transendothelial resistance assay","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus siRNA with specific MT dynamics and barrier function readout; single lab, multiple orthogonal methods","pmids":["20952690"],"is_preprint":false},{"year":2012,"finding":"In the heart, Yotiao assembles a macromolecular IKs signaling complex containing PKA, PP1, PDE4D3, AC9, and the KCNQ1-KCNE1 channel; AC9 is the only Yotiao-interacting AC isoform expressed in cardiac myocytes, and AC9 association with the complex sensitizes PKA phosphorylation of KCNQ1 to β-adrenergic stimulation. Addition of the AC9 N-terminus disrupts AC activity associated with the IKs-Yotiao complex in transgenic mouse heart.","method":"Co-immunoprecipitation from transgenic mouse heart and guinea pig heart, RT-PCR isoform survey, AC activity assay, peptide competition","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — enzymatic activity assay from native cardiac tissue combined with Co-IP from two species and peptide competition; multiple orthogonal methods","pmids":["22778270"],"is_preprint":false},{"year":2012,"finding":"AKAP9-anchored PDE4D3 generates a centrosomal cAMP microdomain: centrosomal PKA shows a reduced activation threshold due to autophosphorylation of its regulatory subunit at S114 upon AKAP9 binding; disruption of centrosomal PDE4D3 impairs cell cycle progression by accumulating cells in prophase.","method":"FRET-based real-time cAMP imaging, displacement of centrosomal PDE4D3, cell cycle analysis by flow cytometry","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — live-cell FRET imaging combined with functional cell cycle readout; clean mechanistic dissection; single lab, two orthogonal methods","pmids":["22908311"],"is_preprint":false},{"year":2005,"finding":"CG-NAP/AKAP450 redistributes from centrosome/Golgi to microtubules in trailing extensions of LFA-1-stimulated T cells; it forms a physical complex with LFA-1, tubulin, and PKCβ/δ isoenzymes, and is critically required for T cell polarization and migration induced by LFA-1 but not fibronectin (β1 integrin).","method":"In situ immunoprecipitation, immunofluorescence co-localization, GFP-tagged dominant-negative construct, T cell migration assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — novel in situ Co-IP plus dominant-negative functional assay; single lab","pmids":["16339516"],"is_preprint":false},{"year":2002,"finding":"TACC4 interacts with AKAP350 at the centrosome in interphase via its C-terminal coiled-coil region; AKAP350 sequesters TACC4 to the centrosome in interphase, while a distinct N-terminal domain of TACC4 mediates spindle localization in mitosis. Overexpression of spindle-targeting TACC4 increases the proportion of cells in prometaphase.","method":"Yeast two-hybrid, co-localization immunofluorescence, truncation analysis, cell cycle analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — yeast two-hybrid domain mapping with co-localization and functional overexpression phenotype; single lab","pmids":["12015314"],"is_preprint":false},{"year":2017,"finding":"In differentiated muscle cells (myotubes), Nesprin-1α recruits Akap450 to the nuclear envelope independently of kinesin; Akap450 (but not Pericentrin or Pcm1) is required for microtubule nucleation from the nuclear envelope, and this MT nucleation activity is required for nuclear spreading and positioning in myotubes. This mechanism is disrupted in congenital muscular dystrophy patient myotubes carrying a SYNE1 nonsense mutation.","method":"BioID proximity labeling, siRNA knockdown, live-cell MT nucleation assay, computer simulation, immunofluorescence","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — proximity labeling to identify interaction, siRNA loss-of-function with specific MT nucleation and nuclear positioning readouts, validated in patient cells; multiple orthogonal methods","pmids":["28966089"],"is_preprint":false},{"year":2020,"finding":"In cardiomyocytes, AKAP6 acts as an adaptor linking Nesprin-1α to AKAP9 (and Pericentrin) at the nuclear envelope MTOC via spectrin repeats; AKAP6 and AKAP9 form a protein platform tethering the Golgi to the nucleus, and both Golgi and nuclear envelope exhibit MTOC activity utilizing AKAP9. AKAP6 is required for formation and activity of the nuclear envelope MTOC.","method":"Co-immunoprecipitation, immunofluorescence, siRNA/shRNA knockdown, MT nucleation assay, ectopic expression in epithelial cells","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple Co-IP and functional knockdown experiments; single lab but multiple orthogonal methods","pmids":["33295871"],"is_preprint":false},{"year":2013,"finding":"Akap9 disruption in mice causes infertility through failure of Sertoli cell maturation: Sertoli cells continue expressing immaturity markers (AMH, thyroid hormone receptor α) and fail to express maturation marker p27(Kip1); gap and tight junctions essential for the blood-testis barrier are disrupted, with mislocalized connexin43 and ZO-1.","method":"Three Akap9 mouse alleles (loss-of-function), immunofluorescence, Western blot, histology","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic loss-of-function in three alleles with defined cellular phenotypes; single lab","pmids":["23608191"],"is_preprint":false},{"year":2017,"finding":"AKAP350 recruits EB1 to the spindle poles; decreased AKAP350 expression reduces EB1 levels at spindle poles and astral microtubules, causes defective spindle alignment in 3D epithelial cysts with abnormal lumen, and EB1 overexpression rescues the spindle orientation defect. Specific delocalization of the AKAP350/EB1 complex from the centrosome phenocopies AKAP350 knockdown.","method":"siRNA knockdown, 3D organotypic culture, immunofluorescence, EB1 overexpression rescue, dominant-negative delocalization construct","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown with rescue experiment and dominant-negative phenocopy; single lab, multiple methods","pmids":["29097729"],"is_preprint":false},{"year":2015,"finding":"AKAP350 recruits CIP4 to the centrosome; decreased AKAP350 or CIP4 expression, or inhibition of the CIP4-AKAP350 interaction, impairs formation of the nucleus-centrosome-Golgi front-back axis and directional cell migration. Centrosome positioning (but not nuclear movement) is specifically affected.","method":"siRNA knockdown, dominant-negative CIP4-binding domain, immunofluorescence, wound-healing and migration assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function and interaction-disruption with specific functional readouts; single lab, multiple orthogonal approaches","pmids":["26208639"],"is_preprint":false},{"year":2015,"finding":"AKAP9-deficient T cells (T cell-specific deletion) exhibit reduced microtubule-dependent TCR recycling to the cell surface, impairing TCR re-activation by non-classical antigen-presenting cells; this leads to increased T cell egress from inflamed tissues and protection from organ damage in inflammatory disease models.","method":"Conditional T cell-specific Akap9 knockout mice, TCR surface recycling assay, in vivo inflammatory disease models, flow cytometry","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined mechanistic readout (TCR recycling) and in vivo phenotype; multiple orthogonal methods","pmids":["26680259"],"is_preprint":false},{"year":2013,"finding":"AKAP350 at the centrosome facilitates the initiation of DNA synthesis by scaffolding Cdk2 to the centrosome; an AKAP350 C-terminal domain increases centrosomal Cdk2 levels and phosphorylation of nucleophosmin (a Cdk2 centrosomal substrate marking G1/S transition), whereas AKAP350 knockdown inhibits G1/S transition and DNA synthesis.","method":"siRNA knockdown, overexpression of C-terminal domain, BrdU incorporation, nucleophosmin phosphorylation assay, centrosome fractionation","journal":"Cellular logistics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function combined with gain-of-function domain construct and specific centrosomal substrate readout; single lab","pmids":["24475373"],"is_preprint":false},{"year":2020,"finding":"AKAP350 localizes p150(Glued) (dynactin component) to the spindle poles, facilitating p150(Glued)/EB1 interaction at these structures; AKAP350 depletion reduces p150(Glued) at astral microtubules and impairs elongation of astral microtubules during anaphase.","method":"siRNA knockdown, co-immunoprecipitation, immunofluorescence, astral MT length measurement","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — siRNA knockdown with Co-IP and specific MT readout; single lab, two orthogonal methods","pmids":["32841682"],"is_preprint":false},{"year":2024,"finding":"Yotiao decreases ER calcium content by suppressing store-operated calcium entry (SOCE) through Orai1; this effect requires AC9 (which increases cAMP upon Yotiao interaction) and involves Yotiao acting on the Orai1 C-terminus, but does not require IP3R1, PKA, PP1, or AC2.","method":"ER Ca2+ imaging, Yotiao truncation constructs, knockout cells, pharmacological tools, SOCE assay","journal":"Cell calcium","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional Ca2+ assay with KO and truncation mapping; single lab, multiple orthogonal approaches","pmids":["38781694"],"is_preprint":false},{"year":2025,"finding":"Purified full-length AKAP350 forms polydisperse fibrillar clusters (~50 nm) with fibrous outgrowths; cryo-EM revealed fibers reconstructing as double-stranded DNA, confirmed by DNA sequencing. AKAP350 co-purifies with endogenous PKA, CEP170, CDK5RAP2, and DNA-binding proteins NFIB and nucleolin; NFIB and nucleolin pull-down was reduced by DNase-I treatment (indicating DNA-mediated interaction), whereas centrosomal protein pull-downs were not affected by DNase-I.","method":"Cryo-EM, cryo-ET, mass spectrometry, DNA sequencing, pull-down with DNase-I treatment, purification of full-length protein from human cells","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct cryo-EM/cryo-ET structural analysis of purified full-length protein plus mass spectrometry and functional pull-down with enzymatic perturbation; single lab but multiple rigorous orthogonal methods","pmids":["40154916"],"is_preprint":false},{"year":2025,"finding":"PDE4DIP coordinates with AKAP9 to enhance Golgi localization and stability of PKA RIIα; depletion of PDE4DIP mislocalizes RIIα from the Golgi and leads to its degradation, compromising RIIα's negative regulatory effect on PKA signaling.","method":"Co-immunoprecipitation, siRNA knockdown, immunofluorescence localization of PKA RIIα, Western blot for RIIα stability","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP combined with knockdown and localization/stability readout; single lab","pmids":["39905234"],"is_preprint":false}],"current_model":"AKAP9 encodes a large family of scaffolding proteins (including AKAP450/CG-NAP and the shorter isoform Yotiao) that serve as multi-valent anchoring platforms at the centrosome and Golgi apparatus, where they recruit and organize PKA (via an amphipathic RII-binding helix), PP1, PP2A, PKCε, CK1δ/ε, PDE4D3, cyclin E-Cdk2, Ran, γ-TuRC (via GCP2/GCP3), AC isoforms (particularly AC9), CIP4, EB1, and dynein/dynactin to control microtubule nucleation and dynamics, cell cycle progression (G1/S transition and centriole duplication), Golgi structure maintenance, and directional cell migration; in neurons and cardiomyocytes the Yotiao isoform additionally forms a macromolecular IKs channel complex (KCNQ1/KCNE1/PKA/PP1/PDE4D3/AC9) that is essential for sympathetic regulation of cardiac repolarization, while also scaffolding InsP3R1 and NMDA receptors to couple cAMP and Ca²⁺ signaling at synapses."},"narrative":{"mechanistic_narrative":"AKAP9 encodes large multi-valent scaffolding proteins (AKAP450/CG-NAP/AKAP350 and the shorter neuronal/cardiac isoform Yotiao) that nucleate signalling complexes at the centrosome, Golgi apparatus, and nuclear envelope to organize microtubules and compartmentalize cAMP signalling [PMID:10202149, PMID:10358086, PMID:9915845, PMID:12221128, PMID:19242490]. The protein is targeted to the centrosome by a C-terminal PACT domain and to the Golgi by an adjacent, separable Golgi-targeting motif, and recruits PKA type II via an RII-binding amphipathic helix [PMID:11263498, PMID:12163481, PMID:10202149, PMID:10358086, PMID:9915845]. At microtubule-organizing centers it anchors the γ-tubulin ring complex through GCP2/GCP3, tethers Ran, and recruits dynein-dynactin (p150Glued), CK1δ/ε, PKCε, EB1, and CIP4, thereby driving microtubule nucleation from the centrosome, Golgi, and nuclear envelope and supporting nuclear positioning, spindle orientation, and directional cell migration [PMID:12221128, PMID:14517334, PMID:12270714, PMID:10945988, PMID:17352745, PMID:29097729, PMID:26208639, PMID:28966089, PMID:33295871]. AKAP9 couples this scaffolding to cell-cycle control, recruiting cyclin E-Cdk2 and concentrating a PDE4D3-shaped cAMP microdomain that lowers the activation threshold of centrosomal PKA to license centriole duplication and the G1/S transition [PMID:12808041, PMID:15670215, PMID:22908311, PMID:24475373]. In neurons and cardiomyocytes the Yotiao isoform assembles a macromolecular IKs channel complex (KCNQ1/KCNE1 with PKA, PP1, PDE4D3, and AC9) and is itself a PKA substrate at Ser-43, integrating β-adrenergic input into channel gating, while also scaffolding InsP3R1 and NMDA receptor NR1 to couple cAMP and Ca²⁺ signalling [PMID:15528278, PMID:16002409, PMID:22778270, PMID:14982933, PMID:9482789]. A paracentric inversion of 7q creates an oncogenic AKAP9-BRAF fusion with constitutive kinase activity found in radiation-induced papillary thyroid carcinoma [PMID:15630448].","teleology":[{"year":1998,"claim":"Established the first molecular role for an AKAP9 product by showing the Yotiao isoform physically links the NMDA receptor NR1 subunit to the postsynaptic apparatus, defining it as a synaptic scaffold.","evidence":"Yeast two-hybrid plus reciprocal Co-IP from brain and heterologous cells, with fractionation and NMJ co-localization","pmids":["9482789"],"confidence":"High","gaps":["Did not define the full receptor complex or downstream signalling enzymes recruited","No structural basis for the C1 exon-dependent interaction"]},{"year":1999,"claim":"Identified the centrosomal/Golgi scaffold AKAP450/CG-NAP and its capacity to anchor PKA-RII alongside phosphatases PP1 and PP2A, establishing the protein as a multi-enzyme anchoring platform.","evidence":"RII overlay screening, reciprocal Co-IP, immunofluorescence, and mutation of the RII-binding helix in HeLa cells; plus Yotiao RII Kd measurement","pmids":["10202149","10358086","9915845","10618500"],"confidence":"High","gaps":["Did not establish functional consequence of kinase/phosphatase anchoring","Stoichiometry and spatial organization of the bound enzymes unresolved"]},{"year":2002,"claim":"Mapped distinct C-terminal targeting modules (PACT for centrosome, a separate Golgi motif) and showed AKAP450 anchors γ-TuRC via GCP2/GCP3 to drive microtubule nucleation, linking localization to MTOC function.","evidence":"GFP chimera mapping, dominant-negative displacement, Co-IP, and antibody inhibition of nucleation from isolated centrosomes; plus CK1δ/ε, CLIC, TACC4 partner mapping","pmids":["11263498","12163481","12221128","12270714","12163479","12015314"],"confidence":"High","gaps":["Did not resolve how γ-TuRC recruitment is regulated through the cell cycle","Functional roles of CLIC and TACC4 binding remained correlative"]},{"year":2003,"claim":"Demonstrated that centrosomal AKAP450 is required for microtubule regrowth/anchoring and for cell-cycle progression, tethering Ran and PKA and controlling cytokinesis, ploidy, and centriole duplication.","evidence":"Dominant-negative displacement of the C-terminal targeting domain with IF, immuno-EM, fractionation, and flow-cytometry cell-cycle analysis in HeLa and RPE1 cells","pmids":["14517334","12808041"],"confidence":"High","gaps":["Did not separate scaffolding of MT machinery from PKA anchoring in producing the phenotypes","Mechanism coupling Ran delocalization to anchoring defect unclear"]},{"year":2004,"claim":"Defined Yotiao as the organizer of a cardiac/neuronal IKs and InsP3R1 signalling complex, recruiting PKA and PP1 to channels and exerting direct allosteric control over gating.","evidence":"Patch-clamp electrophysiology, channel mutagenesis, Co-IP from brain, and LIZ-motif domain mapping","pmids":["15528278","14982933"],"confidence":"High","gaps":["Did not yet identify the upstream cAMP-generating and degrading enzymes in the cardiac complex","Allosteric gating mechanism not structurally defined"]},{"year":2005,"claim":"Showed Yotiao is itself a PKA substrate (Ser-43) whose phosphorylation is required for the cAMP response of the IKs channel, and that the centrosomal domain recruits cyclin E-Cdk2 to drive centrosome amplification.","evidence":"Phospho-specific antibody and S43A mutagenesis with electrophysiology; Co-IP of cyclin-Cdk2 kinase activity and dominant-negative cyclin E with centrosome counting","pmids":["16002409","15670215"],"confidence":"High","gaps":["Did not establish how scaffold phosphorylation alters complex architecture","Physiological trigger for cyclin E-Cdk2 recruitment to the scaffold undefined"]},{"year":2005,"claim":"Connected AKAP9 to disease and to immune cell migration: a 7q inversion generates a transforming AKAP9-BRAF fusion in thyroid carcinoma, and the scaffold relocates to migrating T-cell extensions in complex with LFA-1, tubulin, and PKC.","evidence":"Molecular cloning, kinase activity and NIH3T3 transformation assays, tumor sequencing; in situ Co-IP and T-cell migration assay","pmids":["15630448","16339516"],"confidence":"High","gaps":["AKAP9-BRAF fusion describes a chimeric oncoprotein, not wild-type AKAP9 function","Mechanism of LFA-1-specific scaffold relocation not resolved"]},{"year":2009,"claim":"Established AKAP450 as the essential nucleator of Golgi-derived microtubules, recruited to the cis-Golgi in a GM130-dependent, MT-independent manner.","evidence":"siRNA depletion with live-cell MT regrowth assays, brefeldin A treatment, and IF","pmids":["19242490"],"confidence":"High","gaps":["Did not define how GM130 docking is coordinated with centrosomal pools","Functional consequence of Golgi MTs for trafficking not addressed here"]},{"year":2010,"claim":"Extended scaffold function to endothelial barrier control, showing AKAP9 binds Epac1 and is required for Epac1-stimulated microtubule growth and integrin-mediated barrier strengthening.","evidence":"Co-IP, siRNA, live MT dynamics imaging, and transendothelial resistance assay","pmids":["20952690"],"confidence":"Medium","gaps":["Single lab; reciprocal validation of the Epac1 interaction limited","Direct vs indirect nature of the AKAP9-Epac1 association unresolved"]},{"year":2012,"claim":"Resolved the full cAMP regulatory architecture of AKAP9 complexes: a cardiac IKs complex incorporating PDE4D3 and AC9 to sensitize PKA phosphorylation, and a centrosomal PDE4D3-shaped cAMP microdomain that lowers the PKA activation threshold for cell-cycle progression.","evidence":"Co-IP from transgenic/guinea-pig heart, RT-PCR isoform survey, AC activity and peptide competition; FRET cAMP imaging and cell-cycle analysis","pmids":["22778270","22908311"],"confidence":"High","gaps":["Did not define spatial extent of the microdomains in vivo","Crosstalk between centrosomal and Golgi cAMP pools unexplored"]},{"year":2013,"claim":"Demonstrated organismal and cell-cycle roles in vivo, showing Akap9 loss causes infertility via failed Sertoli cell maturation and blood-testis barrier disruption, and that the centrosomal scaffold drives G1/S by tethering Cdk2.","evidence":"Three Akap9 mouse loss-of-function alleles with histology/IF; siRNA, C-terminal domain overexpression, BrdU, and nucleophosmin phosphorylation assays","pmids":["23608191","24475373"],"confidence":"Medium","gaps":["Did not connect junctional defects to a specific AKAP9-anchored enzyme","Tissue specificity of the Cdk2-scaffolding role unclear"]},{"year":2015,"claim":"Defined the directional-migration and immune functions of the scaffold: AKAP350 recruits CIP4 to position the centrosome in the nucleus-centrosome-Golgi axis, and AKAP9 supports microtubule-dependent TCR recycling controlling T-cell egress in inflammation.","evidence":"siRNA, dominant-negative interaction-disruption, wound-healing assays; conditional T-cell Akap9 knockout with TCR recycling assay and in vivo disease models","pmids":["26208639","26680259"],"confidence":"High","gaps":["Did not establish whether MT nucleation or anchoring underlies the migration defect","Link between TCR recycling kinetics and tissue egress mechanistically incomplete"]},{"year":2017,"claim":"Identified a nuclear-envelope MTOC role, showing Nesprin-1α recruits Akap450 to the nuclear envelope to nucleate microtubules required for myonuclear positioning, a pathway disrupted in SYNE1-mutant muscular dystrophy myotubes.","evidence":"BioID proximity labeling, siRNA, live MT nucleation assays, and patient-cell validation","pmids":["28966089"],"confidence":"High","gaps":["Did not define the adaptor bridging Nesprin-1α to AKAP450","Whether nuclear-envelope nucleation uses γ-TuRC as at the centrosome unstated"]},{"year":2020,"claim":"Filled in the nuclear-envelope MTOC adaptor and refined spindle-pole mechanics, identifying AKAP6 as the Nesprin-1α-to-AKAP9 linker tethering Golgi to nucleus, and showing AKAP350 localizes p150Glued to spindle poles for astral MT elongation.","evidence":"Co-IP, siRNA/shRNA, MT nucleation and ectopic-expression assays; Co-IP and astral MT length measurement","pmids":["33295871","32841682"],"confidence":"Medium","gaps":["Single-lab observations; reciprocal in vivo validation limited","Regulation of switching between centrosomal and nuclear-envelope MTOCs unresolved"]},{"year":2024,"claim":"Expanded Yotiao's calcium-signalling role, showing it suppresses store-operated calcium entry through Orai1 via AC9-dependent cAMP, independently of IP3R1, PKA, and PP1.","evidence":"ER Ca2+ and SOCE imaging with truncation constructs, knockout cells, and pharmacology","pmids":["38781694"],"confidence":"Medium","gaps":["Single lab; direct Yotiao-Orai1 contact not structurally confirmed","Physiological context of SOCE suppression unaddressed"]},{"year":2025,"claim":"Provided the first structural and DNA-association data for full-length AKAP350 and refined Golgi PKA regulation, revealing fibrillar clusters that scaffold double-stranded DNA with NFIB/nucleolin, and showing PDE4DIP stabilizes Golgi-localized PKA RIIα.","evidence":"Cryo-EM/cryo-ET, mass spectrometry, DNA sequencing, DNase-I pull-downs; Co-IP, siRNA, and RIIα localization/stability assays","pmids":["40154916","39905234"],"confidence":"High","gaps":["Functional significance of the DNA-associated fibrillar assembly unknown","How PDE4DIP and AKAP9 jointly stabilize RIIα not mechanistically resolved"]},{"year":null,"claim":"How the centrosomal, Golgi, nuclear-envelope, and channel-complex pools of AKAP9 are differentially specified and dynamically regulated within a single cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated model of isoform- and localization-specific complex assembly","No high-resolution structure of the full-length scaffold with its bound enzymes","Physiological role of the DNA-associated fibrillar state undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2,4,9,10,21]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[4,5,8,18,28,32]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[12,22,33,35]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[34]}],"localization":[{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[0,1,4,6,19,22,31]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[0,8,15,16,17,18,29]},{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[25,26]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,10,23]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[6,19,22,31]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,10,12,21,22]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[4,5,8,25,26]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[23,30]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[14,25]}],"complexes":["IKs channel complex (KCNQ1/KCNE1/PKA/PP1/PDE4D3/AC9)","γ-tubulin ring complex (γ-TuRC)","nuclear envelope MTOC","NMDA receptor-PKA postsynaptic complex"],"partners":["PRKAR2A","KCNQ1","PDE4D","ADCY9","GM130","DCTN1","ITPR1","GRIN1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q99996","full_name":"A-kinase anchor protein 9","aliases":["A-kinase anchor protein 350 kDa","AKAP 350","hgAKAP 350","A-kinase anchor protein 450 kDa","AKAP 450","AKAP 120-like protein","Centrosome- and Golgi-localized PKN-associated protein","CG-NAP","Protein hyperion","Protein kinase A-anchoring protein 9","PRKA9","Protein yotiao"],"length_aa":3907,"mass_kda":453.0,"function":"Scaffolding protein that assembles several protein kinases and phosphatases on the centrosome and Golgi apparatus. Required to maintain the integrity of the Golgi apparatus (PubMed:10202149, PubMed:15047863). Required for microtubule nucleation at the cis-side of the Golgi apparatus (PubMed:15047863, PubMed:19242490). Required for association of the centrosomes with the poles of the bipolar mitotic spindle during metaphase (PubMed:25657325). In complex with PDE4DIP isoform 13/MMG8/SMYLE, recruits CAMSAP2 to the Golgi apparatus and tethers non-centrosomal minus-end microtubules to the Golgi, an important step for polarized cell movement (PubMed:27666745, PubMed:28814570). In complex with PDE4DIP isoform 13/MMG8/SMYLE, EB1/MAPRE1 and CDK5RAP2, contributes to microtubules nucleation and extension also from the centrosome to the cell periphery (PubMed:29162697) Associated with the N-methyl-D-aspartate receptor and is specifically found in the neuromuscular junction (NMJ) as well as in neuronal synapses, suggesting a role in the organization of postsynaptic specializations","subcellular_location":"Golgi apparatus; Cytoplasm; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome","url":"https://www.uniprot.org/uniprotkb/Q99996/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AKAP9","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":"CLIP1","stoichiometry":0.2},{"gene":"DYNLL1","stoichiometry":0.2},{"gene":"DYNLL2","stoichiometry":0.2},{"gene":"FXR1","stoichiometry":0.2},{"gene":"IPO5","stoichiometry":0.2},{"gene":"PRKACA","stoichiometry":0.2},{"gene":"SCYL2","stoichiometry":0.2},{"gene":"STK25","stoichiometry":0.2},{"gene":"STK4","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/AKAP9","total_profiled":1310},"omim":[{"mim_id":"617398","title":"FK506-BINDING PROTEIN 15; FKBP15","url":"https://www.omim.org/entry/617398"},{"mim_id":"616475","title":"CENTROSOMAL PROTEIN, 72-KD; CEP72","url":"https://www.omim.org/entry/616475"},{"mim_id":"614071","title":"MYOCARDIAL ZONULA ADHERENS PROTEIN; MYZAP","url":"https://www.omim.org/entry/614071"},{"mim_id":"611820","title":"LONG QT SYNDROME 11; LQT11","url":"https://www.omim.org/entry/611820"},{"mim_id":"607542","title":"POTASSIUM CHANNEL, VOLTAGE-GATED, KQT-LIKE SUBFAMILY, MEMBER 1; KCNQ1","url":"https://www.omim.org/entry/607542"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Golgi apparatus","reliability":"Enhanced"},{"location":"Vesicles","reliability":"Additional"},{"location":"Centrosome","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/AKAP9"},"hgnc":{"alias_symbol":["KIAA0803","AKAP350","AKAP450","CG-NAP","YOTIAO","HYPERION","PRKA9","MU-RMS-40.16A","PPP1R45","LQT11"],"prev_symbol":[]},"alphafold":{"accession":"Q99996","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99996","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q99996-4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q99996-4-F1-predicted_aligned_error_v6.png","plddt_mean":64.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AKAP9","jax_strain_url":"https://www.jax.org/strain/search?query=AKAP9"},"sequence":{"accession":"Q99996","fasta_url":"https://rest.uniprot.org/uniprotkb/Q99996.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q99996/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99996"}},"corpus_meta":[{"pmid":"15630448","id":"PMC_15630448","title":"Oncogenic AKAP9-BRAF fusion is a novel 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cells. The protein localizes to centrosome throughout the cell cycle, the midbody at telophase, and the Golgi apparatus at interphase.\",\n      \"method\": \"RII overlay screening, immunoprecipitation, immunofluorescence, mutation analysis of RII-binding site\",\n      \"journal\": \"The EMBO journal / The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP and direct binding assays replicated across multiple labs (PMID:10202149 and PMID:10358086), with mutation analysis identifying the RII-binding site\",\n      \"pmids\": [\"10202149\", \"10358086\", \"9915845\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The C-terminal ~90 amino acid PACT domain of AKAP450 is necessary and sufficient for centrosomal targeting; fusion of this domain to a reporter confers centrosomal localization, overexpression displaces endogenous pericentrin, and the isolated C-terminal domain associates with calmodulin when isolated from transfected cells.\",\n      \"method\": \"GFP fusion reporter localization, overexpression displacement assay, calmodulin association from transfected cells\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct functional mapping of targeting domain with reporter, dominant-negative displacement, and binding assay; replicated conceptually across AKAP450 and pericentrin\",\n      \"pmids\": [\"11263498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Yotiao (a splice variant/isoform encoded by AKAP9) was identified as a binding partner of the NR1 subunit of the NMDA receptor in a C1 exon-dependent manner; yotiao co-immunoprecipitates with NR1 from heterologous cells and from rat brain, fractionates with postsynaptic density and cytoskeletal proteins, and colocalizes with NR1 at neuromuscular junctions.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation from brain and heterologous cells, immunofluorescence co-localization, subcellular fractionation\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid confirmed by reciprocal Co-IP from brain tissue and heterologous cells, multiple orthogonal methods\",\n      \"pmids\": [\"9482789\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Yotiao interacts with PKA regulatory subunit RII via an RII-binding site constituted by amino acids 1452–1469, with a Kd of 50–90 nM in vitro; a stable complex of Yotiao, RIIβ, and NR1 was immunoprecipitated from whole rat brain.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding assay with purified C-terminal Yotiao fragment, co-immunoprecipitation from rat brain\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro binding with Kd measurement plus reciprocal Co-IP from brain; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"10618500\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CG-NAP/AKAP450 anchors the gamma-tubulin ring complex (γ-TuRC) at the centrosome: its N-terminal region associates with γ-TuRC indirectly by binding GCP2 and/or GCP3, while its C-terminal region interacts with calmodulin. Antibody inhibition of CG-NAP (or kendrin, or both combined) moderately to strongly inhibits microtubule nucleation from isolated centrosomes.\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins, yeast two-hybrid for calmodulin interaction, antibody inhibition of microtubule nucleation from isolated centrosomes\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro nucleation assay with antibody inhibition plus Co-IP; replicated with two scaffold proteins and multiple γ-TuRC components\",\n      \"pmids\": [\"12221128\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"A fraction of the small GTPase Ran is tightly associated with the centrosome via AKAP450; when AKAP450 is delocalized from the centrosome, Ran is also delocalized, and microtubule regrowth and anchoring are impaired despite persistent γ-tubulin association with the centrosome.\",\n      \"method\": \"Immunofluorescence, immunoelectron microscopy, biochemical fractionation, dominant-negative AKAP450 displacement\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (IF, IEM, biochemistry) and functional consequence (MT regrowth defect); single lab but rigorous\",\n      \"pmids\": [\"14517334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Displacement of endogenous AKAP450 from centrosomes (by overexpression of its C-terminal centrosome-targeting domain) delocalizes centrosomal PKA type IIα, impairs cytokinesis, increases ploidy in HeLa cells, arrests diploid RPE1 cells in G1, and interrupts centriole duplication.\",\n      \"method\": \"Overexpression of dominant-negative C-terminal domain, immunofluorescence, flow cytometry cell cycle analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean dominant-negative perturbation with defined phenotypes in two cell lines, multiple readouts\",\n      \"pmids\": [\"12808041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CG-NAP/AKAP450 interacts with CK1δ and CK1ε (but not other CK1 isoforms) via a 182 amino acid fragment; this fragment co-immunoprecipitates with CK1δ/ε from mammalian cells, co-localizes with endogenous CK1δ at the centrosome, and when targeted to the plasma membrane is sufficient to re-localize CK1δ to the membrane, establishing CG-NAP as the centrosomal anchor for CK1δ/ε.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, immunofluorescence co-localization, membrane-targeting re-localization experiment\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by Co-IP from mammalian cells plus functional re-localization experiment; multiple orthogonal methods\",\n      \"pmids\": [\"12270714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"AKAP450 is required for microtubule nucleation at the Golgi apparatus: depletion of AKAP450 abolishes Golgi MT nucleation, and depletion of the cis-Golgi protein GM130 disorganizes the AKAP450 network and impairs MT nucleation. AKAP450 binds the cis-side of the Golgi in an MT-independent, GM130-dependent manner.\",\n      \"method\": \"siRNA depletion, live-cell MT regrowth assay, brefeldin A treatment, immunofluorescence\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean siRNA loss-of-function with specific MT nucleation readout, identification of GM130-dependent recruitment mechanism, replicated with pharmacological perturbation\",\n      \"pmids\": [\"19242490\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Yotiao interacts with the type 1 InsP3 receptor (InsP3R1) via the leucine/isoleucine zipper (LIZ) motif in the InsP3R1 coupling domain and the fourth LIZ motif in AKAP9/Yotiao; this interaction mediates PKA-InsP3R1 association in brain, is isoform-specific (type 1 only), and promotes association of InsP3R1 with the NR1 NMDA receptor as well as indirect association with PP1.\",\n      \"method\": \"Biochemical co-immunoprecipitation from brain, in vitro binding, domain mapping with LIZ mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical experiments in one study including domain mapping and isoform specificity; Co-IP from brain tissue\",\n      \"pmids\": [\"14982933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Yotiao (AKAP9) directly associates with the IKs potassium channel complex (KCNQ1/KCNE1), recruits PKA and PP1 to the channel, and exerts direct allosteric effects on channel gating that are distinct from its role in coordinating PKA phosphorylation — demonstrated by studying channels mutated to simulate phosphorylation.\",\n      \"method\": \"Electrophysiology (patch-clamp), co-immunoprecipitation, channel mutagenesis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — electrophysiological functional assay combined with biochemical Co-IP; replicated and extended in subsequent publications\",\n      \"pmids\": [\"15528278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Yotiao itself is a PKA substrate: Ser-43 in the N-terminus of Yotiao is phosphorylated by PKA in response to β-adrenergic receptor stimulation. Alanine substitution of Ser-43 abolishes PKA phosphorylation of Yotiao and markedly diminishes the functional (voltage-dependent activation and kinetics) response of the IKs channel to cAMP, without preventing PKA phosphorylation of KCNQ1 or KCNQ1 binding to Yotiao.\",\n      \"method\": \"Phospho-specific antibody, mutagenesis (S43A), β-adrenergic stimulation, electrophysiology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — site-directed mutagenesis identifying specific PKA phosphorylation site combined with functional electrophysiology; clean separation of effects\",\n      \"pmids\": [\"16002409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Yotiao (AKAP9) directly interacts with adenylyl cyclase (AC) isoforms 1, 2, 3, and 9 but not AC5 or AC6; it inhibits AC2 and AC3 enzymatic activity but has no effect on AC1 or AC9. The N-terminus of AC2 binds directly to amino acids 808–957 of Yotiao. Disruption of Yotiao-AC interactions increases brain AC activity by ~40%, establishing Yotiao as a direct regulator of cAMP production.\",\n      \"method\": \"Co-immunoprecipitation, enzymological AC activity assay, domain mapping with truncations, peptide competition\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro enzymatic assay combined with Co-IP domain mapping and peptide competition; multiple orthogonal methods in one study\",\n      \"pmids\": [\"18772391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"CG-NAP anchors hypophosphorylated/immature PKCε at the Golgi/centrosome area via direct binding to PKCε's catalytic domain; sufficiently phosphorylated (mature) PKCε does not bind CG-NAP. Phosphorylation-site mutants (T566A or S729A) of PKCε co-localize with CG-NAP at Golgi/centrosome, while wild-type PKCε distributes in cytosol upon maturation.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assay, pulse-chase, mutagenesis, immunofluorescence\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro binding with mutagenesis plus pulse-chase and immunofluorescence; multiple orthogonal methods establishing phosphorylation-dependent binding\",\n      \"pmids\": [\"10945988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"AKAP9-BRAF fusion protein results from paracentric inversion of chromosome 7q, creating an in-frame fusion of AKAP9 exons 1–8 with BRAF exons 9–18. The fusion protein retains the BRAF kinase domain but lacks the autoinhibitory N-terminal portion, displays elevated kinase activity, and transforms NIH3T3 cells; it was preferentially found in radiation-induced papillary thyroid carcinomas.\",\n      \"method\": \"Molecular cloning, kinase activity assay, NIH3T3 transformation assay, PCR/sequencing of tumor samples\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — kinase activity measurement plus functional transformation assay; multiple methods in single rigorous study\",\n      \"pmids\": [\"15630448\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"AKAP350A contains a distinct Golgi apparatus targeting motif between amino acids 3259 and 3307 that is functionally distinguishable from the adjacent centrosomal PACT domain (amino acids 3308–3324); GFP chimeras of the carboxyl-terminal regions defined these two non-overlapping targeting domains.\",\n      \"method\": \"GFP chimeric construct localization, brefeldin A treatment, immunofluorescence\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct domain mapping with GFP fusions and pharmacological confirmation; single lab, multiple constructs\",\n      \"pmids\": [\"12163481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"AKAP350 associates with all CLIC family members via a 133 amino acid domain; specifically, CLIC5B (a novel CLIC isoform) co-localizes and co-immunoprecipitates with AKAP350 at the Golgi apparatus, and this association is disrupted by brefeldin A treatment.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, GFP targeting constructs, immunofluorescence, brefeldin A treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — yeast two-hybrid confirmed by Co-IP from cells and co-localization; single lab\",\n      \"pmids\": [\"12163479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"AKAP350 interacts with CIP4 (and structurally related proteins FBP17, FBP17b) via yeast two-hybrid and pull-down. CIP4 is phosphorylated by PKA in vitro, and forskolin stimulates CIP4 phosphorylation in situ. Disruption of the CIP4-AKAP350 interaction or AKAP350 knockdown by RNAi leads to changes in Golgi structure.\",\n      \"method\": \"Yeast two-hybrid, GST pull-down, in vitro PKA phosphorylation assay, RNAi knockdown, immunofluorescence of Golgi morphology\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay plus pull-down plus RNAi functional readout; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"15047863\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CG-NAP is recruited to the Golgi apparatus via interaction with the dynein-dynactin complex: CG-NAP possesses two microtubule-binding domains, co-immunoprecipitates with dynactin subunit p150(Glued), and the p150(Glued)-binding region of CG-NAP when targeted to mitochondria recruits mitochondria to the pericentriolar area. Overexpression of this region causes Golgi fragmentation similar to dynamitin overexpression.\",\n      \"method\": \"Co-immunoprecipitation, microtubule co-sedimentation, mitochondria-targeting re-localization, overexpression/dominant-negative, immunofluorescence\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional re-localization assay and dominant-negative; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"17352745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The centrosome-targeting region of CG-NAP (CG-NAP/D) causes centrosome amplification by recruiting cyclin E-cdk2 to centrosomes; CG-NAP/D co-immunoprecipitates active cyclin-cdk complexes (histone H1 kinase activity), centrosome fractions from CG-NAP/D cells have increased cdk2, and amplification is suppressed by a mutant cyclin E unable to bind cdk2.\",\n      \"method\": \"Overexpression of targeting domain, centrosome counting, co-immunoprecipitation of kinase activity, dominant-negative cyclin E, immunofluorescence\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase activity measurement combined with domain-dependent localization and genetic epistasis with cyclin E mutant; single lab\",\n      \"pmids\": [\"15670215\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"AKAP9 interacts with Epac1 and facilitates microtubule polymerization in endothelial cells; AKAP9 silencing abolishes Epac1-stimulated microtubule growth and the ability of Epac1 activation to enhance barrier function via integrin adhesion at cell-cell contacts, despite intact Rap1 activation, cortical actin, and VE-cadherin adhesion.\",\n      \"method\": \"siRNA knockdown, live-cell microtubule dynamics imaging, co-immunoprecipitation (Epac1-AKAP9), transendothelial resistance assay\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus siRNA with specific MT dynamics and barrier function readout; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"20952690\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In the heart, Yotiao assembles a macromolecular IKs signaling complex containing PKA, PP1, PDE4D3, AC9, and the KCNQ1-KCNE1 channel; AC9 is the only Yotiao-interacting AC isoform expressed in cardiac myocytes, and AC9 association with the complex sensitizes PKA phosphorylation of KCNQ1 to β-adrenergic stimulation. Addition of the AC9 N-terminus disrupts AC activity associated with the IKs-Yotiao complex in transgenic mouse heart.\",\n      \"method\": \"Co-immunoprecipitation from transgenic mouse heart and guinea pig heart, RT-PCR isoform survey, AC activity assay, peptide competition\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — enzymatic activity assay from native cardiac tissue combined with Co-IP from two species and peptide competition; multiple orthogonal methods\",\n      \"pmids\": [\"22778270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"AKAP9-anchored PDE4D3 generates a centrosomal cAMP microdomain: centrosomal PKA shows a reduced activation threshold due to autophosphorylation of its regulatory subunit at S114 upon AKAP9 binding; disruption of centrosomal PDE4D3 impairs cell cycle progression by accumulating cells in prophase.\",\n      \"method\": \"FRET-based real-time cAMP imaging, displacement of centrosomal PDE4D3, cell cycle analysis by flow cytometry\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell FRET imaging combined with functional cell cycle readout; clean mechanistic dissection; single lab, two orthogonal methods\",\n      \"pmids\": [\"22908311\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CG-NAP/AKAP450 redistributes from centrosome/Golgi to microtubules in trailing extensions of LFA-1-stimulated T cells; it forms a physical complex with LFA-1, tubulin, and PKCβ/δ isoenzymes, and is critically required for T cell polarization and migration induced by LFA-1 but not fibronectin (β1 integrin).\",\n      \"method\": \"In situ immunoprecipitation, immunofluorescence co-localization, GFP-tagged dominant-negative construct, T cell migration assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — novel in situ Co-IP plus dominant-negative functional assay; single lab\",\n      \"pmids\": [\"16339516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"TACC4 interacts with AKAP350 at the centrosome in interphase via its C-terminal coiled-coil region; AKAP350 sequesters TACC4 to the centrosome in interphase, while a distinct N-terminal domain of TACC4 mediates spindle localization in mitosis. Overexpression of spindle-targeting TACC4 increases the proportion of cells in prometaphase.\",\n      \"method\": \"Yeast two-hybrid, co-localization immunofluorescence, truncation analysis, cell cycle analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — yeast two-hybrid domain mapping with co-localization and functional overexpression phenotype; single lab\",\n      \"pmids\": [\"12015314\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In differentiated muscle cells (myotubes), Nesprin-1α recruits Akap450 to the nuclear envelope independently of kinesin; Akap450 (but not Pericentrin or Pcm1) is required for microtubule nucleation from the nuclear envelope, and this MT nucleation activity is required for nuclear spreading and positioning in myotubes. This mechanism is disrupted in congenital muscular dystrophy patient myotubes carrying a SYNE1 nonsense mutation.\",\n      \"method\": \"BioID proximity labeling, siRNA knockdown, live-cell MT nucleation assay, computer simulation, immunofluorescence\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity labeling to identify interaction, siRNA loss-of-function with specific MT nucleation and nuclear positioning readouts, validated in patient cells; multiple orthogonal methods\",\n      \"pmids\": [\"28966089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In cardiomyocytes, AKAP6 acts as an adaptor linking Nesprin-1α to AKAP9 (and Pericentrin) at the nuclear envelope MTOC via spectrin repeats; AKAP6 and AKAP9 form a protein platform tethering the Golgi to the nucleus, and both Golgi and nuclear envelope exhibit MTOC activity utilizing AKAP9. AKAP6 is required for formation and activity of the nuclear envelope MTOC.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, siRNA/shRNA knockdown, MT nucleation assay, ectopic expression in epithelial cells\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple Co-IP and functional knockdown experiments; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"33295871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Akap9 disruption in mice causes infertility through failure of Sertoli cell maturation: Sertoli cells continue expressing immaturity markers (AMH, thyroid hormone receptor α) and fail to express maturation marker p27(Kip1); gap and tight junctions essential for the blood-testis barrier are disrupted, with mislocalized connexin43 and ZO-1.\",\n      \"method\": \"Three Akap9 mouse alleles (loss-of-function), immunofluorescence, Western blot, histology\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic loss-of-function in three alleles with defined cellular phenotypes; single lab\",\n      \"pmids\": [\"23608191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"AKAP350 recruits EB1 to the spindle poles; decreased AKAP350 expression reduces EB1 levels at spindle poles and astral microtubules, causes defective spindle alignment in 3D epithelial cysts with abnormal lumen, and EB1 overexpression rescues the spindle orientation defect. Specific delocalization of the AKAP350/EB1 complex from the centrosome phenocopies AKAP350 knockdown.\",\n      \"method\": \"siRNA knockdown, 3D organotypic culture, immunofluorescence, EB1 overexpression rescue, dominant-negative delocalization construct\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown with rescue experiment and dominant-negative phenocopy; single lab, multiple methods\",\n      \"pmids\": [\"29097729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"AKAP350 recruits CIP4 to the centrosome; decreased AKAP350 or CIP4 expression, or inhibition of the CIP4-AKAP350 interaction, impairs formation of the nucleus-centrosome-Golgi front-back axis and directional cell migration. Centrosome positioning (but not nuclear movement) is specifically affected.\",\n      \"method\": \"siRNA knockdown, dominant-negative CIP4-binding domain, immunofluorescence, wound-healing and migration assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function and interaction-disruption with specific functional readouts; single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"26208639\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"AKAP9-deficient T cells (T cell-specific deletion) exhibit reduced microtubule-dependent TCR recycling to the cell surface, impairing TCR re-activation by non-classical antigen-presenting cells; this leads to increased T cell egress from inflamed tissues and protection from organ damage in inflammatory disease models.\",\n      \"method\": \"Conditional T cell-specific Akap9 knockout mice, TCR surface recycling assay, in vivo inflammatory disease models, flow cytometry\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined mechanistic readout (TCR recycling) and in vivo phenotype; multiple orthogonal methods\",\n      \"pmids\": [\"26680259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"AKAP350 at the centrosome facilitates the initiation of DNA synthesis by scaffolding Cdk2 to the centrosome; an AKAP350 C-terminal domain increases centrosomal Cdk2 levels and phosphorylation of nucleophosmin (a Cdk2 centrosomal substrate marking G1/S transition), whereas AKAP350 knockdown inhibits G1/S transition and DNA synthesis.\",\n      \"method\": \"siRNA knockdown, overexpression of C-terminal domain, BrdU incorporation, nucleophosmin phosphorylation assay, centrosome fractionation\",\n      \"journal\": \"Cellular logistics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function combined with gain-of-function domain construct and specific centrosomal substrate readout; single lab\",\n      \"pmids\": [\"24475373\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"AKAP350 localizes p150(Glued) (dynactin component) to the spindle poles, facilitating p150(Glued)/EB1 interaction at these structures; AKAP350 depletion reduces p150(Glued) at astral microtubules and impairs elongation of astral microtubules during anaphase.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, immunofluorescence, astral MT length measurement\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — siRNA knockdown with Co-IP and specific MT readout; single lab, two orthogonal methods\",\n      \"pmids\": [\"32841682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Yotiao decreases ER calcium content by suppressing store-operated calcium entry (SOCE) through Orai1; this effect requires AC9 (which increases cAMP upon Yotiao interaction) and involves Yotiao acting on the Orai1 C-terminus, but does not require IP3R1, PKA, PP1, or AC2.\",\n      \"method\": \"ER Ca2+ imaging, Yotiao truncation constructs, knockout cells, pharmacological tools, SOCE assay\",\n      \"journal\": \"Cell calcium\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional Ca2+ assay with KO and truncation mapping; single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"38781694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Purified full-length AKAP350 forms polydisperse fibrillar clusters (~50 nm) with fibrous outgrowths; cryo-EM revealed fibers reconstructing as double-stranded DNA, confirmed by DNA sequencing. AKAP350 co-purifies with endogenous PKA, CEP170, CDK5RAP2, and DNA-binding proteins NFIB and nucleolin; NFIB and nucleolin pull-down was reduced by DNase-I treatment (indicating DNA-mediated interaction), whereas centrosomal protein pull-downs were not affected by DNase-I.\",\n      \"method\": \"Cryo-EM, cryo-ET, mass spectrometry, DNA sequencing, pull-down with DNase-I treatment, purification of full-length protein from human cells\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct cryo-EM/cryo-ET structural analysis of purified full-length protein plus mass spectrometry and functional pull-down with enzymatic perturbation; single lab but multiple rigorous orthogonal methods\",\n      \"pmids\": [\"40154916\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PDE4DIP coordinates with AKAP9 to enhance Golgi localization and stability of PKA RIIα; depletion of PDE4DIP mislocalizes RIIα from the Golgi and leads to its degradation, compromising RIIα's negative regulatory effect on PKA signaling.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, immunofluorescence localization of PKA RIIα, Western blot for RIIα stability\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP combined with knockdown and localization/stability readout; single lab\",\n      \"pmids\": [\"39905234\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AKAP9 encodes a large family of scaffolding proteins (including AKAP450/CG-NAP and the shorter isoform Yotiao) that serve as multi-valent anchoring platforms at the centrosome and Golgi apparatus, where they recruit and organize PKA (via an amphipathic RII-binding helix), PP1, PP2A, PKCε, CK1δ/ε, PDE4D3, cyclin E-Cdk2, Ran, γ-TuRC (via GCP2/GCP3), AC isoforms (particularly AC9), CIP4, EB1, and dynein/dynactin to control microtubule nucleation and dynamics, cell cycle progression (G1/S transition and centriole duplication), Golgi structure maintenance, and directional cell migration; in neurons and cardiomyocytes the Yotiao isoform additionally forms a macromolecular IKs channel complex (KCNQ1/KCNE1/PKA/PP1/PDE4D3/AC9) that is essential for sympathetic regulation of cardiac repolarization, while also scaffolding InsP3R1 and NMDA receptors to couple cAMP and Ca²⁺ signaling at synapses.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AKAP9 encodes large multi-valent scaffolding proteins (AKAP450/CG-NAP/AKAP350 and the shorter neuronal/cardiac isoform Yotiao) that nucleate signalling complexes at the centrosome, Golgi apparatus, and nuclear envelope to organize microtubules and compartmentalize cAMP signalling [#0, #4, #8]. The protein is targeted to the centrosome by a C-terminal PACT domain and to the Golgi by an adjacent, separable Golgi-targeting motif, and recruits PKA type II via an RII-binding amphipathic helix [#1, #15, #0]. At microtubule-organizing centers it anchors the \\u03b3-tubulin ring complex through GCP2/GCP3, tethers Ran, and recruits dynein-dynactin (p150Glued), CK1\\u03b4/\\u03b5, PKC\\u03b5, EB1, and CIP4, thereby driving microtubule nucleation from the centrosome, Golgi, and nuclear envelope and supporting nuclear positioning, spindle orientation, and directional cell migration [#4, #5, #7, #13, #18, #28, #29, #25, #26]. AKAP9 couples this scaffolding to cell-cycle control, recruiting cyclin E-Cdk2 and concentrating a PDE4D3-shaped cAMP microdomain that lowers the activation threshold of centrosomal PKA to license centriole duplication and the G1/S transition [#6, #19, #22, #31]. In neurons and cardiomyocytes the Yotiao isoform assembles a macromolecular IKs channel complex (KCNQ1/KCNE1 with PKA, PP1, PDE4D3, and AC9) and is itself a PKA substrate at Ser-43, integrating \\u03b2-adrenergic input into channel gating, while also scaffolding InsP3R1 and NMDA receptor NR1 to couple cAMP and Ca\\u00b2\\u207a signalling [#10, #11, #21, #9, #2]. A paracentric inversion of 7q creates an oncogenic AKAP9-BRAF fusion with constitutive kinase activity found in radiation-induced papillary thyroid carcinoma [#14].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established the first molecular role for an AKAP9 product by showing the Yotiao isoform physically links the NMDA receptor NR1 subunit to the postsynaptic apparatus, defining it as a synaptic scaffold.\",\n      \"evidence\": \"Yeast two-hybrid plus reciprocal Co-IP from brain and heterologous cells, with fractionation and NMJ co-localization\",\n      \"pmids\": [\"9482789\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the full receptor complex or downstream signalling enzymes recruited\", \"No structural basis for the C1 exon-dependent interaction\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Identified the centrosomal/Golgi scaffold AKAP450/CG-NAP and its capacity to anchor PKA-RII alongside phosphatases PP1 and PP2A, establishing the protein as a multi-enzyme anchoring platform.\",\n      \"evidence\": \"RII overlay screening, reciprocal Co-IP, immunofluorescence, and mutation of the RII-binding helix in HeLa cells; plus Yotiao RII Kd measurement\",\n      \"pmids\": [\"10202149\", \"10358086\", \"9915845\", \"10618500\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish functional consequence of kinase/phosphatase anchoring\", \"Stoichiometry and spatial organization of the bound enzymes unresolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mapped distinct C-terminal targeting modules (PACT for centrosome, a separate Golgi motif) and showed AKAP450 anchors \\u03b3-TuRC via GCP2/GCP3 to drive microtubule nucleation, linking localization to MTOC function.\",\n      \"evidence\": \"GFP chimera mapping, dominant-negative displacement, Co-IP, and antibody inhibition of nucleation from isolated centrosomes; plus CK1\\u03b4/\\u03b5, CLIC, TACC4 partner mapping\",\n      \"pmids\": [\"11263498\", \"12163481\", \"12221128\", \"12270714\", \"12163479\", \"12015314\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how \\u03b3-TuRC recruitment is regulated through the cell cycle\", \"Functional roles of CLIC and TACC4 binding remained correlative\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstrated that centrosomal AKAP450 is required for microtubule regrowth/anchoring and for cell-cycle progression, tethering Ran and PKA and controlling cytokinesis, ploidy, and centriole duplication.\",\n      \"evidence\": \"Dominant-negative displacement of the C-terminal targeting domain with IF, immuno-EM, fractionation, and flow-cytometry cell-cycle analysis in HeLa and RPE1 cells\",\n      \"pmids\": [\"14517334\", \"12808041\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not separate scaffolding of MT machinery from PKA anchoring in producing the phenotypes\", \"Mechanism coupling Ran delocalization to anchoring defect unclear\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined Yotiao as the organizer of a cardiac/neuronal IKs and InsP3R1 signalling complex, recruiting PKA and PP1 to channels and exerting direct allosteric control over gating.\",\n      \"evidence\": \"Patch-clamp electrophysiology, channel mutagenesis, Co-IP from brain, and LIZ-motif domain mapping\",\n      \"pmids\": [\"15528278\", \"14982933\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not yet identify the upstream cAMP-generating and degrading enzymes in the cardiac complex\", \"Allosteric gating mechanism not structurally defined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed Yotiao is itself a PKA substrate (Ser-43) whose phosphorylation is required for the cAMP response of the IKs channel, and that the centrosomal domain recruits cyclin E-Cdk2 to drive centrosome amplification.\",\n      \"evidence\": \"Phospho-specific antibody and S43A mutagenesis with electrophysiology; Co-IP of cyclin-Cdk2 kinase activity and dominant-negative cyclin E with centrosome counting\",\n      \"pmids\": [\"16002409\", \"15670215\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish how scaffold phosphorylation alters complex architecture\", \"Physiological trigger for cyclin E-Cdk2 recruitment to the scaffold undefined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Connected AKAP9 to disease and to immune cell migration: a 7q inversion generates a transforming AKAP9-BRAF fusion in thyroid carcinoma, and the scaffold relocates to migrating T-cell extensions in complex with LFA-1, tubulin, and PKC.\",\n      \"evidence\": \"Molecular cloning, kinase activity and NIH3T3 transformation assays, tumor sequencing; in situ Co-IP and T-cell migration assay\",\n      \"pmids\": [\"15630448\", \"16339516\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"AKAP9-BRAF fusion describes a chimeric oncoprotein, not wild-type AKAP9 function\", \"Mechanism of LFA-1-specific scaffold relocation not resolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Established AKAP450 as the essential nucleator of Golgi-derived microtubules, recruited to the cis-Golgi in a GM130-dependent, MT-independent manner.\",\n      \"evidence\": \"siRNA depletion with live-cell MT regrowth assays, brefeldin A treatment, and IF\",\n      \"pmids\": [\"19242490\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how GM130 docking is coordinated with centrosomal pools\", \"Functional consequence of Golgi MTs for trafficking not addressed here\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Extended scaffold function to endothelial barrier control, showing AKAP9 binds Epac1 and is required for Epac1-stimulated microtubule growth and integrin-mediated barrier strengthening.\",\n      \"evidence\": \"Co-IP, siRNA, live MT dynamics imaging, and transendothelial resistance assay\",\n      \"pmids\": [\"20952690\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; reciprocal validation of the Epac1 interaction limited\", \"Direct vs indirect nature of the AKAP9-Epac1 association unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved the full cAMP regulatory architecture of AKAP9 complexes: a cardiac IKs complex incorporating PDE4D3 and AC9 to sensitize PKA phosphorylation, and a centrosomal PDE4D3-shaped cAMP microdomain that lowers the PKA activation threshold for cell-cycle progression.\",\n      \"evidence\": \"Co-IP from transgenic/guinea-pig heart, RT-PCR isoform survey, AC activity and peptide competition; FRET cAMP imaging and cell-cycle analysis\",\n      \"pmids\": [\"22778270\", \"22908311\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define spatial extent of the microdomains in vivo\", \"Crosstalk between centrosomal and Golgi cAMP pools unexplored\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated organismal and cell-cycle roles in vivo, showing Akap9 loss causes infertility via failed Sertoli cell maturation and blood-testis barrier disruption, and that the centrosomal scaffold drives G1/S by tethering Cdk2.\",\n      \"evidence\": \"Three Akap9 mouse loss-of-function alleles with histology/IF; siRNA, C-terminal domain overexpression, BrdU, and nucleophosmin phosphorylation assays\",\n      \"pmids\": [\"23608191\", \"24475373\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not connect junctional defects to a specific AKAP9-anchored enzyme\", \"Tissue specificity of the Cdk2-scaffolding role unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined the directional-migration and immune functions of the scaffold: AKAP350 recruits CIP4 to position the centrosome in the nucleus-centrosome-Golgi axis, and AKAP9 supports microtubule-dependent TCR recycling controlling T-cell egress in inflammation.\",\n      \"evidence\": \"siRNA, dominant-negative interaction-disruption, wound-healing assays; conditional T-cell Akap9 knockout with TCR recycling assay and in vivo disease models\",\n      \"pmids\": [\"26208639\", \"26680259\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish whether MT nucleation or anchoring underlies the migration defect\", \"Link between TCR recycling kinetics and tissue egress mechanistically incomplete\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified a nuclear-envelope MTOC role, showing Nesprin-1\\u03b1 recruits Akap450 to the nuclear envelope to nucleate microtubules required for myonuclear positioning, a pathway disrupted in SYNE1-mutant muscular dystrophy myotubes.\",\n      \"evidence\": \"BioID proximity labeling, siRNA, live MT nucleation assays, and patient-cell validation\",\n      \"pmids\": [\"28966089\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the adaptor bridging Nesprin-1\\u03b1 to AKAP450\", \"Whether nuclear-envelope nucleation uses \\u03b3-TuRC as at the centrosome unstated\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Filled in the nuclear-envelope MTOC adaptor and refined spindle-pole mechanics, identifying AKAP6 as the Nesprin-1\\u03b1-to-AKAP9 linker tethering Golgi to nucleus, and showing AKAP350 localizes p150Glued to spindle poles for astral MT elongation.\",\n      \"evidence\": \"Co-IP, siRNA/shRNA, MT nucleation and ectopic-expression assays; Co-IP and astral MT length measurement\",\n      \"pmids\": [\"33295871\", \"32841682\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab observations; reciprocal in vivo validation limited\", \"Regulation of switching between centrosomal and nuclear-envelope MTOCs unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Expanded Yotiao's calcium-signalling role, showing it suppresses store-operated calcium entry through Orai1 via AC9-dependent cAMP, independently of IP3R1, PKA, and PP1.\",\n      \"evidence\": \"ER Ca2+ and SOCE imaging with truncation constructs, knockout cells, and pharmacology\",\n      \"pmids\": [\"38781694\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; direct Yotiao-Orai1 contact not structurally confirmed\", \"Physiological context of SOCE suppression unaddressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided the first structural and DNA-association data for full-length AKAP350 and refined Golgi PKA regulation, revealing fibrillar clusters that scaffold double-stranded DNA with NFIB/nucleolin, and showing PDE4DIP stabilizes Golgi-localized PKA RII\\u03b1.\",\n      \"evidence\": \"Cryo-EM/cryo-ET, mass spectrometry, DNA sequencing, DNase-I pull-downs; Co-IP, siRNA, and RII\\u03b1 localization/stability assays\",\n      \"pmids\": [\"40154916\", \"39905234\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional significance of the DNA-associated fibrillar assembly unknown\", \"How PDE4DIP and AKAP9 jointly stabilize RII\\u03b1 not mechanistically resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the centrosomal, Golgi, nuclear-envelope, and channel-complex pools of AKAP9 are differentially specified and dynamically regulated within a single cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated model of isoform- and localization-specific complex assembly\", \"No high-resolution structure of the full-length scaffold with its bound enzymes\", \"Physiological role of the DNA-associated fibrillar state undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2, 4, 9, 10, 21]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [4, 5, 8, 18, 28, 32]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [12, 22, 33, 35]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [34]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [0, 1, 4, 6, 19, 22, 31]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0, 8, 15, 16, 17, 18, 29]},\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [25, 26]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 10, 23]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [6, 19, 22, 31]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 10, 12, 21, 22]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [4, 5, 8, 25, 26]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [23, 30]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [14, 25]}\n    ],\n    \"complexes\": [\n      \"IKs channel complex (KCNQ1/KCNE1/PKA/PP1/PDE4D3/AC9)\",\n      \"\\u03b3-tubulin ring complex (\\u03b3-TuRC)\",\n      \"nuclear envelope MTOC\",\n      \"NMDA receptor-PKA postsynaptic complex\"\n    ],\n    \"partners\": [\n      \"PRKAR2A\",\n      \"KCNQ1\",\n      \"PDE4D\",\n      \"ADCY9\",\n      \"GM130\",\n      \"DCTN1\",\n      \"ITPR1\",\n      \"GRIN1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}