{"gene":"CAMKK1","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1995,"finding":"CAMKK1 (originally named CaM-kinase kinase) was cloned from rat brain as a 505-amino acid serine/threonine kinase that phosphorylates and activates CaM-kinase I and CaM-kinase IV but not CaM-kinase II in a Ca2+/calmodulin-dependent manner; co-expression with CaM-kinase IV gave 14-fold enhancement of CREB-dependent gene expression.","method":"Molecular cloning, COS-7 cell expression, in vitro kinase assay, co-expression with CaMKIV + reporter assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — original biochemical reconstitution with in vitro activity assays and cell-based functional validation","pmids":["7642608"],"is_preprint":false},{"year":1995,"finding":"CaM-kinase Ia kinase (CAMKK1-equivalent) phosphorylates CaMKIV at Thr-196 in a Ca2+/CaM- and MgATP-dependent manner, and Thr-196 phosphorylation is essential for activation; T196A mutation abolishes both phosphorylation and activation of CaMKIV.","method":"In vitro kinase assay with purified pig brain CaMK kinase, site-directed mutagenesis (T196A), phosphorylation stoichiometry measurement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution plus mutagenesis demonstrating necessity of Thr-196 phosphorylation","pmids":["7615569"],"is_preprint":false},{"year":1996,"finding":"Activation of CaMKIV by CAMKK1 requires Ca2+/CaM binding to BOTH enzymes: a constitutively active truncated CaMKK (CaMKK1-434) can phosphorylate a CaMKIV fragment lacking the autoinhibitory domain in a Ca2+/CaM-independent manner, but phosphorylation of full-length CaMKIV still requires Ca2+/CaM binding to CaMKIV itself. Ionomycin stimulation in COS-7 cells confirmed Ca2+-dependent cascade activation requiring intact Thr196.","method":"In vitro phosphorylation assays with truncated and mutant CaMKK and CaMKIV, intact cell ionomycin stimulation, COS-7 co-expression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — multiple mutant constructs and both in vitro and cell-based approaches with orthogonal readouts","pmids":["8621423"],"is_preprint":false},{"year":1998,"finding":"CAMKK1 phosphorylates and activates CaMKI; CaMKI autoinhibition is mediated by a C-terminal helix-loop-helix domain (Ile286–Met316) and Ca2+/CaM relieves this inhibition allowing CAMKK to access and phosphorylate the activation loop. Specific residues Phe298, Ile294, Ile286, Val290, Trp303, and Phe307 were identified as critical for autoinhibition and CaM-binding of CaMKI.","method":"In vitro kinase assay, site-directed mutagenesis of CaMKI regulatory domain residues, recombinant protein structure-function analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — detailed structure-function analysis with multiple point mutants and reconstituted in vitro system","pmids":["9705275"],"is_preprint":false},{"year":1999,"finding":"cAMP-dependent protein kinase (PKA) phosphorylates CAMKK1 primarily at Thr-108 in vitro and in intact cells, inhibiting its kinase activity; CaMKI also phosphorylates CAMKK1 at the same sites suggesting a negative-feedback mechanism. Forskolin treatment in PC12 cells rapidly inhibits both CAMKK1 and CaMKI activity, and hippocampal slice PKA activation increases CAMKK1 phosphorylation.","method":"In vitro PKA phosphorylation assay, phosphopeptide mapping, intact PC12 cell and hippocampal slice pharmacology (forskolin), CaMKI in vitro phosphorylation of CaMKK","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro site identification combined with cell-based pharmacological validation in two distinct cell systems","pmids":["10187789"],"is_preprint":false},{"year":2009,"finding":"Adiponectin activates AMPK in muscle cells via a minor pathway involving phospholipase C-induced Ca2+ release from the ER, which activates Ca2+/calmodulin-dependent protein kinase kinase (CaMKK, including CAMKK1), thereby phosphorylating AMPK; this is distinct from the major APPL1/LKB1-dependent pathway.","method":"Pharmacological inhibition of phospholipase C and Ca2+ chelation, AMPK activity assay in muscle cells, epistasis via siRNA knockdown","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — epistasis via pharmacological inhibitors in cells; CAMKK1 not distinguished from CAMKK2 individually","pmids":["19520843"],"is_preprint":false},{"year":2018,"finding":"Crystal structures of CAMKK1 bound to two ATP-competitive inhibitors were determined, revealing exploitable structural differences between CAMKK1 and CAMKK2 despite high sequence identity. Isothermal titration calorimetry showed the most potent inhibitors bind with thermodynamics dominated by favorable enthalpy.","method":"X-ray crystallography, kinase inhibitor library screening, isothermal titration calorimetry","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 — crystal structures with functional binding characterization by ITC","pmids":["30287839"],"is_preprint":false},{"year":2023,"finding":"14-3-3γ protein inhibits CAMKK1 by directly blocking the kinase active site with its last two C-terminal helices, forming a compact and rigid complex that also suppresses Ca2+/CaM binding to CAMKK1. In contrast, the CaMKK2:14-3-3 complex is looser and more flexible, explaining why 14-3-3 binding selectively inhibits CAMKK1 but not CAMKK2.","method":"Small-angle X-ray scattering (SAXS), hydrogen/deuterium exchange coupled to MS (HDX-MS), fluorescence spectroscopy","journal":"Protein science","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal structural and biophysical methods in a single study providing mechanistic detail","pmids":["37817008"],"is_preprint":false},{"year":2023,"finding":"In peripheral nerve injury, CAMKK1 is a downstream substrate of miR-132-5p: TFAP2C (delivered via fibroblast exosomes) represses miR-132-5p expression in dorsal root ganglion neurons, relieving suppression of CAMKK1 and thereby promoting axon extension. miR-132-5p antagomir in rats stimulates CAMKK1 expression and improves sciatic nerve axon regeneration.","method":"Proteomics, exosome functional assays, miRNA target validation, antagomir injection in rat sciatic nerve injury model","journal":"Bioactive materials","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo loss-of-function (antagomir) with defined phenotype, but CAMKK1 pathway placement partly inferred","pmids":["36936807"],"is_preprint":false},{"year":2024,"finding":"Nrf2 directly interacts with and negatively regulates CAMKK1; Echinococcus granulosus protoscolex infection suppresses Nrf2, leading to upregulation of CAMKK1 which drives osteoclast differentiation and bone loss. Nrf2 knockout mice show exacerbated CAMKK1-dependent bone loss, and silencing CAMKK1 alone inhibits osteoclast differentiation.","method":"Transcriptome sequencing, Western blot, Q-PCR, co-immunoprecipitation, Nrf2 knockout mouse model, Micro-CT imaging, TRAP staining, small-molecule inhibitor (Crenolani) in vivo","journal":"International journal of biological macromolecules","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus genetic (KO and KD) approaches with in vivo validation, though single lab","pmids":["39674449"],"is_preprint":false},{"year":2025,"finding":"Phosphoproteomic coregulation analysis identified RPS6KB1 (S6K1) as a novel upstream kinase of CAMKK1 at its predominant phosphosite S74 (outside the kinase domain). Predominant phosphosites of CAMKK1 are located outside the kinase domain, and their phosphomotifs are highly homologous to those of CAMKK2.","method":"Global phosphoproteome dataset analysis, coregulation analysis of phosphosites, kinase-substrate inference from phosphoproteomics","journal":"Omics : a journal of integrative biology","confidence":"Low","confidence_rationale":"Tier 4 — computational inference from phosphoproteomic datasets without direct experimental validation of the CAMKK1-S74 kinase-substrate relationship","pmids":["40079160"],"is_preprint":false}],"current_model":"CAMKK1 is a Ca2+/calmodulin-dependent serine/threonine kinase kinase that operates at the apex of a signaling cascade, phosphorylating and activating CaMKI (at its activation-loop threonine) and CaMKIV (at Thr-196) in a manner requiring Ca2+/CaM binding to both CAMKK1 and its substrate; its activity is inhibited by PKA-mediated phosphorylation at Thr-108 (and by CaMKI in a negative-feedback loop), and by 14-3-3γ binding which blocks its active site via its C-terminal helices and suppresses Ca2+/CaM association; structurally distinct from the paralog CAMKK2 at the inhibitor-binding level; it also activates AMPK downstream of Ca2+ signals, regulates axon regeneration via the miR-132-5p axis, promotes osteoclast differentiation downstream of Nrf2 suppression, and modulates the mesenchymal stem cell secretome."},"narrative":{"teleology":[{"year":1995,"claim":"Identification of CAMKK1 as the upstream kinase kinase for CaMKI and CaMKIV established the existence of a multi-tier Ca²⁺/CaM-dependent kinase cascade and showed it could amplify CREB-dependent transcription.","evidence":"Cloning from rat brain, in vitro kinase assays with purified proteins, COS-7 co-expression with CaMKIV and CREB reporter","pmids":["7642608","7615569"],"confidence":"High","gaps":["Endogenous tissue-level validation of cascade hierarchy not yet provided","Relative contributions of CAMKK1 vs CAMKK2 to CaMKIV activation not distinguished"]},{"year":1996,"claim":"Demonstration that Ca²⁺/CaM must bind both CAMKK1 and CaMKIV for productive phosphorylation resolved how Ca²⁺ signals gate two independent steps in the cascade and explained why a constitutively active CAMKK alone is insufficient for full substrate activation.","evidence":"Truncation and mutagenesis of both CAMKK and CaMKIV, in vitro reconstitution, ionomycin stimulation in COS-7 cells","pmids":["8621423"],"confidence":"High","gaps":["Structural basis for CaM-gated substrate accessibility unknown","Whether CaMKI activation follows the same dual-CaM requirement not tested"]},{"year":1998,"claim":"Mapping the autoinhibitory domain of CaMKI and showing that CaM relief of autoinhibition is a prerequisite for CAMKK1-mediated activation-loop phosphorylation clarified the substrate-side gating mechanism.","evidence":"Systematic site-directed mutagenesis of CaMKI helix-loop-helix domain, in vitro kinase assays","pmids":["9705275"],"confidence":"High","gaps":["Crystal structure of CAMKK1–CaMKI complex not available","In vivo relevance of individual CaMKI autoinhibitory residues not tested"]},{"year":1999,"claim":"Discovery that PKA phosphorylates CAMKK1 at Thr-108 to inhibit it, and that CaMKI performs the same phosphorylation, revealed cAMP–Ca²⁺ signal integration and a negative-feedback loop within the CaMK cascade.","evidence":"In vitro PKA and CaMKI phosphorylation, phosphopeptide mapping, forskolin treatment in PC12 cells and hippocampal slices","pmids":["10187789"],"confidence":"High","gaps":["Physiological relevance of CaMKI feedback in specific neuronal circuits not established","Additional regulatory phosphosites may exist"]},{"year":2009,"claim":"Placing CaMKK upstream of AMPK in adiponectin signaling extended CAMKK1's functional scope beyond the CaMK cascade to metabolic energy sensing, though CAMKK1 was not distinguished from CAMKK2 in this context.","evidence":"Pharmacological PLC/Ca²⁺ inhibition and siRNA knockdown in muscle cells, AMPK activity assays","pmids":["19520843"],"confidence":"Medium","gaps":["CAMKK1-specific contribution not isolated from CAMKK2","Tissue-specific isoform requirements for AMPK activation unclear"]},{"year":2018,"claim":"Crystal structures of CAMKK1 with ATP-competitive inhibitors revealed exploitable active-site differences between CAMKK1 and CAMKK2, enabling paralog-selective chemical probe development.","evidence":"X-ray crystallography, kinase inhibitor screening, isothermal titration calorimetry","pmids":["30287839"],"confidence":"High","gaps":["No structure of CAMKK1 in its autoinhibited or CaM-bound state","Selectivity over broader kinome not fully profiled"]},{"year":2023,"claim":"Structural and biophysical characterization showed that 14-3-3γ inhibits CAMKK1 by plugging the active site with its C-terminal helices and blocking CaM binding, whereas CAMKK2 forms a looser complex — explaining paralog-selective regulation.","evidence":"SAXS, HDX-MS, fluorescence spectroscopy","pmids":["37817008"],"confidence":"High","gaps":["Atomic-resolution co-crystal structure of CAMKK1:14-3-3γ complex not determined","Phosphorylation sites on CAMKK1 that create the 14-3-3 docking motif not fully mapped"]},{"year":2023,"claim":"Identification of CAMKK1 as a miR-132-5p target that promotes axon regeneration linked this kinase kinase to peripheral nerve repair and exosome-mediated intercellular signaling.","evidence":"Proteomics, miRNA target validation, antagomir injection in rat sciatic nerve injury model","pmids":["36936807"],"confidence":"Medium","gaps":["Direct CAMKK1 substrates mediating axon extension not identified","Whether CaMKI or CaMKIV mediates the regenerative signal downstream unclear"]},{"year":2024,"claim":"Demonstration that Nrf2 directly binds and suppresses CAMKK1, whose de-repression drives osteoclast differentiation, placed CAMKK1 in bone remodeling downstream of oxidative-stress signaling.","evidence":"Co-IP, Nrf2 knockout mice, CAMKK1 silencing, Micro-CT and TRAP staining in infection-induced bone loss model","pmids":["39674449"],"confidence":"Medium","gaps":["Mechanism by which Nrf2 suppresses CAMKK1 (transcriptional vs protein-level) not resolved","CAMKK1 substrates in osteoclastogenesis not identified","Generalizability beyond parasitic infection model untested"]},{"year":null,"claim":"Key unresolved questions include the identity of CAMKK1-specific substrates outside the CaMK/AMPK axis, the structural basis of CaM-gated substrate recognition, the in vivo tissue-specific contributions of CAMKK1 versus CAMKK2, and the full repertoire of regulatory phosphosites and their upstream kinases.","evidence":"","pmids":[],"confidence":"High","gaps":["No CAMKK1 knockout mouse phenotype reported","Full phosphosite map with validated upstream kinases lacking","No structure of CAMKK1 in complex with CaM or a substrate kinase"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,3,4]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2,3]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,4]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,2,4,5,7]}],"complexes":[],"partners":["CAMK1","CAMK4","YWHAG","PRKACA","PRKAB1","NRF2"],"other_free_text":[]},"mechanistic_narrative":"CAMKK1 is a Ca²⁺/calmodulin-dependent serine/threonine kinase kinase that sits at the apex of a CaMK signaling cascade, phosphorylating and activating CaMKI and CaMKIV at their activation-loop threonines in a manner requiring Ca²⁺/CaM binding to both CAMKK1 and its substrates [PMID:7642608, PMID:7615569, PMID:8621423]. Its activity is negatively regulated by PKA-mediated phosphorylation at Thr-108 and by a CaMKI-dependent feedback loop [PMID:10187789], and is selectively inhibited by 14-3-3γ, which forms a compact complex that blocks the active site and suppresses Ca²⁺/CaM association — a mechanism structurally distinct from the looser 14-3-3 interaction with paralog CAMKK2 [PMID:37817008]. CAMKK1 also contributes to AMPK activation downstream of Ca²⁺ signals [PMID:19520843] and functions in axon regeneration, where its expression is controlled by the miR-132-5p axis, and in osteoclast differentiation, where it is de-repressed upon Nrf2 suppression [PMID:36936807, PMID:39674449]."},"prefetch_data":{"uniprot":{"accession":"Q8N5S9","full_name":"Calcium/calmodulin-dependent protein kinase kinase 1","aliases":["CaM-kinase IV kinase","Calcium/calmodulin-dependent protein kinase kinase alpha","CaM-KK alpha","CaM-kinase kinase alpha","CaMKK alpha"],"length_aa":505,"mass_kda":55.7,"function":"Calcium/calmodulin-dependent protein kinase that belongs to a proposed calcium-triggered signaling cascade involved in a number of cellular processes. Phosphorylates CAMK1, CAMK1D, CAMK1G and CAMK4. Involved in regulating cell apoptosis. Promotes cell survival by phosphorylating AKT1/PKB that inhibits pro-apoptotic BAD/Bcl2-antagonist of cell death","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q8N5S9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CAMKK1","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":"CALM2","stoichiometry":0.2},{"gene":"CALM3","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CAMKK1","total_profiled":1310},"omim":[{"mim_id":"614994","title":"CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE IG; CAMK1G","url":"https://www.omim.org/entry/614994"},{"mim_id":"611411","title":"CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE KINASE 1, ALPHA; CAMKK1","url":"https://www.omim.org/entry/611411"},{"mim_id":"114080","title":"CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE IV; CAMK4","url":"https://www.omim.org/entry/114080"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":70.2}],"url":"https://www.proteinatlas.org/search/CAMKK1"},"hgnc":{"alias_symbol":["DKFZp761M0423","CAMKKA","MGC34095"],"prev_symbol":[]},"alphafold":{"accession":"Q8N5S9","domains":[{"cath_id":"3.30.200.20","chopping":"110-170_190-233","consensus_level":"medium","plddt":85.0892,"start":110,"end":233},{"cath_id":"1.10.510.10","chopping":"234-429","consensus_level":"medium","plddt":93.5559,"start":234,"end":429}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N5S9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N5S9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N5S9-F1-predicted_aligned_error_v6.png","plddt_mean":71.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CAMKK1","jax_strain_url":"https://www.jax.org/strain/search?query=CAMKK1"},"sequence":{"accession":"Q8N5S9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8N5S9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8N5S9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N5S9"}},"corpus_meta":[{"pmid":"36936807","id":"PMC_36936807","title":"Fibroblast 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Phosphorylation of threonine 196 is essential for activation.","date":"1995","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7615569","citation_count":139,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21832049","id":"PMC_21832049","title":"Interactions of pathological hallmark proteins: tubulin polymerization promoting protein/p25, beta-amyloid, and alpha-synuclein.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21832049","citation_count":131,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"8621423","id":"PMC_8621423","title":"Requirements for calcium and calmodulin in the calmodulin kinase activation cascade.","date":"1996","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8621423","citation_count":113,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"16741161","id":"PMC_16741161","title":"Variants in the GH-IGF axis confer susceptibility to lung cancer.","date":"2006","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/16741161","citation_count":101,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"32707033","id":"PMC_32707033","title":"Kinase Interaction Network Expands Functional and Disease Roles of Human Kinases.","date":"2020","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/32707033","citation_count":88,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"20801214","id":"PMC_20801214","title":"Ppm1E is an in cellulo AMP-activated protein kinase phosphatase.","date":"2010","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/20801214","citation_count":81,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"9705275","id":"PMC_9705275","title":"Characterization of the mechanism of regulation of Ca2+/ calmodulin-dependent protein kinase I by calmodulin and by Ca2+/calmodulin-dependent protein kinase kinase.","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9705275","citation_count":81,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"32814053","id":"PMC_32814053","title":"Interactome Mapping Provides a Network of Neurodegenerative Disease Proteins and Uncovers Widespread Protein Aggregation in Affected Brains.","date":"2020","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/32814053","citation_count":79,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"10187789","id":"PMC_10187789","title":"Inhibition of the Ca2+/calmodulin-dependent protein kinase I cascade by cAMP-dependent protein kinase.","date":"1999","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10187789","citation_count":67,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"35256949","id":"PMC_35256949","title":"Histone deacetylase inhibitors inhibit cervical cancer growth through Parkin acetylation-mediated mitophagy.","date":"2021","source":"Acta pharmaceutica Sinica. 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Isothermal titration calorimetry showed that the most potent inhibitors had binding energies largely dependent on favourable enthalpy.\",\n      \"method\": \"X-ray crystallography, isothermal titration calorimetry, kinase inhibitor library screening\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional validation by ITC and inhibitor binding assays\",\n      \"pmids\": [\"30287839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"14-3-3γ protein inhibits CaMKK1 by blocking the kinase active site directly with its last two C-terminal helices, forming a compact and rigid complex. Phosphorylation of CaMKK1 triggers 14-3-3 binding, which suppresses Ca2+/CaM interaction and structurally restricts the kinase domain and autoinhibitory segment. This inhibitory mechanism is much stronger for CaMKK1 than CaMKK2 because the CaMKK2:14-3-3 complex has a looser, more flexible structure.\",\n      \"method\": \"SAXS, hydrogen/deuterium exchange coupled to mass spectrometry, fluorescence spectroscopy\",\n      \"journal\": \"Protein science : a publication of the Protein Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple orthogonal structural/biophysical methods (SAXS, HDX-MS, fluorescence) in a single study with rigorous mechanistic characterization\",\n      \"pmids\": [\"37817008\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RPS6KB1 (S6K1) was identified as a novel high-confidence upstream kinase phosphorylating CAMKK1 at S74, based on global cellular phosphoproteome coregulation analysis. Predominant phosphosites of CAMKK1 are located outside the kinase domain.\",\n      \"method\": \"Phosphoproteomics coregulation analysis, kinase-substrate association inference from global datasets\",\n      \"journal\": \"Omics : a journal of integrative biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 — computational inference from phosphoproteomics datasets, no direct in vitro kinase assay confirming RPS6KB1→CAMKK1 S74\",\n      \"pmids\": [\"40079160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Nrf2 directly interacts with and regulates CAMKK1 expression; Nrf2 knockdown upregulates CAMKK1, which in turn promotes osteoclast differentiation. Silencing CAMKK1 alone inhibits osteoclast differentiation. This pathway is activated during Echinococcus granulosus infection-induced bone loss.\",\n      \"method\": \"Co-immunoprecipitation, high-throughput sequencing, Western blot, siRNA knockdown, Nrf2 knockout mouse model, Micro-CT imaging, TRAP staining\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP and KO mouse with defined cellular phenotype, but single lab and the upstream regulatory relationship relies partly on sequencing inference\",\n      \"pmids\": [\"39674449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CAMKK1 is a downstream target of miR-132-5p in dorsal root ganglion neurons; relief of miR-132-5p repression (via antagomir) increases CAMKK1 expression and promotes axon extension and regeneration in rat sciatic nerve injury models. TFAP2C in fibroblast-derived exosomes represses miR-132-5p, thereby derepressing CAMKK1.\",\n      \"method\": \"miRNA antagomir in vivo, axon extension assays, sciatic nerve injury rat model, exosomal protein identification\",\n      \"journal\": \"Bioactive materials\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — in vivo loss-of-function (antagomir) with defined axon regeneration phenotype and pathway placement, single lab\",\n      \"pmids\": [\"36936807\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Overexpression of CAMKK1 in mesenchymal stem cells alters their secretome (conditioned medium), leading to increased vascular density, decreased scar formation, and improved cardiac function after acute myocardial infarction. Direct overexpression of CAMKK1 in infarcted cardiac tissue also improved ejection fraction and decreased infarct size.\",\n      \"method\": \"CAMKK1 overexpression in MSCs, conditioned medium injection, plasmid-based in vivo overexpression, echocardiography, histology\",\n      \"journal\": \"Stem cells translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — overexpression with defined in vivo phenotypes but no detailed molecular pathway placement for secretome regulation\",\n      \"pmids\": [\"28688176\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CAMKK1 is a Ca2+/calmodulin-activated serine/threonine kinase that phosphorylates and activates downstream kinases (CaMKI, CaMKIV, and PKB/Akt); its activity is suppressed by phosphorylation-induced binding of 14-3-3γ, which occludes the kinase active site via its C-terminal helices and blocks Ca2+/CaM access, while upstream regulation includes miR-132-5p-mediated translational repression and Nrf2-dependent transcriptional control, placing CAMKK1 in pathways governing neuronal axon regeneration, osteoclast differentiation, and cardiac tissue repair.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries from KEPT papers only.\n\n**Classification:**\n\nRetrieved papers:\n1. PMID:36936807 — KEEP (CAMKK1 as downstream target of miR-132-5p in axon regeneration)\n2. PMID:40079160 — KEEP (phosphosite analysis of CAMKK1 and CAMKK2)\n3. PMID:30287839 — KEEP (crystal structure of CAMKK1)\n4. PMID:28688176 — KEEP (CAMKK1 role in MSC secretome)\n5. PMID:23737288 — EXCLUDE (GWAS/association study, no mechanism)\n6. PMID:38009718 — EXCLUDE (mostly about CaMKK2; inhibitors for CaMKK2)\n7. PMID:37817008 — KEEP (14-3-3 inhibits CaMKK1 structurally)\n8. PMID:33155526 — EXCLUDE (computational/virtual screening only)\n9. PMID:39674449 — KEEP (Nrf2 regulates CAMKK1 in osteoclast differentiation)\n10. PMID:34165505 — EXCLUDE (genetic association study)\n11. PMID:40965347 — EXCLUDE (expression correlation, no direct mechanism)\n\nGene2pubmed curated papers:\n- PMID:12477932 — EXCLUDE (cDNA sequencing project)\n- PMID:28514442 — EXCLUDE (large-scale interactome, not CAMKK1-specific mechanism)\n- PMID:26496610 — EXCLUDE (large-scale interactome, not CAMKK1-specific)\n- PMID:22939624 — EXCLUDE (HSP90 client survey, not CAMKK1-specific)\n- PMID:15489334 — EXCLUDE (MGC cDNA project)\n- PMID:21873635 — EXCLUDE (GO annotation methodology)\n- PMID:35271311 — EXCLUDE (large-scale localization atlas)\n- PMID:15324660 — EXCLUDE (14-3-3 proteomics survey, not CAMKK1-specific)\n- PMID:24255178 — EXCLUDE (Hippo pathway, not CAMKK1-specific)\n- PMID:18695047 — EXCLUDE (CaMKIα/Drp1, not CAMKK1)\n- PMID:7642608 — KEEP (original cloning and characterization of CaM-KK = CAMKK1)\n- PMID:19247474 — EXCLUDE (GWAS smoking behavior)\n- PMID:19520843 — KEEP (Ca2+/calmodulin-dependent protein kinase kinase activates AMPK via Ca2+ pathway)\n- PMID:11230166 — EXCLUDE (cDNA sequencing project)\n- PMID:7615569 — KEEP (CaMK kinase activates CaMKIV via Thr196 phosphorylation)\n- PMID:8621423 — KEEP (requirements for Ca2+/CaM in CaMKK cascade)\n- PMID:16741161 — EXCLUDE (GWAS association study)\n- PMID:32707033 — EXCLUDE (large kinase interactome, not CAMKK1-specific)\n- PMID:20801214 — EXCLUDE (Ppm1E/AMPK phosphatase, not CAMKK1-specific)\n- PMID:9705275 — KEEP (CaMKI regulation by CaMKK)\n- PMID:21832049 — EXCLUDE (TPPP/p25, alias collision)\n- PMID:10187789 — KEEP (PKA inhibits CaMKK via phosphorylation)\n- PMID:35256949 — EXCLUDE (Parkin/HDAC, not CAMKK1)\n- PMID:36931259 — EXCLUDE (14-3-3 chaperone function, large-scale)\n- PMID:20106900 — EXCLUDE (genetic association study)\n- PMID:20453000 — EXCLUDE (genetic association study)\n- PMID:33277362 — EXCLUDE (UBQLN2/ALS proteomics)\n- PMID:23444366 — EXCLUDE (TRIM32/14-3-3, not CAMKK1)\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"CAMKK1 (originally named CaM-kinase kinase) was cloned from rat brain as a 505-amino acid serine/threonine kinase that phosphorylates and activates CaM-kinase I and CaM-kinase IV but not CaM-kinase II in a Ca2+/calmodulin-dependent manner; co-expression with CaM-kinase IV gave 14-fold enhancement of CREB-dependent gene expression.\",\n      \"method\": \"Molecular cloning, COS-7 cell expression, in vitro kinase assay, co-expression with CaMKIV + reporter assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — original biochemical reconstitution with in vitro activity assays and cell-based functional validation\",\n      \"pmids\": [\"7642608\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"CaM-kinase Ia kinase (CAMKK1-equivalent) phosphorylates CaMKIV at Thr-196 in a Ca2+/CaM- and MgATP-dependent manner, and Thr-196 phosphorylation is essential for activation; T196A mutation abolishes both phosphorylation and activation of CaMKIV.\",\n      \"method\": \"In vitro kinase assay with purified pig brain CaMK kinase, site-directed mutagenesis (T196A), phosphorylation stoichiometry measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution plus mutagenesis demonstrating necessity of Thr-196 phosphorylation\",\n      \"pmids\": [\"7615569\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Activation of CaMKIV by CAMKK1 requires Ca2+/CaM binding to BOTH enzymes: a constitutively active truncated CaMKK (CaMKK1-434) can phosphorylate a CaMKIV fragment lacking the autoinhibitory domain in a Ca2+/CaM-independent manner, but phosphorylation of full-length CaMKIV still requires Ca2+/CaM binding to CaMKIV itself. Ionomycin stimulation in COS-7 cells confirmed Ca2+-dependent cascade activation requiring intact Thr196.\",\n      \"method\": \"In vitro phosphorylation assays with truncated and mutant CaMKK and CaMKIV, intact cell ionomycin stimulation, COS-7 co-expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple mutant constructs and both in vitro and cell-based approaches with orthogonal readouts\",\n      \"pmids\": [\"8621423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"CAMKK1 phosphorylates and activates CaMKI; CaMKI autoinhibition is mediated by a C-terminal helix-loop-helix domain (Ile286–Met316) and Ca2+/CaM relieves this inhibition allowing CAMKK to access and phosphorylate the activation loop. Specific residues Phe298, Ile294, Ile286, Val290, Trp303, and Phe307 were identified as critical for autoinhibition and CaM-binding of CaMKI.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis of CaMKI regulatory domain residues, recombinant protein structure-function analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — detailed structure-function analysis with multiple point mutants and reconstituted in vitro system\",\n      \"pmids\": [\"9705275\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"cAMP-dependent protein kinase (PKA) phosphorylates CAMKK1 primarily at Thr-108 in vitro and in intact cells, inhibiting its kinase activity; CaMKI also phosphorylates CAMKK1 at the same sites suggesting a negative-feedback mechanism. Forskolin treatment in PC12 cells rapidly inhibits both CAMKK1 and CaMKI activity, and hippocampal slice PKA activation increases CAMKK1 phosphorylation.\",\n      \"method\": \"In vitro PKA phosphorylation assay, phosphopeptide mapping, intact PC12 cell and hippocampal slice pharmacology (forskolin), CaMKI in vitro phosphorylation of CaMKK\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro site identification combined with cell-based pharmacological validation in two distinct cell systems\",\n      \"pmids\": [\"10187789\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Adiponectin activates AMPK in muscle cells via a minor pathway involving phospholipase C-induced Ca2+ release from the ER, which activates Ca2+/calmodulin-dependent protein kinase kinase (CaMKK, including CAMKK1), thereby phosphorylating AMPK; this is distinct from the major APPL1/LKB1-dependent pathway.\",\n      \"method\": \"Pharmacological inhibition of phospholipase C and Ca2+ chelation, AMPK activity assay in muscle cells, epistasis via siRNA knockdown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis via pharmacological inhibitors in cells; CAMKK1 not distinguished from CAMKK2 individually\",\n      \"pmids\": [\"19520843\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Crystal structures of CAMKK1 bound to two ATP-competitive inhibitors were determined, revealing exploitable structural differences between CAMKK1 and CAMKK2 despite high sequence identity. Isothermal titration calorimetry showed the most potent inhibitors bind with thermodynamics dominated by favorable enthalpy.\",\n      \"method\": \"X-ray crystallography, kinase inhibitor library screening, isothermal titration calorimetry\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structures with functional binding characterization by ITC\",\n      \"pmids\": [\"30287839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"14-3-3γ protein inhibits CAMKK1 by directly blocking the kinase active site with its last two C-terminal helices, forming a compact and rigid complex that also suppresses Ca2+/CaM binding to CAMKK1. In contrast, the CaMKK2:14-3-3 complex is looser and more flexible, explaining why 14-3-3 binding selectively inhibits CAMKK1 but not CAMKK2.\",\n      \"method\": \"Small-angle X-ray scattering (SAXS), hydrogen/deuterium exchange coupled to MS (HDX-MS), fluorescence spectroscopy\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal structural and biophysical methods in a single study providing mechanistic detail\",\n      \"pmids\": [\"37817008\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In peripheral nerve injury, CAMKK1 is a downstream substrate of miR-132-5p: TFAP2C (delivered via fibroblast exosomes) represses miR-132-5p expression in dorsal root ganglion neurons, relieving suppression of CAMKK1 and thereby promoting axon extension. miR-132-5p antagomir in rats stimulates CAMKK1 expression and improves sciatic nerve axon regeneration.\",\n      \"method\": \"Proteomics, exosome functional assays, miRNA target validation, antagomir injection in rat sciatic nerve injury model\",\n      \"journal\": \"Bioactive materials\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo loss-of-function (antagomir) with defined phenotype, but CAMKK1 pathway placement partly inferred\",\n      \"pmids\": [\"36936807\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Nrf2 directly interacts with and negatively regulates CAMKK1; Echinococcus granulosus protoscolex infection suppresses Nrf2, leading to upregulation of CAMKK1 which drives osteoclast differentiation and bone loss. Nrf2 knockout mice show exacerbated CAMKK1-dependent bone loss, and silencing CAMKK1 alone inhibits osteoclast differentiation.\",\n      \"method\": \"Transcriptome sequencing, Western blot, Q-PCR, co-immunoprecipitation, Nrf2 knockout mouse model, Micro-CT imaging, TRAP staining, small-molecule inhibitor (Crenolani) in vivo\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus genetic (KO and KD) approaches with in vivo validation, though single lab\",\n      \"pmids\": [\"39674449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Phosphoproteomic coregulation analysis identified RPS6KB1 (S6K1) as a novel upstream kinase of CAMKK1 at its predominant phosphosite S74 (outside the kinase domain). Predominant phosphosites of CAMKK1 are located outside the kinase domain, and their phosphomotifs are highly homologous to those of CAMKK2.\",\n      \"method\": \"Global phosphoproteome dataset analysis, coregulation analysis of phosphosites, kinase-substrate inference from phosphoproteomics\",\n      \"journal\": \"Omics : a journal of integrative biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 — computational inference from phosphoproteomic datasets without direct experimental validation of the CAMKK1-S74 kinase-substrate relationship\",\n      \"pmids\": [\"40079160\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CAMKK1 is a Ca2+/calmodulin-dependent serine/threonine kinase kinase that operates at the apex of a signaling cascade, phosphorylating and activating CaMKI (at its activation-loop threonine) and CaMKIV (at Thr-196) in a manner requiring Ca2+/CaM binding to both CAMKK1 and its substrate; its activity is inhibited by PKA-mediated phosphorylation at Thr-108 (and by CaMKI in a negative-feedback loop), and by 14-3-3γ binding which blocks its active site via its C-terminal helices and suppresses Ca2+/CaM association; structurally distinct from the paralog CAMKK2 at the inhibitor-binding level; it also activates AMPK downstream of Ca2+ signals, regulates axon regeneration via the miR-132-5p axis, promotes osteoclast differentiation downstream of Nrf2 suppression, and modulates the mesenchymal stem cell secretome.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CAMKK1 is a Ca2+/calmodulin-dependent serine/threonine kinase whose activity is negatively regulated by phosphorylation-dependent binding of 14-3-3γ, which forms a compact, rigid complex that occludes the kinase active site and blocks Ca2+/CaM access [PMID:37817008]. Crystal structures of CAMKK1 with ATP-competitive inhibitors reveal exploitable structural differences from CAMKK2 despite high sequence identity, providing a basis for isoform-selective targeting [PMID:30287839]. CAMKK1 expression is repressed by miR-132-5p in dorsal root ganglion neurons, where its derepression promotes axon regeneration after sciatic nerve injury [PMID:36936807], and is negatively regulated by Nrf2, with CAMKK1 upregulation promoting osteoclast differentiation during pathological bone loss [PMID:39674449].\",\n  \"teleology\": [\n    {\n      \"year\": 2017,\n      \"claim\": \"Establishing that CAMKK1 has a functional role beyond canonical CaMK cascade signaling, overexpression in mesenchymal stem cells and cardiac tissue demonstrated that CAMKK1 activity can remodel the cellular secretome and improve cardiac repair outcomes, linking the kinase to paracrine tissue repair programs.\",\n      \"evidence\": \"CAMKK1 overexpression in MSCs and direct cardiac injection in a myocardial infarction model with echocardiographic and histological readouts\",\n      \"pmids\": [\"28688176\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Downstream signaling pathway connecting CAMKK1 to secretome changes was not identified\",\n        \"Specific substrates mediating the cardioprotective effect are unknown\",\n        \"Overexpression approach does not confirm requirement of endogenous CAMKK1\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Determination of CAMKK1 crystal structures with ATP-competitive inhibitors resolved the active-site architecture and revealed small but druggable differences from CAMKK2, establishing the structural basis for isoform-selective inhibitor design.\",\n      \"evidence\": \"X-ray crystallography of CAMKK1–inhibitor complexes with ITC-based binding thermodynamics\",\n      \"pmids\": [\"30287839\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No co-crystal with a physiological substrate or CaM to reveal activation-state conformations\",\n        \"Selectivity of identified inhibitors was not validated in cellular assays\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Biophysical dissection of the CAMKK1:14-3-3γ complex revealed the molecular mechanism of kinase inhibition: phosphorylation-dependent 14-3-3γ binding positions its C-terminal helices directly over the CAMKK1 active site, forming a rigid complex that simultaneously blocks substrate access and Ca2+/CaM binding, explaining isoform-specific regulation since the analogous CAMKK2 complex is looser.\",\n      \"evidence\": \"SAXS, hydrogen/deuterium exchange mass spectrometry, and fluorescence spectroscopy on reconstituted complexes\",\n      \"pmids\": [\"37817008\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Identity of the kinase(s) responsible for the phosphorylation events that prime 14-3-3 binding in cells is not fully established\",\n        \"Functional consequence of 14-3-3 release in a specific physiological context was not tested\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"CAMKK1 was placed downstream of miR-132-5p in dorsal root ganglion neurons, showing that relief of translational repression increases CAMKK1 protein and is sufficient to promote axon extension and regeneration in vivo, thereby linking CAMKK1 to neuronal repair.\",\n      \"evidence\": \"miR-132-5p antagomir in rat sciatic nerve injury model with axon extension assays and exosomal pathway analysis\",\n      \"pmids\": [\"36936807\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct binding of miR-132-5p to CAMKK1 3′UTR was not validated by luciferase reporter assay in this study\",\n        \"Downstream substrates of CAMKK1 mediating axon regeneration are uncharacterized\",\n        \"Single lab finding\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Nrf2 was identified as a transcriptional repressor of CAMKK1; its loss leads to CAMKK1 upregulation that drives osteoclast differentiation, connecting CAMKK1 to bone remodeling and pathological bone loss.\",\n      \"evidence\": \"Co-immunoprecipitation, Nrf2 knockout mouse, siRNA knockdown, micro-CT and TRAP staining in an infection-induced bone loss model\",\n      \"pmids\": [\"39674449\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether Nrf2 directly binds the CAMKK1 promoter (e.g., ChIP) was not shown\",\n        \"Downstream effectors of CAMKK1 in osteoclast differentiation (CaMKI, CaMKIV, or others) are unidentified\",\n        \"Single lab, context limited to Echinococcus infection model\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The physiological substrates and signaling outputs of CAMKK1 in specific tissue contexts (neuronal regeneration, osteoclast differentiation, cardiac repair) remain poorly defined, and the upstream kinase(s) that phosphorylate CAMKK1 to prime 14-3-3 binding have not been confirmed by direct biochemical assays.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No in vitro kinase assay confirming a specific upstream kinase for 14-3-3-priming phosphorylation sites\",\n        \"Context-specific substrate usage (CaMKI vs CaMKIV vs Akt) not mapped in neuronal, bone, or cardiac settings\",\n        \"No activation-state structure of CAMKK1 bound to Ca2+/CaM or a substrate\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 4, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"YWHAG\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"CAMKK1 is a Ca²⁺/calmodulin-dependent serine/threonine kinase kinase that sits at the apex of a CaMK signaling cascade, phosphorylating and activating CaMKI and CaMKIV at their activation-loop threonines in a manner requiring Ca²⁺/CaM binding to both CAMKK1 and its substrates [PMID:7642608, PMID:7615569, PMID:8621423]. Its activity is negatively regulated by PKA-mediated phosphorylation at Thr-108 and by a CaMKI-dependent feedback loop [PMID:10187789], and is selectively inhibited by 14-3-3γ, which forms a compact complex that blocks the active site and suppresses Ca²⁺/CaM association — a mechanism structurally distinct from the looser 14-3-3 interaction with paralog CAMKK2 [PMID:37817008]. CAMKK1 also contributes to AMPK activation downstream of Ca²⁺ signals [PMID:19520843] and functions in axon regeneration, where its expression is controlled by the miR-132-5p axis, and in osteoclast differentiation, where it is de-repressed upon Nrf2 suppression [PMID:36936807, PMID:39674449].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Identification of CAMKK1 as the upstream kinase kinase for CaMKI and CaMKIV established the existence of a multi-tier Ca²⁺/CaM-dependent kinase cascade and showed it could amplify CREB-dependent transcription.\",\n      \"evidence\": \"Cloning from rat brain, in vitro kinase assays with purified proteins, COS-7 co-expression with CaMKIV and CREB reporter\",\n      \"pmids\": [\"7642608\", \"7615569\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous tissue-level validation of cascade hierarchy not yet provided\", \"Relative contributions of CAMKK1 vs CAMKK2 to CaMKIV activation not distinguished\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Demonstration that Ca²⁺/CaM must bind both CAMKK1 and CaMKIV for productive phosphorylation resolved how Ca²⁺ signals gate two independent steps in the cascade and explained why a constitutively active CAMKK alone is insufficient for full substrate activation.\",\n      \"evidence\": \"Truncation and mutagenesis of both CAMKK and CaMKIV, in vitro reconstitution, ionomycin stimulation in COS-7 cells\",\n      \"pmids\": [\"8621423\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for CaM-gated substrate accessibility unknown\", \"Whether CaMKI activation follows the same dual-CaM requirement not tested\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Mapping the autoinhibitory domain of CaMKI and showing that CaM relief of autoinhibition is a prerequisite for CAMKK1-mediated activation-loop phosphorylation clarified the substrate-side gating mechanism.\",\n      \"evidence\": \"Systematic site-directed mutagenesis of CaMKI helix-loop-helix domain, in vitro kinase assays\",\n      \"pmids\": [\"9705275\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crystal structure of CAMKK1–CaMKI complex not available\", \"In vivo relevance of individual CaMKI autoinhibitory residues not tested\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Discovery that PKA phosphorylates CAMKK1 at Thr-108 to inhibit it, and that CaMKI performs the same phosphorylation, revealed cAMP–Ca²⁺ signal integration and a negative-feedback loop within the CaMK cascade.\",\n      \"evidence\": \"In vitro PKA and CaMKI phosphorylation, phosphopeptide mapping, forskolin treatment in PC12 cells and hippocampal slices\",\n      \"pmids\": [\"10187789\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance of CaMKI feedback in specific neuronal circuits not established\", \"Additional regulatory phosphosites may exist\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Placing CaMKK upstream of AMPK in adiponectin signaling extended CAMKK1's functional scope beyond the CaMK cascade to metabolic energy sensing, though CAMKK1 was not distinguished from CAMKK2 in this context.\",\n      \"evidence\": \"Pharmacological PLC/Ca²⁺ inhibition and siRNA knockdown in muscle cells, AMPK activity assays\",\n      \"pmids\": [\"19520843\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CAMKK1-specific contribution not isolated from CAMKK2\", \"Tissue-specific isoform requirements for AMPK activation unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Crystal structures of CAMKK1 with ATP-competitive inhibitors revealed exploitable active-site differences between CAMKK1 and CAMKK2, enabling paralog-selective chemical probe development.\",\n      \"evidence\": \"X-ray crystallography, kinase inhibitor screening, isothermal titration calorimetry\",\n      \"pmids\": [\"30287839\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of CAMKK1 in its autoinhibited or CaM-bound state\", \"Selectivity over broader kinome not fully profiled\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Structural and biophysical characterization showed that 14-3-3γ inhibits CAMKK1 by plugging the active site with its C-terminal helices and blocking CaM binding, whereas CAMKK2 forms a looser complex — explaining paralog-selective regulation.\",\n      \"evidence\": \"SAXS, HDX-MS, fluorescence spectroscopy\",\n      \"pmids\": [\"37817008\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution co-crystal structure of CAMKK1:14-3-3γ complex not determined\", \"Phosphorylation sites on CAMKK1 that create the 14-3-3 docking motif not fully mapped\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of CAMKK1 as a miR-132-5p target that promotes axon regeneration linked this kinase kinase to peripheral nerve repair and exosome-mediated intercellular signaling.\",\n      \"evidence\": \"Proteomics, miRNA target validation, antagomir injection in rat sciatic nerve injury model\",\n      \"pmids\": [\"36936807\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct CAMKK1 substrates mediating axon extension not identified\", \"Whether CaMKI or CaMKIV mediates the regenerative signal downstream unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstration that Nrf2 directly binds and suppresses CAMKK1, whose de-repression drives osteoclast differentiation, placed CAMKK1 in bone remodeling downstream of oxidative-stress signaling.\",\n      \"evidence\": \"Co-IP, Nrf2 knockout mice, CAMKK1 silencing, Micro-CT and TRAP staining in infection-induced bone loss model\",\n      \"pmids\": [\"39674449\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which Nrf2 suppresses CAMKK1 (transcriptional vs protein-level) not resolved\", \"CAMKK1 substrates in osteoclastogenesis not identified\", \"Generalizability beyond parasitic infection model untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the identity of CAMKK1-specific substrates outside the CaMK/AMPK axis, the structural basis of CaM-gated substrate recognition, the in vivo tissue-specific contributions of CAMKK1 versus CAMKK2, and the full repertoire of regulatory phosphosites and their upstream kinases.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No CAMKK1 knockout mouse phenotype reported\", \"Full phosphosite map with validated upstream kinases lacking\", \"No structure of CAMKK1 in complex with CaM or a substrate kinase\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2, 4, 5, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CAMK1\",\n      \"CAMK4\",\n      \"YWHAG\",\n      \"PRKACA\",\n      \"PRKAB1\",\n      \"NRF2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}