{"gene":"CNKSR2","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1999,"finding":"CNKSR2 (MAGUIN-1) was identified as a novel neuronal scaffold protein that interacts via its C-terminal PDZ-binding motif with the PDZ domains of PSD-95/SAP90 and S-SCAM. MAGUIN-1 localizes to the plasma membrane via its pleckstrin homology (PH) domain and C-terminal region, and is co-immunoprecipitated with PSD-95/SAP90 and S-SCAM from rat crude synaptosome, indicating it is a component of the postsynaptic density complex.","method":"Co-immunoprecipitation from rat synaptosome, domain-deletion localization experiments, yeast two-hybrid","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP from native brain tissue plus domain mapping, replicated across multiple interaction partners","pmids":["10207009"],"is_preprint":false},{"year":2002,"finding":"MAGUIN-1/CNKSR2 bridges Densin-180 and PSD-95 by forming a ternary complex: the PDZ domain of Densin-180 binds the C-terminal PDZ-binding motif of MAGUIN-1, while the first PDZ domain of PSD-95 binds MAGUIN-1 separately. Densin-180 cannot associate with PSD-95 in the absence of MAGUIN-1, establishing CNKSR2 as an obligatory linker between these two synaptic scaffold proteins. MAGUIN-1 also forms dimers/multimers via its C-terminal leucine-rich region.","method":"Co-immunoprecipitation from rat brain, yeast two-hybrid domain mapping, transfected cell ternary complex assays, co-localization in hippocampal neurons","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP from native brain plus domain-deletion analysis and ternary complex reconstitution in transfected cells","pmids":["12390249"],"is_preprint":false},{"year":2003,"finding":"The human CNKSR2 protein (CNK2A and splice variant CNK2B) interacts with the Ras effector proteins RAF1 (mapped to the regulatory and kinase domains of Raf and the C-terminal half of CNK2) and with Ral signaling components including Ral GTPase and the RalGDS family member Rlf (mapped to the GEF domain of Rlf). CNK2 is phosphorylated in a MAPK-dependent manner in vivo and localizes to both membrane and cytoplasmic fractions, with full-length CNK2 at the lateral plasma membrane in MDCK cells.","method":"Co-immunoprecipitation, GST pulldown domain mapping, subcellular fractionation, confocal localization in MDCK cells","journal":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal binding assays with domain mapping, plus subcellular localization with functional context","pmids":["14597674"],"is_preprint":false},{"year":2004,"finding":"CNK2/CNKSR2 is required for NGF- but not EGF-induced ERK activation in neuronal precursor cells, demonstrating stimulus-specific scaffold function. CNK2 additionally couples NGF signaling to membrane/cytoskeletal remodeling through a separate, essential contribution, indicating that it integrates multiple regulatory pathways needed for neuronal differentiation.","method":"siRNA knockdown in PC12 cells, ERK activation assays (immunoblot), morphological readouts of cytoskeletal remodeling","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — loss-of-function knockdown with two distinct, orthogonal phenotypic readouts (ERK signaling and cytoskeletal remodeling) and stimulus specificity controls","pmids":["15028221"],"is_preprint":false},{"year":2008,"finding":"The SAM domain of Drosophila CNK (the ortholog of CNKSR2) forms a 1:1 heterodimer with the SAM domain of HYP, as determined by X-ray crystal structure. This specific SAM-SAM dimerization mode is essential for RAF kinase signaling in vivo and facilitates recruitment of KSR to form the CNK/HYP/KSR regulatory complex.","method":"X-ray crystallography, in vitro mutagenesis, in vivo Drosophila signaling rescue assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — crystal structure combined with mutagenesis and in vivo functional validation","pmids":["18287031"],"is_preprint":false},{"year":2011,"finding":"Loss-of-function deletion of the CNKSR2 gene (removing 15 of 21 exons) causes X-linked intellectual disability with epilepsy, establishing CNKSR2 as a causative gene for NS-XLID. High brain expression and postsynaptic density localization support its role in RAS/MAPK-dependent synaptic signal transduction.","method":"Copy number analysis (array CGH), gene expression profiling, known PSD localization data","journal":"Molecular syndromology","confidence":"Medium","confidence_rationale":"Tier 3 — human deletion mapping with supporting expression/localization evidence but no direct functional experiment in this paper","pmids":["22511892"],"is_preprint":false},{"year":2014,"finding":"CNKSR2 (CNK2) interacts constitutively with Vilse/ARHGAP39 via the WW domains of Vilse and a proline-rich motif in CNK2. CNK2 complexes are enriched for Rac/Cdc42 signaling components (Rac1, α/β-PIX, GIT1/2, PAK3/4, cytohesins). CNK2 acts as a spatial modulator of Rac GTP/GDP cycling during dendritic spine morphogenesis; disruption of the CNK2–Vilse interaction impairs spine formation by unbalancing RacGDP/GTP levels.","method":"Mass spectrometry interactome of endogenous CNK2, co-immunoprecipitation, domain-deletion mutagenesis, shRNA knockdown and rescue experiments in hippocampal neurons, spine morphology quantification","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — endogenous MS interactome plus reciprocal Co-IP, domain mapping, and loss-of-function rescue in primary neurons with defined morphological readout","pmids":["24656827"],"is_preprint":false},{"year":2014,"finding":"Smurf2 E3 ubiquitin ligase physically interacts with CNKSR2 and regulates AKT-dependent cell proliferation and invasion of breast cancer cells through the PI3K-PTEN-AKT-FoxO3a pathway in a CNKSR2-dependent manner. Silencing Smurf2 downregulates CNKSR2 and suppresses breast cancer cell growth.","method":"siRNA knockdown, co-immunoprecipitation, cell proliferation/invasion assays, cell cycle analysis, western blotting","journal":"Cell division","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP interaction and pathway epistasis by KD, single lab, single set of methods","pmids":["25191523"],"is_preprint":false},{"year":2018,"finding":"Smurf2 physically associates with CNKSR2 (confirmed by co-immunoprecipitation, indirect immunofluorescence, and surface plasmon resonance) and acts as a positive regulator of CNKSR2 stability: Smurf2 ubiquitinates CNKSR2 but protects it from proteasomal degradation. Smurf2 knockdown leads to enhanced polyubiquitination and proteasomal degradation of CNKSR2, reducing breast cancer cell proliferation and clonogenic survival.","method":"Co-immunoprecipitation, surface plasmon resonance, indirect immunofluorescence, ubiquitination assays, proteasome inhibitor experiments, siRNA knockdown","journal":"BMC cancer","confidence":"High","confidence_rationale":"Tier 1-2 — three independent methods (Co-IP, SPR, IF) confirming interaction, plus biochemical ubiquitination and proteasomal degradation assays","pmids":["29534682"],"is_preprint":false},{"year":2020,"finding":"CNK2/CNKSR2 modulates the size of the postsynaptic density (PSD) in neurons and directs the subcellular localization of the regulatory kinase TNIK to dendritic spines. Both CNK2 and TNIK localize postsynaptically in hippocampal neurons; CNK2 expression is critical for maintaining TNIK at appropriate levels and location, establishing a functional scaffold–kinase relationship.","method":"Immunofluorescence co-localization with synaptic markers, overexpression and knockdown experiments in neurons, PSD size morphometry","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2-3 — localization with functional consequence (PSD size, TNIK localization), single lab","pmids":["32235845"],"is_preprint":false},{"year":2021,"finding":"Cnksr2 knockout mice display spontaneous electrographic seizures, increased neural activity, anxiety, impaired learning/memory, and progressive loss of ultrasonic vocalizations. In vivo quantitative proteomics shows that Cnksr2 anchors key binding partners at synapses, and its loss significantly alters the synaptic proteome including proteins implicated in epilepsy. Cnksr2 is localized at both excitatory and inhibitory postsynapses in cortical, striatal, and cerebellar regions.","method":"Cnksr2 KO mouse model, EEG, behavioral assays, in vivo quantitative proteomics, immunolocalization","journal":"The Journal of neuroscience : the official journal of the Society for Neuroscience","confidence":"High","confidence_rationale":"Tier 2 — KO mouse with multiple orthogonal phenotypic readouts plus in vivo proteomics defining synaptic organization role","pmids":["34580165"],"is_preprint":false},{"year":2021,"finding":"CNKSR2 forms a molecular complex with the ARF-GEF CYTH2 in the hippocampal dentate gyrus, and CYTH2 binding prevents proteasomal degradation of CNKSR2. Silencing either CNKSR2 or CYTH2 in vivo causes abnormal positioning of granule cell precursors at the granule cell layer–hilus boundary with characteristics of immature neurons, demonstrating that the CNKSR2–CYTH2 complex is necessary for dentate granule cell development and differentiation.","method":"Co-immunoprecipitation, shRNA in vivo knockdown in mouse via stereotaxic injection, immunohistochemistry, cell positioning quantification","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP plus in vivo loss-of-function with defined cellular differentiation phenotype","pmids":["34800437"],"is_preprint":false},{"year":2023,"finding":"CNK2/CNKSR2 promotes cancer cell migration by coupling the pro-metastatic receptor tyrosine kinase AXL to ARF6 GTPase activation. AXL signaling drives PI3K-dependent recruitment of CNK2 to the plasma membrane; CNK2 then stimulates ARF6 by associating with cytohesin ARF-GEFs and a novel adaptor SAMD12. ARF6-GTP coordinates RAC1 activation and RHOA inhibition to generate motile forces. Genetic ablation of CNK2 or SAMD12 reduces metastasis in a mouse xenograft model.","method":"Co-immunoprecipitation, proximity ligation assay, GTPase activity assays, shRNA/CRISPR ablation, live-cell migration assays, mouse xenograft metastasis model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal biochemical assays plus in vivo xenograft validation, two independent genetic ablations","pmids":["37322019"],"is_preprint":false},{"year":2023,"finding":"s-Afadin (a splice variant of afadin) preferentially binds MAGUIN/CNKSR2 over l-afadin in vivo and in vitro. Genetic ablation of MAGUIN impairs PSD-95 localization and AMPA receptor surface accumulation in cultured hippocampal neurons and reduces postsynaptic glutamatergic responses (without affecting presynaptic release), establishing CNKSR2 as a regulator of PSD-95-dependent AMPA receptor surface localization.","method":"Co-immunoprecipitation in vivo and in vitro, MAGUIN KO neurons, surface AMPA receptor immunostaining, whole-cell patch-clamp electrophysiology","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — KO neurons with electrophysiology and biochemical surface receptor assays, multiple orthogonal methods","pmids":["36803960"],"is_preprint":false},{"year":2023,"finding":"CNKSR2 functions downstream of retinoic acid (RA) signaling in the chick forebrain roof plate, where its knockdown phenocopies RA signaling inhibition (defective invagination, reduced cell proliferation, altered patterning). CNKSR2 exerts these effects by modulating the Ras/Raf/MEK signaling pathway, as demonstrated by epistasis experiments.","method":"In ovo siRNA knockdown in chick embryo, morphological analysis, BrdU proliferation assay, pharmacological MEK inhibition epistasis","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2-3 — in vivo knockdown with phenotypic and pathway epistasis, single organism model","pmids":["36734326"],"is_preprint":false},{"year":2024,"finding":"CNKSR2 interacts with the microtubule motor protein DYNC1H1 and the centrosome marker CEP290, and localizes to centrosomes in neurons. Downregulation of CNKSR2 in Neuro-2A cells alters expression of numerous centrosomal genes and impairs centrosome-related functions including cell size, cell proliferation, and motility, revealing a previously unknown cytoplasmic/centrosomal role for CNKSR2.","method":"Immunoprecipitation–mass spectrometry, bioinformatic enrichment analysis, co-localization immunofluorescence, CNKSR2 siRNA knockdown with centrosome functional assays","journal":"Neural regeneration research","confidence":"Medium","confidence_rationale":"Tier 2-3 — IP-MS interactome plus co-localization and loss-of-function phenotyping, single lab","pmids":["39359098"],"is_preprint":false},{"year":2025,"finding":"SAMD12 (and its homolog SAMD10) binds CNKSR1/2/3 scaffold proteins with exceptionally high affinity and quantitatively displaces MAP4K kinases (MAP4K4, MINK1, TNIK) from CNKSR2 complexes. CNKSR2 acts as both a scaffold and an activator of TNIK during neuronal synapse development; ectopic SAMD12 expression alters synapse development by inhibiting TNIK activity through its dissociation from CNKSR2.","method":"Biochemical binding assays (affinity measurements), competitive displacement assays, co-immunoprecipitation, ectopic expression in neurons with synapse morphology readout","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1-2 — quantitative binding assays with competitive displacement plus functional neuronal assays establishing scaffold-activator role","pmids":["40010432"],"is_preprint":false},{"year":2024,"finding":"CNKSR2 interacts with RSK kinases through a DDVF-like short linear motif (SLiM), using the same docking interface exploited by viral and bacterial pathogen proteins. This interaction was confirmed by co-immunoprecipitation, and the RSK docking site appears to participate in negative feedback regulation of the RAS-ERK MAPK pathway.","method":"AlphaFold docking prediction, co-immunoprecipitation, RSK docking-site mutagenesis with ERK activation readout","journal":"bioRxiv (preprint)","confidence":"Low","confidence_rationale":"Tier 3 — single Co-IP in preprint, computational prediction-guided, limited mechanistic follow-up specifically for CNKSR2","pmids":["bio_10.1101_2024.08.08.607128"],"is_preprint":true},{"year":2024,"finding":"Conditional deletion of Cnksr2 specifically in excitatory neurons recapitulates anxiety and ultrasonic vocalization (USV) deficits seen in global KO mice. Further restriction of Cnksr2 deletion to excitatory neurons of the anterior cingulate cortex (ACC) is sufficient to produce USV impairments, dissociating the communication deficit from seizure and anxiety phenotypes and identifying the ACC excitatory neuron population as the critical locus for CNKSR2-dependent vocal communication.","method":"Conditional (Cre-lox) neuronal subtype-specific KO mice, EEG, behavioral assays (USV, anxiety, learning), stereotaxic viral Cre delivery to ACC","journal":"eNeuro","confidence":"High","confidence_rationale":"Tier 2 — cell-type-specific conditional KO with multiple behavioral readouts and regional dissection of phenotype","pmids":["39694826"],"is_preprint":false}],"current_model":"CNKSR2 (CNK2/MAGUIN) is a multidomain synaptic scaffold protein that organizes postsynaptic density complexes by bridging PSD-95/S-SCAM with Densin-180, anchors and activates the kinase TNIK, modulates Rac GTP/GDP cycling via its constitutive interaction with the RacGAP Vilse/ARHGAP39 to control dendritic spine morphogenesis, stabilizes AMPA receptor surface localization through a PSD-95-dependent mechanism, promotes NGF-selective ERK signaling and neuronal differentiation, couples AXL receptor signaling to ARF6/RAC1-driven cancer cell migration via cytohesin GEFs and SAMD12, is stabilized by CYTH2 binding and by Smurf2-mediated non-degradative ubiquitination, and is required in anterior cingulate cortex excitatory neurons for vocal communication, with its loss causing epilepsy-aphasia syndrome phenotypes in mice and humans."},"narrative":{"teleology":[{"year":1999,"claim":"Establishing CNKSR2 as a synaptic scaffold: discovery of its PDZ-binding motif-mediated interactions with PSD-95 and S-SCAM placed it within the postsynaptic density complex, answering how this novel neuronal protein integrates into synaptic architecture.","evidence":"Co-immunoprecipitation from rat synaptosomes, yeast two-hybrid, domain-deletion localization in transfected cells","pmids":["10207009"],"confidence":"High","gaps":["No functional consequence of disrupting these interactions was tested","Endogenous stoichiometry unknown"]},{"year":2002,"claim":"Defining CNKSR2 as an obligatory molecular bridge: demonstration that CNKSR2 is required to link Densin-180 to PSD-95 in a ternary complex resolved how these two PSD proteins associate and revealed CNKSR2's non-redundant scaffolding role.","evidence":"Ternary complex reconstitution in transfected cells, Co-IP from rat brain, domain mapping","pmids":["12390249"],"confidence":"High","gaps":["Functional consequence of disrupting the ternary complex in neurons not tested","Multimerization role unexplored"]},{"year":2003,"claim":"Connecting CNKSR2 to the Ras/MAPK cascade: identification of interactions with RAF1, Ral GTPase, and RalGDS-family GEFs established CNKSR2 as a Ras-effector scaffold, extending its function beyond static PSD scaffolding to active signaling pathway organization.","evidence":"Co-IP, GST pulldown domain mapping, MAPK-dependent phosphorylation, subcellular fractionation in MDCK cells","pmids":["14597674"],"confidence":"High","gaps":["Endogenous Ras-pathway scaffolding not shown in neurons at this stage","Specific Ras isoform selectivity not determined"]},{"year":2004,"claim":"Demonstrating stimulus-specific signaling: siRNA knockdown revealed CNKSR2 is required for NGF- but not EGF-induced ERK activation, answering whether CNKSR2 provides pathway selectivity and linking its scaffold function to neuronal differentiation.","evidence":"siRNA knockdown in PC12 cells, ERK activation immunoblots, morphological cytoskeletal readouts","pmids":["15028221"],"confidence":"High","gaps":["Mechanism of stimulus selectivity not defined","Whether selectivity applies in primary neurons untested"]},{"year":2008,"claim":"Structural basis for SAM-domain-mediated complex assembly: the crystal structure of the Drosophila CNK SAM domain bound to HYP revealed the heterodimeric interface critical for RAF signaling, providing the first atomic-level insight into how CNK-family scaffolds nucleate kinase complexes.","evidence":"X-ray crystallography, mutagenesis, in vivo Drosophila rescue assays","pmids":["18287031"],"confidence":"High","gaps":["Human CNKSR2 SAM domain structure not solved","Whether the same heterodimer mode operates with mammalian partners remains inferential"]},{"year":2014,"claim":"Revealing CNKSR2 as a Rac-GTP/GDP cycling modulator: the constitutive CNKSR2–Vilse/ARHGAP39 interaction and its requirement for dendritic spine morphogenesis answered how CNKSR2 spatially regulates small GTPase signaling at spines, unifying its scaffold and morphogenesis roles.","evidence":"Endogenous IP-MS interactome, domain-deletion mutagenesis, shRNA knockdown and rescue in hippocampal neurons, spine morphometry","pmids":["24656827"],"confidence":"High","gaps":["Whether Vilse-independent CNK2 functions contribute to spine formation not dissected","Temporal dynamics of Rac cycling at spines not measured"]},{"year":2018,"claim":"Defining CNKSR2 protein stability regulation: Smurf2 was shown to ubiquitinate CNKSR2 in a non-degradative manner that protects it from proteasomal turnover, establishing a post-translational mechanism controlling CNKSR2 abundance relevant to cancer cell proliferation.","evidence":"Co-IP, surface plasmon resonance, ubiquitination assays, proteasome inhibitor experiments in breast cancer cells","pmids":["29534682"],"confidence":"High","gaps":["Ubiquitin chain type not characterized","Whether this stabilization mechanism operates in neurons unknown"]},{"year":2021,"claim":"In vivo loss-of-function phenotyping: Cnksr2 knockout mice displayed seizures, vocalization loss, and synaptic proteome remodeling, directly establishing CNKSR2 as essential for neural circuit function and modeling the human epilepsy-aphasia syndrome.","evidence":"Cnksr2 KO mouse, EEG, behavioral assays, quantitative synaptic proteomics, immunolocalization at excitatory and inhibitory synapses","pmids":["34580165"],"confidence":"High","gaps":["Causal contribution of individual synaptic protein changes not delineated","Inhibitory synapse-specific role of CNKSR2 not functionally tested"]},{"year":2021,"claim":"CYTH2-dependent stabilization and dentate gyrus development: CYTH2 binding prevents CNKSR2 proteasomal degradation and the complex is required for granule cell differentiation, revealing a second stability-control mechanism and a neurodevelopmental role beyond synapse organization.","evidence":"Co-IP, in vivo shRNA knockdown via stereotaxic injection in mouse, immunohistochemistry, cell positioning analysis","pmids":["34800437"],"confidence":"High","gaps":["How CYTH2 binding blocks CNKSR2 degradation mechanistically is unresolved","Relationship to Smurf2-mediated stabilization not explored"]},{"year":2023,"claim":"CNKSR2 controls AMPA receptor surface expression: genetic ablation of CNKSR2 impaired PSD-95 localization and reduced surface AMPA receptors and postsynaptic responses, answering a longstanding question about the functional consequence of the PSD-95–CNKSR2 scaffold interaction for glutamatergic transmission.","evidence":"MAGUIN KO neurons, surface AMPA receptor immunostaining, whole-cell patch-clamp electrophysiology","pmids":["36803960"],"confidence":"High","gaps":["Specific AMPA receptor subunit dependence not determined","Whether inhibitory receptor trafficking is also affected untested"]},{"year":2023,"claim":"Cancer migration mechanism: CNKSR2 was shown to couple AXL receptor signaling to ARF6/RAC1-driven migration via cytohesin GEFs and SAMD12, with ablation reducing metastasis in vivo, answering how CNKSR2 scaffold function is co-opted in cancer.","evidence":"Co-IP, proximity ligation assay, GTPase activity assays, CRISPR/shRNA ablation, mouse xenograft metastasis model","pmids":["37322019"],"confidence":"High","gaps":["Applicability beyond the tested cancer cell lines not established","Whether neuronal CNKSR2–cytohesin signaling uses the same AXL-dependent mechanism unknown"]},{"year":2025,"claim":"SAMD12 as a competitive regulator of CNKSR2–TNIK complexes: quantitative binding assays showed SAMD12 displaces MAP4K kinases (including TNIK) from CNKSR2, and ectopic SAMD12 disrupts synapse development, establishing CNKSR2 as both a scaffold and an activator of TNIK and identifying SAMD12 as a natural antagonist.","evidence":"Biochemical affinity measurements, competitive displacement assays, Co-IP, neuronal synapse morphology readouts","pmids":["40010432"],"confidence":"High","gaps":["Endogenous SAMD12 expression levels in brain regions not quantified","Whether SAMD12 regulation is developmentally dynamic remains unknown"]},{"year":2024,"claim":"Circuit-level dissection: conditional deletion in ACC excitatory neurons sufficed to reproduce vocalization deficits, dissociating the communication phenotype from seizures and anxiety and identifying the critical neuronal population for CNKSR2-dependent vocal communication.","evidence":"Cre-lox conditional KO mice, stereotaxic viral Cre delivery to ACC, EEG, behavioral assays","pmids":["39694826"],"confidence":"High","gaps":["Molecular mechanism downstream of CNKSR2 loss in ACC neurons not defined","Whether restoration of CNKSR2 in ACC can rescue established deficits untested"]},{"year":null,"claim":"Several major mechanistic questions remain: (1) the structural basis of human CNKSR2 scaffold assembly is unknown; (2) how the two protein-stability mechanisms (Smurf2 and CYTH2) are coordinated is unresolved; (3) the specific contribution of CNKSR2 at inhibitory synapses has not been functionally dissected; and (4) whether CNKSR2's centrosomal role is relevant in vivo remains unvalidated.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal or cryo-EM structure of mammalian CNKSR2","Smurf2 vs CYTH2 stabilization interplay untested","Inhibitory synapse function of CNKSR2 not explored beyond localization"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,6,9,12,16]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,2,12]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[15]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,3,4,6,12,14]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[0,1,6,9,10,13,18]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[3,11,14]}],"complexes":["PSD-95/Densin-180/CNKSR2 ternary complex","CNKSR2–Vilse/ARHGAP39 complex","CNKSR2–CYTH2 complex"],"partners":["DLG4","ARHGAP39","TNIK","CYTH2","SAMD12","SMURF2","RAF1","LRFN1"],"other_free_text":[]},"mechanistic_narrative":"CNKSR2 is a multidomain scaffold protein that organizes signaling complexes at neuronal synapses and in cancer cell migration by bridging Ras/MAPK pathway components, small GTPase regulators, and postsynaptic density proteins. At excitatory synapses, CNKSR2 links PSD-95 and Densin-180 into ternary complexes, recruits and activates the kinase TNIK, spatially modulates Rac GTP/GDP cycling through its constitutive partner ARHGAP39/Vilse, and stabilizes AMPA receptor surface expression via PSD-95, thereby controlling dendritic spine morphogenesis and postsynaptic glutamatergic transmission [PMID:10207009, PMID:12390249, PMID:24656827, PMID:36803960, PMID:40010432]. In non-neuronal contexts, CNKSR2 couples AXL receptor signaling to ARF6/RAC1-driven migration through cytohesin GEFs and the adaptor SAMD12, and its stability is maintained by Smurf2-mediated non-degradative ubiquitination and by CYTH2 binding [PMID:37322019, PMID:29534682, PMID:34800437]. Loss-of-function mutations in CNKSR2 cause X-linked intellectual disability with seizures and language impairment (epilepsy–aphasia spectrum), and conditional deletion in anterior cingulate cortex excitatory neurons is sufficient to produce vocal communication deficits [PMID:22511892, PMID:34580165, PMID:39694826]."},"prefetch_data":{"uniprot":{"accession":"Q8WXI2","full_name":"Connector enhancer of kinase suppressor of ras 2","aliases":["CNK homolog protein 2","CNK2"],"length_aa":1034,"mass_kda":117.5,"function":"May function as an adapter protein or regulator of Ras signaling pathways","subcellular_location":"Cytoplasm; Membrane","url":"https://www.uniprot.org/uniprotkb/Q8WXI2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CNKSR2","classification":"Not Classified","n_dependent_lines":7,"n_total_lines":1208,"dependency_fraction":0.005794701986754967},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CNKSR2","total_profiled":1310},"omim":[{"mim_id":"619000","title":"INTELLECTUAL DEVELOPMENTAL DISORDER WITH SEIZURES AND LANGUAGE DELAY; IDDSELD","url":"https://www.omim.org/entry/619000"},{"mim_id":"301008","title":"INTELLECTUAL DEVELOPMENTAL DISORDER, X-LINKED, SYNDROMIC, HOUGE TYPE; MRXSHG","url":"https://www.omim.org/entry/301008"},{"mim_id":"300724","title":"CONNECTOR ENHANCER OF KINASE SUPPRESSOR OF RAS 2; CNKSR2","url":"https://www.omim.org/entry/300724"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Golgi apparatus","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Approved"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"brain","ntpm":35.0},{"tissue":"retina","ntpm":16.6}],"url":"https://www.proteinatlas.org/search/CNKSR2"},"hgnc":{"alias_symbol":["KIAA0902","CNK2","KSR2"],"prev_symbol":[]},"alphafold":{"accession":"Q8WXI2","domains":[{"cath_id":"1.10.150.50","chopping":"8-196","consensus_level":"medium","plddt":81.5315,"start":8,"end":196},{"cath_id":"2.30.42.10","chopping":"211-294","consensus_level":"high","plddt":80.2987,"start":211,"end":294},{"cath_id":"2.30.29.30","chopping":"564-678","consensus_level":"high","plddt":80.5527,"start":564,"end":678},{"cath_id":"-","chopping":"977-1000_1017-1034","consensus_level":"medium","plddt":64.5298,"start":977,"end":1034}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WXI2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WXI2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WXI2-F1-predicted_aligned_error_v6.png","plddt_mean":57.34},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CNKSR2","jax_strain_url":"https://www.jax.org/strain/search?query=CNKSR2"},"sequence":{"accession":"Q8WXI2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8WXI2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8WXI2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WXI2"}},"corpus_meta":[{"pmid":"24209692","id":"PMC_24209692","title":"KSR2 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Serie III, Sciences de la vie","url":"https://pubmed.ncbi.nlm.nih.gov/7757816","citation_count":70,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"10207009","id":"PMC_10207009","title":"MAGUIN, a novel neuronal membrane-associated guanylate kinase-interacting protein.","date":"1999","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10207009","citation_count":66,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"35044719","id":"PMC_35044719","title":"Proteome-scale mapping of binding sites in the unstructured regions of the human proteome.","date":"2022","source":"Molecular systems biology","url":"https://pubmed.ncbi.nlm.nih.gov/35044719","citation_count":61,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12390249","id":"PMC_12390249","title":"Densin-180, a synaptic protein, links to PSD-95 through its direct interaction with MAGUIN-1.","date":"2002","source":"Genes to cells : devoted to molecular & cellular mechanisms","url":"https://pubmed.ncbi.nlm.nih.gov/12390249","citation_count":60,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15342556","id":"PMC_15342556","title":"Sequence comparison of human and mouse genes reveals a homologous block structure in the promoter regions.","date":"2004","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/15342556","citation_count":57,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"16637659","id":"PMC_16637659","title":"Uncovering quantitative protein interaction networks for mouse PDZ domains using protein microarrays.","date":"2006","source":"Journal of the American Chemical Society","url":"https://pubmed.ncbi.nlm.nih.gov/16637659","citation_count":54,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"14597674","id":"PMC_14597674","title":"Human homologue of Drosophila CNK interacts with Ras effector proteins Raf and Rlf.","date":"2003","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/14597674","citation_count":50,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"36138187","id":"PMC_36138187","title":"NUDT21 limits CD19 levels through alternative mRNA polyadenylation in B cell acute lymphoblastic leukemia.","date":"2022","source":"Nature immunology","url":"https://pubmed.ncbi.nlm.nih.gov/36138187","citation_count":46,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"19260141","id":"PMC_19260141","title":"Genome-wide association study of biochemical traits in Korcula Island, Croatia.","date":"2009","source":"Croatian medical journal","url":"https://pubmed.ncbi.nlm.nih.gov/19260141","citation_count":42,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"18287031","id":"PMC_18287031","title":"CNK and HYP form a discrete dimer by their SAM domains to mediate RAF kinase signaling.","date":"2008","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/18287031","citation_count":41,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"35337019","id":"PMC_35337019","title":"A BioID-Derived Proximity Interactome for SARS-CoV-2 Proteins.","date":"2022","source":"Viruses","url":"https://pubmed.ncbi.nlm.nih.gov/35337019","citation_count":37,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"36115835","id":"PMC_36115835","title":"Quantitative fragmentomics allow affinity mapping of interactomes.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/36115835","citation_count":33,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"35559673","id":"PMC_35559673","title":"Genome-wide CRISPR screens using isogenic cells reveal vulnerabilities conferred by loss of tumor suppressors.","date":"2022","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/35559673","citation_count":28,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"25754917","id":"PMC_25754917","title":"CNKSR2 deletions: a novel cause of X-linked intellectual disability and seizures.","date":"2015","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/25754917","citation_count":27,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"27956147","id":"PMC_27956147","title":"Systematic Identification of Oncogenic EGFR Interaction Partners.","date":"2016","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/27956147","citation_count":21,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":30082,"output_tokens":6429,"usd":0.093341},"stage2":{"model":"claude-opus-4-6","input_tokens":10047,"output_tokens":4252,"usd":0.234802},"total_usd":0.633917,"stage1_batch_id":"msgbatch_011kfMfE81o6CTcyhVUqumuW","stage2_batch_id":"msgbatch_01DTputS6WNkH745x1nVSoBo","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":42544,"output_tokens":5778,"usd":0.107151},"round2_rules_fired":"R2","round2_stage2":{"model":"claude-opus-4-6","input_tokens":9033,"output_tokens":3490,"usd":0.198623}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"KSR2 (CNKSR2) interacts with and modulates the activity of AMPK, regulating AMPK-dependent glucose uptake and fatty acid oxidation in mouse embryonic fibroblasts and glycolysis in neuronal cell lines; disruption of KSR2 in vivo impairs AMPK-regulated fatty acid oxidation and thermogenesis.\",\n      \"method\": \"Co-immunoprecipitation, KO mouse model (ksr2-/-), metabolic assays in MEFs and neuronal cell lines, hyperinsulinemic-euglycemic clamp\",\n      \"journal\": \"Cell metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal interaction confirmed, multiple orthogonal methods, in vivo KO with defined metabolic phenotypes, replicated in multiple cell types\",\n      \"pmids\": [\"19883615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Calcineurin (protein phosphatase) selectively interacts with KSR2 (but not KSR1) and dephosphorylates KSR2 on specific sites in response to Ca2+ signals, regulating KSR2 localization and activity; KSR2 uniquely contributes to Ca2+-mediated ERK signaling in pancreatic beta-cells and neuroblastoma cells.\",\n      \"method\": \"Proteomics (mass spectrometry-based binding partner comparison), co-immunoprecipitation, phosphosite mapping, KSR2 depletion with ERK activation readout in INS1 and NG108 cells\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — proteomics plus co-IP plus phosphosite identification plus functional KD with defined signaling readout in multiple cell lines\",\n      \"pmids\": [\"19560418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CNK2/CNKSR2 is required for NGF- but not EGF-induced ERK activation in neuronal precursor cells, and makes a separate essential contribution to NGF signaling-driven membrane/cytoskeletal remodeling, coupling NGF signal propagation to multiple downstream regulatory cascades driving cell differentiation.\",\n      \"method\": \"Loss-of-function (RNAi/dominant negative), epistasis with NGF vs EGF signaling, ERK activation assays, morphological readouts\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — pathway epistasis with specific stimulus discrimination, multiple readouts (ERK activation and cytoskeletal remodeling)\",\n      \"pmids\": [\"15028221\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CNK2/CNKSR2 interacts constitutively with Vilse/ARHGAP39 (a RacGAP) via Vilse WW domains and a proline motif in CNK2; CNK2 complexes are enriched for Rac/Cdc42 signaling components including Rac1, α-PIX, β-PIX, GIT1, GIT2, PAK3, PAK4, and cytohesins. CNK2 acts as a spatial modulator of Rac cycling during dendritic spine morphogenesis, with the Vilse interaction critical for maintaining RacGDP/GTP balance required for spine formation.\",\n      \"method\": \"Mass spectrometry of endogenous CNK2 interactome, co-IP, mutant analysis, protein depletion and rescue experiments, spine morphogenesis assays in hippocampal neurons\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — endogenous MS interactome, constitutive interaction domain mapping, functional rescue experiments with specific spine morphogenesis readout\",\n      \"pmids\": [\"24656827\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"KSR2 acts as a scaffold for the RAF-MEK-ERK kinase module with distinct isoform specificity: KSR2 preferentially mediates A-RAF signaling while KSR1 transduces signals from c-RAF; the KSR2 interactome includes ~100 associated proteins, with ~43 recruited after TNF-α stimulation.\",\n      \"method\": \"Functional proteomics (co-immunoprecipitation coupled with mass spectrometry) in HEK-293 cells under basal and TNF-α-stimulated conditions\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single proteomics study, RAF isoform specificity inferred from interactome, no direct kinase reconstitution\",\n      \"pmids\": [\"19563921\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"KSR2 promotes ERK signaling and anchorage-independent growth; its effect on tumor cell energy homeostasis is mediated through AMPK signaling (not MAPK). Constitutive AMPK activation complements metabolic but not mitogenic KSR2 functions; a KSR2 mutant unable to interact with ERK retains transformed phenotype, demonstrating MAPK signaling is dispensable for transformation in these cells.\",\n      \"method\": \"KSR1-/- MEFs with Ras(V12) expression, RNAi knockdown, AMPK constitutive activation, KSR2 ERK-interaction mutant, colony formation and metabolic capacity assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal genetic approaches (KO, RNAi, separation-of-function mutant) with defined mechanistic dissection of ERK vs AMPK pathways\",\n      \"pmids\": [\"22801368\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"KSR2 rare variants identified in obese humans disrupt signaling through the Raf-MEK-ERK pathway and impair cellular fatty acid oxidation and glucose oxidation in transfected cells; these effects can be ameliorated by metformin.\",\n      \"method\": \"Human sequencing, functional assays in transfected cells (ERK signaling, fatty acid oxidation, glucose oxidation), metformin treatment\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — human genetics combined with functional cell-based assays with multiple metabolic readouts and pharmacological rescue\",\n      \"pmids\": [\"24209692\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"KSR2-associated calcineurin is required for store-operated calcium entry (SOCE); KSR2 deficiency affects STIM1/ORAI1 puncta formation and is correlated with cytoskeleton disorganization. Blocking calcineurin activity impairs STIM1/ORAI1 puncta-like formation in a KSR2-dependent manner.\",\n      \"method\": \"ksr2-/- lymphocytes and fibroblasts, shKSR2-depleted cells, Ca2+ imaging, STIM1/ORAI1 puncta formation assays, cytoskeleton analysis, calcineurin inhibitors\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO and KD with mechanistic dissection using calcineurin inhibitors, but single lab study\",\n      \"pmids\": [\"24672054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"KSR2 expression in the brain is required for regulation of energy balance and adaptive thermogenesis; brain-specific KSR2 knockout (Nes-Cre) recapitulates obesity and glucose intolerance of global KO, but unlike global KO mice, brain-specific KO mice respond normally to leptin and AICAR, indicating a separate KSR2-dependent mechanism in other tissues modulates AMPK sensitivity.\",\n      \"method\": \"Conditional KO (Nestin-Cre × KSR2fl/fl), metabolic phenotyping, leptin and AICAR tolerance tests\",\n      \"journal\": \"Molecular metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — tissue-specific conditional KO with physiological epistasis defining brain-specific vs peripheral KSR2 functions\",\n      \"pmids\": [\"28180061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"KSR2 functions cell non-autonomously to regulate GH-stimulated IGF-1 expression in neonatal mouse liver; ksr2-/- mice show reduced JAK2 and STAT5 phosphorylation in liver but not skeletal muscle after GH injection, yet isolated primary hepatocytes from ksr2-/- mice show normal GH-stimulated STAT5 phosphorylation, suggesting an indirect (cell non-autonomous) mechanism.\",\n      \"method\": \"ksr2-/- mice, GH injection with JAK2/STAT5 phosphorylation measurement, primary hepatocyte isolation, adenoviral IGF-1 rescue, fgf21-/-;ksr2-/- double KO\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo vs in vitro discrepancy establishes cell non-autonomy, multiple genetic tools used, but mechanism not fully resolved\",\n      \"pmids\": [\"27561547\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Smurf2 E3 ubiquitin ligase physically associates with CNKSR2 and modulates PI3K-PTEN-AKT-FoxO3a pathway activity via CNKSR2; Smurf2 silencing reduces CNKSR2 levels and downstream AKT-dependent cell proliferation and invasion.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation (implied), Western blotting, cell proliferation/invasion assays, rescue experiments\",\n      \"journal\": \"Cell division\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, limited mechanistic detail on direct interaction, but multiple functional readouts and pathway placement via knockdown\",\n      \"pmids\": [\"25191523\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Smurf2 E3 ubiquitin ligase directly binds CNKSR2 (confirmed by co-immunoprecipitation and surface plasmon resonance) and paradoxically stabilizes CNKSR2 by protecting it from proteasomal degradation; Smurf2 knockdown results in enhanced polyubiquitination and proteasomal degradation of CNKSR2.\",\n      \"method\": \"Co-immunoprecipitation, indirect immunofluorescence, surface plasmon resonance (SPR), ubiquitination assays, proteasome inhibition, protein stability assays\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — interaction confirmed by orthogonal methods (Co-IP + SPR), ubiquitination and proteasomal degradation directly demonstrated, single lab\",\n      \"pmids\": [\"29534682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CNKSR2 forms a molecular complex with CYTH2 (a GEF for ARF GTPases); CYTH2 binding prevents proteasomal degradation of CNKSR2. Knockdown of either CNKSR2 or CYTH2 in mouse hippocampal granule cell precursors results in abnormal localization of transduced cells and characteristics of immature granule cells, indicating their roles in dentate granule cell development.\",\n      \"method\": \"Co-immunoprecipitation, in vivo shRNA knockdown in mouse granule cell precursors, protein stability assays, immunofluorescence, neuronal morphology analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP interaction, in vivo KD with defined developmental phenotype, protein stability mechanism, single lab\",\n      \"pmids\": [\"34800437\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CNK2/CNKSR2 modulates postsynaptic density (PSD) size and influences subcellular localization of the regulatory kinase TNIK in neurons; CNK2 and TNIK co-localize at postsynaptic sites, and CNK2 directs TNIK to the correct subcellular location at appropriate levels.\",\n      \"method\": \"Co-immunoprecipitation to identify TNIK as CNK2 interactor, immunofluorescence with synaptic markers, CNK2 depletion/overexpression with TNIK localization readout, PSD morphology analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — novel interaction identified, localization data with functional consequence, single lab\",\n      \"pmids\": [\"32235845\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cnksr2 KO mice exhibit spontaneous electrographic seizures and increased neural activity; Cnksr2 is localized at both excitatory and inhibitory postsynapses. Proteomics of Cnksr2 KO synapses reveals that Cnksr2 anchors key binding partners at synapses and its loss results in significant alterations of the synaptic proteome including proteins implicated in epilepsy.\",\n      \"method\": \"Cnksr2 KO mouse model, in vivo electrophysiology (EEG), behavioral testing, subcellular fractionation, in vivo quantitative proteomics of synaptic preparations\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — novel KO model with electrophysiology, subcellular localization, and quantitative synaptic proteomics providing mechanistic insight\",\n      \"pmids\": [\"34580165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CNK2/CNKSR2 promotes cancer cell migration by coupling the receptor tyrosine kinase AXL to ARF6 GTPase activation; AXL signaling induces PI3K-dependent recruitment of CNK2 to the plasma membrane, where CNK2 stimulates ARF6 by associating with cytohesin ARF GEFs and the adaptor SAMD12. ARF6-GTP coordinates RAC1 activation and RHOA inhibition to drive motility; genetic ablation of CNK2 or SAMD12 reduces metastasis in mouse xenograft model.\",\n      \"method\": \"Genetic ablation (CRISPR), co-immunoprecipitation, PI3K inhibition, ARF6/RAC1/RHOA activity assays, xenograft metastasis model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic pathway dissection with multiple co-IPs, GTPase activity assays, PI3K epistasis, and in vivo metastasis validation\",\n      \"pmids\": [\"37322019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"s-Afadin binds MAGUIN/CNKSR2 (preferentially over l-afadin) in vivo and in vitro; genetic ablation of MAGUIN impairs PSD-95 localization and AMPA receptor surface accumulation in hippocampal neurons, and reduces postsynaptic response to glutamate without affecting presynaptic glutamate release.\",\n      \"method\": \"Co-immunoprecipitation in vivo and in vitro, genetic MAGUIN KO, immunofluorescence, electrophysiology (mEPSC recording), surface biotinylation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct binding demonstrated in vivo and in vitro, KO with electrophysiological and biochemical readouts of postsynaptic function\",\n      \"pmids\": [\"36803960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SAMD12 binds CNKSR2 with exceptionally strong affinity and can quantitatively displace MAP4K kinases (TNIK, MAP4K4, MINK1) from the CNKSR2 scaffold; CNKSR2 acts as both a scaffold and an activator of TNIK during neuronal synapse development. Ectopic SAMD12 expression alters synapse development by inhibiting TNIK activity through dissociation from CNKSR2.\",\n      \"method\": \"Biochemical binding/competition assays, co-immunoprecipitation, ectopic expression experiments, synapse development assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic competition assay establishing displacement mechanism, functional synapse development readout, single lab\",\n      \"pmids\": [\"40010432\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"KSR2 regulates bone formation by influencing adipocyte differentiation at the expense of osteoblasts in the bone marrow; osteoblast-specific conditional deletion of KSR2 reveals cell-autonomous regulation of bone formation by KSR2, independent of its hypothalamic feeding regulatory role.\",\n      \"method\": \"Global Ksr2 KO mice, pair-feeding experiments, osteoblast-specific conditional KO, micro-CT, bone histomorphometry\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditional cell-type-specific KO establishes autonomous function, but mechanism of adipocyte-osteoblast fate switching not molecularly resolved\",\n      \"pmids\": [\"36342465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CNKSR2 interacts with microtubule protein DYNC1H1 and centrosome marker CEP290; CNKSR2 co-localizes with centrosomal structures. Downregulation of CNKSR2 in neuroblastoma cells causes significant changes in centrosomal gene expression and affects centrosome-related functions including cell size, shape, proliferation, and motility.\",\n      \"method\": \"Immunoprecipitation coupled with high-resolution LC-MS, co-localization analysis, CNKSR2 knockdown in Neuro 2A cells, bioinformatic analysis of interactome enrichment\",\n      \"journal\": \"Neural regeneration research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — MS-identified interactions confirmed by co-localization, functional KD with centrosome-related readouts, single lab\",\n      \"pmids\": [\"39359098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CNKSR2 is a downstream mediator of retinoic acid (RA) signaling in the chick forebrain roof plate; knockdown of CNKSR2 phenocopies RA signaling inhibition and affects forebrain invagination, cell proliferation, and patterning by modulating the Ras/Raf/MEK signaling pathway.\",\n      \"method\": \"Chick embryo knockdown (in ovo RNAi), RA signaling inhibition epistasis, Ras/Raf/MEK signaling assays, morphological readouts\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — avian ortholog model, epistasis placing CNKSR2 downstream of RA and upstream of Ras/Raf/MEK with defined developmental phenotype\",\n      \"pmids\": [\"36734326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"KSR2 interacts with 14-3-3ζ (phospho-serine binding protein); elevated 14-3-3ζ increases KSR2 protein levels, and co-overexpression of both causes hyperactivated MAPK signaling and resistance to sorafenib in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, co-overexpression and knockdown experiments, MAPK signaling assays, flow cytometry for apoptosis/drug resistance\",\n      \"journal\": \"Biomarker research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP interaction confirmed, functional consequence of co-expression on MAPK and drug resistance defined, single lab\",\n      \"pmids\": [\"35468812\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"KSR2 competes with CRBN for binding to the K52 site of AMPKα1, inhibiting CRL4A E3 ubiquitin ligase complex-mediated K48-linked polyubiquitination and proteasomal degradation of AMPKα1; this stabilizes AMPKα1 and activates AMPK signaling to maintain glycolytic balance in endothelial cells with anti-inflammatory and anti-apoptotic effects.\",\n      \"method\": \"Co-immunoprecipitation, CRISPR/Cas9 KO, endothelial-specific KSR2 overexpression (AAV9), ubiquitination assays, site-specific competition binding assays\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mechanistic site-specific competition binding identified, ubiquitination demonstrated, multiple mouse models with atherosclerosis readout, multiple orthogonal methods\",\n      \"pmids\": [\"41424849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CNKSR2 interacts with RSK kinases through a DDVF-like short linear motif (SLiM); this interaction uses a similar interface as that employed by viral/bacterial proteins to hijack RSKs. CNKSR2 acts upstream of RSK in the RAS-ERK MAPK pathway, potentially providing negative feedback to the ERK pathway via RSK.\",\n      \"method\": \"Co-immunoprecipitation, AlphaFold docking prediction, SLiM-based screening\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP confirmation plus computational docking, preprint, functional consequence indirect\",\n      \"pmids\": [\"bio_10.1101_2024.08.08.607128\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cnksr2 conditional deletion specifically from excitatory neurons, and more specifically from excitatory neurons of the Anterior Cingulate Cortex (ACC), leads to loss of ultrasonic vocalizations (USVs) in mice, dissociating the communication impairment from seizures or anxiety phenotypes caused by broader Cnksr2 loss.\",\n      \"method\": \"Cell-type-specific conditional Cnksr2 KO (Cre-dependent), ultrasonic vocalization assays, EEG, behavioral testing\",\n      \"journal\": \"eNeuro\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional cell-type and region-specific KO establishing mechanistic dissociation of distinct phenotypes with defined neuronal circuit\",\n      \"pmids\": [\"39694826\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CNKSR2/CNK2 is a multi-domain scaffold protein that operates at multiple signaling nodes: it facilitates Raf-MEK-ERK cascade activation (with A-RAF selectivity distinct from KSR1), couples Ca2+ signals to ERK via selective calcineurin binding and dephosphorylation, regulates AMPK activity to control energy metabolism and fatty acid oxidation, stabilizes AMPKα1 by competing with the CRL4A ubiquitin ligase for K52 binding, couples AXL receptor signaling to ARF6-dependent RAC1/RHOA regulation for cell motility, and at neuronal synapses acts as a postsynaptic scaffold that modulates PSD organization, AMPA receptor surface localization, Rac cycling during dendritic spine morphogenesis (via Vilse/ARHGAP39), and TNIK kinase localization, with its protein stability regulated by CYTH2 binding and Smurf2-dependent ubiquitination.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries only from KEEP papers.\n\n**Classification:**\n\nPapers about CNKSR2/CNK2/MAGUIN (canonical protein): KEEP — [5], [10], [11], [12], [13], [15], [16], [17], [18], [19], [22], [23], [24], [25], [29], [34], [36], [37], [38], [48], [50], [53], [56] from retrieved; [17], [19], [22], [25] from curated\n\nPapers about KSR2 (kinase suppressor of Ras 2, a DIFFERENT gene — KSR2 vs CNKSR2): EXCLUDE — [1], [2], [3], [6], [7], [14], [20], [21], [26], [27], [31], [35], [40], [41], [42], [51], [52], [55] from retrieved\n\nPapers about cNK-2 peptide (chicken NK-lysin): EXCLUDE — [8]\n\nPapers about Chlamydomonas CNK2 kinase (ciliary length): EXCLUDE — [9]\n\nPapers about C. elegans ksr-2: EXCLUDE — [4]\n\nClinical/genetic reports with no mechanistic data: EXCLUDE — [28], [30], [32], [33], [39], [44], [45], [46], [47], [49]\n\nLarge-scale genomics/proteomics with CNKSR2 as incidental hit: EXCLUDE from curated — [1]-[16], [18], [20], [23]-[24], [26]-[28], [30] (most curated papers)\n\nCurated KEEP: [17] MAGUIN paper (Yao 1999), [19] Densin-180 paper (Ohtakara 2002), [22] CNK2A human homologue (Lanigan 2003), [25] CNK/HYP SAM domain structure (Rajakulendran 2008)\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"CNKSR2 (MAGUIN-1) was identified as a novel neuronal scaffold protein that interacts via its C-terminal PDZ-binding motif with the PDZ domains of PSD-95/SAP90 and S-SCAM. MAGUIN-1 localizes to the plasma membrane via its pleckstrin homology (PH) domain and C-terminal region, and is co-immunoprecipitated with PSD-95/SAP90 and S-SCAM from rat crude synaptosome, indicating it is a component of the postsynaptic density complex.\",\n      \"method\": \"Co-immunoprecipitation from rat synaptosome, domain-deletion localization experiments, yeast two-hybrid\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP from native brain tissue plus domain mapping, replicated across multiple interaction partners\",\n      \"pmids\": [\"10207009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"MAGUIN-1/CNKSR2 bridges Densin-180 and PSD-95 by forming a ternary complex: the PDZ domain of Densin-180 binds the C-terminal PDZ-binding motif of MAGUIN-1, while the first PDZ domain of PSD-95 binds MAGUIN-1 separately. Densin-180 cannot associate with PSD-95 in the absence of MAGUIN-1, establishing CNKSR2 as an obligatory linker between these two synaptic scaffold proteins. MAGUIN-1 also forms dimers/multimers via its C-terminal leucine-rich region.\",\n      \"method\": \"Co-immunoprecipitation from rat brain, yeast two-hybrid domain mapping, transfected cell ternary complex assays, co-localization in hippocampal neurons\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP from native brain plus domain-deletion analysis and ternary complex reconstitution in transfected cells\",\n      \"pmids\": [\"12390249\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The human CNKSR2 protein (CNK2A and splice variant CNK2B) interacts with the Ras effector proteins RAF1 (mapped to the regulatory and kinase domains of Raf and the C-terminal half of CNK2) and with Ral signaling components including Ral GTPase and the RalGDS family member Rlf (mapped to the GEF domain of Rlf). CNK2 is phosphorylated in a MAPK-dependent manner in vivo and localizes to both membrane and cytoplasmic fractions, with full-length CNK2 at the lateral plasma membrane in MDCK cells.\",\n      \"method\": \"Co-immunoprecipitation, GST pulldown domain mapping, subcellular fractionation, confocal localization in MDCK cells\",\n      \"journal\": \"FASEB journal : official publication of the Federation of American Societies for Experimental Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal binding assays with domain mapping, plus subcellular localization with functional context\",\n      \"pmids\": [\"14597674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CNK2/CNKSR2 is required for NGF- but not EGF-induced ERK activation in neuronal precursor cells, demonstrating stimulus-specific scaffold function. CNK2 additionally couples NGF signaling to membrane/cytoskeletal remodeling through a separate, essential contribution, indicating that it integrates multiple regulatory pathways needed for neuronal differentiation.\",\n      \"method\": \"siRNA knockdown in PC12 cells, ERK activation assays (immunoblot), morphological readouts of cytoskeletal remodeling\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function knockdown with two distinct, orthogonal phenotypic readouts (ERK signaling and cytoskeletal remodeling) and stimulus specificity controls\",\n      \"pmids\": [\"15028221\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The SAM domain of Drosophila CNK (the ortholog of CNKSR2) forms a 1:1 heterodimer with the SAM domain of HYP, as determined by X-ray crystal structure. This specific SAM-SAM dimerization mode is essential for RAF kinase signaling in vivo and facilitates recruitment of KSR to form the CNK/HYP/KSR regulatory complex.\",\n      \"method\": \"X-ray crystallography, in vitro mutagenesis, in vivo Drosophila signaling rescue assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure combined with mutagenesis and in vivo functional validation\",\n      \"pmids\": [\"18287031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Loss-of-function deletion of the CNKSR2 gene (removing 15 of 21 exons) causes X-linked intellectual disability with epilepsy, establishing CNKSR2 as a causative gene for NS-XLID. High brain expression and postsynaptic density localization support its role in RAS/MAPK-dependent synaptic signal transduction.\",\n      \"method\": \"Copy number analysis (array CGH), gene expression profiling, known PSD localization data\",\n      \"journal\": \"Molecular syndromology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — human deletion mapping with supporting expression/localization evidence but no direct functional experiment in this paper\",\n      \"pmids\": [\"22511892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CNKSR2 (CNK2) interacts constitutively with Vilse/ARHGAP39 via the WW domains of Vilse and a proline-rich motif in CNK2. CNK2 complexes are enriched for Rac/Cdc42 signaling components (Rac1, α/β-PIX, GIT1/2, PAK3/4, cytohesins). CNK2 acts as a spatial modulator of Rac GTP/GDP cycling during dendritic spine morphogenesis; disruption of the CNK2–Vilse interaction impairs spine formation by unbalancing RacGDP/GTP levels.\",\n      \"method\": \"Mass spectrometry interactome of endogenous CNK2, co-immunoprecipitation, domain-deletion mutagenesis, shRNA knockdown and rescue experiments in hippocampal neurons, spine morphology quantification\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — endogenous MS interactome plus reciprocal Co-IP, domain mapping, and loss-of-function rescue in primary neurons with defined morphological readout\",\n      \"pmids\": [\"24656827\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Smurf2 E3 ubiquitin ligase physically interacts with CNKSR2 and regulates AKT-dependent cell proliferation and invasion of breast cancer cells through the PI3K-PTEN-AKT-FoxO3a pathway in a CNKSR2-dependent manner. Silencing Smurf2 downregulates CNKSR2 and suppresses breast cancer cell growth.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, cell proliferation/invasion assays, cell cycle analysis, western blotting\",\n      \"journal\": \"Cell division\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP interaction and pathway epistasis by KD, single lab, single set of methods\",\n      \"pmids\": [\"25191523\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Smurf2 physically associates with CNKSR2 (confirmed by co-immunoprecipitation, indirect immunofluorescence, and surface plasmon resonance) and acts as a positive regulator of CNKSR2 stability: Smurf2 ubiquitinates CNKSR2 but protects it from proteasomal degradation. Smurf2 knockdown leads to enhanced polyubiquitination and proteasomal degradation of CNKSR2, reducing breast cancer cell proliferation and clonogenic survival.\",\n      \"method\": \"Co-immunoprecipitation, surface plasmon resonance, indirect immunofluorescence, ubiquitination assays, proteasome inhibitor experiments, siRNA knockdown\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — three independent methods (Co-IP, SPR, IF) confirming interaction, plus biochemical ubiquitination and proteasomal degradation assays\",\n      \"pmids\": [\"29534682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CNK2/CNKSR2 modulates the size of the postsynaptic density (PSD) in neurons and directs the subcellular localization of the regulatory kinase TNIK to dendritic spines. Both CNK2 and TNIK localize postsynaptically in hippocampal neurons; CNK2 expression is critical for maintaining TNIK at appropriate levels and location, establishing a functional scaffold–kinase relationship.\",\n      \"method\": \"Immunofluorescence co-localization with synaptic markers, overexpression and knockdown experiments in neurons, PSD size morphometry\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — localization with functional consequence (PSD size, TNIK localization), single lab\",\n      \"pmids\": [\"32235845\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cnksr2 knockout mice display spontaneous electrographic seizures, increased neural activity, anxiety, impaired learning/memory, and progressive loss of ultrasonic vocalizations. In vivo quantitative proteomics shows that Cnksr2 anchors key binding partners at synapses, and its loss significantly alters the synaptic proteome including proteins implicated in epilepsy. Cnksr2 is localized at both excitatory and inhibitory postsynapses in cortical, striatal, and cerebellar regions.\",\n      \"method\": \"Cnksr2 KO mouse model, EEG, behavioral assays, in vivo quantitative proteomics, immunolocalization\",\n      \"journal\": \"The Journal of neuroscience : the official journal of the Society for Neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with multiple orthogonal phenotypic readouts plus in vivo proteomics defining synaptic organization role\",\n      \"pmids\": [\"34580165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CNKSR2 forms a molecular complex with the ARF-GEF CYTH2 in the hippocampal dentate gyrus, and CYTH2 binding prevents proteasomal degradation of CNKSR2. Silencing either CNKSR2 or CYTH2 in vivo causes abnormal positioning of granule cell precursors at the granule cell layer–hilus boundary with characteristics of immature neurons, demonstrating that the CNKSR2–CYTH2 complex is necessary for dentate granule cell development and differentiation.\",\n      \"method\": \"Co-immunoprecipitation, shRNA in vivo knockdown in mouse via stereotaxic injection, immunohistochemistry, cell positioning quantification\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus in vivo loss-of-function with defined cellular differentiation phenotype\",\n      \"pmids\": [\"34800437\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CNK2/CNKSR2 promotes cancer cell migration by coupling the pro-metastatic receptor tyrosine kinase AXL to ARF6 GTPase activation. AXL signaling drives PI3K-dependent recruitment of CNK2 to the plasma membrane; CNK2 then stimulates ARF6 by associating with cytohesin ARF-GEFs and a novel adaptor SAMD12. ARF6-GTP coordinates RAC1 activation and RHOA inhibition to generate motile forces. Genetic ablation of CNK2 or SAMD12 reduces metastasis in a mouse xenograft model.\",\n      \"method\": \"Co-immunoprecipitation, proximity ligation assay, GTPase activity assays, shRNA/CRISPR ablation, live-cell migration assays, mouse xenograft metastasis model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal biochemical assays plus in vivo xenograft validation, two independent genetic ablations\",\n      \"pmids\": [\"37322019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"s-Afadin (a splice variant of afadin) preferentially binds MAGUIN/CNKSR2 over l-afadin in vivo and in vitro. Genetic ablation of MAGUIN impairs PSD-95 localization and AMPA receptor surface accumulation in cultured hippocampal neurons and reduces postsynaptic glutamatergic responses (without affecting presynaptic release), establishing CNKSR2 as a regulator of PSD-95-dependent AMPA receptor surface localization.\",\n      \"method\": \"Co-immunoprecipitation in vivo and in vitro, MAGUIN KO neurons, surface AMPA receptor immunostaining, whole-cell patch-clamp electrophysiology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — KO neurons with electrophysiology and biochemical surface receptor assays, multiple orthogonal methods\",\n      \"pmids\": [\"36803960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CNKSR2 functions downstream of retinoic acid (RA) signaling in the chick forebrain roof plate, where its knockdown phenocopies RA signaling inhibition (defective invagination, reduced cell proliferation, altered patterning). CNKSR2 exerts these effects by modulating the Ras/Raf/MEK signaling pathway, as demonstrated by epistasis experiments.\",\n      \"method\": \"In ovo siRNA knockdown in chick embryo, morphological analysis, BrdU proliferation assay, pharmacological MEK inhibition epistasis\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — in vivo knockdown with phenotypic and pathway epistasis, single organism model\",\n      \"pmids\": [\"36734326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CNKSR2 interacts with the microtubule motor protein DYNC1H1 and the centrosome marker CEP290, and localizes to centrosomes in neurons. Downregulation of CNKSR2 in Neuro-2A cells alters expression of numerous centrosomal genes and impairs centrosome-related functions including cell size, cell proliferation, and motility, revealing a previously unknown cytoplasmic/centrosomal role for CNKSR2.\",\n      \"method\": \"Immunoprecipitation–mass spectrometry, bioinformatic enrichment analysis, co-localization immunofluorescence, CNKSR2 siRNA knockdown with centrosome functional assays\",\n      \"journal\": \"Neural regeneration research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — IP-MS interactome plus co-localization and loss-of-function phenotyping, single lab\",\n      \"pmids\": [\"39359098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SAMD12 (and its homolog SAMD10) binds CNKSR1/2/3 scaffold proteins with exceptionally high affinity and quantitatively displaces MAP4K kinases (MAP4K4, MINK1, TNIK) from CNKSR2 complexes. CNKSR2 acts as both a scaffold and an activator of TNIK during neuronal synapse development; ectopic SAMD12 expression alters synapse development by inhibiting TNIK activity through its dissociation from CNKSR2.\",\n      \"method\": \"Biochemical binding assays (affinity measurements), competitive displacement assays, co-immunoprecipitation, ectopic expression in neurons with synapse morphology readout\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — quantitative binding assays with competitive displacement plus functional neuronal assays establishing scaffold-activator role\",\n      \"pmids\": [\"40010432\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CNKSR2 interacts with RSK kinases through a DDVF-like short linear motif (SLiM), using the same docking interface exploited by viral and bacterial pathogen proteins. This interaction was confirmed by co-immunoprecipitation, and the RSK docking site appears to participate in negative feedback regulation of the RAS-ERK MAPK pathway.\",\n      \"method\": \"AlphaFold docking prediction, co-immunoprecipitation, RSK docking-site mutagenesis with ERK activation readout\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP in preprint, computational prediction-guided, limited mechanistic follow-up specifically for CNKSR2\",\n      \"pmids\": [\"bio_10.1101_2024.08.08.607128\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Conditional deletion of Cnksr2 specifically in excitatory neurons recapitulates anxiety and ultrasonic vocalization (USV) deficits seen in global KO mice. Further restriction of Cnksr2 deletion to excitatory neurons of the anterior cingulate cortex (ACC) is sufficient to produce USV impairments, dissociating the communication deficit from seizure and anxiety phenotypes and identifying the ACC excitatory neuron population as the critical locus for CNKSR2-dependent vocal communication.\",\n      \"method\": \"Conditional (Cre-lox) neuronal subtype-specific KO mice, EEG, behavioral assays (USV, anxiety, learning), stereotaxic viral Cre delivery to ACC\",\n      \"journal\": \"eNeuro\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific conditional KO with multiple behavioral readouts and regional dissection of phenotype\",\n      \"pmids\": [\"39694826\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CNKSR2 (CNK2/MAGUIN) is a multidomain synaptic scaffold protein that organizes postsynaptic density complexes by bridging PSD-95/S-SCAM with Densin-180, anchors and activates the kinase TNIK, modulates Rac GTP/GDP cycling via its constitutive interaction with the RacGAP Vilse/ARHGAP39 to control dendritic spine morphogenesis, stabilizes AMPA receptor surface localization through a PSD-95-dependent mechanism, promotes NGF-selective ERK signaling and neuronal differentiation, couples AXL receptor signaling to ARF6/RAC1-driven cancer cell migration via cytohesin GEFs and SAMD12, is stabilized by CYTH2 binding and by Smurf2-mediated non-degradative ubiquitination, and is required in anterior cingulate cortex excitatory neurons for vocal communication, with its loss causing epilepsy-aphasia syndrome phenotypes in mice and humans.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CNKSR2 (also known as CNK2/KSR2/MAGUIN) is a multi-domain scaffold protein that integrates multiple signaling cascades in metabolic regulation, neuronal development, and cell motility. In the Raf-MEK-ERK pathway, CNKSR2 shows A-RAF selectivity and couples stimulus-specific signals—including NGF, retinoic acid, and Ca²⁺/calcineurin—to ERK activation, while independently scaffolding AMPK to regulate fatty acid oxidation, glucose metabolism, and energy homeostasis; brain-specific knockout recapitulates obesity and glucose intolerance, and CNKSR2 stabilizes AMPKα1 by competing with the CRL4A ubiquitin ligase at K52 [PMID:19883615, PMID:19560418, PMID:15028221, PMID:28180061, PMID:41424849]. At neuronal synapses, CNKSR2 localizes to both excitatory and inhibitory postsynaptic densities where it organizes PSD size, anchors TNIK kinase, recruits the RacGAP Vilse/ARHGAP39 to regulate Rac cycling during spine morphogenesis, and promotes AMPA receptor surface accumulation through interaction with s-Afadin; Cnksr2 knockout mice exhibit spontaneous seizures and altered synaptic proteomes, and region-specific deletion from anterior cingulate cortex excitatory neurons selectively ablates ultrasonic vocalizations [PMID:24656827, PMID:34580165, PMID:36803960, PMID:39694826]. CNKSR2 also couples AXL receptor tyrosine kinase signaling to ARF6-dependent RAC1/RHOA regulation via cytohesin GEFs and SAMD12, driving cancer cell migration and metastasis [PMID:37322019]. Rare CNKSR2 variants in humans are associated with obesity and impaired fatty acid and glucose oxidation that can be ameliorated by metformin [PMID:24209692].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing CNK2 as a stimulus-selective scaffold: the question of whether CNK2 participates in MAPK signaling in neurons was answered by showing it is required for NGF- but not EGF-induced ERK activation and also contributes to cytoskeletal remodeling independently of ERK, establishing CNK2 as a multi-output scaffold with receptor selectivity.\",\n      \"evidence\": \"RNAi/dominant-negative in neuronal precursor cells with NGF vs EGF epistasis and morphological readouts\",\n      \"pmids\": [\"15028221\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of upstream receptor-proximal coupling mechanism unknown\", \"Whether the cytoskeletal remodeling function operates through distinct domains was not resolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Three studies simultaneously expanded CNKSR2 from a simple MAPK scaffold to a multi-pathway integrator: (1) proteomics revealed A-RAF selectivity distinguishing it from KSR1, (2) calcineurin was identified as a selective CNKSR2 phosphatase coupling Ca²⁺ to ERK, and (3) CNKSR2 was shown to interact with and modulate AMPK, regulating fatty acid oxidation and thermogenesis in vivo.\",\n      \"evidence\": \"Functional proteomics in HEK-293 cells; phosphosite mapping and calcineurin co-IP in INS1/NG108 cells; KSR2 KO mice with metabolic phenotyping and MEF assays\",\n      \"pmids\": [\"19563921\", \"19560418\", \"19883615\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for A-RAF vs c-RAF selectivity not determined\", \"Calcineurin dephosphorylation sites mapped but functional consequence of individual sites not dissected\", \"Molecular interface between CNKSR2 and AMPK not resolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"The question of whether CNKSR2's metabolic and mitogenic functions are separable was resolved: a separation-of-function mutant unable to bind ERK still supported transformation, while constitutive AMPK activation complemented metabolic but not mitogenic functions, establishing AMPK and MAPK as parallel independent outputs of the CNKSR2 scaffold.\",\n      \"evidence\": \"ERK-interaction mutant, constitutive AMPK activation, and RNAi in KSR1-/- MEFs with Ras(V12)\",\n      \"pmids\": [\"22801368\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether AMPK-dependent energy metabolic reprogramming is sufficient for tumorigenesis in vivo not tested\", \"Domain(s) mediating AMPK interaction not mapped\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"CNKSR2's metabolic role was extended to human disease: rare KSR2 variants in obese individuals disrupted both Raf-MEK-ERK signaling and fatty acid/glucose oxidation in cells, and these defects were ameliorated by metformin, linking CNKSR2 to human obesity.\",\n      \"evidence\": \"Human sequencing, functional assays in transfected cells with metabolic readouts, metformin rescue\",\n      \"pmids\": [\"24209692\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Penetrance and effect size of individual variants in population not established\", \"Whether metformin acts through AMPK-dependent or -independent mechanisms downstream of CNKSR2 not clarified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"CNKSR2's neuronal function was mechanistically defined: it constitutively binds the RacGAP Vilse/ARHGAP39 and nucleates a complex containing Rac/Cdc42 signaling components (α-PIX, β-PIX, GIT1/2, PAK3/4, cytohesins) to spatially regulate Rac GTP/GDP cycling during dendritic spine morphogenesis; simultaneously, the KSR2-calcineurin axis was shown to regulate store-operated calcium entry via STIM1/ORAI1.\",\n      \"evidence\": \"Endogenous CNK2 interactome MS in neurons, domain mapping, spine morphogenesis rescue; KSR2 KO lymphocytes/fibroblasts with Ca²⁺ imaging and STIM1/ORAI1 puncta assays\",\n      \"pmids\": [\"24656827\", \"24672054\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Vilse recruitment is spatially regulated at individual spines not resolved\", \"Whether SOCE regulation is relevant in neurons specifically not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"CNKSR2 protein stability was linked to the Smurf2 ubiquitin ligase, which paradoxically stabilizes CNKSR2 by protecting it from proteasomal degradation rather than targeting it for destruction, and modulates downstream PI3K-AKT signaling.\",\n      \"evidence\": \"Co-IP, SPR binding, ubiquitination and proteasome inhibition assays in cancer cells\",\n      \"pmids\": [\"25191523\", \"29534682\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Smurf2 blocks a specific E3 ligase or acts through non-catalytic shielding is unclear\", \"In vivo relevance of Smurf2-CNKSR2 axis not tested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Brain-specific conditional KO demonstrated that hypothalamic/neuronal CNKSR2 is sufficient for obesity and glucose intolerance, but peripheral CNKSR2 independently modulates leptin and AICAR (AMPK) sensitivity, mechanistically separating central and peripheral metabolic functions.\",\n      \"evidence\": \"Nestin-Cre conditional KO with metabolic phenotyping, leptin and AICAR tolerance tests\",\n      \"pmids\": [\"28180061\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which peripheral tissue(s) mediate the AMPK-sensitivity arm not identified\", \"Neuronal circuit mediating hypothalamic energy regulation not defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"CNKSR2 was established as a postsynaptic scaffold that controls the subcellular localization and levels of the regulatory kinase TNIK at synapses, providing a mechanism for spatial kinase regulation at the PSD.\",\n      \"evidence\": \"Co-IP, immunofluorescence with synaptic markers, CNK2 depletion/overexpression with TNIK localization readout\",\n      \"pmids\": [\"32235845\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether TNIK mislocalization mediates specific CNKSR2-loss phenotypes (seizures, spine defects) not determined\", \"Direct vs indirect TNIK interaction not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Two advances: (1) CYTH2 was identified as a CNKSR2 binding partner that stabilizes CNKSR2 and is co-required for dentate granule cell development, and (2) Cnksr2 KO mice were shown to exhibit spontaneous seizures with altered synaptic proteomes, placing CNKSR2 at both excitatory and inhibitory postsynapses as a master organizer of the synaptic protein landscape.\",\n      \"evidence\": \"Co-IP and protein stability assays with in vivo shRNA in hippocampus; Cnksr2 KO mice with EEG, behavioral testing, subcellular fractionation, and quantitative synaptic proteomics\",\n      \"pmids\": [\"34800437\", \"34580165\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether seizure phenotype arises from excitatory, inhibitory, or combined postsynaptic dysfunction not resolved\", \"Mechanism by which CYTH2 prevents CNKSR2 degradation not molecularly defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"CNKSR2's roles were extended to bone biology (osteoblast-specific KO reveals cell-autonomous regulation of bone formation by influencing adipocyte-osteoblast fate) and to hepatocellular carcinoma (14-3-3ζ binding stabilizes CNKSR2 and hyperactivates MAPK, conferring sorafenib resistance).\",\n      \"evidence\": \"Osteoblast-specific conditional KO with micro-CT and histomorphometry; Co-IP with 14-3-3ζ, MAPK assays, and drug resistance assays in HCC cells\",\n      \"pmids\": [\"36342465\", \"35468812\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism of adipocyte-osteoblast fate switching by CNKSR2 unknown\", \"Whether 14-3-3ζ-CNKSR2 axis operates in non-cancer settings not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Two studies defined CNKSR2's roles in cell motility and synaptic function: (1) CNKSR2 couples AXL RTK to ARF6-dependent RAC1/RHOA regulation via cytohesin GEFs and SAMD12 to drive cancer cell migration and metastasis, and (2) CNKSR2 (as MAGUIN) interacts with s-Afadin and is required for PSD-95 localization and AMPA receptor surface accumulation at hippocampal synapses; additionally, CNKSR2 was placed downstream of retinoic acid signaling in chick forebrain patterning.\",\n      \"evidence\": \"CRISPR ablation, ARF6/RAC1/RHOA activity assays, xenograft metastasis model; MAGUIN KO with mEPSC recording and surface biotinylation; chick in ovo RNAi with RA epistasis\",\n      \"pmids\": [\"37322019\", \"36803960\", \"36734326\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the AXL-CNKSR2-ARF6 axis is active in normal neuronal physiology not explored\", \"How s-Afadin-CNKSR2 interaction feeds into AMPA receptor trafficking machinery not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Region- and cell-type-specific Cnksr2 deletion from excitatory neurons of the anterior cingulate cortex selectively ablated ultrasonic vocalizations without causing seizures or anxiety, dissociating communication deficits from other Cnksr2-loss phenotypes and identifying a specific neural circuit requirement.\",\n      \"evidence\": \"Cre-dependent conditional KO with cell-type and region specificity, USV assays, EEG, behavioral testing\",\n      \"pmids\": [\"39694826\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Synaptic mechanism underlying USV loss in ACC neurons not defined\", \"Whether this maps to a specific CNKSR2 signaling output (MAPK, Rac, TNIK) unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"SAMD12 was shown to bind CNKSR2 with very high affinity and quantitatively displace MAP4K kinases (TNIK, MAP4K4, MINK1), revealing a competitive switching mechanism on the CNKSR2 scaffold that regulates TNIK activity during synapse development; concurrently, CNKSR2 was shown to stabilize AMPKα1 by competing with the CRL4A ubiquitin ligase at K52, directly linking CNKSR2 scaffolding to AMPK protein homeostasis.\",\n      \"evidence\": \"Biochemical competition/displacement assays, synapse development assays; site-specific competition binding, ubiquitination assays, endothelial-specific KSR2 overexpression with atherosclerosis models\",\n      \"pmids\": [\"40010432\", \"41424849\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo confirmation of SAMD12-TNIK competition at endogenous synapses not yet shown\", \"Whether K52-dependent AMPK stabilization operates in neurons not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: how CNKSR2's multiple scaffold functions (MAPK, AMPK, Rac/ARF6, TNIK) are coordinated in single cells; the structural basis for multi-partner engagement; the relative contribution of each signaling arm to the epilepsy, obesity, and neurodevelopmental phenotypes; and whether CNKSR2 loss-of-function constitutes a human Mendelian neurodevelopmental disorder with defined genotype-phenotype relationships.\",\n      \"evidence\": \"Gaps across the literature\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length structure or cryo-EM of CNKSR2 in complex with partners\", \"Relative contribution of MAPK vs AMPK vs Rac arms to seizure phenotype undefined\", \"Human genetic evidence for CNKSR2-linked neurodevelopmental disorder not in this timeline\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2, 3, 5, 15, 17]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 5, 22]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [15]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [19]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 4, 5, 6, 15, 20, 22]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 5, 6, 22]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [3, 13, 14, 16, 17, 24]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [12, 20, 24]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"ARHGAP39\",\n      \"TNIK\",\n      \"CYTH2\",\n      \"SAMD12\",\n      \"ARAF\",\n      \"AMPK\",\n      \"SMURF2\",\n      \"YWHAZ\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"CNKSR2 is a multidomain scaffold protein that organizes signaling complexes at neuronal synapses and in cancer cell migration by bridging Ras/MAPK pathway components, small GTPase regulators, and postsynaptic density proteins. At excitatory synapses, CNKSR2 links PSD-95 and Densin-180 into ternary complexes, recruits and activates the kinase TNIK, spatially modulates Rac GTP/GDP cycling through its constitutive partner ARHGAP39/Vilse, and stabilizes AMPA receptor surface expression via PSD-95, thereby controlling dendritic spine morphogenesis and postsynaptic glutamatergic transmission [PMID:10207009, PMID:12390249, PMID:24656827, PMID:36803960, PMID:40010432]. In non-neuronal contexts, CNKSR2 couples AXL receptor signaling to ARF6/RAC1-driven migration through cytohesin GEFs and the adaptor SAMD12, and its stability is maintained by Smurf2-mediated non-degradative ubiquitination and by CYTH2 binding [PMID:37322019, PMID:29534682, PMID:34800437]. Loss-of-function mutations in CNKSR2 cause X-linked intellectual disability with seizures and language impairment (epilepsy–aphasia spectrum), and conditional deletion in anterior cingulate cortex excitatory neurons is sufficient to produce vocal communication deficits [PMID:22511892, PMID:34580165, PMID:39694826].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Establishing CNKSR2 as a synaptic scaffold: discovery of its PDZ-binding motif-mediated interactions with PSD-95 and S-SCAM placed it within the postsynaptic density complex, answering how this novel neuronal protein integrates into synaptic architecture.\",\n      \"evidence\": \"Co-immunoprecipitation from rat synaptosomes, yeast two-hybrid, domain-deletion localization in transfected cells\",\n      \"pmids\": [\"10207009\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No functional consequence of disrupting these interactions was tested\", \"Endogenous stoichiometry unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defining CNKSR2 as an obligatory molecular bridge: demonstration that CNKSR2 is required to link Densin-180 to PSD-95 in a ternary complex resolved how these two PSD proteins associate and revealed CNKSR2's non-redundant scaffolding role.\",\n      \"evidence\": \"Ternary complex reconstitution in transfected cells, Co-IP from rat brain, domain mapping\",\n      \"pmids\": [\"12390249\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of disrupting the ternary complex in neurons not tested\", \"Multimerization role unexplored\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Connecting CNKSR2 to the Ras/MAPK cascade: identification of interactions with RAF1, Ral GTPase, and RalGDS-family GEFs established CNKSR2 as a Ras-effector scaffold, extending its function beyond static PSD scaffolding to active signaling pathway organization.\",\n      \"evidence\": \"Co-IP, GST pulldown domain mapping, MAPK-dependent phosphorylation, subcellular fractionation in MDCK cells\",\n      \"pmids\": [\"14597674\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous Ras-pathway scaffolding not shown in neurons at this stage\", \"Specific Ras isoform selectivity not determined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrating stimulus-specific signaling: siRNA knockdown revealed CNKSR2 is required for NGF- but not EGF-induced ERK activation, answering whether CNKSR2 provides pathway selectivity and linking its scaffold function to neuronal differentiation.\",\n      \"evidence\": \"siRNA knockdown in PC12 cells, ERK activation immunoblots, morphological cytoskeletal readouts\",\n      \"pmids\": [\"15028221\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of stimulus selectivity not defined\", \"Whether selectivity applies in primary neurons untested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Structural basis for SAM-domain-mediated complex assembly: the crystal structure of the Drosophila CNK SAM domain bound to HYP revealed the heterodimeric interface critical for RAF signaling, providing the first atomic-level insight into how CNK-family scaffolds nucleate kinase complexes.\",\n      \"evidence\": \"X-ray crystallography, mutagenesis, in vivo Drosophila rescue assays\",\n      \"pmids\": [\"18287031\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Human CNKSR2 SAM domain structure not solved\", \"Whether the same heterodimer mode operates with mammalian partners remains inferential\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Revealing CNKSR2 as a Rac-GTP/GDP cycling modulator: the constitutive CNKSR2–Vilse/ARHGAP39 interaction and its requirement for dendritic spine morphogenesis answered how CNKSR2 spatially regulates small GTPase signaling at spines, unifying its scaffold and morphogenesis roles.\",\n      \"evidence\": \"Endogenous IP-MS interactome, domain-deletion mutagenesis, shRNA knockdown and rescue in hippocampal neurons, spine morphometry\",\n      \"pmids\": [\"24656827\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Vilse-independent CNK2 functions contribute to spine formation not dissected\", \"Temporal dynamics of Rac cycling at spines not measured\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defining CNKSR2 protein stability regulation: Smurf2 was shown to ubiquitinate CNKSR2 in a non-degradative manner that protects it from proteasomal turnover, establishing a post-translational mechanism controlling CNKSR2 abundance relevant to cancer cell proliferation.\",\n      \"evidence\": \"Co-IP, surface plasmon resonance, ubiquitination assays, proteasome inhibitor experiments in breast cancer cells\",\n      \"pmids\": [\"29534682\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain type not characterized\", \"Whether this stabilization mechanism operates in neurons unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"In vivo loss-of-function phenotyping: Cnksr2 knockout mice displayed seizures, vocalization loss, and synaptic proteome remodeling, directly establishing CNKSR2 as essential for neural circuit function and modeling the human epilepsy-aphasia syndrome.\",\n      \"evidence\": \"Cnksr2 KO mouse, EEG, behavioral assays, quantitative synaptic proteomics, immunolocalization at excitatory and inhibitory synapses\",\n      \"pmids\": [\"34580165\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Causal contribution of individual synaptic protein changes not delineated\", \"Inhibitory synapse-specific role of CNKSR2 not functionally tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"CYTH2-dependent stabilization and dentate gyrus development: CYTH2 binding prevents CNKSR2 proteasomal degradation and the complex is required for granule cell differentiation, revealing a second stability-control mechanism and a neurodevelopmental role beyond synapse organization.\",\n      \"evidence\": \"Co-IP, in vivo shRNA knockdown via stereotaxic injection in mouse, immunohistochemistry, cell positioning analysis\",\n      \"pmids\": [\"34800437\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CYTH2 binding blocks CNKSR2 degradation mechanistically is unresolved\", \"Relationship to Smurf2-mediated stabilization not explored\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"CNKSR2 controls AMPA receptor surface expression: genetic ablation of CNKSR2 impaired PSD-95 localization and reduced surface AMPA receptors and postsynaptic responses, answering a longstanding question about the functional consequence of the PSD-95–CNKSR2 scaffold interaction for glutamatergic transmission.\",\n      \"evidence\": \"MAGUIN KO neurons, surface AMPA receptor immunostaining, whole-cell patch-clamp electrophysiology\",\n      \"pmids\": [\"36803960\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific AMPA receptor subunit dependence not determined\", \"Whether inhibitory receptor trafficking is also affected untested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Cancer migration mechanism: CNKSR2 was shown to couple AXL receptor signaling to ARF6/RAC1-driven migration via cytohesin GEFs and SAMD12, with ablation reducing metastasis in vivo, answering how CNKSR2 scaffold function is co-opted in cancer.\",\n      \"evidence\": \"Co-IP, proximity ligation assay, GTPase activity assays, CRISPR/shRNA ablation, mouse xenograft metastasis model\",\n      \"pmids\": [\"37322019\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Applicability beyond the tested cancer cell lines not established\", \"Whether neuronal CNKSR2–cytohesin signaling uses the same AXL-dependent mechanism unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"SAMD12 as a competitive regulator of CNKSR2–TNIK complexes: quantitative binding assays showed SAMD12 displaces MAP4K kinases (including TNIK) from CNKSR2, and ectopic SAMD12 disrupts synapse development, establishing CNKSR2 as both a scaffold and an activator of TNIK and identifying SAMD12 as a natural antagonist.\",\n      \"evidence\": \"Biochemical affinity measurements, competitive displacement assays, Co-IP, neuronal synapse morphology readouts\",\n      \"pmids\": [\"40010432\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous SAMD12 expression levels in brain regions not quantified\", \"Whether SAMD12 regulation is developmentally dynamic remains unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Circuit-level dissection: conditional deletion in ACC excitatory neurons sufficed to reproduce vocalization deficits, dissociating the communication phenotype from seizures and anxiety and identifying the critical neuronal population for CNKSR2-dependent vocal communication.\",\n      \"evidence\": \"Cre-lox conditional KO mice, stereotaxic viral Cre delivery to ACC, EEG, behavioral assays\",\n      \"pmids\": [\"39694826\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism downstream of CNKSR2 loss in ACC neurons not defined\", \"Whether restoration of CNKSR2 in ACC can rescue established deficits untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Several major mechanistic questions remain: (1) the structural basis of human CNKSR2 scaffold assembly is unknown; (2) how the two protein-stability mechanisms (Smurf2 and CYTH2) are coordinated is unresolved; (3) the specific contribution of CNKSR2 at inhibitory synapses has not been functionally dissected; and (4) whether CNKSR2's centrosomal role is relevant in vivo remains unvalidated.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal or cryo-EM structure of mammalian CNKSR2\", \"Smurf2 vs CYTH2 stabilization interplay untested\", \"Inhibitory synapse function of CNKSR2 not explored beyond localization\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 6, 9, 12, 16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 2, 12]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3, 4, 6, 12, 14]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [0, 1, 6, 9, 10, 13, 18]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [3, 11, 14]}\n    ],\n    \"complexes\": [\n      \"PSD-95/Densin-180/CNKSR2 ternary complex\",\n      \"CNKSR2–Vilse/ARHGAP39 complex\",\n      \"CNKSR2–CYTH2 complex\"\n    ],\n    \"partners\": [\n      \"DLG4\",\n      \"ARHGAP39\",\n      \"TNIK\",\n      \"CYTH2\",\n      \"SAMD12\",\n      \"SMURF2\",\n      \"RAF1\",\n      \"LRFN1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}