{"gene":"CNKSR1","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1998,"finding":"Drosophila CNK (connector enhancer of KSR) physically interacts with RAF through its C-terminal portion and is required for RAS signaling upstream of or in parallel to RAF; CNK contains multiple protein-protein interaction domains and localizes to cell-cell contact regions","method":"Genetic screen (modifier of ksr phenotype), co-immunoprecipitation, subcellular localization","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — genetic screen combined with direct binding and localization in founding paper with high citation count","pmids":["9814705"],"is_preprint":false},{"year":1999,"finding":"The N-terminal portion of CNK cooperates with RAS (specifically the RAS(V12G37) effector loop mutant that activates the RAL pathway), while the C-terminal portion regulates RAF in the MAPK pathway, suggesting CNK functions in more than one pathway downstream of RAS","method":"Transfection/overexpression in Drosophila eye, epistasis with RAS effector loop mutants","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — multiple effector loop mutant epistasis experiments with defined pathway readouts","pmids":["10557308"],"is_preprint":false},{"year":2003,"finding":"Drosophila CNK has bimodal (antagonistic) properties toward RAF: the N-terminal SAM and CRIC domains are essential for RAF activation, while a C-terminal bipartite RAF-inhibitory region (RIR) inhibits RAF catalytic function; together these prevent signaling leakage in the absence of signal but enable potent RAF activation upon stimulation","method":"Transfection experiments, RNAi-based rescue assay in Drosophila S2 cells, domain deletion/mutation analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — multiple domain-swap and mutagenesis experiments with functional readout, replicated by RNAi rescue","pmids":["14517245"],"is_preprint":false},{"year":2004,"finding":"Human CNK1 (CNKSR1) interacts with RhoA in a GTP-dependent manner through its PH domain; CNK1 depletion by RNAi inhibits Rho-induced gene expression via serum response factor but not Rho-induced stress fiber formation; CNK1 also associates with Rhophilin and RalGDS, linking Rho and Ras pathways","method":"Yeast two-hybrid, co-immunoprecipitation, RNAi knockdown, reporter assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP combined with RNAi knockdown with defined transcriptional phenotype","pmids":["14749388"],"is_preprint":false},{"year":2004,"finding":"CNK1 binds directly to RASSF1A (and RASSF1C) and this interaction mediates association of CNK1 with MST1/MST2 kinases; co-expression of CNK1 with RASSF1A greatly augments CNK1-induced apoptosis; CNK1-induced apoptosis is suppressed by dominant-negative MST1/MST2","method":"Co-immunoprecipitation, yeast two-hybrid, overexpression/apoptosis assays, dominant-negative suppression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — direct binding demonstrated by pulldown/CoIP with multiple functional validation experiments","pmids":["15075335"],"is_preprint":false},{"year":2005,"finding":"CNK1 acts as a scaffold mediating Src-dependent tyrosine phosphorylation and activation of Raf-1; CNK1 binds pre-activated Raf-1 and activated Src, forming a trimeric complex; siRNA knockdown of endogenous CNK1 interferes with Src-dependent ERK activation","method":"Co-immunoprecipitation, siRNA knockdown, kinase activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — trimeric complex demonstrated by CoIP, loss-of-function by siRNA with defined ERK activation phenotype","pmids":["15845549"],"is_preprint":false},{"year":2005,"finding":"CNK1 interacts with Net1 or p115RhoGEF (Rho-specific GEFs) and with MLK2 and MKK7 (JNK cascade kinases); CNK1 acts cooperatively with these GEFs to activate JNK MAP kinase but not other Rho-mediated pathways; endogenous CNK1 is required for serum/S1P-stimulated Rho-dependent JNK activation in HeLa cells","method":"Co-immunoprecipitation, siRNA knockdown, reporter assays, kinase activation assays","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — CoIP with multiple binding partners, RNAi knockdown with defined JNK pathway phenotype","pmids":["15753034"],"is_preprint":false},{"year":2005,"finding":"In Drosophila, the Src42 tyrosine kinase associates in an RTK-dependent manner with a conserved region of CNK located C-terminal to the RAF-inhibitory region (RIR), and this association counteracts the repressive RIR effect on RAF; derepression by Src42 depends on its SH3/SH2 domains but not its catalytic activity; several cnk loss-of-function alleles have mutations that impair Src42 binding","method":"Genetic allele mapping, co-immunoprecipitation, dominant-negative/kinase-dead Src constructs","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — multiple cnk allele analysis combined with binding domain mapping and catalytic-dead controls","pmids":["15660123"],"is_preprint":false},{"year":2005,"finding":"CNK1 binds the angiotensin II type 2 (AT2) receptor through its SAM and CRIC domains; the interaction is strengthened by a Ras-regulated linker region; endogenous CNK1 and AT2 receptor co-precipitate from mouse heart extracts","method":"Co-immunoprecipitation, domain mapping, pulldown from tissue extracts","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 — single-lab Co-IP with domain mapping but limited functional follow-up","pmids":["16289034"],"is_preprint":false},{"year":2006,"finding":"A KSR/CNK complex mediated by a novel SAM domain-containing protein HYP (Hyphen) regulates RAS-dependent RAF activation in Drosophila; KSR induces RAF activation via its kinase-like domain independently of its scaffolding property or kinase activity; KSR is recruited to RAF prior to signal activation by CNK/HYP","method":"Genetic epistasis, co-immunoprecipitation, RNAi in Drosophila S2 cells, domain-function analysis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — multi-approach including genetics, CoIP, and domain dissection across multiple experiments","pmids":["16600912"],"is_preprint":false},{"year":2008,"finding":"Crystal structure of the SAM domain of CNK in complex with the SAM domain of HYP reveals a 1:1 heterodimer with single-junction interaction mode; mutational analysis shows this specific dimerization is essential for RAF signaling and facilitates recruitment of KSR to form the CNK/HYP/KSR regulatory complex","method":"X-ray crystallography, in vitro and in vivo mutational analysis, co-immunoprecipitation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional validation by in vitro and in vivo mutagenesis","pmids":["18287031"],"is_preprint":false},{"year":2010,"finding":"The major binding partners of CNK1 scaffold are cytohesins (Arf GEFs); CNK1 interacts constitutively with the coiled-coil domain of cytohesins via its C-terminal region; CNK1/cytohesin interaction facilitates membrane recruitment of cytohesin-2 following insulin stimulation and promotes PI3K/AKT cascade activation by generating PIP2-rich microenvironment for IRS1 recruitment","method":"Mass spectrometry interactome, co-immunoprecipitation, protein depletion/rescue, membrane fractionation","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — MS interactome validated by CoIP, depletion/rescue with defined signaling phenotypes","pmids":["20634316"],"is_preprint":false},{"year":2010,"finding":"CNK1 interacts with AKT and knockdown of CNK1 decreases AKT activity in breast cancer cells; CNK1 controls AKT-dependent phosphorylation of FoxO, and CNK1-induced proliferation is blocked by FoxO overexpression; CNK1 promotes NF-κB2 p100 processing to p52 and nuclear localization, driving MMP-9/MT1-MMP expression and cell invasion","method":"Co-immunoprecipitation, siRNA knockdown, reporter assays, proliferation/invasion assays","journal":"Oncogene / Molecular cancer research","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods across two papers from the same lab establishing AKT and NF-κB scaffolding roles","pmids":["20383191","20197385"],"is_preprint":false},{"year":2014,"finding":"GEF-H1 acts as an adaptor linking PP2A B' subunits to KSR-1, mediating dephosphorylation of KSR-1 S392 and activation of MAPK signaling; this constitutes a positive feedback loop for the RAS/MAPK pathway independent of GEF-H1's RhoGEF activity","method":"Co-immunoprecipitation, phosphorylation assays, siRNA knockdown, xenograft models","journal":"Cancer cell","confidence":"High","confidence_rationale":"Tier 2 — CoIP demonstrating trimeric complex, phosphorylation assays, and in vivo validation","pmids":["24525234"],"is_preprint":false},{"year":2014,"finding":"EphrinB1 interacts with CNK1 in an EphB receptor-independent manner; co-transfection of ephrinB1 with CNK1 increases JNK phosphorylation; CNK1 overexpression alone activates RhoA, but both ephrinB1 and CNK1 are required for JNK phosphorylation; CNK1 depletion abrogates ephrinB1-mediated cell migration and JNK activation; adhesion to fibronectin or Src activity increases ephrinB1/CNK1 binding","method":"Co-immunoprecipitation, siRNA knockdown, kinase activation assays, cell migration assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods with defined mechanistic phenotypes","pmids":["24825906"],"is_preprint":false},{"year":2016,"finding":"AKT phosphorylates CNKSR1/CNK1 at Ser22 within the SAM domain, triggering SAM domain-dependent oligomerization; oligomeric CNK1 increases affinity for active AKT (positive feedback); phosphorylation of Thr8 within the SAM domain by EGF signaling prevents AKT binding and antagonizes CNK1-mediated AKT signaling","method":"Phosphorylation site mapping (mass spectrometry), mutagenesis, co-immunoprecipitation, oligomerization assays","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"High","confidence_rationale":"Tier 1-2 — phosphorylation site identified by MS, validated by mutagenesis with functional readouts","pmids":["27769899"],"is_preprint":false},{"year":2015,"finding":"PDGF induces phosphorylation of CNK1 at Tyr26; Src induces phosphorylation at Tyr519 and Tyr665; phosphorylation of Tyr519 recruits CNK1 to the nucleus; additional Tyr26 phosphorylation enables SRE-dependent gene expression; phosphorylation-deficient mutants promote MMP14 promoter activity; PDGF-stimulated Src recruits CNK1 to plasma membrane","method":"Phosphorylation site mapping, phosphomimetic/deficient mutants, subcellular localization, reporter assays","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 — phosphosite mapping with mutagenesis and localization data, single-lab study","pmids":["26319181"],"is_preprint":false},{"year":2017,"finding":"ERK signaling induces acetylation of CNK1 at Lys414 (in the PH domain) by the acetyltransferase CBP; SIRT2 deacetylates CNK1; acetylation drives membrane localization of CNK1 in growth factor-stimulated cells and promotes interaction with CRAF, stimulating ERK-dependent cell proliferation and migration; constitutively acetylated or membrane-anchored CNK1 mutants constitutively activate this positive feedback","method":"Mass spectrometry (acetylation site ID), mutagenesis, co-immunoprecipitation, subcellular fractionation/live imaging, proliferation/migration assays","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1-2 — PTM identified by MS, writer (CBP) and eraser (SIRT2) identified by CoIP, mutagenesis validates functional consequence","pmids":["28819643"],"is_preprint":false},{"year":2021,"finding":"CNKSR1 serves as a scaffold that interacts with RhoB-GTP (identified via human protein arrays) and with PTPRH (protein tyrosine phosphatase receptor type H) at the plasma membrane; when RhoB is degraded by CUL3/KCTD10, CNKSR1 binds PTPRH and inactivates its EGFR phosphatase activity; accumulation of RhoB-GTP displaces PTPRH from CNKSR1, releasing active PTPRH to dephosphorylate/suppress EGFR and HER2","method":"Protein array binding screen, co-immunoprecipitation, siRNA knockdown, membrane fractionation","journal":"Life science alliance","confidence":"High","confidence_rationale":"Tier 2 — protein array identification of interactors validated by CoIP, mechanistic model supported by depletion experiments with defined EGFR phosphorylation readout","pmids":["34187934"],"is_preprint":false},{"year":2019,"finding":"The PH domain of CNK1 (CNKSR1) binds PtdIns(4,5)P2 with higher affinity than PtdIns(3,4,5)P3 and mediates plasma membrane colocalization with mutant KRAS; a small molecule (PHT-7.3) that binds selectively to the CNK1 PH domain prevents its plasma membrane colocalization with mutant KRAS and blocks mutant-KRAS but not wild-type KRAS cancer cell growth","method":"In vitro lipid binding assay, molecular modeling, cell imaging, pharmacological inhibition, cell growth assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — lipid binding assay combined with small molecule inhibitor and imaging, single-lab study","pmids":["31040156"],"is_preprint":false},{"year":2023,"finding":"MEK inhibition in CNKSR1-high cancer cells induces translocation of CNKSR1 to the plasma membrane where it interacts with and stabilizes phosphorylated AKT, activating PI3K/AKT signaling as an adaptive resistance mechanism; this is associated with reduced FoxO1 nuclear translocation","method":"siRNA knockdown, co-immunoprecipitation, subcellular fractionation, pharmacological inhibition, in vivo xenograft","journal":"Molecular cancer research : MCR","confidence":"Medium","confidence_rationale":"Tier 2 — CoIP and fractionation with defined signaling phenotype, single-lab study","pmids":["36790955"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM structure of the minimal Drosophila KSR-MEK-CNK-HYP complex reveals a ring-like arrangement; CNK simultaneously engages both KSR and MEK, stabilizing their binary interaction; CNK-HYP scaffolding complex promotes RAF activation by enhancing KSR-MEK interaction, which in turn drives RAF-KSR heterodimerization and RAF catalytic activation","method":"Cryo-EM structure determination, in vitro binding assays, mutagenesis, functional RAF activation assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure with in vitro reconstitution and mutagenesis validating functional model","pmids":["38388830"],"is_preprint":false},{"year":2018,"finding":"CNKSR1 (human KSR1 ortholog context): CNKSR1 loss-of-function frameshift mutation causes syndromic autosomal recessive intellectual disability; RNAi knockdown of cnk (CNKSR1 ortholog) in Drosophila brain causes defects in eye and mushroom body structures","method":"Next-generation sequencing, RT-PCR/western blot (patient lymphoblastoid cells), RNAi in Drosophila","journal":"American journal of medical genetics. Part B, Neuropsychiatric genetics","confidence":"Medium","confidence_rationale":"Tier 2-3 — human loss-of-function genetics plus Drosophila RNAi with neurological phenotype, limited mechanistic detail","pmids":["30450701"],"is_preprint":false},{"year":2003,"finding":"Human CNK2 (CNK2A and splice variant CNK2B) interacts with Raf through the Raf regulatory and kinase domains as well as the C-terminal half of CNK2; CNK2 also interacts with Ral GTPase and RalGDS family member Rlf via the GEF domain; CNK2 localizes to lateral plasma membrane in MDCK cells","method":"Co-immunoprecipitation, domain mapping, subcellular fractionation/imaging","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP with domain mapping, single-lab study","pmids":["14597674"],"is_preprint":false},{"year":2016,"finding":"Optogenetic clustering of CNK1 reveals that low-intensity light/low EGF drives CNK1-RAF interaction and ERK-dependent differentiation, while higher intensity/higher EGF drives active AKT binding to CNK1 which phosphorylates and inhibits RAF; CNK1 acts as a molecular switch controlling differentiation vs. proliferation decisions","method":"Optogenetic clustering (light-controlled oligomerization), co-immunoprecipitation, kinase activity assays, cell fate assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — novel optogenetic approach with CoIP, single-lab study","pmids":["27901111"],"is_preprint":false},{"year":2004,"finding":"Leukocyte-specific protein 1 (LSP1) associates with KSR, MEK1 and ERK2, and targets them to peripheral actin filaments; LSP1-associated MEK1 is activated by anti-IgM in a PKCβI-dependent manner; this places CNKSR1/KSR as part of a cytoskeletal signaling complex","method":"Co-immunoprecipitation, confocal microscopy, kinase activation assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP and imaging demonstrating cytoskeletal localization with partial functional readout","pmids":["15090600"],"is_preprint":false}],"current_model":"CNKSR1 (connector enhancer of kinase suppressor of Ras 1) functions as a multidomain scaffold protein that integrates signals from multiple GTPases (RAS, RhoA, RhoB, RalGDS) by simultaneously binding RAF, MEK, KSR, and cytohesins, regulating the RAF-MEK-ERK MAPK cascade through bimodal control of RAF activity (with an N-terminal activation region and C-terminal inhibitory element), coordinating JNK pathway activation downstream of Rho GEFs, mediating insulin/AKT/PI3K signaling via cytohesin interactions, controlling EGFR/HER2 phosphorylation through RhoB-GTP-gated recruitment of the PTPRH phosphatase, and serving as a positive feedback node via ERK-induced acetylation and AKT-induced SAM domain oligomerization that drive its membrane localization and sustained signaling."},"narrative":{"teleology":[{"year":1998,"claim":"The founding discovery that CNK physically interacts with RAF and is genetically required for RAS signaling established it as a scaffold linking RAS pathway activation to RAF regulation.","evidence":"Genetic modifier screen of ksr in Drosophila eye combined with co-immunoprecipitation and subcellular localization","pmids":["9814705"],"confidence":"High","gaps":["No mammalian ortholog characterized","Mechanism by which CNK activates RAF unknown","Binding domains on RAF not mapped"]},{"year":1999,"claim":"Epistasis with RAS effector-loop mutants revealed that CNK operates in more than one RAS-effector branch — the N-terminus cooperates with the RAL pathway while the C-terminus regulates RAF — expanding CNK's role beyond simple RAF scaffolding.","evidence":"Overexpression with RAS effector loop mutants in Drosophila eye development","pmids":["10557308"],"confidence":"High","gaps":["RAL-branch mechanism not defined at the molecular level","Whether the two branches function independently or coordinately was unclear"]},{"year":2003,"claim":"Domain dissection uncovered bimodal control of RAF: N-terminal SAM/CRIC domains activate RAF while a C-terminal RIR inhibits it, explaining how CNK prevents basal signaling yet enables stimulus-dependent activation.","evidence":"Domain deletion/mutation analysis with RNAi rescue in Drosophila S2 cells","pmids":["14517245"],"confidence":"High","gaps":["Identity of the signal that relieves RIR-mediated inhibition unknown","No structural information on SAM/CRIC activation mechanism"]},{"year":2004,"claim":"Identification of GTP-dependent RhoA binding via the PH domain, plus interactions with Rhophilin and RalGDS, established human CNK1 as a node integrating Rho and Ras pathways in transcriptional control (SRF-dependent genes) and apoptosis (via RASSF1A/MST1).","evidence":"Yeast two-hybrid, co-immunoprecipitation, RNAi knockdown, and reporter assays in mammalian cells","pmids":["14749388","15075335"],"confidence":"High","gaps":["Direct structural basis of RhoA-PH domain interaction unresolved","In vivo relevance of RASSF1A-CNK1-MST1 apoptotic axis not tested in animal models"]},{"year":2005,"claim":"Multiple studies converged to show that CNK1 scaffolds distinct MAPK modules — Src/Raf-1/ERK and Rho-GEF/MLK2/MKK7/JNK — and that Src binding to a conserved C-terminal region relieves the RAF-inhibitory region, answering the key question of how RIR-mediated repression is derepressed.","evidence":"Co-immunoprecipitation of trimeric Src-CNK1-Raf-1 complex, siRNA knockdown with ERK/JNK readouts in HeLa cells, Drosophila genetic allele mapping of Src42 binding site","pmids":["15845549","15753034","15660123"],"confidence":"High","gaps":["Whether Src derepression requires catalytic activity or only SH2/SH3 docking in mammalian cells untested","Structural basis of JNK-branch scaffold assembly unknown"]},{"year":2006,"claim":"Discovery of the adaptor protein HYP (Hyphen/AVVRK) as the missing link that bridges CNK's SAM domain to KSR explained how CNK recruits KSR to RAF prior to signal activation.","evidence":"Genetic epistasis, co-immunoprecipitation, and RNAi in Drosophila S2 cells","pmids":["16600912"],"confidence":"High","gaps":["Mammalian HYP ortholog function not yet validated","Stoichiometry of the CNK-HYP-KSR complex unknown"]},{"year":2008,"claim":"The crystal structure of the CNK-SAM/HYP-SAM heterodimer revealed a single-junction 1:1 interaction mode, and mutagenesis confirmed that this interface is essential for RAF signaling, providing the first atomic-resolution view of the scaffolding mechanism.","evidence":"X-ray crystallography with in vivo and in vitro mutagenesis validation","pmids":["18287031"],"confidence":"High","gaps":["Full-length complex structure unavailable","Whether SAM-SAM interaction is regulated by post-translational modifications unknown"]},{"year":2010,"claim":"Mass spectrometry-based interactomics identified cytohesins as the major CNK1 binding partners and showed that CNK1 scaffolds cytohesin-2 membrane recruitment to activate PI3K/AKT signaling upon insulin stimulation, broadening CNK1's role beyond MAPK to metabolic signaling.","evidence":"Mass spectrometry interactome, co-immunoprecipitation, depletion/rescue, membrane fractionation","pmids":["20634316"],"confidence":"High","gaps":["Whether CNK1-cytohesin interaction is conserved in all insulin-responsive tissues not tested","Relative contribution of CNK1 vs. other cytohesin scaffolds undetermined"]},{"year":2010,"claim":"CNK1 was shown to interact with AKT and drive AKT-dependent FoxO phosphorylation and NF-κB2 processing, linking scaffold function to pro-proliferative and pro-invasive transcriptional programs in breast cancer cells.","evidence":"Co-immunoprecipitation, siRNA knockdown, reporter assays, invasion assays in breast cancer cell lines","pmids":["20383191","20197385"],"confidence":"High","gaps":["Whether CNK1-AKT interaction is direct or mediated by cytohesins not fully resolved","In vivo tumor model validation limited"]},{"year":2016,"claim":"AKT-dependent phosphorylation of Ser22 in the SAM domain was found to trigger CNK1 oligomerization and create a positive feedback loop with AKT, while optogenetic clustering revealed that CNK1 oligomerization state switches cell fate between differentiation (ERK) and proliferation (AKT).","evidence":"Phosphorylation site mapping by mass spectrometry, mutagenesis, optogenetic clustering with cell fate readouts","pmids":["27769899","27901111"],"confidence":"High","gaps":["Structural basis of SAM-dependent oligomerization upon Ser22 phosphorylation unknown","Optogenetic findings require validation under endogenous expression levels"]},{"year":2017,"claim":"ERK-induced acetylation of Lys414 in the PH domain by CBP (reversed by SIRT2) was shown to drive CNK1 membrane recruitment and CRAF interaction, establishing a post-translational positive feedback loop sustaining ERK signaling.","evidence":"Mass spectrometry acetylation site identification, mutagenesis, co-immunoprecipitation, subcellular fractionation and live imaging","pmids":["28819643"],"confidence":"High","gaps":["Whether acetylation and phosphorylation feedbacks operate simultaneously or sequentially not determined","In vivo acetylation dynamics not measured"]},{"year":2018,"claim":"A homozygous frameshift loss-of-function mutation in CNKSR1 was identified as the cause of syndromic autosomal recessive intellectual disability, establishing a human disease role and confirming CNK function in neuronal development.","evidence":"Next-generation sequencing in patient family, RT-PCR/western blot in lymphoblastoid cells, RNAi in Drosophila brain","pmids":["30450701"],"confidence":"Medium","gaps":["Single family reported; replication in additional kindreds needed","Precise neuronal pathway disrupted in patients not identified","Mouse knockout model not reported"]},{"year":2019,"claim":"The PH domain was shown to preferentially bind PtdIns(4,5)P2, mediating colocalization with mutant KRAS; a small-molecule inhibitor of the PH domain (PHT-7.3) selectively blocked mutant-KRAS-driven cell growth, establishing CNK1 as a druggable target.","evidence":"In vitro lipid binding assay, molecular modeling, pharmacological inhibition and cell growth assays","pmids":["31040156"],"confidence":"Medium","gaps":["No in vivo efficacy data for PHT-7.3","Selectivity across PH domain-containing proteins not fully profiled"]},{"year":2021,"claim":"A RhoB-GTP-gated mechanism was uncovered in which CNKSR1 sequesters PTPRH phosphatase, keeping EGFR/HER2 phosphorylated; RhoB-GTP accumulation displaces PTPRH from CNKSR1, enabling EGFR dephosphorylation — revealing a non-MAPK scaffolding function.","evidence":"Protein array binding screen, co-immunoprecipitation, siRNA knockdown with EGFR phosphorylation readout","pmids":["34187934"],"confidence":"High","gaps":["Whether this mechanism operates at physiological CNKSR1 expression levels not confirmed","Structural basis of RhoB-GTP displacing PTPRH from CNKSR1 unknown"]},{"year":2023,"claim":"MEK inhibitor treatment was found to induce CNKSR1 membrane translocation and stabilization of phospho-AKT, revealing CNKSR1 as a mediator of adaptive resistance to targeted MAPK pathway therapy in cancer.","evidence":"siRNA knockdown, co-immunoprecipitation, subcellular fractionation, in vivo xenograft models","pmids":["36790955"],"confidence":"Medium","gaps":["Patient-level validation of CNKSR1 as resistance biomarker lacking","Mechanism triggering CNKSR1 membrane translocation upon MEK inhibition not defined"]},{"year":2024,"claim":"Cryo-EM structure of the KSR-MEK-CNK-HYP complex revealed a ring-like architecture in which CNK simultaneously contacts both KSR and MEK, resolving how the scaffold stabilizes the KSR-MEK binary interaction that drives RAF-KSR heterodimerization and RAF activation.","evidence":"Cryo-EM at near-atomic resolution with in vitro reconstitution and mutagenesis-based functional validation","pmids":["38388830"],"confidence":"High","gaps":["Structure determined with Drosophila proteins; human complex structure not yet solved","How post-translational modifications remodel the ring complex is unknown","RAS-GTP-induced conformational changes within the assembled complex not captured"]},{"year":null,"claim":"Key open questions include the full-length structure of mammalian CNKSR1 in complex with its partners, how multiple post-translational modifications (phosphorylation, acetylation) are integrated temporally to direct pathway switching, and the precise neuronal mechanism underlying CNKSR1-linked intellectual disability.","evidence":"","pmids":[],"confidence":"High","gaps":["No mammalian full-length CNKSR1 complex structure","Temporal coordination of acetylation and phosphorylation feedbacks unresolved","Conditional knockout mouse models not reported","Role of CNKSR1 in neuronal signaling circuits mechanistically undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2,5,6,9,11,21]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[19]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,18,24]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,11,16,17,19,20]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[17]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,3,5,6,7,9,11,12,15,17,18,21,24]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[4]}],"complexes":["KSR-MEK-CNK-HYP complex","CNK1-cytohesin signaling complex"],"partners":["KSR1","RAF1","CYTH2","RHOA","RHOB","PTPRH","AKT1","SRC"],"other_free_text":[]},"mechanistic_narrative":"CNKSR1 is a multidomain scaffold protein that orchestrates signal transduction downstream of RAS, Rho GTPases, and receptor tyrosine kinases by assembling pathway-specific signaling complexes at the plasma membrane. Through its SAM domain heterodimer with HYP/AVVRK and direct contacts with both KSR and MEK, CNKSR1 forms a ring-like complex that promotes RAF-KSR heterodimerization and RAF catalytic activation, while a C-terminal inhibitory region (RIR) prevents basal signaling leakage and is counteracted by Src binding [PMID:14517245, PMID:38388830, PMID:15660123]. Beyond the RAF-MEK-ERK cascade, CNKSR1 scaffolds Rho GEFs with MLK2/MKK7 to activate JNK signaling, interacts with cytohesins to promote insulin-stimulated PI3K/AKT activation, and gates EGFR/HER2 dephosphorylation by sequestering the phosphatase PTPRH until RhoB-GTP displaces it [PMID:15753034, PMID:20634316, PMID:34187934]. Loss-of-function frameshift mutation in CNKSR1 causes syndromic autosomal recessive intellectual disability [PMID:30450701]."},"prefetch_data":{"uniprot":{"accession":"Q969H4","full_name":"Connector enhancer of kinase suppressor of ras 1","aliases":["CNK homolog protein 1","CNK1","hCNK1","Connector enhancer of KSR-like"],"length_aa":720,"mass_kda":79.7,"function":"May function as an adapter protein or regulator of Ras signaling pathways","subcellular_location":"Cytoplasm; Membrane","url":"https://www.uniprot.org/uniprotkb/Q969H4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CNKSR1","classification":"Not 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biology","url":"https://pubmed.ncbi.nlm.nih.gov/20600897","citation_count":3,"is_preprint":false},{"pmid":"36790955","id":"PMC_36790955","title":"Scaffolding Protein Connector Enhancer of Kinase Suppressor of Ras 1 (CNKSR1) Regulates MAPK Inhibition Responsiveness in Pancreas Cancer via Crosstalk with AKT Signaling.","date":"2023","source":"Molecular cancer research : MCR","url":"https://pubmed.ncbi.nlm.nih.gov/36790955","citation_count":2,"is_preprint":false},{"pmid":"35525547","id":"PMC_35525547","title":"Conformational control and regulation of the pseudokinase KSR via small molecule binding interactions.","date":"2022","source":"Methods in enzymology","url":"https://pubmed.ncbi.nlm.nih.gov/35525547","citation_count":2,"is_preprint":false},{"pmid":"18729764","id":"PMC_18729764","title":"Knockout serum replacement (KSR) has a suppressive effect on Sendai virus-mediated transduction of cynomolgus ES cells.","date":"2008","source":"Cloning and stem cells","url":"https://pubmed.ncbi.nlm.nih.gov/18729764","citation_count":2,"is_preprint":false},{"pmid":"39399787","id":"PMC_39399787","title":"G.3.2 is a novel allele of the gene connector enhancer of ksr ( cnk ) in Drosophila melanogaster.","date":"2024","source":"microPublication biology","url":"https://pubmed.ncbi.nlm.nih.gov/39399787","citation_count":1,"is_preprint":false},{"pmid":"38628722","id":"PMC_38628722","title":"Identification of CNKSR1 as a biomarker for \"cold\" tumor microenvironment in lung adenocarcinoma: An integrative analysis based on a novel workflow.","date":"2024","source":"Heliyon","url":"https://pubmed.ncbi.nlm.nih.gov/38628722","citation_count":1,"is_preprint":false},{"pmid":"33514974","id":"PMC_33514974","title":"On the CN-K coordination modes in Kn[M6-n(CN)6]·xH2O: first evidence of CN-K electron-deficient bonding.","date":"2021","source":"Dalton transactions (Cambridge, England : 2003)","url":"https://pubmed.ncbi.nlm.nih.gov/33514974","citation_count":1,"is_preprint":false},{"pmid":"40832204","id":"PMC_40832204","title":"Gene model for the ortholog of cnk in Drosophila erecta.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/40832204","citation_count":0,"is_preprint":false},{"pmid":"21114928","id":"PMC_21114928","title":"[Construction, expression and purification of kinase suppressor of Ras, KSR].","date":"2007","source":"Zhongguo fei ai za zhi = Chinese journal of lung cancer","url":"https://pubmed.ncbi.nlm.nih.gov/21114928","citation_count":0,"is_preprint":false},{"pmid":"21144295","id":"PMC_21144295","title":"[Construction of eukaryotic expression vectors of carboxyl terminus and amino terminus of kinase suppressor of Ras (KSR) and their expression in 293T cell line].","date":"2006","source":"Zhongguo fei ai za zhi = Chinese journal of lung cancer","url":"https://pubmed.ncbi.nlm.nih.gov/21144295","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.08.06.668985","title":"Gene model for the ortholog of <i>cnk</i> in <i>Drosophila erecta</i>","date":"2025-08-12","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.06.668985","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.11.18.689017","title":"Chemical proteomics decrypts the kinases that shape the dynamic human phosphoproteome","date":"2025-11-18","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.18.689017","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.08.03.668294","title":"Quantitative Membrane Binding Assays Reveal an Inhibitory Role for the BRAF-Specific Region in CRD and Lipid 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localization\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic screen combined with direct binding and localization in founding paper with high citation count\",\n      \"pmids\": [\"9814705\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The N-terminal portion of CNK cooperates with RAS (specifically the RAS(V12G37) effector loop mutant that activates the RAL pathway), while the C-terminal portion regulates RAF in the MAPK pathway, suggesting CNK functions in more than one pathway downstream of RAS\",\n      \"method\": \"Transfection/overexpression in Drosophila eye, epistasis with RAS effector loop mutants\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple effector loop mutant epistasis experiments with defined pathway readouts\",\n      \"pmids\": [\"10557308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Drosophila CNK has bimodal (antagonistic) properties toward RAF: the N-terminal SAM and CRIC domains are essential for RAF activation, while a C-terminal bipartite RAF-inhibitory region (RIR) inhibits RAF catalytic function; together these prevent signaling leakage in the absence of signal but enable potent RAF activation upon stimulation\",\n      \"method\": \"Transfection experiments, RNAi-based rescue assay in Drosophila S2 cells, domain deletion/mutation analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple domain-swap and mutagenesis experiments with functional readout, replicated by RNAi rescue\",\n      \"pmids\": [\"14517245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human CNK1 (CNKSR1) interacts with RhoA in a GTP-dependent manner through its PH domain; CNK1 depletion by RNAi inhibits Rho-induced gene expression via serum response factor but not Rho-induced stress fiber formation; CNK1 also associates with Rhophilin and RalGDS, linking Rho and Ras pathways\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, RNAi knockdown, reporter assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP combined with RNAi knockdown with defined transcriptional phenotype\",\n      \"pmids\": [\"14749388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CNK1 binds directly to RASSF1A (and RASSF1C) and this interaction mediates association of CNK1 with MST1/MST2 kinases; co-expression of CNK1 with RASSF1A greatly augments CNK1-induced apoptosis; CNK1-induced apoptosis is suppressed by dominant-negative MST1/MST2\",\n      \"method\": \"Co-immunoprecipitation, yeast two-hybrid, overexpression/apoptosis assays, dominant-negative suppression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct binding demonstrated by pulldown/CoIP with multiple functional validation experiments\",\n      \"pmids\": [\"15075335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CNK1 acts as a scaffold mediating Src-dependent tyrosine phosphorylation and activation of Raf-1; CNK1 binds pre-activated Raf-1 and activated Src, forming a trimeric complex; siRNA knockdown of endogenous CNK1 interferes with Src-dependent ERK activation\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, kinase activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — trimeric complex demonstrated by CoIP, loss-of-function by siRNA with defined ERK activation phenotype\",\n      \"pmids\": [\"15845549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CNK1 interacts with Net1 or p115RhoGEF (Rho-specific GEFs) and with MLK2 and MKK7 (JNK cascade kinases); CNK1 acts cooperatively with these GEFs to activate JNK MAP kinase but not other Rho-mediated pathways; endogenous CNK1 is required for serum/S1P-stimulated Rho-dependent JNK activation in HeLa cells\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, reporter assays, kinase activation assays\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — CoIP with multiple binding partners, RNAi knockdown with defined JNK pathway phenotype\",\n      \"pmids\": [\"15753034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"In Drosophila, the Src42 tyrosine kinase associates in an RTK-dependent manner with a conserved region of CNK located C-terminal to the RAF-inhibitory region (RIR), and this association counteracts the repressive RIR effect on RAF; derepression by Src42 depends on its SH3/SH2 domains but not its catalytic activity; several cnk loss-of-function alleles have mutations that impair Src42 binding\",\n      \"method\": \"Genetic allele mapping, co-immunoprecipitation, dominant-negative/kinase-dead Src constructs\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple cnk allele analysis combined with binding domain mapping and catalytic-dead controls\",\n      \"pmids\": [\"15660123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CNK1 binds the angiotensin II type 2 (AT2) receptor through its SAM and CRIC domains; the interaction is strengthened by a Ras-regulated linker region; endogenous CNK1 and AT2 receptor co-precipitate from mouse heart extracts\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, pulldown from tissue extracts\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single-lab Co-IP with domain mapping but limited functional follow-up\",\n      \"pmids\": [\"16289034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"A KSR/CNK complex mediated by a novel SAM domain-containing protein HYP (Hyphen) regulates RAS-dependent RAF activation in Drosophila; KSR induces RAF activation via its kinase-like domain independently of its scaffolding property or kinase activity; KSR is recruited to RAF prior to signal activation by CNK/HYP\",\n      \"method\": \"Genetic epistasis, co-immunoprecipitation, RNAi in Drosophila S2 cells, domain-function analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multi-approach including genetics, CoIP, and domain dissection across multiple experiments\",\n      \"pmids\": [\"16600912\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Crystal structure of the SAM domain of CNK in complex with the SAM domain of HYP reveals a 1:1 heterodimer with single-junction interaction mode; mutational analysis shows this specific dimerization is essential for RAF signaling and facilitates recruitment of KSR to form the CNK/HYP/KSR regulatory complex\",\n      \"method\": \"X-ray crystallography, in vitro and in vivo mutational analysis, co-immunoprecipitation\",\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 with functional validation by in vitro and in vivo mutagenesis\",\n      \"pmids\": [\"18287031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The major binding partners of CNK1 scaffold are cytohesins (Arf GEFs); CNK1 interacts constitutively with the coiled-coil domain of cytohesins via its C-terminal region; CNK1/cytohesin interaction facilitates membrane recruitment of cytohesin-2 following insulin stimulation and promotes PI3K/AKT cascade activation by generating PIP2-rich microenvironment for IRS1 recruitment\",\n      \"method\": \"Mass spectrometry interactome, co-immunoprecipitation, protein depletion/rescue, membrane fractionation\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — MS interactome validated by CoIP, depletion/rescue with defined signaling phenotypes\",\n      \"pmids\": [\"20634316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CNK1 interacts with AKT and knockdown of CNK1 decreases AKT activity in breast cancer cells; CNK1 controls AKT-dependent phosphorylation of FoxO, and CNK1-induced proliferation is blocked by FoxO overexpression; CNK1 promotes NF-κB2 p100 processing to p52 and nuclear localization, driving MMP-9/MT1-MMP expression and cell invasion\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, reporter assays, proliferation/invasion assays\",\n      \"journal\": \"Oncogene / Molecular cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods across two papers from the same lab establishing AKT and NF-κB scaffolding roles\",\n      \"pmids\": [\"20383191\", \"20197385\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GEF-H1 acts as an adaptor linking PP2A B' subunits to KSR-1, mediating dephosphorylation of KSR-1 S392 and activation of MAPK signaling; this constitutes a positive feedback loop for the RAS/MAPK pathway independent of GEF-H1's RhoGEF activity\",\n      \"method\": \"Co-immunoprecipitation, phosphorylation assays, siRNA knockdown, xenograft models\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — CoIP demonstrating trimeric complex, phosphorylation assays, and in vivo validation\",\n      \"pmids\": [\"24525234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"EphrinB1 interacts with CNK1 in an EphB receptor-independent manner; co-transfection of ephrinB1 with CNK1 increases JNK phosphorylation; CNK1 overexpression alone activates RhoA, but both ephrinB1 and CNK1 are required for JNK phosphorylation; CNK1 depletion abrogates ephrinB1-mediated cell migration and JNK activation; adhesion to fibronectin or Src activity increases ephrinB1/CNK1 binding\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, kinase activation assays, cell migration assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods with defined mechanistic phenotypes\",\n      \"pmids\": [\"24825906\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"AKT phosphorylates CNKSR1/CNK1 at Ser22 within the SAM domain, triggering SAM domain-dependent oligomerization; oligomeric CNK1 increases affinity for active AKT (positive feedback); phosphorylation of Thr8 within the SAM domain by EGF signaling prevents AKT binding and antagonizes CNK1-mediated AKT signaling\",\n      \"method\": \"Phosphorylation site mapping (mass spectrometry), mutagenesis, co-immunoprecipitation, oligomerization assays\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — phosphorylation site identified by MS, validated by mutagenesis with functional readouts\",\n      \"pmids\": [\"27769899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PDGF induces phosphorylation of CNK1 at Tyr26; Src induces phosphorylation at Tyr519 and Tyr665; phosphorylation of Tyr519 recruits CNK1 to the nucleus; additional Tyr26 phosphorylation enables SRE-dependent gene expression; phosphorylation-deficient mutants promote MMP14 promoter activity; PDGF-stimulated Src recruits CNK1 to plasma membrane\",\n      \"method\": \"Phosphorylation site mapping, phosphomimetic/deficient mutants, subcellular localization, reporter assays\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — phosphosite mapping with mutagenesis and localization data, single-lab study\",\n      \"pmids\": [\"26319181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ERK signaling induces acetylation of CNK1 at Lys414 (in the PH domain) by the acetyltransferase CBP; SIRT2 deacetylates CNK1; acetylation drives membrane localization of CNK1 in growth factor-stimulated cells and promotes interaction with CRAF, stimulating ERK-dependent cell proliferation and migration; constitutively acetylated or membrane-anchored CNK1 mutants constitutively activate this positive feedback\",\n      \"method\": \"Mass spectrometry (acetylation site ID), mutagenesis, co-immunoprecipitation, subcellular fractionation/live imaging, proliferation/migration assays\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — PTM identified by MS, writer (CBP) and eraser (SIRT2) identified by CoIP, mutagenesis validates functional consequence\",\n      \"pmids\": [\"28819643\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CNKSR1 serves as a scaffold that interacts with RhoB-GTP (identified via human protein arrays) and with PTPRH (protein tyrosine phosphatase receptor type H) at the plasma membrane; when RhoB is degraded by CUL3/KCTD10, CNKSR1 binds PTPRH and inactivates its EGFR phosphatase activity; accumulation of RhoB-GTP displaces PTPRH from CNKSR1, releasing active PTPRH to dephosphorylate/suppress EGFR and HER2\",\n      \"method\": \"Protein array binding screen, co-immunoprecipitation, siRNA knockdown, membrane fractionation\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — protein array identification of interactors validated by CoIP, mechanistic model supported by depletion experiments with defined EGFR phosphorylation readout\",\n      \"pmids\": [\"34187934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The PH domain of CNK1 (CNKSR1) binds PtdIns(4,5)P2 with higher affinity than PtdIns(3,4,5)P3 and mediates plasma membrane colocalization with mutant KRAS; a small molecule (PHT-7.3) that binds selectively to the CNK1 PH domain prevents its plasma membrane colocalization with mutant KRAS and blocks mutant-KRAS but not wild-type KRAS cancer cell growth\",\n      \"method\": \"In vitro lipid binding assay, molecular modeling, cell imaging, pharmacological inhibition, cell growth assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — lipid binding assay combined with small molecule inhibitor and imaging, single-lab study\",\n      \"pmids\": [\"31040156\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MEK inhibition in CNKSR1-high cancer cells induces translocation of CNKSR1 to the plasma membrane where it interacts with and stabilizes phosphorylated AKT, activating PI3K/AKT signaling as an adaptive resistance mechanism; this is associated with reduced FoxO1 nuclear translocation\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, subcellular fractionation, pharmacological inhibition, in vivo xenograft\",\n      \"journal\": \"Molecular cancer research : MCR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CoIP and fractionation with defined signaling phenotype, single-lab study\",\n      \"pmids\": [\"36790955\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM structure of the minimal Drosophila KSR-MEK-CNK-HYP complex reveals a ring-like arrangement; CNK simultaneously engages both KSR and MEK, stabilizing their binary interaction; CNK-HYP scaffolding complex promotes RAF activation by enhancing KSR-MEK interaction, which in turn drives RAF-KSR heterodimerization and RAF catalytic activation\",\n      \"method\": \"Cryo-EM structure determination, in vitro binding assays, mutagenesis, functional RAF activation assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure with in vitro reconstitution and mutagenesis validating functional model\",\n      \"pmids\": [\"38388830\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CNKSR1 (human KSR1 ortholog context): CNKSR1 loss-of-function frameshift mutation causes syndromic autosomal recessive intellectual disability; RNAi knockdown of cnk (CNKSR1 ortholog) in Drosophila brain causes defects in eye and mushroom body structures\",\n      \"method\": \"Next-generation sequencing, RT-PCR/western blot (patient lymphoblastoid cells), RNAi in Drosophila\",\n      \"journal\": \"American journal of medical genetics. Part B, Neuropsychiatric genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — human loss-of-function genetics plus Drosophila RNAi with neurological phenotype, limited mechanistic detail\",\n      \"pmids\": [\"30450701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Human CNK2 (CNK2A and splice variant CNK2B) interacts with Raf through the Raf regulatory and kinase domains as well as the C-terminal half of CNK2; CNK2 also interacts with Ral GTPase and RalGDS family member Rlf via the GEF domain; CNK2 localizes to lateral plasma membrane in MDCK cells\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, subcellular fractionation/imaging\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP with domain mapping, single-lab study\",\n      \"pmids\": [\"14597674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Optogenetic clustering of CNK1 reveals that low-intensity light/low EGF drives CNK1-RAF interaction and ERK-dependent differentiation, while higher intensity/higher EGF drives active AKT binding to CNK1 which phosphorylates and inhibits RAF; CNK1 acts as a molecular switch controlling differentiation vs. proliferation decisions\",\n      \"method\": \"Optogenetic clustering (light-controlled oligomerization), co-immunoprecipitation, kinase activity assays, cell fate assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — novel optogenetic approach with CoIP, single-lab study\",\n      \"pmids\": [\"27901111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Leukocyte-specific protein 1 (LSP1) associates with KSR, MEK1 and ERK2, and targets them to peripheral actin filaments; LSP1-associated MEK1 is activated by anti-IgM in a PKCβI-dependent manner; this places CNKSR1/KSR as part of a cytoskeletal signaling complex\",\n      \"method\": \"Co-immunoprecipitation, confocal microscopy, kinase activation assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and imaging demonstrating cytoskeletal localization with partial functional readout\",\n      \"pmids\": [\"15090600\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CNKSR1 (connector enhancer of kinase suppressor of Ras 1) functions as a multidomain scaffold protein that integrates signals from multiple GTPases (RAS, RhoA, RhoB, RalGDS) by simultaneously binding RAF, MEK, KSR, and cytohesins, regulating the RAF-MEK-ERK MAPK cascade through bimodal control of RAF activity (with an N-terminal activation region and C-terminal inhibitory element), coordinating JNK pathway activation downstream of Rho GEFs, mediating insulin/AKT/PI3K signaling via cytohesin interactions, controlling EGFR/HER2 phosphorylation through RhoB-GTP-gated recruitment of the PTPRH phosphatase, and serving as a positive feedback node via ERK-induced acetylation and AKT-induced SAM domain oligomerization that drive its membrane localization and sustained signaling.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CNKSR1 is a multidomain scaffold protein that orchestrates signal transduction downstream of RAS, Rho GTPases, and receptor tyrosine kinases by assembling pathway-specific signaling complexes at the plasma membrane. Through its SAM domain heterodimer with HYP/AVVRK and direct contacts with both KSR and MEK, CNKSR1 forms a ring-like complex that promotes RAF-KSR heterodimerization and RAF catalytic activation, while a C-terminal inhibitory region (RIR) prevents basal signaling leakage and is counteracted by Src binding [PMID:14517245, PMID:38388830, PMID:15660123]. Beyond the RAF-MEK-ERK cascade, CNKSR1 scaffolds Rho GEFs with MLK2/MKK7 to activate JNK signaling, interacts with cytohesins to promote insulin-stimulated PI3K/AKT activation, and gates EGFR/HER2 dephosphorylation by sequestering the phosphatase PTPRH until RhoB-GTP displaces it [PMID:15753034, PMID:20634316, PMID:34187934]. Loss-of-function frameshift mutation in CNKSR1 causes syndromic autosomal recessive intellectual disability [PMID:30450701].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"The founding discovery that CNK physically interacts with RAF and is genetically required for RAS signaling established it as a scaffold linking RAS pathway activation to RAF regulation.\",\n      \"evidence\": \"Genetic modifier screen of ksr in Drosophila eye combined with co-immunoprecipitation and subcellular localization\",\n      \"pmids\": [\"9814705\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No mammalian ortholog characterized\", \"Mechanism by which CNK activates RAF unknown\", \"Binding domains on RAF not mapped\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Epistasis with RAS effector-loop mutants revealed that CNK operates in more than one RAS-effector branch — the N-terminus cooperates with the RAL pathway while the C-terminus regulates RAF — expanding CNK's role beyond simple RAF scaffolding.\",\n      \"evidence\": \"Overexpression with RAS effector loop mutants in Drosophila eye development\",\n      \"pmids\": [\"10557308\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RAL-branch mechanism not defined at the molecular level\", \"Whether the two branches function independently or coordinately was unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Domain dissection uncovered bimodal control of RAF: N-terminal SAM/CRIC domains activate RAF while a C-terminal RIR inhibits it, explaining how CNK prevents basal signaling yet enables stimulus-dependent activation.\",\n      \"evidence\": \"Domain deletion/mutation analysis with RNAi rescue in Drosophila S2 cells\",\n      \"pmids\": [\"14517245\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the signal that relieves RIR-mediated inhibition unknown\", \"No structural information on SAM/CRIC activation mechanism\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identification of GTP-dependent RhoA binding via the PH domain, plus interactions with Rhophilin and RalGDS, established human CNK1 as a node integrating Rho and Ras pathways in transcriptional control (SRF-dependent genes) and apoptosis (via RASSF1A/MST1).\",\n      \"evidence\": \"Yeast two-hybrid, co-immunoprecipitation, RNAi knockdown, and reporter assays in mammalian cells\",\n      \"pmids\": [\"14749388\", \"15075335\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct structural basis of RhoA-PH domain interaction unresolved\", \"In vivo relevance of RASSF1A-CNK1-MST1 apoptotic axis not tested in animal models\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Multiple studies converged to show that CNK1 scaffolds distinct MAPK modules — Src/Raf-1/ERK and Rho-GEF/MLK2/MKK7/JNK — and that Src binding to a conserved C-terminal region relieves the RAF-inhibitory region, answering the key question of how RIR-mediated repression is derepressed.\",\n      \"evidence\": \"Co-immunoprecipitation of trimeric Src-CNK1-Raf-1 complex, siRNA knockdown with ERK/JNK readouts in HeLa cells, Drosophila genetic allele mapping of Src42 binding site\",\n      \"pmids\": [\"15845549\", \"15753034\", \"15660123\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Src derepression requires catalytic activity or only SH2/SH3 docking in mammalian cells untested\", \"Structural basis of JNK-branch scaffold assembly unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Discovery of the adaptor protein HYP (Hyphen/AVVRK) as the missing link that bridges CNK's SAM domain to KSR explained how CNK recruits KSR to RAF prior to signal activation.\",\n      \"evidence\": \"Genetic epistasis, co-immunoprecipitation, and RNAi in Drosophila S2 cells\",\n      \"pmids\": [\"16600912\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mammalian HYP ortholog function not yet validated\", \"Stoichiometry of the CNK-HYP-KSR complex unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"The crystal structure of the CNK-SAM/HYP-SAM heterodimer revealed a single-junction 1:1 interaction mode, and mutagenesis confirmed that this interface is essential for RAF signaling, providing the first atomic-resolution view of the scaffolding mechanism.\",\n      \"evidence\": \"X-ray crystallography with in vivo and in vitro mutagenesis validation\",\n      \"pmids\": [\"18287031\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length complex structure unavailable\", \"Whether SAM-SAM interaction is regulated by post-translational modifications unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Mass spectrometry-based interactomics identified cytohesins as the major CNK1 binding partners and showed that CNK1 scaffolds cytohesin-2 membrane recruitment to activate PI3K/AKT signaling upon insulin stimulation, broadening CNK1's role beyond MAPK to metabolic signaling.\",\n      \"evidence\": \"Mass spectrometry interactome, co-immunoprecipitation, depletion/rescue, membrane fractionation\",\n      \"pmids\": [\"20634316\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CNK1-cytohesin interaction is conserved in all insulin-responsive tissues not tested\", \"Relative contribution of CNK1 vs. other cytohesin scaffolds undetermined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"CNK1 was shown to interact with AKT and drive AKT-dependent FoxO phosphorylation and NF-κB2 processing, linking scaffold function to pro-proliferative and pro-invasive transcriptional programs in breast cancer cells.\",\n      \"evidence\": \"Co-immunoprecipitation, siRNA knockdown, reporter assays, invasion assays in breast cancer cell lines\",\n      \"pmids\": [\"20383191\", \"20197385\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CNK1-AKT interaction is direct or mediated by cytohesins not fully resolved\", \"In vivo tumor model validation limited\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"AKT-dependent phosphorylation of Ser22 in the SAM domain was found to trigger CNK1 oligomerization and create a positive feedback loop with AKT, while optogenetic clustering revealed that CNK1 oligomerization state switches cell fate between differentiation (ERK) and proliferation (AKT).\",\n      \"evidence\": \"Phosphorylation site mapping by mass spectrometry, mutagenesis, optogenetic clustering with cell fate readouts\",\n      \"pmids\": [\"27769899\", \"27901111\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of SAM-dependent oligomerization upon Ser22 phosphorylation unknown\", \"Optogenetic findings require validation under endogenous expression levels\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"ERK-induced acetylation of Lys414 in the PH domain by CBP (reversed by SIRT2) was shown to drive CNK1 membrane recruitment and CRAF interaction, establishing a post-translational positive feedback loop sustaining ERK signaling.\",\n      \"evidence\": \"Mass spectrometry acetylation site identification, mutagenesis, co-immunoprecipitation, subcellular fractionation and live imaging\",\n      \"pmids\": [\"28819643\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether acetylation and phosphorylation feedbacks operate simultaneously or sequentially not determined\", \"In vivo acetylation dynamics not measured\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"A homozygous frameshift loss-of-function mutation in CNKSR1 was identified as the cause of syndromic autosomal recessive intellectual disability, establishing a human disease role and confirming CNK function in neuronal development.\",\n      \"evidence\": \"Next-generation sequencing in patient family, RT-PCR/western blot in lymphoblastoid cells, RNAi in Drosophila brain\",\n      \"pmids\": [\"30450701\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single family reported; replication in additional kindreds needed\", \"Precise neuronal pathway disrupted in patients not identified\", \"Mouse knockout model not reported\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The PH domain was shown to preferentially bind PtdIns(4,5)P2, mediating colocalization with mutant KRAS; a small-molecule inhibitor of the PH domain (PHT-7.3) selectively blocked mutant-KRAS-driven cell growth, establishing CNK1 as a druggable target.\",\n      \"evidence\": \"In vitro lipid binding assay, molecular modeling, pharmacological inhibition and cell growth assays\",\n      \"pmids\": [\"31040156\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vivo efficacy data for PHT-7.3\", \"Selectivity across PH domain-containing proteins not fully profiled\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A RhoB-GTP-gated mechanism was uncovered in which CNKSR1 sequesters PTPRH phosphatase, keeping EGFR/HER2 phosphorylated; RhoB-GTP accumulation displaces PTPRH from CNKSR1, enabling EGFR dephosphorylation — revealing a non-MAPK scaffolding function.\",\n      \"evidence\": \"Protein array binding screen, co-immunoprecipitation, siRNA knockdown with EGFR phosphorylation readout\",\n      \"pmids\": [\"34187934\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this mechanism operates at physiological CNKSR1 expression levels not confirmed\", \"Structural basis of RhoB-GTP displacing PTPRH from CNKSR1 unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"MEK inhibitor treatment was found to induce CNKSR1 membrane translocation and stabilization of phospho-AKT, revealing CNKSR1 as a mediator of adaptive resistance to targeted MAPK pathway therapy in cancer.\",\n      \"evidence\": \"siRNA knockdown, co-immunoprecipitation, subcellular fractionation, in vivo xenograft models\",\n      \"pmids\": [\"36790955\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Patient-level validation of CNKSR1 as resistance biomarker lacking\", \"Mechanism triggering CNKSR1 membrane translocation upon MEK inhibition not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Cryo-EM structure of the KSR-MEK-CNK-HYP complex revealed a ring-like architecture in which CNK simultaneously contacts both KSR and MEK, resolving how the scaffold stabilizes the KSR-MEK binary interaction that drives RAF-KSR heterodimerization and RAF activation.\",\n      \"evidence\": \"Cryo-EM at near-atomic resolution with in vitro reconstitution and mutagenesis-based functional validation\",\n      \"pmids\": [\"38388830\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure determined with Drosophila proteins; human complex structure not yet solved\", \"How post-translational modifications remodel the ring complex is unknown\", \"RAS-GTP-induced conformational changes within the assembled complex not captured\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include the full-length structure of mammalian CNKSR1 in complex with its partners, how multiple post-translational modifications (phosphorylation, acetylation) are integrated temporally to direct pathway switching, and the precise neuronal mechanism underlying CNKSR1-linked intellectual disability.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No mammalian full-length CNKSR1 complex structure\", \"Temporal coordination of acetylation and phosphorylation feedbacks unresolved\", \"Conditional knockout mouse models not reported\", \"Role of CNKSR1 in neuronal signaling circuits mechanistically undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2, 5, 6, 9, 11, 21]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [19]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 18, 24]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 11, 16, 17, 19, 20]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 3, 5, 6, 7, 9, 11, 12, 15, 17, 18, 21, 24]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"complexes\": [\n      \"KSR-MEK-CNK-HYP complex\",\n      \"CNK1-cytohesin signaling complex\"\n    ],\n    \"partners\": [\n      \"KSR1\",\n      \"RAF1\",\n      \"CYTH2\",\n      \"RHOA\",\n      \"RHOB\",\n      \"PTPRH\",\n      \"AKT1\",\n      \"SRC\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}