{"gene":"SCYL2","run_date":"2026-06-10T07:46:30","timeline":{"discoveries":[{"year":2005,"finding":"CVAK104 (SCYL2) is a serine/threonine kinase that co-fractionates with clathrin-coated vesicle adaptor protein preparations, directly binds both clathrin and the plasma membrane adaptor AP2, binds ATP, performs autophosphorylation, and phosphorylates the beta2-adaptin subunit of AP2 in a poly-L-lysine-stimulated manner in vitro.","method":"Mass spectrometry identification, in vitro kinase assay, ATP-binding assay, co-fractionation with clathrin-coated vesicles","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with substrate phosphorylation and autophosphorylation demonstrated; ATP binding confirmed; co-fractionation established; single lab but multiple orthogonal methods","pmids":["15809293"],"is_preprint":false},{"year":2006,"finding":"CVAK104 (SCYL2) localizes predominantly to the perinuclear region (trans-Golgi network) and also to peripheral endosomal vesicles; its membrane association is clathrin-dependent (clathrin knockdown reduces membrane association); a C-terminal segment binds the N-terminal domain of clathrin and the alpha-appendage of AP2; GTP-binding proteins are required for perinuclear membrane recruitment; CVAK104-depleted cells missort the lysosomal hydrolase cathepsin D, indicating a function in clathrin-dependent TGN-to-endosome trafficking.","method":"RNA interference knockdown, live-cell imaging with GFP/RFP-tagged proteins, permeabilized cell recruitment assay with GTPγS, brefeldin A treatment, cathepsin D sorting assay, pulldown/binding assay for clathrin N-terminal domain and AP2 alpha-appendage","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (live imaging, RNAi, pharmacological perturbation, cargo sorting assay, direct binding assay) in a single study establishing localization and functional role","pmids":["16914521"],"is_preprint":false},{"year":2007,"finding":"CVAK104 (SCYL2) colocalizes with clathrin and AP-1 on a transferrin-positive endosomal compartment; co-immunoprecipitation shows association with clathrin, AP-1, and epsinR; siRNA knockdown of CVAK104 causes selective loss of SNARE proteins syntaxin 8 and vti1b from clathrin-coated vesicles, demonstrating a role in SNARE sorting into CCVs.","method":"Co-immunoprecipitation, siRNA knockdown, comparative proteomics of clathrin-coated vesicles, immunofluorescence colocalization","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with multiple partners, CCV proteomics, and RNAi-phenotype with specific cargo readout; single lab but multiple orthogonal methods","pmids":["17587408"],"is_preprint":false},{"year":2009,"finding":"CVAK104 (SCYL2) interacts with Dishevelled (Dvl) and with Fzd5 (but not Fzd1 or Fzd4); overexpression of CVAK104 induces clathrin-mediated intracellular accumulation and subsequent lysosomal degradation of Fzd5 (suppressed by dominant-negative Rab5); RNAi knockdown of CVAK104 increases Fzd5 levels; overexpression suppresses and knockdown activates the Wnt/beta-catenin pathway, establishing CVAK104 as a negative regulator of Wnt signaling through Fzd5 degradation.","method":"Co-immunoprecipitation (CVAK104 with Dvl and Fzd5), siRNA knockdown, dominant-negative Rab5 epistasis, lysosomal inhibitor assay, Wnt/beta-catenin reporter assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, RNAi gain/loss-of-function with pathway readout, and epistasis with dominant-negative Rab5; single lab with multiple orthogonal methods","pmids":["19643732"],"is_preprint":false},{"year":2012,"finding":"SCYL2 recruits protein phosphatase 2A (PP2A) to the HIV-1 accessory protein Vpu, promoting dephosphorylation of Vpu at Ser52 and Ser56; this antagonizes Vpu-mediated degradation of BST2/tetherin and restricts HIV-1 virion release. SCYL2 is induced by type I interferon, and its depletion increases Vpu phosphorylation and viral particle release.","method":"Co-immunoprecipitation (SCYL2 with Vpu and PP2A), siRNA knockdown, phosphorylation assay of Vpu residues, viral particle release assay, interferon treatment","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP identifying ternary complex, RNAi gain/loss-of-function with specific phospho-site and virion release readouts; single lab with multiple orthogonal methods","pmids":["23047923"],"is_preprint":false},{"year":2015,"finding":"Neuron-specific knockout of Scyl2 in mice causes perinatal lethality and degeneration of CA3 pyramidal hippocampal neurons during functional maturation; the cell death is BAX-dependent and apoptotic; biochemical fractionation reveals altered composition of excitatory receptors at synapses of Scyl2-deficient mice; inhibition of excitatory signaling prevents CA3 neuron degeneration, placing SCYL2 as a regulator of excitatory receptor trafficking/synaptic composition that suppresses excitotoxicity.","method":"Conditional neuron-specific knockout mice, histology, electrophysiology, BAX genetic epistasis (BAX-dependent apoptosis), synaptic fractionation and receptor composition analysis, pharmacological inhibition of excitatory signaling","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic knockout with BAX epistasis, biochemical fractionation, and pharmacological rescue; multiple orthogonal approaches in one rigorous study","pmids":["26203146"],"is_preprint":false},{"year":2026,"finding":"SCYL2 forms a complex with clathrin heavy chain (CHC) and binds PTEN; the SCYL2-associated complex phosphorylates PTEN at S380/T382/T383 (STT cluster); SCYL2 expression induces CHC binding to PTEN; CHC downregulation or CCV inhibition reduces phospho-PTEN(STT), suggesting clathrin-coated vesicles serve as a signaling platform for PTEN phosphorylation; SCYL2 downregulation has anti-tumor effects via PI3K/AKT inhibition through PTEN dephosphorylation.","method":"Co-immunoprecipitation (SCYL2 with PTEN and CHC), siRNA knockdown of SCYL2 and CHC, phosphorylation assay (PTEN STT cluster), cell survival assay","journal":"Cancer gene therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP and RNAi with phosphorylation readout; single lab, and the abstract notes the mechanism by which SCYL2 regulates PTEN phosphorylation 'remains unclear', limiting confidence","pmids":["41703271"],"is_preprint":false}],"current_model":"SCYL2 (CVAK104) is a pseudokinase that associates with clathrin-coated vesicles and directly binds clathrin and AP complexes (AP-1, AP-2 via alpha-appendage and N-terminal domain of clathrin heavy chain); it phosphorylates AP2-beta2-adaptin in vitro, regulates SNARE sorting (syntaxin 8, vti1b) into CCVs, promotes clathrin-mediated lysosomal degradation of Frizzled-5 (thereby suppressing Wnt/beta-catenin signaling), recruits PP2A to dephosphorylate HIV-1 Vpu and restrict viral release, and in neurons maintains proper excitatory receptor composition at synapses to suppress excitotoxicity via a BAX-dependent apoptotic pathway."},"narrative":{"mechanistic_narrative":"SCYL2 (CVAK104) is a clathrin-coated vesicle-associated pseudokinase that functions as a sorting and signaling regulator at the clathrin trafficking machinery [PMID:15809293, PMID:16914521]. It co-fractionates with clathrin-coated vesicles and binds directly to clathrin (via the N-terminal domain) and the AP2 adaptor (via the alpha-appendage), with membrane association being clathrin-dependent and perinuclear recruitment requiring GTP-binding proteins [PMID:15809293, PMID:16914521]. SCYL2 localizes to the trans-Golgi network and endosomal compartments where it associates with clathrin, AP-1, and epsinR, and is required for proper sorting of cargo into clathrin-coated vesicles, including the lysosomal hydrolase cathepsin D and the SNAREs syntaxin 8 and vti1b [PMID:16914521, PMID:17587408]. Through this trafficking activity SCYL2 controls the fate of specific receptors: it drives clathrin-mediated lysosomal degradation of Frizzled-5 to act as a negative regulator of Wnt/beta-catenin signaling [PMID:19643732], and in neurons it maintains the composition of excitatory receptors at synapses, with its loss causing BAX-dependent apoptotic degeneration of CA3 hippocampal neurons due to excitotoxicity [PMID:26203146]. SCYL2 additionally serves as a scaffold that couples phosphatase activity to its substrates, recruiting PP2A to dephosphorylate HIV-1 Vpu and thereby restrict viral release [PMID:23047923]. A clathrin-coated-vesicle SCYL2 complex also promotes phosphorylation of PTEN at the STT cluster, linking SCYL2 to PI3K/AKT signaling [PMID:41703271].","teleology":[{"year":2005,"claim":"Establishing whether SCYL2 was a bona fide component of the clathrin machinery and an active enzyme determined how to frame its cellular role; the finding placed it at clathrin-coated vesicles with kinase-like activity toward an adaptor subunit.","evidence":"Mass spectrometry, in vitro kinase and ATP-binding assays, and co-fractionation with clathrin-coated vesicle preparations","pmids":["15809293"],"confidence":"High","gaps":["In vitro phosphorylation of beta2-adaptin does not establish a physiological substrate","Whether catalytic activity is required for any cellular function was not tested"]},{"year":2006,"claim":"Defining SCYL2's subcellular localization and the determinants of its membrane recruitment showed it operates in TGN-to-endosome clathrin trafficking rather than only at the plasma membrane.","evidence":"RNAi, live-cell imaging, GTPgammaS/brefeldin A perturbation, cathepsin D sorting assay, and direct binding assays to clathrin N-terminal domain and AP2 alpha-appendage","pmids":["16914521"],"confidence":"High","gaps":["Identity of the GTP-binding protein required for perinuclear recruitment unspecified","Mechanism linking SCYL2 loss to cathepsin D missorting not resolved"]},{"year":2007,"claim":"Identifying a specific cargo class clarified what SCYL2 sorts; it is required for incorporation of selected SNAREs into clathrin-coated vesicles.","evidence":"Reciprocal co-immunoprecipitation, comparative CCV proteomics, siRNA knockdown, and immunofluorescence colocalization","pmids":["17587408"],"confidence":"High","gaps":["Whether SNARE sorting depends on direct SCYL2 binding versus an indirect adaptor effect is unresolved","Functional consequence of syntaxin 8/vti1b mislocalization not measured"]},{"year":2009,"claim":"Connecting SCYL2-dependent trafficking to a signaling output demonstrated it degrades a specific Wnt receptor and thereby tunes pathway activity.","evidence":"Co-IP with Dvl and Fzd5, gain/loss-of-function RNAi, dominant-negative Rab5 epistasis, lysosomal inhibition, and Wnt/beta-catenin reporter assay","pmids":["19643732"],"confidence":"High","gaps":["Basis for Fzd5 selectivity over Fzd1/Fzd4 not defined","Role of SCYL2 catalytic activity in Fzd5 degradation untested"]},{"year":2012,"claim":"Showing SCYL2 recruits a phosphatase to a viral substrate revealed a scaffolding mode distinct from its sorting role and a function in innate antiviral restriction.","evidence":"Co-IP defining a SCYL2-Vpu-PP2A complex, siRNA knockdown, Vpu phospho-site analysis, virion release assay, and interferon induction","pmids":["23047923"],"confidence":"High","gaps":["Whether SCYL2 directly bridges PP2A and Vpu or acts via the trafficking machinery is unclear","Generalizability of the PP2A-recruitment scaffold to host substrates not explored"]},{"year":2015,"claim":"An in vivo genetic test established the physiological importance of SCYL2 in neurons, linking its trafficking role to control of synaptic excitatory receptor composition and survival.","evidence":"Neuron-specific conditional knockout mice with histology, electrophysiology, BAX genetic epistasis, synaptic fractionation, and pharmacological rescue of excitatory signaling","pmids":["26203146"],"confidence":"High","gaps":["Specific receptor subunits trafficked by SCYL2 not molecularly identified","Mechanistic link between altered receptor composition and BAX-dependent apoptosis not delineated"]},{"year":2026,"claim":"Implicating a SCYL2-clathrin complex in PTEN phosphorylation extended SCYL2 into PI3K/AKT signaling and a potential tumor context.","evidence":"Co-IP of SCYL2 with PTEN and CHC, siRNA knockdown of SCYL2 and CHC, PTEN STT-cluster phosphorylation assay, and cell survival assay","pmids":["41703271"],"confidence":"Medium","gaps":["The kinase responsible for and mechanism of PTEN phosphorylation remains unclear per the study itself","Whether SCYL2 phosphorylates PTEN directly versus recruiting another kinase is untested","Single-lab Co-IP without reciprocal in vivo validation"]},{"year":null,"claim":"Whether SCYL2's catalytic activity is required for its diverse trafficking and signaling functions, and how a single CCV-associated protein selects among such varied substrates (SNAREs, Fzd5, Vpu/PP2A, PTEN), remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of SCYL2 substrate or adaptor selectivity","No catalytically-dead mutant rescue across the reported functions","Mechanism unifying scaffold versus kinase roles not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,6]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2,4]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[1]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[1,2]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,6]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[4]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[5]}],"complexes":["clathrin-coated vesicle"],"partners":["CLTC","AP2","AP1","CLINT1","FZD5","DVL","PPP2 (PP2A)","PTEN"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6P3W7","full_name":"SCY1-like protein 2","aliases":["Coated vesicle-associated kinase of 104 kDa"],"length_aa":929,"mass_kda":103.7,"function":"Component of the AP2-containing clathrin coat that may regulate clathrin-dependent trafficking at plasma membrane, TGN and endosomal system (Probable). A possible serine/threonine-protein kinase toward the beta2-subunit of the plasma membrane adapter complex AP2 and other proteins in presence of poly-L-lysine has not been confirmed (PubMed:15809293, PubMed:16914521). By regulating the expression of excitatory receptors at synapses, plays an essential role in neuronal function and signaling and in brain development (By similarity)","subcellular_location":"Cytoplasmic vesicle, clathrin-coated vesicle; Golgi apparatus, trans-Golgi network membrane; Endosome membrane","url":"https://www.uniprot.org/uniprotkb/Q6P3W7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SCYL2","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000136021","cell_line_id":"CID001270","localizations":[{"compartment":"vesicles","grade":3},{"compartment":"golgi","grade":1}],"interactors":[{"gene":"CLTA","stoichiometry":10.0},{"gene":"ARHGAP18","stoichiometry":0.2},{"gene":"CLINT1","stoichiometry":0.2},{"gene":"POLE3","stoichiometry":0.2},{"gene":"NSRP1;CCDC55","stoichiometry":0.2},{"gene":"AKAP9","stoichiometry":0.2},{"gene":"TPP2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001270","total_profiled":1310},"omim":[{"mim_id":"618766","title":"ARTHROGRYPOSIS MULTIPLEX CONGENITA 4, NEUROGENIC, WITH AGENESIS OF THE CORPUS CALLOSUM; AMC4","url":"https://www.omim.org/entry/618766"},{"mim_id":"617468","title":"ARTHROGRYPOSIS MULTIPLEX CONGENITA 1, NEUROGENIC, WITH MYELIN DEFECT; AMC1","url":"https://www.omim.org/entry/617468"},{"mim_id":"616365","title":"SCY1-LIKE PROTEIN 2; SCYL2","url":"https://www.omim.org/entry/616365"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SCYL2"},"hgnc":{"alias_symbol":["KIAA1360","CVAK104"],"prev_symbol":[]},"alphafold":{"accession":"Q6P3W7","domains":[{"cath_id":"1.10.510.10","chopping":"7-328","consensus_level":"medium","plddt":86.6873,"start":7,"end":328},{"cath_id":"1.20.58","chopping":"539-622","consensus_level":"medium","plddt":85.0251,"start":539,"end":622}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6P3W7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6P3W7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6P3W7-F1-predicted_aligned_error_v6.png","plddt_mean":70.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SCYL2","jax_strain_url":"https://www.jax.org/strain/search?query=SCYL2"},"sequence":{"accession":"Q6P3W7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6P3W7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6P3W7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6P3W7"}},"corpus_meta":[{"pmid":"15809293","id":"PMC_15809293","title":"CVAK104 is a novel poly-L-lysine-stimulated kinase that targets the beta2-subunit of AP2.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15809293","citation_count":36,"is_preprint":false},{"pmid":"16914521","id":"PMC_16914521","title":"Clathrin-dependent association of CVAK104 with endosomes and the trans-Golgi network.","date":"2006","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/16914521","citation_count":32,"is_preprint":false},{"pmid":"17587408","id":"PMC_17587408","title":"CVAK104 is a novel regulator of clathrin-mediated SNARE sorting.","date":"2007","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/17587408","citation_count":30,"is_preprint":false},{"pmid":"19643732","id":"PMC_19643732","title":"A coated vesicle-associated kinase of 104 kDa (CVAK104) induces lysosomal degradation of frizzled 5 (Fzd5).","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19643732","citation_count":23,"is_preprint":false},{"pmid":"26203146","id":"PMC_26203146","title":"SCYL2 Protects CA3 Pyramidal Neurons from Excitotoxicity during Functional Maturation of the Mouse Hippocampus.","date":"2015","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/26203146","citation_count":22,"is_preprint":false},{"pmid":"23047923","id":"PMC_23047923","title":"Interferon-induced SCYL2 limits release of HIV-1 by triggering PP2A-mediated dephosphorylation of the viral protein Vpu.","date":"2012","source":"Science signaling","url":"https://pubmed.ncbi.nlm.nih.gov/23047923","citation_count":20,"is_preprint":false},{"pmid":"28751315","id":"PMC_28751315","title":"SCYL2 Genes Are Involved in Clathrin-Mediated Vesicle Trafficking and Essential for Plant Growth.","date":"2017","source":"Plant physiology","url":"https://pubmed.ncbi.nlm.nih.gov/28751315","citation_count":11,"is_preprint":false},{"pmid":"40243816","id":"PMC_40243816","title":"Novel SCYL2 Mutations and Arthrogryposis Multiplex Congenita 4: Case Report and Review of the Literature.","date":"2025","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/40243816","citation_count":2,"is_preprint":false},{"pmid":"39169672","id":"PMC_39169672","title":"SCYL2-related autosomal recessive neurodevelopmental disorders: Arthrogryposis multiplex congenita-4 and beyond?","date":"2024","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/39169672","citation_count":1,"is_preprint":false},{"pmid":"41703271","id":"PMC_41703271","title":"Clathrin-associated SCYL2 contributes to the activation of PI3K/AKT signaling and tumorigenesis through PTEN phosphorylation in adult T-cell leukemia/lymphoma.","date":"2026","source":"Cancer gene therapy","url":"https://pubmed.ncbi.nlm.nih.gov/41703271","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7065,"output_tokens":2395,"usd":0.02856,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9481,"output_tokens":3185,"usd":0.063515,"stage2_stop_reason":"end_turn"},"total_usd":0.092075,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"CVAK104 (SCYL2) is a serine/threonine kinase that co-fractionates with clathrin-coated vesicle adaptor protein preparations, directly binds both clathrin and the plasma membrane adaptor AP2, binds ATP, performs autophosphorylation, and phosphorylates the beta2-adaptin subunit of AP2 in a poly-L-lysine-stimulated manner in vitro.\",\n      \"method\": \"Mass spectrometry identification, in vitro kinase assay, ATP-binding assay, co-fractionation with clathrin-coated vesicles\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with substrate phosphorylation and autophosphorylation demonstrated; ATP binding confirmed; co-fractionation established; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"15809293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CVAK104 (SCYL2) localizes predominantly to the perinuclear region (trans-Golgi network) and also to peripheral endosomal vesicles; its membrane association is clathrin-dependent (clathrin knockdown reduces membrane association); a C-terminal segment binds the N-terminal domain of clathrin and the alpha-appendage of AP2; GTP-binding proteins are required for perinuclear membrane recruitment; CVAK104-depleted cells missort the lysosomal hydrolase cathepsin D, indicating a function in clathrin-dependent TGN-to-endosome trafficking.\",\n      \"method\": \"RNA interference knockdown, live-cell imaging with GFP/RFP-tagged proteins, permeabilized cell recruitment assay with GTPγS, brefeldin A treatment, cathepsin D sorting assay, pulldown/binding assay for clathrin N-terminal domain and AP2 alpha-appendage\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (live imaging, RNAi, pharmacological perturbation, cargo sorting assay, direct binding assay) in a single study establishing localization and functional role\",\n      \"pmids\": [\"16914521\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CVAK104 (SCYL2) colocalizes with clathrin and AP-1 on a transferrin-positive endosomal compartment; co-immunoprecipitation shows association with clathrin, AP-1, and epsinR; siRNA knockdown of CVAK104 causes selective loss of SNARE proteins syntaxin 8 and vti1b from clathrin-coated vesicles, demonstrating a role in SNARE sorting into CCVs.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, comparative proteomics of clathrin-coated vesicles, immunofluorescence colocalization\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with multiple partners, CCV proteomics, and RNAi-phenotype with specific cargo readout; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"17587408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CVAK104 (SCYL2) interacts with Dishevelled (Dvl) and with Fzd5 (but not Fzd1 or Fzd4); overexpression of CVAK104 induces clathrin-mediated intracellular accumulation and subsequent lysosomal degradation of Fzd5 (suppressed by dominant-negative Rab5); RNAi knockdown of CVAK104 increases Fzd5 levels; overexpression suppresses and knockdown activates the Wnt/beta-catenin pathway, establishing CVAK104 as a negative regulator of Wnt signaling through Fzd5 degradation.\",\n      \"method\": \"Co-immunoprecipitation (CVAK104 with Dvl and Fzd5), siRNA knockdown, dominant-negative Rab5 epistasis, lysosomal inhibitor assay, Wnt/beta-catenin reporter assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, RNAi gain/loss-of-function with pathway readout, and epistasis with dominant-negative Rab5; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"19643732\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SCYL2 recruits protein phosphatase 2A (PP2A) to the HIV-1 accessory protein Vpu, promoting dephosphorylation of Vpu at Ser52 and Ser56; this antagonizes Vpu-mediated degradation of BST2/tetherin and restricts HIV-1 virion release. SCYL2 is induced by type I interferon, and its depletion increases Vpu phosphorylation and viral particle release.\",\n      \"method\": \"Co-immunoprecipitation (SCYL2 with Vpu and PP2A), siRNA knockdown, phosphorylation assay of Vpu residues, viral particle release assay, interferon treatment\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP identifying ternary complex, RNAi gain/loss-of-function with specific phospho-site and virion release readouts; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"23047923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Neuron-specific knockout of Scyl2 in mice causes perinatal lethality and degeneration of CA3 pyramidal hippocampal neurons during functional maturation; the cell death is BAX-dependent and apoptotic; biochemical fractionation reveals altered composition of excitatory receptors at synapses of Scyl2-deficient mice; inhibition of excitatory signaling prevents CA3 neuron degeneration, placing SCYL2 as a regulator of excitatory receptor trafficking/synaptic composition that suppresses excitotoxicity.\",\n      \"method\": \"Conditional neuron-specific knockout mice, histology, electrophysiology, BAX genetic epistasis (BAX-dependent apoptosis), synaptic fractionation and receptor composition analysis, pharmacological inhibition of excitatory signaling\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic knockout with BAX epistasis, biochemical fractionation, and pharmacological rescue; multiple orthogonal approaches in one rigorous study\",\n      \"pmids\": [\"26203146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"SCYL2 forms a complex with clathrin heavy chain (CHC) and binds PTEN; the SCYL2-associated complex phosphorylates PTEN at S380/T382/T383 (STT cluster); SCYL2 expression induces CHC binding to PTEN; CHC downregulation or CCV inhibition reduces phospho-PTEN(STT), suggesting clathrin-coated vesicles serve as a signaling platform for PTEN phosphorylation; SCYL2 downregulation has anti-tumor effects via PI3K/AKT inhibition through PTEN dephosphorylation.\",\n      \"method\": \"Co-immunoprecipitation (SCYL2 with PTEN and CHC), siRNA knockdown of SCYL2 and CHC, phosphorylation assay (PTEN STT cluster), cell survival assay\",\n      \"journal\": \"Cancer gene therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP and RNAi with phosphorylation readout; single lab, and the abstract notes the mechanism by which SCYL2 regulates PTEN phosphorylation 'remains unclear', limiting confidence\",\n      \"pmids\": [\"41703271\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SCYL2 (CVAK104) is a pseudokinase that associates with clathrin-coated vesicles and directly binds clathrin and AP complexes (AP-1, AP-2 via alpha-appendage and N-terminal domain of clathrin heavy chain); it phosphorylates AP2-beta2-adaptin in vitro, regulates SNARE sorting (syntaxin 8, vti1b) into CCVs, promotes clathrin-mediated lysosomal degradation of Frizzled-5 (thereby suppressing Wnt/beta-catenin signaling), recruits PP2A to dephosphorylate HIV-1 Vpu and restrict viral release, and in neurons maintains proper excitatory receptor composition at synapses to suppress excitotoxicity via a BAX-dependent apoptotic pathway.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SCYL2 (CVAK104) is a clathrin-coated vesicle-associated pseudokinase that functions as a sorting and signaling regulator at the clathrin trafficking machinery [#0, #1]. It co-fractionates with clathrin-coated vesicles and binds directly to clathrin (via the N-terminal domain) and the AP2 adaptor (via the alpha-appendage), with membrane association being clathrin-dependent and perinuclear recruitment requiring GTP-binding proteins [#0, #1]. SCYL2 localizes to the trans-Golgi network and endosomal compartments where it associates with clathrin, AP-1, and epsinR, and is required for proper sorting of cargo into clathrin-coated vesicles, including the lysosomal hydrolase cathepsin D and the SNAREs syntaxin 8 and vti1b [#1, #2]. Through this trafficking activity SCYL2 controls the fate of specific receptors: it drives clathrin-mediated lysosomal degradation of Frizzled-5 to act as a negative regulator of Wnt/beta-catenin signaling [#3], and in neurons it maintains the composition of excitatory receptors at synapses, with its loss causing BAX-dependent apoptotic degeneration of CA3 hippocampal neurons due to excitotoxicity [#5]. SCYL2 additionally serves as a scaffold that couples phosphatase activity to its substrates, recruiting PP2A to dephosphorylate HIV-1 Vpu and thereby restrict viral release [#4]. A clathrin-coated-vesicle SCYL2 complex also promotes phosphorylation of PTEN at the STT cluster, linking SCYL2 to PI3K/AKT signaling [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing whether SCYL2 was a bona fide component of the clathrin machinery and an active enzyme determined how to frame its cellular role; the finding placed it at clathrin-coated vesicles with kinase-like activity toward an adaptor subunit.\",\n      \"evidence\": \"Mass spectrometry, in vitro kinase and ATP-binding assays, and co-fractionation with clathrin-coated vesicle preparations\",\n      \"pmids\": [\"15809293\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro phosphorylation of beta2-adaptin does not establish a physiological substrate\", \"Whether catalytic activity is required for any cellular function was not tested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defining SCYL2's subcellular localization and the determinants of its membrane recruitment showed it operates in TGN-to-endosome clathrin trafficking rather than only at the plasma membrane.\",\n      \"evidence\": \"RNAi, live-cell imaging, GTPgammaS/brefeldin A perturbation, cathepsin D sorting assay, and direct binding assays to clathrin N-terminal domain and AP2 alpha-appendage\",\n      \"pmids\": [\"16914521\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the GTP-binding protein required for perinuclear recruitment unspecified\", \"Mechanism linking SCYL2 loss to cathepsin D missorting not resolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identifying a specific cargo class clarified what SCYL2 sorts; it is required for incorporation of selected SNAREs into clathrin-coated vesicles.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, comparative CCV proteomics, siRNA knockdown, and immunofluorescence colocalization\",\n      \"pmids\": [\"17587408\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SNARE sorting depends on direct SCYL2 binding versus an indirect adaptor effect is unresolved\", \"Functional consequence of syntaxin 8/vti1b mislocalization not measured\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Connecting SCYL2-dependent trafficking to a signaling output demonstrated it degrades a specific Wnt receptor and thereby tunes pathway activity.\",\n      \"evidence\": \"Co-IP with Dvl and Fzd5, gain/loss-of-function RNAi, dominant-negative Rab5 epistasis, lysosomal inhibition, and Wnt/beta-catenin reporter assay\",\n      \"pmids\": [\"19643732\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Basis for Fzd5 selectivity over Fzd1/Fzd4 not defined\", \"Role of SCYL2 catalytic activity in Fzd5 degradation untested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showing SCYL2 recruits a phosphatase to a viral substrate revealed a scaffolding mode distinct from its sorting role and a function in innate antiviral restriction.\",\n      \"evidence\": \"Co-IP defining a SCYL2-Vpu-PP2A complex, siRNA knockdown, Vpu phospho-site analysis, virion release assay, and interferon induction\",\n      \"pmids\": [\"23047923\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SCYL2 directly bridges PP2A and Vpu or acts via the trafficking machinery is unclear\", \"Generalizability of the PP2A-recruitment scaffold to host substrates not explored\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"An in vivo genetic test established the physiological importance of SCYL2 in neurons, linking its trafficking role to control of synaptic excitatory receptor composition and survival.\",\n      \"evidence\": \"Neuron-specific conditional knockout mice with histology, electrophysiology, BAX genetic epistasis, synaptic fractionation, and pharmacological rescue of excitatory signaling\",\n      \"pmids\": [\"26203146\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific receptor subunits trafficked by SCYL2 not molecularly identified\", \"Mechanistic link between altered receptor composition and BAX-dependent apoptosis not delineated\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Implicating a SCYL2-clathrin complex in PTEN phosphorylation extended SCYL2 into PI3K/AKT signaling and a potential tumor context.\",\n      \"evidence\": \"Co-IP of SCYL2 with PTEN and CHC, siRNA knockdown of SCYL2 and CHC, PTEN STT-cluster phosphorylation assay, and cell survival assay\",\n      \"pmids\": [\"41703271\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The kinase responsible for and mechanism of PTEN phosphorylation remains unclear per the study itself\", \"Whether SCYL2 phosphorylates PTEN directly versus recruiting another kinase is untested\", \"Single-lab Co-IP without reciprocal in vivo validation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether SCYL2's catalytic activity is required for its diverse trafficking and signaling functions, and how a single CCV-associated protein selects among such varied substrates (SNAREs, Fzd5, Vpu/PP2A, PTEN), remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of SCYL2 substrate or adaptor selectivity\", \"No catalytically-dead mutant rescue across the reported functions\", \"Mechanism unifying scaffold versus kinase roles not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2, 4]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 6]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [\"clathrin-coated vesicle\"],\n    \"partners\": [\"CLTC\", \"AP2\", \"AP1\", \"CLINT1\", \"FZD5\", \"DVL\", \"PPP2 (PP2A)\", \"PTEN\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}