{"gene":"PLEKHA5","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2011,"finding":"The PH domain of PLEKHA5 specifically interacts with phosphoinositides PI3P, PI4P, PI5P, and PI(3,5)P2, as determined by protein-lipid overlay assays. PLEKHA5 exists in two splice forms (L-PLEKHA5 and S-PLEKHA5) both containing WW and PH domains, and both localize mainly to the cytosol.","method":"Protein-lipid overlay assay, subcellular fractionation, characterization of splice variants","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 1 (direct in vitro binding assay) / Weak — single lab, single method for lipid binding; localization by fractionation without functional consequence linked","pmids":["22037487"],"is_preprint":false},{"year":2014,"finding":"Knockdown of PLEKHA5 decreases viability of brain-tropic melanoma cells (A375Br and YUMUL), inhibits blood-brain barrier transmigration, and inhibits invasion in vitro, establishing a functional role for PLEKHA5 in melanoma brain metastasis. Knockdown did not affect viability of parental A375P cells.","method":"siRNA knockdown, in vitro BBB transmigration assay, invasion assay, cell viability assay","journal":"Clinical Cancer Research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function in two cell lines with multiple functional readouts (viability, transmigration, invasion), single lab","pmids":["25316811"],"is_preprint":false},{"year":2019,"finding":"PLEKHA5 regulates the G1-to-S cell cycle transition in melanoma cells; its knockdown inhibits proliferation coincident with upregulation of PDCD4 protein, while ectopic PLEKHA5 expression has the inverse effect. PLEKHA5 modulation affects PDCD4 protein stability and is coupled with changes in PI3K/AKT/mTOR pathway signaling.","method":"Stable knockdown, ectopic overexpression, cell cycle analysis, Western blot for PDCD4 and PI3K/AKT/mTOR components, xenograft tumor models","journal":"Cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss- and gain-of-function in vitro and in vivo, multiple signaling readouts, single lab","pmids":["31769872"],"is_preprint":false},{"year":2020,"finding":"Plekha5 deficiency in a mouse model of BRCA1-associated breast cancer promotes cancer metastasis to the liver and/or lung, identifying Plekha5 as a tumor metastasis suppressor.","method":"Single-cell whole-exome sequencing (scWES) of mouse/human BRCA1-associated breast cancers combined with functional loss-of-function in vivo metastasis assays","journal":"Nature Communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo loss-of-function with defined metastatic phenotype, single lab but genomic + functional orthogonal methods","pmids":["32978388"],"is_preprint":false},{"year":2021,"finding":"PLEKHA5 (and PLEKHA6) bind to PDZD11 via their WW domains, forming complexes that recruit PDZD11 to distinct plasma membrane localizations. These WW-PLEKHA–PDZD11 complexes are required for efficient anterograde targeting of the Menkes copper ATPase ATP7A to the cell periphery under elevated copper conditions. Pull-down experiments showed WW-PLEKHAs promote PDZD11 interaction with the C-terminus of ATP7A. Loss of WW-PLEKHAs or PDZD11 impairs copper extrusion and elevates intracellular copper.","method":"CRISPR knockout, immunofluorescence microscopy, pull-down assay, copper measurement (bioavailable and total), metallothionein-1 expression assay, cell viability assay","journal":"Molecular Biology of the Cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — CRISPR KO, reciprocal pull-down, multiple orthogonal functional readouts (localization, copper levels, metallothionein, viability), single lab with comprehensive mechanistic coverage","pmids":["34613798"],"is_preprint":false},{"year":2021,"finding":"The WW domain-mediated interaction between PLEKHA5 and PDZD11 is required for their mutual association with cytoplasmic microtubules. The PH domain of PLEKHA5 is required for its localization along the lateral plasma membrane. Distinct subcellular localizations of WW-PLEKHAs arise from cooperative contributions of their WW, PH, and C-terminal domains.","method":"Expression of mutant and chimeric proteins in cultured cells, immunofluorescence microscopy","journal":"Frontiers in Cell and Developmental Biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mutagenesis and chimeric constructs with imaging readouts, single lab, multiple domain combinations tested","pmids":["34568338"],"is_preprint":false},{"year":2021,"finding":"PLEKHA5 is tyrosine-phosphorylated downstream of the Met receptor tyrosine kinase in diffuse-type gastric carcinoma (DGC) cells with Met gene amplification. PLEKHA5 knockdown selectively suppresses growth of Met-amplified DGC cells by inducing apoptosis and dysregulates glycolytic metabolism, leading to JNK pathway activation that promotes apoptosis. PLEKHA5 silencing also abrogates peritoneal dissemination in vivo.","method":"Phosphoproteomic analysis, siRNA knockdown, apoptosis assay, glycolytic metabolism assay, JNK pathway analysis, in vivo peritoneal dissemination model","journal":"Oncogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphoproteomics identifying PTM, loss-of-function with multiple mechanistic readouts (apoptosis, glycolysis, JNK), in vivo validation, single lab","pmids":["33677467"],"is_preprint":false},{"year":2025,"finding":"TNFα activates the PLEKHA5-FCRLA axis in cutaneous melanoma cells. PLEKHA5 knockdown increased ceramide and sphingosine levels while decreasing cholesterol ester and triglyceride levels, implicating PLEKHA5 in regulating neutral lipid storage. FCRLA was identified as a downstream gene of PLEKHA5.","method":"In vitro and in vivo experiments, transcriptomics, proteomics, lipid metabolomics, RNA interference","journal":"Lipids in Health and Disease","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, metabolomics with knockdown but limited mechanistic detail on how PLEKHA5 directly controls lipid metabolism; FCRLA downstream relationship inferred from expression data","pmids":["40462127"],"is_preprint":false},{"year":2025,"finding":"The long isoform of PLEKHA5 (PLEKHA5-L) promotes melanoma cell proliferation and migration. RNA sequencing after PLEKHA5-L manipulation revealed upregulation of oncogenes including HRAS and AKT3, as well as PD-L1 and ABC transporters.","method":"RNA interference, lentiviral overexpression, CCK8 assay, colony formation assay, transwell migration assay, RNA sequencing","journal":"Frontiers in Oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, functional assays with transcriptomic pathway inference but no direct mechanistic validation of HRAS/AKT3 as causal effectors","pmids":["40356756"],"is_preprint":false}],"current_model":"PLEKHA5 is a multi-domain scaffold protein (containing tandem WW domains and a PH domain) that binds phosphoinositides (PI3P, PI4P, PI5P, PI(3,5)P2) through its PH domain, interacts with PDZD11 via its WW domains to form complexes that localize to the lateral plasma membrane and cytoplasmic microtubules, and recruits PDZD11 to direct anterograde trafficking of the Menkes copper ATPase ATP7A to the cell periphery for copper extrusion; in cancer contexts, PLEKHA5 acts downstream of Met receptor signaling (where it is tyrosine-phosphorylated), promotes G1-to-S cell cycle transition by regulating PDCD4 stability and PI3K/AKT/mTOR signaling, and facilitates melanoma brain metastasis by supporting BBB transmigration and cell survival."},"narrative":{"mechanistic_narrative":"PLEKHA5 is a multi-domain scaffold protein that couples phosphoinositide-defined membrane microdomains to protein trafficking and to cell proliferation and metastasis programs. Through its PH domain it binds the phosphoinositides PI3P, PI4P, PI5P, and PI(3,5)P2, and this PH domain directs its localization along the lateral plasma membrane [PMID:22037487, PMID:34568338]. Via tandem WW domains PLEKHA5 binds the adaptor PDZD11, and this interaction both mediates their joint association with cytoplasmic microtubules and recruits PDZD11 to plasma membrane sites [PMID:34568338, PMID:34613798]. Functionally, the WW-PLEKHA–PDZD11 complex promotes PDZD11 binding to the C-terminus of the Menkes copper ATPase ATP7A and is required for efficient anterograde targeting of ATP7A to the cell periphery, so that loss of the complex impairs copper extrusion and elevates intracellular copper [PMID:34613798]. In cancer contexts, PLEKHA5 is tyrosine-phosphorylated downstream of the Met receptor in Met-amplified gastric carcinoma cells, where it supports survival and glycolytic metabolism and restrains JNK-driven apoptosis [PMID:33677467], and it drives the G1-to-S transition in melanoma by destabilizing the tumor suppressor PDCD4 and engaging PI3K/AKT/mTOR signaling [PMID:31769872]. PLEKHA5 is required for melanoma brain-metastatic cell viability, invasion, and blood-brain-barrier transmigration [PMID:25316811], yet acts as a metastasis suppressor in BRCA1-associated breast cancer [PMID:32978388], indicating context-dependent roles in tumor dissemination.","teleology":[{"year":2011,"claim":"Established the biochemical basis for PLEKHA5 membrane association by showing its PH domain reads specific phosphoinositides, defining it as a candidate phosphoinositide-binding scaffold.","evidence":"Protein-lipid overlay assays and subcellular fractionation of two splice variants in vitro","pmids":["22037487"],"confidence":"Medium","gaps":["Lipid binding shown by a single overlay method without cellular validation","No functional consequence of phosphoinositide binding established at this stage","Functional difference between L- and S-isoforms unresolved"]},{"year":2014,"claim":"Provided the first functional role for PLEKHA5, showing it is selectively required for brain-tropic melanoma cell survival, invasion, and BBB transmigration rather than for parental tumor cell viability.","evidence":"siRNA knockdown with in vitro BBB transmigration, invasion, and viability assays in brain-tropic vs parental melanoma lines","pmids":["25316811"],"confidence":"Medium","gaps":["Molecular mechanism linking PLEKHA5 to transmigration not defined","No in vivo brain metastasis confirmation","Effector pathways downstream of PLEKHA5 not identified"]},{"year":2019,"claim":"Connected PLEKHA5 to cell cycle control, showing it promotes the G1-to-S transition by destabilizing PDCD4 and engaging PI3K/AKT/mTOR signaling.","evidence":"Stable knockdown and overexpression with cell cycle analysis, PDCD4/PI3K-AKT-mTOR Western blotting, and xenograft models","pmids":["31769872"],"confidence":"Medium","gaps":["Mechanism by which PLEKHA5 controls PDCD4 protein stability undefined","Whether PI3K/AKT/mTOR changes are direct or secondary unresolved","No direct enzymatic activity assigned to PLEKHA5"]},{"year":2020,"claim":"Revealed context-dependence of PLEKHA5 in metastasis by identifying it as a metastasis suppressor in BRCA1-associated breast cancer, opposite to its pro-metastatic role in melanoma.","evidence":"Single-cell whole-exome sequencing combined with in vivo loss-of-function metastasis assays in mouse/human BRCA1-associated breast cancer","pmids":["32978388"],"confidence":"Medium","gaps":["Mechanism of metastasis suppression not defined","Reconciliation with pro-metastatic melanoma role unexplained","Tissue-specific determinants of opposing roles unknown"]},{"year":2021,"claim":"Defined the core molecular machine: WW-domain binding to PDZD11 builds a complex that, together with PH-domain phosphoinositide reading, recruits the adaptor to membranes and microtubules and directs ATP7A trafficking for copper extrusion.","evidence":"CRISPR knockout, reciprocal pull-downs, immunofluorescence with domain mutants/chimeras, and copper/metallothionein measurements","pmids":["34613798","34568338"],"confidence":"High","gaps":["Structural basis of WW–PDZD11 and PDZD11–ATP7A interactions not resolved","How phosphoinositide binding is regulated in cells unknown","Link between trafficking scaffold function and cancer phenotypes unestablished"]},{"year":2021,"claim":"Placed PLEKHA5 downstream of an oncogenic receptor tyrosine kinase, showing it is Met-phosphorylated and required for survival and metabolic homeostasis of Met-amplified gastric carcinoma cells.","evidence":"Phosphoproteomics, siRNA knockdown, apoptosis and glycolysis assays, JNK analysis, and in vivo peritoneal dissemination model","pmids":["33677467"],"confidence":"Medium","gaps":["Functional consequence of specific tyrosine phosphorylation sites not dissected","Mechanism linking PLEKHA5 to glycolysis and JNK suppression undefined","Whether scaffold/trafficking function underlies the survival role unknown"]},{"year":2025,"claim":"Extended PLEKHA5 function to lipid storage and a TNFα-responsive transcriptional axis in melanoma.","evidence":"RNA interference with transcriptomics, proteomics, and lipid metabolomics in vitro and in vivo","pmids":["40462127"],"confidence":"Low","gaps":["No direct mechanism for how PLEKHA5 controls neutral lipid metabolism","FCRLA downstream relationship inferred from expression data only","Not independently confirmed"]},{"year":2025,"claim":"Attributed isoform-specific oncogenic activity to PLEKHA5-L in melanoma proliferation and migration.","evidence":"RNAi, lentiviral overexpression, proliferation/migration assays, and RNA sequencing","pmids":["40356756"],"confidence":"Low","gaps":["HRAS/AKT3 and other effectors inferred from transcriptomics without causal validation","Molecular basis of isoform-specific activity undefined","Single-lab transcriptomic pathway inference"]},{"year":null,"claim":"How PLEKHA5's defined scaffold/trafficking biochemistry mechanistically produces its divergent, tissue-specific roles in proliferation, survival, and metastasis remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the WW-PLEKHA–PDZD11–ATP7A complex","Mechanism reconciling pro-metastatic vs metastasis-suppressor contexts unknown","Direct link between phosphoinositide binding/trafficking function and cancer signaling not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[4,5]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[4,5]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[5]}],"pathway":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[4]}],"complexes":["WW-PLEKHA–PDZD11 complex"],"partners":["PDZD11","ATP7A","MET"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9HAU0","full_name":"Pleckstrin homology domain-containing family A member 5","aliases":["Phosphoinositol 3-phosphate-binding protein 2","PEPP-2"],"length_aa":1116,"mass_kda":127.5,"function":"","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9HAU0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PLEKHA5","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"MAP4","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PLEKHA5","total_profiled":1310},"omim":[{"mim_id":"607770","title":"PLECKSTRIN HOMOLOGY DOMAIN-CONTAINING PROTEIN, FAMILY A, MEMBER 5; PLEKHA5","url":"https://www.omim.org/entry/607770"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PLEKHA5"},"hgnc":{"alias_symbol":["PEPP2","KIAA1686","FLJ10667"],"prev_symbol":[]},"alphafold":{"accession":"Q9HAU0","domains":[{"cath_id":"2.30.29.30","chopping":"171-269","consensus_level":"high","plddt":91.2081,"start":171,"end":269},{"cath_id":"1.10.287","chopping":"636-780","consensus_level":"high","plddt":90.8208,"start":636,"end":780}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9HAU0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9HAU0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9HAU0-F1-predicted_aligned_error_v6.png","plddt_mean":55.16},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PLEKHA5","jax_strain_url":"https://www.jax.org/strain/search?query=PLEKHA5"},"sequence":{"accession":"Q9HAU0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9HAU0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9HAU0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9HAU0"}},"corpus_meta":[{"pmid":"25316811","id":"PMC_25316811","title":"PLEKHA5 as a Biomarker and Potential Mediator of Melanoma Brain Metastasis.","date":"2014","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/25316811","citation_count":68,"is_preprint":false},{"pmid":"34613798","id":"PMC_34613798","title":"PLEKHA5, PLEKHA6, and PLEKHA7 bind to PDZD11 to target the Menkes ATPase ATP7A to the cell periphery and regulate copper homeostasis.","date":"2021","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/34613798","citation_count":36,"is_preprint":false},{"pmid":"32978388","id":"PMC_32978388","title":"Characterization of BRCA1-deficient premalignant tissues and cancers identifies Plekha5 as a tumor metastasis suppressor.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/32978388","citation_count":26,"is_preprint":false},{"pmid":"22037487","id":"PMC_22037487","title":"Identification and characterization of splicing variants of PLEKHA5 (Plekha5) during brain development.","date":"2011","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/22037487","citation_count":21,"is_preprint":false},{"pmid":"31769872","id":"PMC_31769872","title":"PLEKHA5 regulates tumor growth in metastatic melanoma.","date":"2019","source":"Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/31769872","citation_count":18,"is_preprint":false},{"pmid":"34568338","id":"PMC_34568338","title":"WW, PH and C-Terminal Domains Cooperate to Direct the Subcellular Localizations of PLEKHA5, PLEKHA6 and PLEKHA7.","date":"2021","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/34568338","citation_count":18,"is_preprint":false},{"pmid":"30454882","id":"PMC_30454882","title":"High genetic risk scores of SLIT3, PLEKHA5 and PPP2R2C variants increased insulin resistance and interacted with coffee and caffeine consumption in middle-aged adults.","date":"2018","source":"Nutrition, metabolism, and cardiovascular diseases : NMCD","url":"https://pubmed.ncbi.nlm.nih.gov/30454882","citation_count":18,"is_preprint":false},{"pmid":"33677467","id":"PMC_33677467","title":"PLEKHA5 regulates the survival and peritoneal dissemination of diffuse-type gastric carcinoma cells with Met gene amplification.","date":"2021","source":"Oncogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/33677467","citation_count":14,"is_preprint":false},{"pmid":"40462127","id":"PMC_40462127","title":"TNFα activation of the PLEKHA5-FCRLA axis disturbs lipid metabolism, leading to the progression of cutaneous malignant melanoma.","date":"2025","source":"Lipids in health and disease","url":"https://pubmed.ncbi.nlm.nih.gov/40462127","citation_count":2,"is_preprint":false},{"pmid":"40356756","id":"PMC_40356756","title":"Single-cell transcriptomics and functional validation revealed PLEKHA5-L as a promoter of growth and migration in brain metastatic melanoma cells.","date":"2025","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/40356756","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7383,"output_tokens":2475,"usd":0.029637,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9647,"output_tokens":3179,"usd":0.063855,"stage2_stop_reason":"end_turn"},"total_usd":0.093492,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"The PH domain of PLEKHA5 specifically interacts with phosphoinositides PI3P, PI4P, PI5P, and PI(3,5)P2, as determined by protein-lipid overlay assays. PLEKHA5 exists in two splice forms (L-PLEKHA5 and S-PLEKHA5) both containing WW and PH domains, and both localize mainly to the cytosol.\",\n      \"method\": \"Protein-lipid overlay assay, subcellular fractionation, characterization of splice variants\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 (direct in vitro binding assay) / Weak — single lab, single method for lipid binding; localization by fractionation without functional consequence linked\",\n      \"pmids\": [\"22037487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Knockdown of PLEKHA5 decreases viability of brain-tropic melanoma cells (A375Br and YUMUL), inhibits blood-brain barrier transmigration, and inhibits invasion in vitro, establishing a functional role for PLEKHA5 in melanoma brain metastasis. Knockdown did not affect viability of parental A375P cells.\",\n      \"method\": \"siRNA knockdown, in vitro BBB transmigration assay, invasion assay, cell viability assay\",\n      \"journal\": \"Clinical Cancer Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function in two cell lines with multiple functional readouts (viability, transmigration, invasion), single lab\",\n      \"pmids\": [\"25316811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PLEKHA5 regulates the G1-to-S cell cycle transition in melanoma cells; its knockdown inhibits proliferation coincident with upregulation of PDCD4 protein, while ectopic PLEKHA5 expression has the inverse effect. PLEKHA5 modulation affects PDCD4 protein stability and is coupled with changes in PI3K/AKT/mTOR pathway signaling.\",\n      \"method\": \"Stable knockdown, ectopic overexpression, cell cycle analysis, Western blot for PDCD4 and PI3K/AKT/mTOR components, xenograft tumor models\",\n      \"journal\": \"Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss- and gain-of-function in vitro and in vivo, multiple signaling readouts, single lab\",\n      \"pmids\": [\"31769872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Plekha5 deficiency in a mouse model of BRCA1-associated breast cancer promotes cancer metastasis to the liver and/or lung, identifying Plekha5 as a tumor metastasis suppressor.\",\n      \"method\": \"Single-cell whole-exome sequencing (scWES) of mouse/human BRCA1-associated breast cancers combined with functional loss-of-function in vivo metastasis assays\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo loss-of-function with defined metastatic phenotype, single lab but genomic + functional orthogonal methods\",\n      \"pmids\": [\"32978388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLEKHA5 (and PLEKHA6) bind to PDZD11 via their WW domains, forming complexes that recruit PDZD11 to distinct plasma membrane localizations. These WW-PLEKHA–PDZD11 complexes are required for efficient anterograde targeting of the Menkes copper ATPase ATP7A to the cell periphery under elevated copper conditions. Pull-down experiments showed WW-PLEKHAs promote PDZD11 interaction with the C-terminus of ATP7A. Loss of WW-PLEKHAs or PDZD11 impairs copper extrusion and elevates intracellular copper.\",\n      \"method\": \"CRISPR knockout, immunofluorescence microscopy, pull-down assay, copper measurement (bioavailable and total), metallothionein-1 expression assay, cell viability assay\",\n      \"journal\": \"Molecular Biology of the Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — CRISPR KO, reciprocal pull-down, multiple orthogonal functional readouts (localization, copper levels, metallothionein, viability), single lab with comprehensive mechanistic coverage\",\n      \"pmids\": [\"34613798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The WW domain-mediated interaction between PLEKHA5 and PDZD11 is required for their mutual association with cytoplasmic microtubules. The PH domain of PLEKHA5 is required for its localization along the lateral plasma membrane. Distinct subcellular localizations of WW-PLEKHAs arise from cooperative contributions of their WW, PH, and C-terminal domains.\",\n      \"method\": \"Expression of mutant and chimeric proteins in cultured cells, immunofluorescence microscopy\",\n      \"journal\": \"Frontiers in Cell and Developmental Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mutagenesis and chimeric constructs with imaging readouts, single lab, multiple domain combinations tested\",\n      \"pmids\": [\"34568338\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLEKHA5 is tyrosine-phosphorylated downstream of the Met receptor tyrosine kinase in diffuse-type gastric carcinoma (DGC) cells with Met gene amplification. PLEKHA5 knockdown selectively suppresses growth of Met-amplified DGC cells by inducing apoptosis and dysregulates glycolytic metabolism, leading to JNK pathway activation that promotes apoptosis. PLEKHA5 silencing also abrogates peritoneal dissemination in vivo.\",\n      \"method\": \"Phosphoproteomic analysis, siRNA knockdown, apoptosis assay, glycolytic metabolism assay, JNK pathway analysis, in vivo peritoneal dissemination model\",\n      \"journal\": \"Oncogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphoproteomics identifying PTM, loss-of-function with multiple mechanistic readouts (apoptosis, glycolysis, JNK), in vivo validation, single lab\",\n      \"pmids\": [\"33677467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TNFα activates the PLEKHA5-FCRLA axis in cutaneous melanoma cells. PLEKHA5 knockdown increased ceramide and sphingosine levels while decreasing cholesterol ester and triglyceride levels, implicating PLEKHA5 in regulating neutral lipid storage. FCRLA was identified as a downstream gene of PLEKHA5.\",\n      \"method\": \"In vitro and in vivo experiments, transcriptomics, proteomics, lipid metabolomics, RNA interference\",\n      \"journal\": \"Lipids in Health and Disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, metabolomics with knockdown but limited mechanistic detail on how PLEKHA5 directly controls lipid metabolism; FCRLA downstream relationship inferred from expression data\",\n      \"pmids\": [\"40462127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The long isoform of PLEKHA5 (PLEKHA5-L) promotes melanoma cell proliferation and migration. RNA sequencing after PLEKHA5-L manipulation revealed upregulation of oncogenes including HRAS and AKT3, as well as PD-L1 and ABC transporters.\",\n      \"method\": \"RNA interference, lentiviral overexpression, CCK8 assay, colony formation assay, transwell migration assay, RNA sequencing\",\n      \"journal\": \"Frontiers in Oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, functional assays with transcriptomic pathway inference but no direct mechanistic validation of HRAS/AKT3 as causal effectors\",\n      \"pmids\": [\"40356756\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PLEKHA5 is a multi-domain scaffold protein (containing tandem WW domains and a PH domain) that binds phosphoinositides (PI3P, PI4P, PI5P, PI(3,5)P2) through its PH domain, interacts with PDZD11 via its WW domains to form complexes that localize to the lateral plasma membrane and cytoplasmic microtubules, and recruits PDZD11 to direct anterograde trafficking of the Menkes copper ATPase ATP7A to the cell periphery for copper extrusion; in cancer contexts, PLEKHA5 acts downstream of Met receptor signaling (where it is tyrosine-phosphorylated), promotes G1-to-S cell cycle transition by regulating PDCD4 stability and PI3K/AKT/mTOR signaling, and facilitates melanoma brain metastasis by supporting BBB transmigration and cell survival.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PLEKHA5 is a multi-domain scaffold protein that couples phosphoinositide-defined membrane microdomains to protein trafficking and to cell proliferation and metastasis programs. Through its PH domain it binds the phosphoinositides PI3P, PI4P, PI5P, and PI(3,5)P2, and this PH domain directs its localization along the lateral plasma membrane [#0, #5]. Via tandem WW domains PLEKHA5 binds the adaptor PDZD11, and this interaction both mediates their joint association with cytoplasmic microtubules and recruits PDZD11 to plasma membrane sites [#5, #4]. Functionally, the WW-PLEKHA–PDZD11 complex promotes PDZD11 binding to the C-terminus of the Menkes copper ATPase ATP7A and is required for efficient anterograde targeting of ATP7A to the cell periphery, so that loss of the complex impairs copper extrusion and elevates intracellular copper [#4]. In cancer contexts, PLEKHA5 is tyrosine-phosphorylated downstream of the Met receptor in Met-amplified gastric carcinoma cells, where it supports survival and glycolytic metabolism and restrains JNK-driven apoptosis [#6], and it drives the G1-to-S transition in melanoma by destabilizing the tumor suppressor PDCD4 and engaging PI3K/AKT/mTOR signaling [#2]. PLEKHA5 is required for melanoma brain-metastatic cell viability, invasion, and blood-brain-barrier transmigration [#1], yet acts as a metastasis suppressor in BRCA1-associated breast cancer [#3], indicating context-dependent roles in tumor dissemination.\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Established the biochemical basis for PLEKHA5 membrane association by showing its PH domain reads specific phosphoinositides, defining it as a candidate phosphoinositide-binding scaffold.\",\n      \"evidence\": \"Protein-lipid overlay assays and subcellular fractionation of two splice variants in vitro\",\n      \"pmids\": [\"22037487\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Lipid binding shown by a single overlay method without cellular validation\", \"No functional consequence of phosphoinositide binding established at this stage\", \"Functional difference between L- and S-isoforms unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Provided the first functional role for PLEKHA5, showing it is selectively required for brain-tropic melanoma cell survival, invasion, and BBB transmigration rather than for parental tumor cell viability.\",\n      \"evidence\": \"siRNA knockdown with in vitro BBB transmigration, invasion, and viability assays in brain-tropic vs parental melanoma lines\",\n      \"pmids\": [\"25316811\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism linking PLEKHA5 to transmigration not defined\", \"No in vivo brain metastasis confirmation\", \"Effector pathways downstream of PLEKHA5 not identified\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected PLEKHA5 to cell cycle control, showing it promotes the G1-to-S transition by destabilizing PDCD4 and engaging PI3K/AKT/mTOR signaling.\",\n      \"evidence\": \"Stable knockdown and overexpression with cell cycle analysis, PDCD4/PI3K-AKT-mTOR Western blotting, and xenograft models\",\n      \"pmids\": [\"31769872\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which PLEKHA5 controls PDCD4 protein stability undefined\", \"Whether PI3K/AKT/mTOR changes are direct or secondary unresolved\", \"No direct enzymatic activity assigned to PLEKHA5\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed context-dependence of PLEKHA5 in metastasis by identifying it as a metastasis suppressor in BRCA1-associated breast cancer, opposite to its pro-metastatic role in melanoma.\",\n      \"evidence\": \"Single-cell whole-exome sequencing combined with in vivo loss-of-function metastasis assays in mouse/human BRCA1-associated breast cancer\",\n      \"pmids\": [\"32978388\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of metastasis suppression not defined\", \"Reconciliation with pro-metastatic melanoma role unexplained\", \"Tissue-specific determinants of opposing roles unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined the core molecular machine: WW-domain binding to PDZD11 builds a complex that, together with PH-domain phosphoinositide reading, recruits the adaptor to membranes and microtubules and directs ATP7A trafficking for copper extrusion.\",\n      \"evidence\": \"CRISPR knockout, reciprocal pull-downs, immunofluorescence with domain mutants/chimeras, and copper/metallothionein measurements\",\n      \"pmids\": [\"34613798\", \"34568338\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of WW–PDZD11 and PDZD11–ATP7A interactions not resolved\", \"How phosphoinositide binding is regulated in cells unknown\", \"Link between trafficking scaffold function and cancer phenotypes unestablished\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed PLEKHA5 downstream of an oncogenic receptor tyrosine kinase, showing it is Met-phosphorylated and required for survival and metabolic homeostasis of Met-amplified gastric carcinoma cells.\",\n      \"evidence\": \"Phosphoproteomics, siRNA knockdown, apoptosis and glycolysis assays, JNK analysis, and in vivo peritoneal dissemination model\",\n      \"pmids\": [\"33677467\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of specific tyrosine phosphorylation sites not dissected\", \"Mechanism linking PLEKHA5 to glycolysis and JNK suppression undefined\", \"Whether scaffold/trafficking function underlies the survival role unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended PLEKHA5 function to lipid storage and a TNFα-responsive transcriptional axis in melanoma.\",\n      \"evidence\": \"RNA interference with transcriptomics, proteomics, and lipid metabolomics in vitro and in vivo\",\n      \"pmids\": [\"40462127\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct mechanism for how PLEKHA5 controls neutral lipid metabolism\", \"FCRLA downstream relationship inferred from expression data only\", \"Not independently confirmed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Attributed isoform-specific oncogenic activity to PLEKHA5-L in melanoma proliferation and migration.\",\n      \"evidence\": \"RNAi, lentiviral overexpression, proliferation/migration assays, and RNA sequencing\",\n      \"pmids\": [\"40356756\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"HRAS/AKT3 and other effectors inferred from transcriptomics without causal validation\", \"Molecular basis of isoform-specific activity undefined\", \"Single-lab transcriptomic pathway inference\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PLEKHA5's defined scaffold/trafficking biochemistry mechanistically produces its divergent, tissue-specific roles in proliferation, survival, and metastasis remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the WW-PLEKHA–PDZD11–ATP7A complex\", \"Mechanism reconciling pro-metastatic vs metastasis-suppressor contexts unknown\", \"Direct link between phosphoinositide binding/trafficking function and cancer signaling not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [4, 5]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"complexes\": [\n      \"WW-PLEKHA–PDZD11 complex\"\n    ],\n    \"partners\": [\n      \"PDZD11\",\n      \"ATP7A\",\n      \"MET\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":4,"faith_total":5,"faith_pct":80.0}}