{"gene":"PSKH1","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2000,"finding":"PSKH1 protein autophosphorylates exclusively on serines within its C-terminal region in an intermolecular fashion; autophosphorylation activity is repressed upon addition of Ca2+/calmodulin, suggesting that PSKH1 activity is regulated by Ca2+ concentration in vivo. Immunoisolated PSKH1 does not phosphorylate common kinase substrates in vitro.","method":"In vitro kinase assay with immunoisolated PSKH1; Ca2+/CaM addition; mutational/biochemical analysis","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 1–2 / Weak — in vitro kinase assay with immunoisolated protein, single lab, single study","pmids":["11087665"],"is_preprint":false},{"year":2000,"finding":"PSKH1 localizes to the Golgi apparatus (Brefeldin A-sensitive compartment), centrosomes, and nucleus (speckle-like pattern) in COS-1 cells; centrosomal localization is enhanced during osmotic stress.","method":"Indirect immunofluorescence microscopy of untransfected COS-1 cells with polyclonal antibodies; Brefeldin A treatment","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct localization experiment with functional context (osmotic stress), single lab, single study","pmids":["11087665"],"is_preprint":false},{"year":2002,"finding":"Endogenous PSKH1 localizes to splicing factor compartments (SFCs) in the nucleus; co-expression of SR proteins (ASF/SF2 and SC35) enhances PSKH1-FLAG migration into SFCs. PSKH1 overexpression reorganizes co-expressed SR proteins (SC35 and ASF/SF2) into a more diffuse nuclear pattern, and this redistribution does not require PSKH1 kinase activity. Forced PSKH1 expression stimulates distal splicing of an E1A minigene in HeLa cells. GST-ASF/SF2 is not phosphorylated by PSKH1, indicating indirect action on SR proteins. SFC-association maps to the catalytic kinase domain and C-terminus.","method":"Immunofluorescence microscopy; co-expression assays; E1A minigene splicing assay in HeLa cells; GST pulldown/kinase assay; yeast two-hybrid; domain truncation analysis","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (localization, splicing assay, kinase assay, pulldown), single lab","pmids":["12466556"],"is_preprint":false},{"year":2003,"finding":"PSKH1 is myristoylated on glycine 2 and palmitoylated on cysteine 3. Dual acylation targets PSKH1 to the Golgi apparatus (co-localizing with beta-COP and GM130); myristoylation alone (without palmitoylation) redirects PSKH1 to ER membranes (co-localizing with PDI). The dually acylated N-terminal domain also targets plasma membrane. Expression of a PSKH1 mutant with the C-terminal kinase domain replaced by GFP and Cys3 mutated to Ser causes disassembly of the Golgi apparatus (redistribution of beta-COP and GM130 to diffuse cytoplasm) without affecting tubulin. The first 29 amino acids constitute the minimal Golgi-targeting region.","method":"Biochemical acylation assays; immunofluorescence co-localization with organelle markers (beta-COP, GM130, PDI); immunoelectron microscopy; subcellular fractionation by sucrose gradient; site-directed mutagenesis","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — biochemical acylation assay, immunoelectron microscopy, subcellular fractionation, and mutagenesis in a single study with multiple orthogonal methods","pmids":["14644153"],"is_preprint":false},{"year":2003,"finding":"PSKH1 was identified as an in vitro substrate/target of Chk2 kinase using the GST-peptide approach based on defined phosphorylation consensus sequences.","method":"GST-peptide in vitro kinase assay; mutational analysis of consensus phosphorylation sites","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single in vitro peptide assay, no validation of in vivo relevance, single lab","pmids":["12711320"],"is_preprint":false},{"year":2020,"finding":"Evolutionary analysis reveals that species-level duplications of the canonical PSKH1 kinase led to the appearance of the pseudokinase PSKH2, demonstrating that PSKH1 is a canonical active kinase in the CAMK family whose duplication gave rise to a catalytically inactive paralog.","method":"Comparative sequence analysis and kinome evolutionary analysis","journal":"The FEBS journal","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational/comparative analysis only, no direct biochemical experiment on PSKH1","pmids":["32053275"],"is_preprint":false},{"year":2024,"finding":"Loss-of-function mutations in PSKH1 cause a novel hepatorenal ciliopathy. Recombinant PSKH1 disease variants show loss of catalytic activity in vitro. Patient fibroblasts display abnormally long cilia with aberrant transport. A homozygous Pskh1 mutant mouse recapitulates the human phenotype with abnormally long cilia, establishing that PSKH1 kinase activity is required for normal ciliogenesis and cilia-dependent transport.","method":"In vitro kinase assays of recombinant disease variants; immunofluorescence of patient fibroblast cilia; Pskh1 homozygous mutant mouse model with ciliary phenotype analysis","journal":"Genetics in medicine","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro reconstituted kinase assay of disease variants, patient cell biology, and mouse model with consistent phenotype across multiple independent families","pmids":["39132680"],"is_preprint":false},{"year":2025,"finding":"PSKH1's consensus substrate phosphorylation motif was defined biochemically. Ca2+-Calmodulin (CaM) activates PSKH1 despite the absence of a canonical CaM-binding motif, acting allosterically. Reticulocalbin-3 (an ER-resident Ca2+ sensor of the CREC family) suppresses PSKH1 catalytic activity via allosteric binding. UNC119B directly engages the PSKH1 kinase domain and activates PSKH1 catalytic activity. These represent complementary allosteric regulatory mechanisms that tune PSKH1 activity.","method":"Biochemistry (in vitro kinase assays); mass spectrometry-based substrate motif profiling; protein interaction studies (identification of interactors); domain-level binding/activity assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assays combined with mass spectrometry substrate profiling and protein interaction biochemistry, single lab, multiple orthogonal methods","pmids":["39964718"],"is_preprint":false},{"year":2023,"finding":"PSKH1 knockdown in osteosarcoma cells inhibited proliferation, migration, and invasion, while PSKH1 overexpression promoted proliferation. PSKH1 upregulated phospho-p38 MAPK, and the p38 MAPK inhibitor SB203580 blocked the tumor-promoting effects of PSKH1, placing PSKH1 upstream of p38/MAPK signaling in osteosarcoma cells.","method":"shRNA knockdown and overexpression in OS cell lines; Cell Counting Kit-8, colony formation, wound-healing, Transwell assays; p38 inhibitor SB203580 treatment; in vivo tumor xenograft assay","journal":"Oncology letters","confidence":"Medium","confidence_rationale":"Tier 2–3 / Weak — loss-of-function and gain-of-function with defined cellular phenotypes plus inhibitor epistasis, single lab","pmids":["36936027"],"is_preprint":false},{"year":2012,"finding":"shRNA knockdown of PSKH1 decreased cell growth in both androgen-dependent and castration-resistant prostate cancer cells (LNCaP), identifying PSKH1 as a regulator of prostate cancer cell growth.","method":"Lentiviral shRNA phenotypic screen; cell growth assays in LNCaP prostate cancer cells","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 3 / Weak — phenotypic screen with shRNA knockdown, limited mechanistic follow-up for PSKH1 specifically, single lab","pmids":["22761715"],"is_preprint":false},{"year":2019,"finding":"miR-566 directly targets PSKH1 mRNA (confirmed by luciferase reporter assay), suppressing PSKH1 protein expression; reintroduction of PSKH1 partially reversed the inhibitory effects of miR-566 on CRC cell growth and metastasis, placing PSKH1 downstream of miR-566 in colorectal cancer cell migration and invasion.","method":"Luciferase reporter assay; RT-PCR and western blot; miR-566 overexpression/inhibition; Transwell migration/invasion assays; PSKH1 rescue experiment","journal":"Cancer cell international","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — luciferase reporter confirms direct miRNA targeting, rescue experiment confirms epistasis, multiple methods, single lab","pmids":["31866763"],"is_preprint":false}],"current_model":"PSKH1 is a CAMK-family protein serine kinase that is dually acylated (myristoylated on Gly2 and palmitoylated on Cys3), targeting it to the Golgi apparatus and ER; it localizes to splicing factor compartments and centrosomes, modulates SR protein dynamics and pre-mRNA splicing indirectly, autophosphorylates its own C-terminal serines intermolecularly, and its catalytic activity is allosterically activated by Ca2+-Calmodulin and UNC119B and suppressed by Reticulocalbin-3; loss of PSKH1 kinase activity causes abnormally long cilia and a hepatorenal ciliopathy in humans and mice, and in cancer contexts PSKH1 promotes cell growth partly via p38/MAPK signaling."},"narrative":{"mechanistic_narrative":"PSKH1 is a calmodulin-regulated protein serine kinase of the CAMK family whose subcellular targeting and catalytic output are tightly controlled to support membrane organization and ciliary function [PMID:11087665, PMID:14644153, PMID:39964718]. The enzyme autophosphorylates on C-terminal serines in an intermolecular reaction and is allosterically tuned by calcium-sensing partners: Ca2+/calmodulin and UNC119B engage and activate the kinase domain, whereas the ER Ca2+-sensor Reticulocalbin-3 suppresses its activity, even though PSKH1 lacks a canonical CaM-binding motif [PMID:11087665, PMID:39964718]. Dual N-terminal acylation—myristoylation on Gly2 and palmitoylation on Cys3—directs PSKH1 to the Golgi apparatus and, in the absence of palmitoylation, to ER membranes; the integrity of this targeting and the kinase domain is required for normal Golgi structure [PMID:14644153]. PSKH1 also localizes to nuclear splicing factor compartments and centrosomes, where it reorganizes SR proteins and stimulates pre-mRNA splicing of an E1A minigene by a kinase-independent, indirect mechanism that does not involve direct phosphorylation of ASF/SF2 [PMID:11087665, PMID:12466556]. Loss-of-function PSKH1 mutations that abolish catalytic activity cause a hepatorenal ciliopathy in humans, with patient fibroblasts and a homozygous mutant mouse showing abnormally long cilia and aberrant ciliary transport, establishing that PSKH1 kinase activity is required for normal ciliogenesis [PMID:39132680]. In cancer contexts, PSKH1 promotes cell growth and is positioned upstream of p38/MAPK signaling [PMID:36936027].","teleology":[{"year":2000,"claim":"Establishing PSKH1 as an autoregulated, calcium-responsive kinase answered whether and how its catalytic activity is controlled.","evidence":"In vitro kinase assay with immunoisolated PSKH1 and Ca2+/CaM addition","pmids":["11087665"],"confidence":"Medium","gaps":["No physiological substrate identified","Effect of Ca2+/CaM was repression of autophosphorylation, later refined as allosteric activation by other studies"]},{"year":2000,"claim":"Mapping endogenous PSKH1 to Golgi, centrosomes, and nuclear speckles defined the compartments where it might act and linked it to stress responses.","evidence":"Indirect immunofluorescence in COS-1 cells with Brefeldin A and osmotic stress","pmids":["11087665"],"confidence":"Medium","gaps":["Functional consequence at each compartment unresolved","Mechanism of centrosomal enrichment under stress unknown"]},{"year":2002,"claim":"Linking PSKH1 to splicing factor compartments and SR protein dynamics addressed whether it participates in pre-mRNA processing, revealing a kinase-independent, indirect role.","evidence":"Immunofluorescence, SR co-expression, E1A minigene splicing, GST kinase assay, and domain truncation in HeLa cells","pmids":["12466556"],"confidence":"Medium","gaps":["Direct nuclear substrate not identified","Mechanism linking cytoplasmic/Golgi kinase to nuclear SR redistribution unclear","ASF/SF2 is not a direct substrate"]},{"year":2003,"claim":"Identifying dual acylation as the membrane-targeting determinant explained how PSKH1 is positioned at the Golgi and ER and tied it to organelle integrity.","evidence":"Acylation assays, organelle marker co-localization, immunoelectron microscopy, fractionation, and site-directed mutagenesis","pmids":["14644153"],"confidence":"High","gaps":["Whether Golgi disassembly reflects a dominant-negative artifact versus a physiological role is not resolved","Substrates at the Golgi unknown"]},{"year":2003,"claim":"Identification of PSKH1 as an in vitro Chk2 target raised the possibility of upstream phospho-regulation by DNA-damage signaling.","evidence":"GST-peptide in vitro kinase assay with consensus-site mutagenesis","pmids":["12711320"],"confidence":"Low","gaps":["No validation of in vivo phosphorylation by Chk2","Functional consequence unknown"]},{"year":2024,"claim":"Demonstrating that catalytically dead PSKH1 variants cause a hepatorenal ciliopathy established a definitive, kinase-activity-dependent physiological function in ciliogenesis.","evidence":"In vitro kinase assays of disease variants, patient fibroblast cilia imaging, and a homozygous Pskh1 mutant mouse","pmids":["39132680"],"confidence":"High","gaps":["Ciliary substrate(s) of PSKH1 not identified","Molecular link between Golgi/ER localization and cilia phenotype unresolved"]},{"year":2025,"claim":"Defining the substrate motif and the allosteric regulators (Ca2+/CaM, UNC119B activating; Reticulocalbin-3 suppressing) provided the regulatory logic governing when PSKH1 is active.","evidence":"In vitro kinase assays, MS-based substrate motif profiling, and protein interaction biochemistry","pmids":["39964718"],"confidence":"High","gaps":["In vivo substrates matching the motif not validated","Structural basis of non-canonical CaM activation not solved","Cellular context where each regulator dominates unclear"]},{"year":2023,"claim":"Placing PSKH1 upstream of p38/MAPK in osteosarcoma addressed how its catalytic role connects to growth control in cancer.","evidence":"shRNA/overexpression, proliferation/migration/invasion assays, SB203580 epistasis, and xenografts in OS cells","pmids":["36936027"],"confidence":"Medium","gaps":["Direct mechanistic link between PSKH1 kinase activity and p38 activation not defined","Single tumor type"]},{"year":null,"claim":"The bona fide physiological substrates of PSKH1 and the mechanism connecting its membrane localization to cilia and splicing phenotypes remain unknown.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No validated in vivo substrate","No structural model of the active kinase or its regulatory complexes","Unclear how a Golgi/ER-anchored kinase controls ciliary length"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,6,7]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,7]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[7]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[1,3]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[3]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[1]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[1,2]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[2]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[6]}],"complexes":[],"partners":["CALM1","UNC119B","RCN3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P11801","full_name":"Serine/threonine-protein kinase H1","aliases":["Protein serine kinase H1","PSK-H1"],"length_aa":424,"mass_kda":48.0,"function":"Serine/threonine protein kinase that may be involved in the regulation of pre-mRNA processing. It may phosphorylate components of nuclear splice factor compartments (SFC), such as non-snRNP splicing factors containing a serine/arginine-rich domain (SR proteins). Reversible phosphorylation of SR proteins may cause their release into the nucleoplasm and change their local concentration, thereby influencing alternative splicing","subcellular_location":"Golgi apparatus; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Nucleus speckle; Endoplasmic reticulum membrane; Cell membrane; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/P11801/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PSKH1","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PSKH1","total_profiled":1310},"omim":[{"mim_id":"620962","title":"CHOLESTASIS, PROGRESSIVE FAMILIAL INTRAHEPATIC, 13; PFIC13","url":"https://www.omim.org/entry/620962"},{"mim_id":"604119","title":"SOLUTE CARRIER FAMILY 12 (POTASSIUM/CHLORIDE TRANSPORTER), MEMBER 4; SLC12A4","url":"https://www.omim.org/entry/604119"},{"mim_id":"211600","title":"CHOLESTASIS, PROGRESSIVE FAMILIAL INTRAHEPATIC, 1; PFIC1","url":"https://www.omim.org/entry/211600"},{"mim_id":"177015","title":"PROTEIN SERINE KINASE H1; PSKH1","url":"https://www.omim.org/entry/177015"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nuclear speckles","reliability":"Supported"},{"location":"Plasma membrane","reliability":"Additional"},{"location":"Primary cilium","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PSKH1"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P11801","domains":[{"cath_id":"3.30.200.20","chopping":"88-171","consensus_level":"high","plddt":92.0461,"start":88,"end":171},{"cath_id":"1.10.510.10","chopping":"177-384_407-420","consensus_level":"high","plddt":86.7985,"start":177,"end":420}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P11801","model_url":"https://alphafold.ebi.ac.uk/files/AF-P11801-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P11801-F1-predicted_aligned_error_v6.png","plddt_mean":74.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PSKH1","jax_strain_url":"https://www.jax.org/strain/search?query=PSKH1"},"sequence":{"accession":"P11801","fasta_url":"https://rest.uniprot.org/uniprotkb/P11801.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P11801/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P11801"}},"corpus_meta":[{"pmid":"8268911","id":"PMC_8268911","title":"A tight cluster of five unrelated human genes on chromosome 16q22.1.","date":"1993","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/8268911","citation_count":85,"is_preprint":false},{"pmid":"11058107","id":"PMC_11058107","title":"Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites for ribozymes.","date":"2000","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/11058107","citation_count":57,"is_preprint":false},{"pmid":"12711320","id":"PMC_12711320","title":"Determination of substrate specificity and putative substrates of Chk2 kinase.","date":"2003","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/12711320","citation_count":46,"is_preprint":false},{"pmid":"22761715","id":"PMC_22761715","title":"Identification of kinases regulating prostate cancer cell growth using an RNAi phenotypic screen.","date":"2012","source":"PloS 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serines within its C-terminal region in an intermolecular fashion; autophosphorylation activity is repressed upon addition of Ca2+/calmodulin, suggesting that PSKH1 activity is regulated by Ca2+ concentration in vivo. Immunoisolated PSKH1 does not phosphorylate common kinase substrates in vitro.\",\n      \"method\": \"In vitro kinase assay with immunoisolated PSKH1; Ca2+/CaM addition; mutational/biochemical analysis\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Weak — in vitro kinase assay with immunoisolated protein, single lab, single study\",\n      \"pmids\": [\"11087665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"PSKH1 localizes to the Golgi apparatus (Brefeldin A-sensitive compartment), centrosomes, and nucleus (speckle-like pattern) in COS-1 cells; centrosomal localization is enhanced during osmotic stress.\",\n      \"method\": \"Indirect immunofluorescence microscopy of untransfected COS-1 cells with polyclonal antibodies; Brefeldin A treatment\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct localization experiment with functional context (osmotic stress), single lab, single study\",\n      \"pmids\": [\"11087665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Endogenous PSKH1 localizes to splicing factor compartments (SFCs) in the nucleus; co-expression of SR proteins (ASF/SF2 and SC35) enhances PSKH1-FLAG migration into SFCs. PSKH1 overexpression reorganizes co-expressed SR proteins (SC35 and ASF/SF2) into a more diffuse nuclear pattern, and this redistribution does not require PSKH1 kinase activity. Forced PSKH1 expression stimulates distal splicing of an E1A minigene in HeLa cells. GST-ASF/SF2 is not phosphorylated by PSKH1, indicating indirect action on SR proteins. SFC-association maps to the catalytic kinase domain and C-terminus.\",\n      \"method\": \"Immunofluorescence microscopy; co-expression assays; E1A minigene splicing assay in HeLa cells; GST pulldown/kinase assay; yeast two-hybrid; domain truncation analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (localization, splicing assay, kinase assay, pulldown), single lab\",\n      \"pmids\": [\"12466556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PSKH1 is myristoylated on glycine 2 and palmitoylated on cysteine 3. Dual acylation targets PSKH1 to the Golgi apparatus (co-localizing with beta-COP and GM130); myristoylation alone (without palmitoylation) redirects PSKH1 to ER membranes (co-localizing with PDI). The dually acylated N-terminal domain also targets plasma membrane. Expression of a PSKH1 mutant with the C-terminal kinase domain replaced by GFP and Cys3 mutated to Ser causes disassembly of the Golgi apparatus (redistribution of beta-COP and GM130 to diffuse cytoplasm) without affecting tubulin. The first 29 amino acids constitute the minimal Golgi-targeting region.\",\n      \"method\": \"Biochemical acylation assays; immunofluorescence co-localization with organelle markers (beta-COP, GM130, PDI); immunoelectron microscopy; subcellular fractionation by sucrose gradient; site-directed mutagenesis\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — biochemical acylation assay, immunoelectron microscopy, subcellular fractionation, and mutagenesis in a single study with multiple orthogonal methods\",\n      \"pmids\": [\"14644153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PSKH1 was identified as an in vitro substrate/target of Chk2 kinase using the GST-peptide approach based on defined phosphorylation consensus sequences.\",\n      \"method\": \"GST-peptide in vitro kinase assay; mutational analysis of consensus phosphorylation sites\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single in vitro peptide assay, no validation of in vivo relevance, single lab\",\n      \"pmids\": [\"12711320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Evolutionary analysis reveals that species-level duplications of the canonical PSKH1 kinase led to the appearance of the pseudokinase PSKH2, demonstrating that PSKH1 is a canonical active kinase in the CAMK family whose duplication gave rise to a catalytically inactive paralog.\",\n      \"method\": \"Comparative sequence analysis and kinome evolutionary analysis\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational/comparative analysis only, no direct biochemical experiment on PSKH1\",\n      \"pmids\": [\"32053275\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Loss-of-function mutations in PSKH1 cause a novel hepatorenal ciliopathy. Recombinant PSKH1 disease variants show loss of catalytic activity in vitro. Patient fibroblasts display abnormally long cilia with aberrant transport. A homozygous Pskh1 mutant mouse recapitulates the human phenotype with abnormally long cilia, establishing that PSKH1 kinase activity is required for normal ciliogenesis and cilia-dependent transport.\",\n      \"method\": \"In vitro kinase assays of recombinant disease variants; immunofluorescence of patient fibroblast cilia; Pskh1 homozygous mutant mouse model with ciliary phenotype analysis\",\n      \"journal\": \"Genetics in medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro reconstituted kinase assay of disease variants, patient cell biology, and mouse model with consistent phenotype across multiple independent families\",\n      \"pmids\": [\"39132680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PSKH1's consensus substrate phosphorylation motif was defined biochemically. Ca2+-Calmodulin (CaM) activates PSKH1 despite the absence of a canonical CaM-binding motif, acting allosterically. Reticulocalbin-3 (an ER-resident Ca2+ sensor of the CREC family) suppresses PSKH1 catalytic activity via allosteric binding. UNC119B directly engages the PSKH1 kinase domain and activates PSKH1 catalytic activity. These represent complementary allosteric regulatory mechanisms that tune PSKH1 activity.\",\n      \"method\": \"Biochemistry (in vitro kinase assays); mass spectrometry-based substrate motif profiling; protein interaction studies (identification of interactors); domain-level binding/activity assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assays combined with mass spectrometry substrate profiling and protein interaction biochemistry, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"39964718\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PSKH1 knockdown in osteosarcoma cells inhibited proliferation, migration, and invasion, while PSKH1 overexpression promoted proliferation. PSKH1 upregulated phospho-p38 MAPK, and the p38 MAPK inhibitor SB203580 blocked the tumor-promoting effects of PSKH1, placing PSKH1 upstream of p38/MAPK signaling in osteosarcoma cells.\",\n      \"method\": \"shRNA knockdown and overexpression in OS cell lines; Cell Counting Kit-8, colony formation, wound-healing, Transwell assays; p38 inhibitor SB203580 treatment; in vivo tumor xenograft assay\",\n      \"journal\": \"Oncology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Weak — loss-of-function and gain-of-function with defined cellular phenotypes plus inhibitor epistasis, single lab\",\n      \"pmids\": [\"36936027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"shRNA knockdown of PSKH1 decreased cell growth in both androgen-dependent and castration-resistant prostate cancer cells (LNCaP), identifying PSKH1 as a regulator of prostate cancer cell growth.\",\n      \"method\": \"Lentiviral shRNA phenotypic screen; cell growth assays in LNCaP prostate cancer cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — phenotypic screen with shRNA knockdown, limited mechanistic follow-up for PSKH1 specifically, single lab\",\n      \"pmids\": [\"22761715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"miR-566 directly targets PSKH1 mRNA (confirmed by luciferase reporter assay), suppressing PSKH1 protein expression; reintroduction of PSKH1 partially reversed the inhibitory effects of miR-566 on CRC cell growth and metastasis, placing PSKH1 downstream of miR-566 in colorectal cancer cell migration and invasion.\",\n      \"method\": \"Luciferase reporter assay; RT-PCR and western blot; miR-566 overexpression/inhibition; Transwell migration/invasion assays; PSKH1 rescue experiment\",\n      \"journal\": \"Cancer cell international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — luciferase reporter confirms direct miRNA targeting, rescue experiment confirms epistasis, multiple methods, single lab\",\n      \"pmids\": [\"31866763\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PSKH1 is a CAMK-family protein serine kinase that is dually acylated (myristoylated on Gly2 and palmitoylated on Cys3), targeting it to the Golgi apparatus and ER; it localizes to splicing factor compartments and centrosomes, modulates SR protein dynamics and pre-mRNA splicing indirectly, autophosphorylates its own C-terminal serines intermolecularly, and its catalytic activity is allosterically activated by Ca2+-Calmodulin and UNC119B and suppressed by Reticulocalbin-3; loss of PSKH1 kinase activity causes abnormally long cilia and a hepatorenal ciliopathy in humans and mice, and in cancer contexts PSKH1 promotes cell growth partly via p38/MAPK signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PSKH1 is a calmodulin-regulated protein serine kinase of the CAMK family whose subcellular targeting and catalytic output are tightly controlled to support membrane organization and ciliary function [#0, #3, #7]. The enzyme autophosphorylates on C-terminal serines in an intermolecular reaction and is allosterically tuned by calcium-sensing partners: Ca2+/calmodulin and UNC119B engage and activate the kinase domain, whereas the ER Ca2+-sensor Reticulocalbin-3 suppresses its activity, even though PSKH1 lacks a canonical CaM-binding motif [#0, #7]. Dual N-terminal acylation—myristoylation on Gly2 and palmitoylation on Cys3—directs PSKH1 to the Golgi apparatus and, in the absence of palmitoylation, to ER membranes; the integrity of this targeting and the kinase domain is required for normal Golgi structure [#3]. PSKH1 also localizes to nuclear splicing factor compartments and centrosomes, where it reorganizes SR proteins and stimulates pre-mRNA splicing of an E1A minigene by a kinase-independent, indirect mechanism that does not involve direct phosphorylation of ASF/SF2 [#1, #2]. Loss-of-function PSKH1 mutations that abolish catalytic activity cause a hepatorenal ciliopathy in humans, with patient fibroblasts and a homozygous mutant mouse showing abnormally long cilia and aberrant ciliary transport, establishing that PSKH1 kinase activity is required for normal ciliogenesis [#6]. In cancer contexts, PSKH1 promotes cell growth and is positioned upstream of p38/MAPK signaling [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing PSKH1 as an autoregulated, calcium-responsive kinase answered whether and how its catalytic activity is controlled.\",\n      \"evidence\": \"In vitro kinase assay with immunoisolated PSKH1 and Ca2+/CaM addition\",\n      \"pmids\": [\"11087665\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No physiological substrate identified\", \"Effect of Ca2+/CaM was repression of autophosphorylation, later refined as allosteric activation by other studies\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Mapping endogenous PSKH1 to Golgi, centrosomes, and nuclear speckles defined the compartments where it might act and linked it to stress responses.\",\n      \"evidence\": \"Indirect immunofluorescence in COS-1 cells with Brefeldin A and osmotic stress\",\n      \"pmids\": [\"11087665\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence at each compartment unresolved\", \"Mechanism of centrosomal enrichment under stress unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Linking PSKH1 to splicing factor compartments and SR protein dynamics addressed whether it participates in pre-mRNA processing, revealing a kinase-independent, indirect role.\",\n      \"evidence\": \"Immunofluorescence, SR co-expression, E1A minigene splicing, GST kinase assay, and domain truncation in HeLa cells\",\n      \"pmids\": [\"12466556\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct nuclear substrate not identified\", \"Mechanism linking cytoplasmic/Golgi kinase to nuclear SR redistribution unclear\", \"ASF/SF2 is not a direct substrate\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identifying dual acylation as the membrane-targeting determinant explained how PSKH1 is positioned at the Golgi and ER and tied it to organelle integrity.\",\n      \"evidence\": \"Acylation assays, organelle marker co-localization, immunoelectron microscopy, fractionation, and site-directed mutagenesis\",\n      \"pmids\": [\"14644153\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Golgi disassembly reflects a dominant-negative artifact versus a physiological role is not resolved\", \"Substrates at the Golgi unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identification of PSKH1 as an in vitro Chk2 target raised the possibility of upstream phospho-regulation by DNA-damage signaling.\",\n      \"evidence\": \"GST-peptide in vitro kinase assay with consensus-site mutagenesis\",\n      \"pmids\": [\"12711320\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No validation of in vivo phosphorylation by Chk2\", \"Functional consequence unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrating that catalytically dead PSKH1 variants cause a hepatorenal ciliopathy established a definitive, kinase-activity-dependent physiological function in ciliogenesis.\",\n      \"evidence\": \"In vitro kinase assays of disease variants, patient fibroblast cilia imaging, and a homozygous Pskh1 mutant mouse\",\n      \"pmids\": [\"39132680\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ciliary substrate(s) of PSKH1 not identified\", \"Molecular link between Golgi/ER localization and cilia phenotype unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defining the substrate motif and the allosteric regulators (Ca2+/CaM, UNC119B activating; Reticulocalbin-3 suppressing) provided the regulatory logic governing when PSKH1 is active.\",\n      \"evidence\": \"In vitro kinase assays, MS-based substrate motif profiling, and protein interaction biochemistry\",\n      \"pmids\": [\"39964718\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo substrates matching the motif not validated\", \"Structural basis of non-canonical CaM activation not solved\", \"Cellular context where each regulator dominates unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Placing PSKH1 upstream of p38/MAPK in osteosarcoma addressed how its catalytic role connects to growth control in cancer.\",\n      \"evidence\": \"shRNA/overexpression, proliferation/migration/invasion assays, SB203580 epistasis, and xenografts in OS cells\",\n      \"pmids\": [\"36936027\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct mechanistic link between PSKH1 kinase activity and p38 activation not defined\", \"Single tumor type\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The bona fide physiological substrates of PSKH1 and the mechanism connecting its membrane localization to cilia and splicing phenotypes remain unknown.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No validated in vivo substrate\", \"No structural model of the active kinase or its regulatory complexes\", \"Unclear how a Golgi/ER-anchored kinase controls ciliary length\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 6, 7]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CALM1\", \"UNC119B\", \"RCN3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":6,"faith_pct":83.33333333333333}}