{"gene":"ANKRD28","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2008,"finding":"ANKRD28 is a regulatory subunit of the PP6 holoenzyme, forming a heterotrimer with the PP6 catalytic subunit and SAPS-domain scaffold subunits (PP6R1 or PP6R3). Tagged ANKRD28 co-precipitated with PP6 but not PP2A or PP4. The C-terminal region of PP6R1 was sufficient to co-precipitate ANKRD28 but not PP6, demonstrating that PP6R1 acts as a scaffold with separate binding regions for PP6 and ANKRD28. Knockdown of PP6R1 or ANKRD28 equivalently enhanced IκBε degradation in response to TNFα, placing ANKRD28 as a functional PP6 co-regulator in the NF-κB pathway.","method":"FLAG co-immunoprecipitation, mass spectrometry, DEAE chromatography/gel filtration, siRNA knockdown with IκBε degradation assay","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, gel filtration, MS identification, functional siRNA knockdown with defined readout, all in one rigorous study","pmids":["18186651"],"is_preprint":false},{"year":2006,"finding":"ANKRD28 (PITK) functions as a PP1 targeting subunit that directs PP1 to nuclear foci to dephosphorylate hnRNP K at S284. PITK is phosphorylated in vivo at S1013 and S1017 flanking its PP1C-binding motif, and this phosphorylation negatively regulates PP1 binding. The phosphomimetic mutant S1013,1017D-PITK showed reduced PP1 binding, whereas constitutively dephosphorylated S1013,1017A-PITK showed increased PP1 binding and more profound hnRNP K dephosphorylation at S284. PITK expression altered transcription of 47 genes, including >14-fold induction of MEK5, in a manner modulated by hnRNP K co-expression.","method":"In vivo phosphorylation site mapping, site-directed mutagenesis, co-immunoprecipitation, PP1 binding assay, Affymetrix microarray","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis of functional phosphorylation sites, binding assay, functional transcription readout, single lab with multiple orthogonal methods","pmids":["16564677"],"is_preprint":false},{"year":2006,"finding":"ANKRD28 (PITK) phosphorylation at S1017 is catalyzed by CaMKIIδ, which promotes subsequent phosphorylation of S1013 by GSK3 in vitro. Phosphorylation state at S1013/S1017 dictates subcellular localization: wildtype and S1013,1017D-PITK show speckled nuclear localization, whereas constitutively dephosphorylated S1013,1017A-PITK displays diffuse cytoplasmic/nuclear localization.","method":"In vitro kinase assay with CaMKIIδ and GSK3, site-directed mutagenesis, fluorescence microscopy of subcellular localization","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay, mutagenesis, direct localization imaging, single lab with multiple orthogonal methods","pmids":["17023142"],"is_preprint":false},{"year":2008,"finding":"ANKRD28 binds the SH3 domain of DOCK180 and competes with ELMO for this interaction. ANKRD28 knockdown reduced HeLa cell migration velocity and altered focal adhesion distribution (Crk, paxillin, p130Cas). Co-expression of ANKRD28 with p130Cas, Crk, and DOCK180 induced hyper-phosphorylation of p130Cas and formation of multiple long cellular processes, distinct from ELMO co-expression which induced lamellipodial protrusion.","method":"Nano-LC/MS/MS co-immunoprecipitation, RNA interference knockdown, live-cell migration assay, fluorescence imaging of focal adhesion proteins, overexpression with p130Cas phosphorylation assay","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — MS-based binding partner identification, reciprocal functional assays (KD and OE), migration velocity measurement, multiple orthogonal methods in single study","pmids":["19118547"],"is_preprint":false},{"year":2016,"finding":"BRCA1 interacts with ANKRD28 in the cytoplasm, identified by yeast two-hybrid screen and confirmed by reciprocal co-immunoprecipitation of overexpressed proteins and endogenous co-IP. The interaction is located in the cytoplasm by proximity ligation assay. The main ANKRD28-binding site on BRCA1 is in its intrinsically disordered scaffold central region. BRCA1 overexpression stabilizes IκBε upon TNFα stimulation, an effect lost with a BRCA1 truncation that cannot interact with ANKRD28, indicating BRCA1 modulates PP6 signaling via ANKRD28.","method":"Yeast two-hybrid screen, reciprocal co-immunoprecipitation, proximity ligation assay, siRNA knockdown, IκBε stabilization assay with truncation mutants","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, proximity ligation, functional domain mapping with truncation mutant, multiple orthogonal methods in single study","pmids":["27026398"],"is_preprint":false},{"year":2007,"finding":"In a patient with MDS/AML, ANKRD28 is fused to NUP98 via a cryptic translocation t(3;5;11)(p25;q35;p15), producing in-frame ANKRD28-NUP98 fusion transcripts. Transient overexpression of ANKRD28-NUP98 in NIH/3T3 cells caused significantly increased focus formation (oncogenic transformation), whereas a C-terminal deletion mutant (ΔC-ANKRD28) did not. ANKRD28-NUP98 localized to the nucleolus and cytoplasm, whereas wildtype ANKRD28 and ΔC-ANKRD28 were exclusively cytoplasmic, indicating the NUP98 fusion alters ANKRD28 subcellular localization.","method":"FISH breakpoint mapping, RT-PCR for fusion transcripts, NIH/3T3 focus formation assay, fluorescence microscopy of subcellular localization","journal":"International journal of hematology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — focus formation assay with deletion mutant, localization imaging, but single lab and clinical case-based discovery","pmids":["17988990"],"is_preprint":false},{"year":2022,"finding":"Rab40c, as part of a Cullin5-based E3 ubiquitin ligase complex (Rab40c/CRL5), binds the PP6 complex and ubiquitylates ANKRD28, targeting it for lysosomal degradation. Rab40c knockout reduces PP6 activity (measured via decreased FAK and MOB1 phosphorylation) and alters focal adhesion number, size, and distribution in migrating MDA-MB-231 cells.","method":"Co-immunoprecipitation, ubiquitylation assay, Rab40c knockout cells, phospho-FAK/MOB1 immunoblotting, focal adhesion imaging","journal":"Life science alliance","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, KO with multiple functional readouts (ubiquitylation, phosphorylation, focal adhesion morphology), single lab with orthogonal methods","pmids":["35512830"],"is_preprint":false},{"year":2024,"finding":"PP6 functions as a heterotrimer composed of PP6c, PP6R (PP6R1/R2/R3), and scaffold subunits including ANKRD28. The PP6c-PP6R3 complex specifically regulates cancer stem cell (CSC) markers in colorectal cancer cells; PP6c knockdown reduced colony-forming ability, in vivo proliferation, and altered expression of stemness-associated genes.","method":"siRNA knockdown, colony formation assay, in vivo xenograft, transcriptome analysis","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2–3 / Weak — KD with defined phenotypic readouts, but ANKRD28's specific contribution within the complex not directly tested; single lab","pmids":["39014521"],"is_preprint":false}],"current_model":"ANKRD28 is a scaffold/regulatory subunit of the PP6 Ser/Thr phosphatase heterotrimer, where it associates with SAPS-domain subunits (PP6R1/R3) and the PP6 catalytic subunit to control PP6 substrate specificity including IκBε stability; it also functions as a PP1 targeting subunit (PITK) that directs PP1 to dephosphorylate hnRNP K at S284, with its own activity regulated by sequential CaMKIIδ- and GSK3-mediated phosphorylation at S1013/S1017 that controls PP1 binding and subcellular localization; additionally, ANKRD28 binds the SH3 domain of DOCK180 to regulate focal adhesion dynamics and Rac1-dependent cell migration, is ubiquitylated by the Rab40c/CRL5 E3 ligase for lysosomal degradation, and is modulated by BRCA1, which stabilizes the ANKRD28–PP6 axis to suppress NF-κB signaling."},"narrative":{"mechanistic_narrative":"ANKRD28 is an ankyrin-repeat regulatory/scaffold subunit that couples protein Ser/Thr phosphatases to specific substrates, thereby shaping signaling outputs in the NF-κB pathway, nuclear transcriptional control, and cell migration [PMID:18186651, PMID:16564677, PMID:19118547]. As part of the PP6 holoenzyme, it forms a heterotrimer with the PP6 catalytic subunit and SAPS-domain scaffolds (PP6R1 or PP6R3), with PP6R1 providing separable binding surfaces for PP6c and ANKRD28; loss of ANKRD28 or PP6R1 accelerates IκBε degradation upon TNFα, defining ANKRD28 as a positive PP6 co-regulator restraining NF-κB activation [PMID:18186651]. Independently, ANKRD28 (PITK) acts as a PP1-targeting subunit that directs PP1 to nuclear foci to dephosphorylate hnRNP K at S284 and reprogram transcription, with its PP1 binding switched off by sequential CaMKIIδ-mediated phosphorylation of S1017 followed by GSK3 phosphorylation of S1013 flanking its PP1C-binding motif, which also governs its speckled-nuclear versus diffuse localization [PMID:16564677, PMID:17023142]. Through its ankyrin repeats it binds the SH3 domain of DOCK180 in competition with ELMO, controlling focal adhesion organization, p130Cas phosphorylation, and Rac1-dependent migration [PMID:19118547], a function further tuned by Rab40c/CRL5-mediated ubiquitylation and lysosomal turnover of ANKRD28 that modulates PP6 activity in migrating cells [PMID:35512830]. BRCA1 binds ANKRD28 in the cytoplasm and stabilizes IκBε through this interaction, linking BRCA1 to suppression of NF-κB signaling via the ANKRD28–PP6 axis [PMID:27026398]. An ANKRD28–NUP98 fusion arising from a cryptic translocation in MDS/AML mislocalizes the protein to the nucleolus and drives oncogenic focus formation in a manner requiring the ANKRD28 C-terminus [PMID:17988990].","teleology":[{"year":2006,"claim":"Established that ANKRD28 (PITK) is a PP1-targeting subunit whose substrate-directing activity toward hnRNP K is controlled by phosphorylation of sites flanking its PP1C-binding motif, answering how a phosphatase is aimed at a specific nuclear substrate.","evidence":"In vivo phosphosite mapping, mutagenesis of S1013/S1017, PP1 binding and hnRNP K dephosphorylation assays, microarray transcription readout in cells","pmids":["16564677"],"confidence":"High","gaps":["Direct structural basis of PP1 docking not resolved","Functional consequence of MEK5 induction for cell physiology not established"]},{"year":2006,"claim":"Identified the upstream kinases governing the ANKRD28 phospho-switch, showing CaMKIIδ primes S1017 for GSK3 phosphorylation of S1013 and that this state dictates nuclear-speckle versus diffuse localization.","evidence":"In vitro kinase assays with CaMKIIδ and GSK3, mutagenesis, fluorescence localization imaging","pmids":["17023142"],"confidence":"High","gaps":["Physiological signal triggering CaMKIIδ/GSK3 cascade not defined","Whether the same phospho-switch regulates PP6-associated functions untested"]},{"year":2007,"claim":"Linked ANKRD28 to leukemogenesis by showing an ANKRD28-NUP98 fusion mislocalizes the protein and confers transforming activity, raising the question of how disrupted ANKRD28 function contributes to malignancy.","evidence":"FISH breakpoint mapping, RT-PCR, NIH/3T3 focus formation with C-terminal deletion, localization imaging","pmids":["17988990"],"confidence":"Medium","gaps":["Single clinical case","Mechanism by which fusion transforms cells not defined","Relationship to ANKRD28's phosphatase-regulatory roles untested"]},{"year":2008,"claim":"Defined ANKRD28 as a regulatory subunit of the PP6 holoenzyme assembled via SAPS-domain scaffolds and showed it restrains NF-κB by stabilizing IκBε, establishing its PP6-pathway role.","evidence":"FLAG Co-IP, mass spectrometry, gel filtration, siRNA knockdown with IκBε degradation assay","pmids":["18186651"],"confidence":"High","gaps":["Whether ANKRD28 confers PP6 substrate specificity directly untested","Stoichiometry and architecture of the heterotrimer not resolved"]},{"year":2008,"claim":"Revealed a PP6-independent role in migration: ANKRD28 binds the DOCK180 SH3 domain in competition with ELMO to shape focal adhesion dynamics and Rac1-dependent motility.","evidence":"Nano-LC/MS/MS Co-IP, RNAi knockdown, live-cell migration assay, focal adhesion imaging, p130Cas phosphorylation assay","pmids":["19118547"],"confidence":"High","gaps":["Whether PP6 phosphatase activity participates in this migration role untested","Mechanism linking ANKRD28-DOCK180 binding to p130Cas hyperphosphorylation unclear"]},{"year":2016,"claim":"Connected BRCA1 to the ANKRD28-PP6 axis, showing cytoplasmic BRCA1 binding stabilizes IκBε and thereby modulates NF-κB signaling through ANKRD28.","evidence":"Yeast two-hybrid, reciprocal endogenous Co-IP, proximity ligation, IκBε stabilization assay with BRCA1 truncation mutants","pmids":["27026398"],"confidence":"High","gaps":["Whether BRCA1 alters PP6 catalytic activity or only complex stability not distinguished","Physiological context where this regulation operates not defined"]},{"year":2022,"claim":"Identified post-translational control of ANKRD28 levels by Rab40c/CRL5-mediated ubiquitylation and lysosomal degradation, tying ANKRD28 turnover to PP6 activity and focal adhesion morphology in migrating cells.","evidence":"Co-IP, ubiquitylation assay, Rab40c knockout, phospho-FAK/MOB1 immunoblotting, focal adhesion imaging in MDA-MB-231","pmids":["35512830"],"confidence":"High","gaps":["Ubiquitylation site(s) on ANKRD28 not mapped","Whether degradation is signal-regulated not established"]},{"year":2024,"claim":"Extended the PP6 holoenzyme to colorectal cancer stem cell control, though the specific contribution of ANKRD28 within the complex was not directly tested.","evidence":"siRNA knockdown of PP6c, colony formation, xenograft proliferation, transcriptome analysis","pmids":["39014521"],"confidence":"Medium","gaps":["ANKRD28's individual role in CSC regulation not assessed","Substrates mediating stemness phenotype unidentified"]},{"year":null,"claim":"How ANKRD28 partitions between its PP6-regulatory, PP1-targeting, and DOCK180-migration functions, and whether a unified structural/regulatory logic governs these roles, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of any ANKRD28-containing complex","Substrate specificity contributions of ANKRD28 not directly demonstrated","Crosstalk between phospho-switch and degradation control unexplored"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,7]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,3]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,2]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,4,5]},{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[5]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,4]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,6]}],"complexes":["PP6 phosphatase holoenzyme","PP1 phosphatase complex (PITK targeting)","Rab40c/CRL5 E3 ubiquitin ligase complex (substrate)"],"partners":["PPP6C","PP6R1","PP6R3","PP1C","HNRNP K","DOCK180","BRCA1","RAB40C"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O15084","full_name":"Serine/threonine-protein phosphatase 6 regulatory ankyrin repeat subunit A","aliases":["Ankyrin repeat domain-containing protein 28","Phosphatase interactor targeting protein hnRNP K","PITK"],"length_aa":1053,"mass_kda":113.0,"function":"Regulatory subunit of protein phosphatase 6 (PP6) that may be involved in the recognition of phosphoprotein substrates. Involved in the PP6-mediated dephosphorylation of NFKBIE opposing its degradation in response to TNF. Selectively inhibits the phosphatase activity of PPP1C. Targets PPP1C to modulate HNRPK phosphorylation. Involved in the PP6-mediated dephosphorylation of MOB1 and induced focal adhesion assembly during cell migration (PubMed:35512830)","subcellular_location":"Nucleus, nucleoplasm; Cytoplasm, cytosol; Cell projection, lamellipodium","url":"https://www.uniprot.org/uniprotkb/O15084/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ANKRD28","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000206560","cell_line_id":"CID001392","localizations":[{"compartment":"cytoplasmic","grade":3}],"interactors":[{"gene":"ARHGEF2","stoichiometry":10.0},{"gene":"PPP6R1","stoichiometry":10.0},{"gene":"PPP6R3","stoichiometry":10.0},{"gene":"PPP6C","stoichiometry":10.0},{"gene":"PPP6R2","stoichiometry":10.0},{"gene":"ANKRD52","stoichiometry":4.0},{"gene":"PRKDC","stoichiometry":0.2},{"gene":"ANKRD44","stoichiometry":0.2},{"gene":"IQSEC1","stoichiometry":0.2},{"gene":"AP2M1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001392","total_profiled":1310},"omim":[{"mim_id":"611122","title":"ANKYRIN REPEAT DOMAIN-CONTAINING PROTEIN 28; ANKRD28","url":"https://www.omim.org/entry/611122"}],"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/ANKRD28"},"hgnc":{"alias_symbol":["KIAA0379","PITK","PP6-ARS-A","PPP1R65","FAP79","CFAP79"],"prev_symbol":[]},"alphafold":{"accession":"O15084","domains":[{"cath_id":"1.25.40.20","chopping":"232-335","consensus_level":"medium","plddt":96.4375,"start":232,"end":335}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O15084","model_url":"https://alphafold.ebi.ac.uk/files/AF-O15084-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O15084-F1-predicted_aligned_error_v6.png","plddt_mean":88.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ANKRD28","jax_strain_url":"https://www.jax.org/strain/search?query=ANKRD28"},"sequence":{"accession":"O15084","fasta_url":"https://rest.uniprot.org/uniprotkb/O15084.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O15084/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O15084"}},"corpus_meta":[{"pmid":"18186651","id":"PMC_18186651","title":"Protein phosphatase 6 regulatory subunits composed of ankyrin repeat domains.","date":"2008","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18186651","citation_count":96,"is_preprint":false},{"pmid":"22491060","id":"PMC_22491060","title":"Genetic and epigenetic analysis of non-small cell lung cancer with NotI-microarrays.","date":"2012","source":"Epigenetics","url":"https://pubmed.ncbi.nlm.nih.gov/22491060","citation_count":62,"is_preprint":false},{"pmid":"24389164","id":"PMC_24389164","title":"Primary urethral clear-cell adenocarcinoma: comprehensive analysis by surgical pathology, cytopathology, and next-generation sequencing.","date":"2014","source":"The American journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/24389164","citation_count":49,"is_preprint":false},{"pmid":"32163900","id":"PMC_32163900","title":"Blockade of interleukin-2-inducible T-cell kinase signaling attenuates acute lung injury in mice through adjustment of pulmonary Th17/Treg immune responses and reduction of oxidative stress.","date":"2020","source":"International immunopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/32163900","citation_count":48,"is_preprint":false},{"pmid":"33007409","id":"PMC_33007409","title":"Inhibition of interleukin-2-inducible T-cell kinase causes reduction in imiquimod-induced psoriasiform inflammation through reduction of Th17 cells and enhancement of Treg cells in mice.","date":"2020","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/33007409","citation_count":41,"is_preprint":false},{"pmid":"19118547","id":"PMC_19118547","title":"Ankyrin repeat domain 28 (ANKRD28), a novel binding partner of DOCK180, promotes cell migration by regulating focal adhesion formation.","date":"2008","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/19118547","citation_count":27,"is_preprint":false},{"pmid":"34224738","id":"PMC_34224738","title":"Targeting of the Tec Kinase ITK Drives Resolution of T Cell-Mediated Colitis and Emerges as Potential Therapeutic Option in Ulcerative Colitis.","date":"2021","source":"Gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/34224738","citation_count":19,"is_preprint":false},{"pmid":"34365077","id":"PMC_34365077","title":"Role of ITK signaling in acute kidney injury in mice: Amelioration of acute kidney injury associated clinical parameters and attenuation of inflammatory transcription factor signaling in CD4+ T cells by ITK inhibition.","date":"2021","source":"International immunopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/34365077","citation_count":19,"is_preprint":false},{"pmid":"17988990","id":"PMC_17988990","title":"A novel gene, ANKRD28 on 3p25, is fused with NUP98 on 11p15 in a cryptic 3-way translocation of t(3;5;11)(p25;q35;p15) in an adult patient with myelodysplastic syndrome/acute myelogenous leukemia.","date":"2007","source":"International journal of hematology","url":"https://pubmed.ncbi.nlm.nih.gov/17988990","citation_count":18,"is_preprint":false},{"pmid":"16564677","id":"PMC_16564677","title":"PITK, a PP1 targeting subunit that modulates the phosphorylation of the transcriptional regulator hnRNP K.","date":"2006","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/16564677","citation_count":15,"is_preprint":false},{"pmid":"27026398","id":"PMC_27026398","title":"BRCA1 affects protein phosphatase 6 signalling through its interaction with ANKRD28.","date":"2016","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/27026398","citation_count":14,"is_preprint":false},{"pmid":"35512830","id":"PMC_35512830","title":"Rab40c regulates focal adhesions and PP6 activity by controlling ANKRD28 ubiquitylation.","date":"2022","source":"Life science alliance","url":"https://pubmed.ncbi.nlm.nih.gov/35512830","citation_count":13,"is_preprint":false},{"pmid":"26502805","id":"PMC_26502805","title":"Polygenic inheritance of cryptorchidism susceptibility in the LE/orl rat.","date":"2015","source":"Molecular human reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/26502805","citation_count":13,"is_preprint":false},{"pmid":"22992777","id":"PMC_22992777","title":"Detection of significant pathways in osteoporosis based on graph clustering.","date":"2012","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/22992777","citation_count":12,"is_preprint":false},{"pmid":"27729219","id":"PMC_27729219","title":"Interleukin-2-inducible T-cell kinase expression and relation to disease severity in systemic lupus erythematosus.","date":"2016","source":"Clinica chimica acta; international journal of clinical chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27729219","citation_count":11,"is_preprint":false},{"pmid":"19458477","id":"PMC_19458477","title":"Regulation of focal adhesion and cell migration by ANKRD28-DOCK180 interaction.","date":"2009","source":"Cell adhesion & 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Tagged ANKRD28 co-precipitated with PP6 but not PP2A or PP4. The C-terminal region of PP6R1 was sufficient to co-precipitate ANKRD28 but not PP6, demonstrating that PP6R1 acts as a scaffold with separate binding regions for PP6 and ANKRD28. Knockdown of PP6R1 or ANKRD28 equivalently enhanced IκBε degradation in response to TNFα, placing ANKRD28 as a functional PP6 co-regulator in the NF-κB pathway.\",\n      \"method\": \"FLAG co-immunoprecipitation, mass spectrometry, DEAE chromatography/gel filtration, siRNA knockdown with IκBε degradation assay\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, gel filtration, MS identification, functional siRNA knockdown with defined readout, all in one rigorous study\",\n      \"pmids\": [\"18186651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ANKRD28 (PITK) functions as a PP1 targeting subunit that directs PP1 to nuclear foci to dephosphorylate hnRNP K at S284. PITK is phosphorylated in vivo at S1013 and S1017 flanking its PP1C-binding motif, and this phosphorylation negatively regulates PP1 binding. The phosphomimetic mutant S1013,1017D-PITK showed reduced PP1 binding, whereas constitutively dephosphorylated S1013,1017A-PITK showed increased PP1 binding and more profound hnRNP K dephosphorylation at S284. PITK expression altered transcription of 47 genes, including >14-fold induction of MEK5, in a manner modulated by hnRNP K co-expression.\",\n      \"method\": \"In vivo phosphorylation site mapping, site-directed mutagenesis, co-immunoprecipitation, PP1 binding assay, Affymetrix microarray\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis of functional phosphorylation sites, binding assay, functional transcription readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"16564677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ANKRD28 (PITK) phosphorylation at S1017 is catalyzed by CaMKIIδ, which promotes subsequent phosphorylation of S1013 by GSK3 in vitro. Phosphorylation state at S1013/S1017 dictates subcellular localization: wildtype and S1013,1017D-PITK show speckled nuclear localization, whereas constitutively dephosphorylated S1013,1017A-PITK displays diffuse cytoplasmic/nuclear localization.\",\n      \"method\": \"In vitro kinase assay with CaMKIIδ and GSK3, site-directed mutagenesis, fluorescence microscopy of subcellular localization\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay, mutagenesis, direct localization imaging, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"17023142\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ANKRD28 binds the SH3 domain of DOCK180 and competes with ELMO for this interaction. ANKRD28 knockdown reduced HeLa cell migration velocity and altered focal adhesion distribution (Crk, paxillin, p130Cas). Co-expression of ANKRD28 with p130Cas, Crk, and DOCK180 induced hyper-phosphorylation of p130Cas and formation of multiple long cellular processes, distinct from ELMO co-expression which induced lamellipodial protrusion.\",\n      \"method\": \"Nano-LC/MS/MS co-immunoprecipitation, RNA interference knockdown, live-cell migration assay, fluorescence imaging of focal adhesion proteins, overexpression with p130Cas phosphorylation assay\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-based binding partner identification, reciprocal functional assays (KD and OE), migration velocity measurement, multiple orthogonal methods in single study\",\n      \"pmids\": [\"19118547\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"BRCA1 interacts with ANKRD28 in the cytoplasm, identified by yeast two-hybrid screen and confirmed by reciprocal co-immunoprecipitation of overexpressed proteins and endogenous co-IP. The interaction is located in the cytoplasm by proximity ligation assay. The main ANKRD28-binding site on BRCA1 is in its intrinsically disordered scaffold central region. BRCA1 overexpression stabilizes IκBε upon TNFα stimulation, an effect lost with a BRCA1 truncation that cannot interact with ANKRD28, indicating BRCA1 modulates PP6 signaling via ANKRD28.\",\n      \"method\": \"Yeast two-hybrid screen, reciprocal co-immunoprecipitation, proximity ligation assay, siRNA knockdown, IκBε stabilization assay with truncation mutants\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, proximity ligation, functional domain mapping with truncation mutant, multiple orthogonal methods in single study\",\n      \"pmids\": [\"27026398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"In a patient with MDS/AML, ANKRD28 is fused to NUP98 via a cryptic translocation t(3;5;11)(p25;q35;p15), producing in-frame ANKRD28-NUP98 fusion transcripts. Transient overexpression of ANKRD28-NUP98 in NIH/3T3 cells caused significantly increased focus formation (oncogenic transformation), whereas a C-terminal deletion mutant (ΔC-ANKRD28) did not. ANKRD28-NUP98 localized to the nucleolus and cytoplasm, whereas wildtype ANKRD28 and ΔC-ANKRD28 were exclusively cytoplasmic, indicating the NUP98 fusion alters ANKRD28 subcellular localization.\",\n      \"method\": \"FISH breakpoint mapping, RT-PCR for fusion transcripts, NIH/3T3 focus formation assay, fluorescence microscopy of subcellular localization\",\n      \"journal\": \"International journal of hematology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — focus formation assay with deletion mutant, localization imaging, but single lab and clinical case-based discovery\",\n      \"pmids\": [\"17988990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Rab40c, as part of a Cullin5-based E3 ubiquitin ligase complex (Rab40c/CRL5), binds the PP6 complex and ubiquitylates ANKRD28, targeting it for lysosomal degradation. Rab40c knockout reduces PP6 activity (measured via decreased FAK and MOB1 phosphorylation) and alters focal adhesion number, size, and distribution in migrating MDA-MB-231 cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assay, Rab40c knockout cells, phospho-FAK/MOB1 immunoblotting, focal adhesion imaging\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, KO with multiple functional readouts (ubiquitylation, phosphorylation, focal adhesion morphology), single lab with orthogonal methods\",\n      \"pmids\": [\"35512830\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PP6 functions as a heterotrimer composed of PP6c, PP6R (PP6R1/R2/R3), and scaffold subunits including ANKRD28. The PP6c-PP6R3 complex specifically regulates cancer stem cell (CSC) markers in colorectal cancer cells; PP6c knockdown reduced colony-forming ability, in vivo proliferation, and altered expression of stemness-associated genes.\",\n      \"method\": \"siRNA knockdown, colony formation assay, in vivo xenograft, transcriptome analysis\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Weak — KD with defined phenotypic readouts, but ANKRD28's specific contribution within the complex not directly tested; single lab\",\n      \"pmids\": [\"39014521\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ANKRD28 is a scaffold/regulatory subunit of the PP6 Ser/Thr phosphatase heterotrimer, where it associates with SAPS-domain subunits (PP6R1/R3) and the PP6 catalytic subunit to control PP6 substrate specificity including IκBε stability; it also functions as a PP1 targeting subunit (PITK) that directs PP1 to dephosphorylate hnRNP K at S284, with its own activity regulated by sequential CaMKIIδ- and GSK3-mediated phosphorylation at S1013/S1017 that controls PP1 binding and subcellular localization; additionally, ANKRD28 binds the SH3 domain of DOCK180 to regulate focal adhesion dynamics and Rac1-dependent cell migration, is ubiquitylated by the Rab40c/CRL5 E3 ligase for lysosomal degradation, and is modulated by BRCA1, which stabilizes the ANKRD28–PP6 axis to suppress NF-κB signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ANKRD28 is an ankyrin-repeat regulatory/scaffold subunit that couples protein Ser/Thr phosphatases to specific substrates, thereby shaping signaling outputs in the NF-\\u03baB pathway, nuclear transcriptional control, and cell migration [#0, #1, #3]. As part of the PP6 holoenzyme, it forms a heterotrimer with the PP6 catalytic subunit and SAPS-domain scaffolds (PP6R1 or PP6R3), with PP6R1 providing separable binding surfaces for PP6c and ANKRD28; loss of ANKRD28 or PP6R1 accelerates I\\u03baB\\u03b5 degradation upon TNF\\u03b1, defining ANKRD28 as a positive PP6 co-regulator restraining NF-\\u03baB activation [#0]. Independently, ANKRD28 (PITK) acts as a PP1-targeting subunit that directs PP1 to nuclear foci to dephosphorylate hnRNP K at S284 and reprogram transcription, with its PP1 binding switched off by sequential CaMKII\\u03b4-mediated phosphorylation of S1017 followed by GSK3 phosphorylation of S1013 flanking its PP1C-binding motif, which also governs its speckled-nuclear versus diffuse localization [#1, #2]. Through its ankyrin repeats it binds the SH3 domain of DOCK180 in competition with ELMO, controlling focal adhesion organization, p130Cas phosphorylation, and Rac1-dependent migration [#3], a function further tuned by Rab40c/CRL5-mediated ubiquitylation and lysosomal turnover of ANKRD28 that modulates PP6 activity in migrating cells [#6]. BRCA1 binds ANKRD28 in the cytoplasm and stabilizes I\\u03baB\\u03b5 through this interaction, linking BRCA1 to suppression of NF-\\u03baB signaling via the ANKRD28\\u2013PP6 axis [#4]. An ANKRD28\\u2013NUP98 fusion arising from a cryptic translocation in MDS/AML mislocalizes the protein to the nucleolus and drives oncogenic focus formation in a manner requiring the ANKRD28 C-terminus [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established that ANKRD28 (PITK) is a PP1-targeting subunit whose substrate-directing activity toward hnRNP K is controlled by phosphorylation of sites flanking its PP1C-binding motif, answering how a phosphatase is aimed at a specific nuclear substrate.\",\n      \"evidence\": \"In vivo phosphosite mapping, mutagenesis of S1013/S1017, PP1 binding and hnRNP K dephosphorylation assays, microarray transcription readout in cells\",\n      \"pmids\": [\"16564677\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct structural basis of PP1 docking not resolved\", \"Functional consequence of MEK5 induction for cell physiology not established\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identified the upstream kinases governing the ANKRD28 phospho-switch, showing CaMKII\\u03b4 primes S1017 for GSK3 phosphorylation of S1013 and that this state dictates nuclear-speckle versus diffuse localization.\",\n      \"evidence\": \"In vitro kinase assays with CaMKII\\u03b4 and GSK3, mutagenesis, fluorescence localization imaging\",\n      \"pmids\": [\"17023142\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological signal triggering CaMKII\\u03b4/GSK3 cascade not defined\", \"Whether the same phospho-switch regulates PP6-associated functions untested\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Linked ANKRD28 to leukemogenesis by showing an ANKRD28-NUP98 fusion mislocalizes the protein and confers transforming activity, raising the question of how disrupted ANKRD28 function contributes to malignancy.\",\n      \"evidence\": \"FISH breakpoint mapping, RT-PCR, NIH/3T3 focus formation with C-terminal deletion, localization imaging\",\n      \"pmids\": [\"17988990\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single clinical case\", \"Mechanism by which fusion transforms cells not defined\", \"Relationship to ANKRD28's phosphatase-regulatory roles untested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined ANKRD28 as a regulatory subunit of the PP6 holoenzyme assembled via SAPS-domain scaffolds and showed it restrains NF-\\u03baB by stabilizing I\\u03baB\\u03b5, establishing its PP6-pathway role.\",\n      \"evidence\": \"FLAG Co-IP, mass spectrometry, gel filtration, siRNA knockdown with I\\u03baB\\u03b5 degradation assay\",\n      \"pmids\": [\"18186651\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ANKRD28 confers PP6 substrate specificity directly untested\", \"Stoichiometry and architecture of the heterotrimer not resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealed a PP6-independent role in migration: ANKRD28 binds the DOCK180 SH3 domain in competition with ELMO to shape focal adhesion dynamics and Rac1-dependent motility.\",\n      \"evidence\": \"Nano-LC/MS/MS Co-IP, RNAi knockdown, live-cell migration assay, focal adhesion imaging, p130Cas phosphorylation assay\",\n      \"pmids\": [\"19118547\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PP6 phosphatase activity participates in this migration role untested\", \"Mechanism linking ANKRD28-DOCK180 binding to p130Cas hyperphosphorylation unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Connected BRCA1 to the ANKRD28-PP6 axis, showing cytoplasmic BRCA1 binding stabilizes I\\u03baB\\u03b5 and thereby modulates NF-\\u03baB signaling through ANKRD28.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal endogenous Co-IP, proximity ligation, I\\u03baB\\u03b5 stabilization assay with BRCA1 truncation mutants\",\n      \"pmids\": [\"27026398\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether BRCA1 alters PP6 catalytic activity or only complex stability not distinguished\", \"Physiological context where this regulation operates not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified post-translational control of ANKRD28 levels by Rab40c/CRL5-mediated ubiquitylation and lysosomal degradation, tying ANKRD28 turnover to PP6 activity and focal adhesion morphology in migrating cells.\",\n      \"evidence\": \"Co-IP, ubiquitylation assay, Rab40c knockout, phospho-FAK/MOB1 immunoblotting, focal adhesion imaging in MDA-MB-231\",\n      \"pmids\": [\"35512830\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitylation site(s) on ANKRD28 not mapped\", \"Whether degradation is signal-regulated not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended the PP6 holoenzyme to colorectal cancer stem cell control, though the specific contribution of ANKRD28 within the complex was not directly tested.\",\n      \"evidence\": \"siRNA knockdown of PP6c, colony formation, xenograft proliferation, transcriptome analysis\",\n      \"pmids\": [\"39014521\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"ANKRD28's individual role in CSC regulation not assessed\", \"Substrates mediating stemness phenotype unidentified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ANKRD28 partitions between its PP6-regulatory, PP1-targeting, and DOCK180-migration functions, and whether a unified structural/regulatory logic governs these roles, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of any ANKRD28-containing complex\", \"Substrate specificity contributions of ANKRD28 not directly demonstrated\", \"Crosstalk between phospho-switch and degradation control unexplored\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 7]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 4, 5]},\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 6]}\n    ],\n    \"complexes\": [\n      \"PP6 phosphatase holoenzyme\",\n      \"PP1 phosphatase complex (PITK targeting)\",\n      \"Rab40c/CRL5 E3 ubiquitin ligase complex (substrate)\"\n    ],\n    \"partners\": [\n      \"PPP6C\",\n      \"PP6R1\",\n      \"PP6R3\",\n      \"PP1C\",\n      \"hnRNP K\",\n      \"DOCK180\",\n      \"BRCA1\",\n      \"Rab40c\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}