{"gene":"PPP1R16B","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2002,"finding":"TIMAP (PPP1R16B) contains five ankyrin repeats, a PP1-interacting domain, and a COOH-terminal CAAX box; a GFP-TIMAP fusion protein localizes to the plasma membrane in a CAAX box-dependent fashion, and TGF-β1 represses TIMAP through a protein synthesis- and histone deacetylase-dependent process.","method":"Immunofluorescence of GFP-fusion protein, Northern blot, domain analysis, TGF-β1 treatment with inhibitor studies","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with CAAX-box dependency shown, and transcriptional repression mechanism characterized; single lab, two orthogonal methods","pmids":["12055102"],"is_preprint":false},{"year":2005,"finding":"TIMAP interacts with the 37/67 kDa laminin receptor (LAMR1) via its fourth ankyrin repeat (amino acids 261–290); this interaction recruits PP1 to LAMR1 and targets PP1-mediated dephosphorylation of LAMR1, as full-length TIMAP (but not the ankyrin-deletion mutant) abolished LAMR1 phosphorylation in cells.","method":"Yeast two-hybrid, co-immunoprecipitation, co-localization at plasma membrane, domain-deletion mutagenesis, in vitro PP1 phosphatase assay","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (yeast two-hybrid, co-IP, in vitro phosphatase assay, deletion mutagenesis) in a single study establishing substrate/mechanism","pmids":["16263087"],"is_preprint":false},{"year":2007,"finding":"TIMAP is phosphorylated by PKA at Ser333/Ser337 and, after PKA priming, by GSK-3β at the same sites; this phosphorylation controls TIMAP association with PP1c and PP1c activity. Abolishing PP1c binding (TIMAPV64A/F66A) causes hyper-phosphorylation of TIMAP, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Reduced PP1c activity (via TIMAP mutants with low PP1c binding) strongly stimulates endothelial cell filopodia formation.","method":"Metabolic 32P-labeling, cell-free kinase assays, site-directed mutagenesis (Ser→Ala, Val/Phe→Ala), phosphomimic substitutions, immunoprecipitation, endothelial cell filopodia imaging","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assays combined with mutagenesis and cellular functional readout; multiple orthogonal methods in a single rigorous study","pmids":["17609201"],"is_preprint":false},{"year":2008,"finding":"Recombinant TIMAP preferentially binds the β-isoform of PP1c (PP1cβ) from pulmonary artery endothelial cells. TIMAP depletion by siRNA attenuates barrier-protective responses and enhances barrier disruption. TIMAP co-immunoprecipitates with moesin and is involved in PKA-mediated moesin dephosphorylation at the cell periphery, contributing to endothelial barrier protection.","method":"Pull-down assay with isoform-specific PP1c, siRNA depletion with transendothelial electrical resistance measurements, immunofluorescent co-localization, co-immunoprecipitation, forskolin/PKA activation experiments","journal":"American journal of physiology. Lung cellular and molecular physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, pull-down isoform selectivity, functional siRNA phenotype with mechanistic follow-up; multiple orthogonal methods, replicated aspects in subsequent papers","pmids":["18586956"],"is_preprint":false},{"year":2011,"finding":"Non-phosphorylated TIMAP binds PP1c with a binding constant Ka = 1.80 × 10⁶ M⁻¹ (surface plasmon resonance). PKA mono-phosphorylation slightly decreases the dissociation rate; double (PKA+GSK3β) phosphorylation of TIMAP does not substantially reduce PP1c binding but strongly attenuates TIMAP's inhibitory effect on PP1c activity toward phospho-moesin substrate (<10% inhibition vs ~60% for non-phosphorylated TIMAP). PKA activation followed by GSK3β activation is required for the barrier-protective effect of forskolin in thrombin-treated endothelial cells.","method":"Surface plasmon resonance binding assay, in vitro thiophosphorylation, phosphatase activity assay with phospho-moesin substrate, specific GSK3β inhibitor in bovine pulmonary endothelial cells","journal":"Biochimie","confidence":"High","confidence_rationale":"Tier 1 / Moderate — quantitative binding constants by SPR, in vitro kinase and phosphatase assays with mutagenesis-equivalent thiophosphorylation; single lab but multiple orthogonal methods","pmids":["21466834"],"is_preprint":false},{"year":2013,"finding":"TIMAP associates with all three PP1c isoforms in vitro but endogenous TIMAP in endothelial cells preferentially co-immunoprecipitates PP1cβ. Structural modeling predicts PP1c C-terminus is buried in the TIMAP ankyrin cluster while the active site remains accessible—consistent with experiments showing PP1c C-terminal phosphorylation by cdk2-cyclinA is masked by TIMAP. TIMAP inhibits PP1c activity toward phosphorylase a (IC₅₀ ~0.4–1.2 nM). TIMAP-bound PP1cβ effectively dephosphorylates MLC2 (direct substrate), while TIMAP inhibits PP1cβ activity toward LAMR1 by masking its phosphorylation sites. TIMAP C-terminal length and Ser333/Ser337 phosphomimic mutations modulate the inhibitory effect.","method":"Co-immunoprecipitation, in vitro phosphatase activity assays (phosphorylase a, MLC2, LAMR1), structural modeling, site-directed mutagenesis, C-terminal truncation variants","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro phosphatase reconstitution with substrate specificity profiling, mutagenesis, and structural modeling; single lab, multiple orthogonal methods","pmids":["23685145"],"is_preprint":false},{"year":2014,"finding":"TIMAP is necessary for Akt-dependent endothelial cell proliferation, survival, and angiogenic sprout formation. TIMAP and PTEN co-localize and co-immunoprecipitate in endothelial cells. TIMAP depletion reduces inhibitory phosphorylation of PTEN at S370 (but not S380/T382/T383), leading to reduced Akt phosphorylation; this effect is rescued by the PTEN inhibitor bpV(phen), placing TIMAP upstream of PTEN/Akt.","method":"siRNA depletion, impedance-based proliferation assay, EdU incorporation, caspase 3 activity, 3D angiogenic sprouting assay, co-immunoprecipitation, co-localization, pharmacological PTEN inhibition rescue","journal":"American journal of physiology. Renal physiology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — epistasis established by rescue with PTEN inhibitor, co-IP of endogenous proteins, multiple functional readouts; single lab with orthogonal methods","pmids":["25007873"],"is_preprint":false},{"year":2015,"finding":"TIMAP directly interacts with eukaryotic elongation factor 1A1 (eEF1A1) via a TD-NEM-like motif in TIMAP. TIMAP regulates membrane localization of eEF1A1 (eEF1A1 disappears from the membrane in TIMAP-depleted cells). ROCK-phosphorylated eEF1A1 is a substrate for the TIMAP-PP1 complex.","method":"Pull-down, LC-MS/MS identification, Far-Western blot, co-immunoprecipitation, subcellular fractionation, siRNA depletion, immunofluorescence","journal":"The international journal of biochemistry & cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal binding assays (pull-down, Far-Western, co-IP) plus functional localization data and substrate identification; single lab","pmids":["26497934"],"is_preprint":false},{"year":2016,"finding":"TGF-β transcriptionally downregulates TIMAP through HDAC3-associated Smad2/3 signaling; HDAC3 is recruited to the Smad binding element on the TIMAP promoter. TIMAP dephosphorylates myosin light chain (MLC) in macrophages, and TGF-β-induced macrophage migration and phagocytosis occur in a TIMAP- and MLC-phosphorylation-dependent manner.","method":"ChIP for HDAC3/Smad2/3 at TIMAP promoter, specific HDAC3 inhibition reversal experiments, TIMAP overexpression, MLC phosphorylation assays in macrophages, migration and phagocytosis assays","journal":"Journal of molecular medicine (Berlin, Germany)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and pharmacological rescue established the HDAC3/Smad mechanism; MLC substrate shown by phosphorylation assay; single lab","pmids":["27709267"],"is_preprint":false},{"year":2016,"finding":"PKCα phosphorylates TIMAP at Ser331 in vitro and in endothelial cells. PKC-phosphorylated TIMAP is enriched at the plasma membrane, but phospho-Ser331 TIMAP shows severely reduced binding to ERM proteins, inhibiting TIMAP-PP1 activity toward phospho-ERM. Phosphomimic S331D TIMAP increases membrane phospho-ERM levels while phosphonull S331A lowers them, indicating Ser331 phosphorylation by PKC inhibits ERM dephosphorylation and slows endothelial barrier recovery.","method":"Co-immunoprecipitation of endogenous PKCα and TIMAP, in vitro PKC kinase assay on recombinant TIMAP, site-directed mutagenesis (S331D/S331A), subcellular fractionation, electric cell-substrate impedance sensing (ECIS)","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay plus mutagenesis with functional cellular readout; single lab, multiple orthogonal methods","pmids":["27939168"],"is_preprint":false},{"year":2016,"finding":"TIMAP-PP1c complex interacts with endothelin converting enzyme-1 (ECE-1); TIMAP depletion reduces TIMAP-PP1c activity toward ECE-1, causing increased ECE-1 at the plasma membrane and elevated endothelin-1 secretion. PKC-phosphorylated ECE-1 is identified as a TIMAP-PP1c substrate.","method":"Co-immunoprecipitation of TIMAP-PP1c with ECE-1, TIMAP siRNA depletion, measurement of ECE-1 membrane levels, endothelin-1 secretion assay, PKC activation experiments","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and functional depletion with defined molecular readout; single lab, two orthogonal methods","pmids":["26806547"],"is_preprint":false},{"year":2016,"finding":"PP1c-TIMAP complex dephosphorylates merlin at Ser518 in endothelial cells. TIMAP N-terminal half (aa 1–290) interacts with the FERM domain of merlin, and PP1c is present in TIMAP-merlin complexes. Without TIMAP, PP1c cannot bind merlin. TIMAP or PP1c depletion increases phospho-Ser518 merlin at the membrane. EBP50 binds unphosphorylated merlin in the nucleus and is required for merlin's nuclear localization.","method":"GST pull-down domain mapping, co-immunoprecipitation, siRNA depletion, subcellular fractionation, immunofluorescence, ECIS wound healing assay","journal":"The international journal of biochemistry & cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — domain-mapped interaction, co-IP of endogenous proteins, functional phosphorylation readout with depletion; single lab, multiple orthogonal methods","pmids":["27871951"],"is_preprint":false},{"year":2019,"finding":"TIMAP inhibits myosin phosphatase activity in endothelial cells by competing with MYPT1 for PP1cβ and blocking the PP1cβ active site. Excess TIMAP reduces MYPT1-PP1cβ association and leads to proteasomal MYPT1 degradation. TIMAP-associated PP1cβ does not interact with microcystin-LR (active-site inhibitor), indicating the active site is blocked when PP1cβ is bound to TIMAP. TIMAP overexpression paradoxically enhances MLC2 phosphorylation by displacing the more active myosin phosphatase MYPT1/PP1cβ.","method":"Co-immunoprecipitation of endogenous proteins, purified recombinant protein interaction assay (GST-TIMAP with His-MLC2), microcystin-LR affinity binding, TIMAP overexpression/knockout mouse, siRNA depletion, proteasome inhibitor experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reconstituted direct protein interaction, active-site probe (microcystin), endogenous co-IP, KO mouse, and cellular functional readout; multiple orthogonal methods","pmids":["31315927"],"is_preprint":false},{"year":2021,"finding":"TIMAP-PP1c forms a complex with laminin receptor (LR) on the cell membrane (LR-TIMAP/PP1c complex). pTyr47-LR determines the stability of this complex. TIMAP/PP1c binding to LR activates PP1c phosphatase activity and regulates LR phosphorylation at Thr125; low phospho-LR stabilizes the complex and maintains endothelial barrier integrity via regulation of VE-cadherin Ser665 phosphorylation.","method":"Co-immunoprecipitation, in vivo gene delivery (AAV), LC-MS/MS phosphosite identification, Western blot for phospho-specific residues, PET perfusion imaging","journal":"EBioMedicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP of endogenous complex and in vivo functional validation; single group, two orthogonal methods","pmids":["33639401"],"is_preprint":false},{"year":2021,"finding":"Phosphorylation of TIMAP at Ser69 by polo-like kinase 4 (PLK4) promotes enrichment of TIMAP at the plasma membrane and increases membrane protrusions, leading to faster wound healing and endothelial cell migration. Ser69 phosphorylation does not affect binding of PP1c, ERM, or RACK1 to TIMAP. Phosphomimic S69D TIMAP showed enhanced membrane localization compared to wild-type.","method":"Site-directed mutagenesis (S69D/S69A), subcellular fractionation, immunofluorescence, pull-down, co-immunoprecipitation with PLK4, ECIS wound healing assay","journal":"Experimental lung research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with functional localization and migration readout; interaction with PLK4 shown by co-IP; single lab","pmids":["34343028"],"is_preprint":false},{"year":2023,"finding":"TIMAP is expressed in SH-SY5Y neuroblastoma cells, is downregulated during neuronal differentiation, and its overexpression inhibits differentiation. During differentiation, TIMAP translocates from the plasma membrane to the nucleus, where its nuclear interactome comprises more than 50 proteins.","method":"qPCR and Western blot for TIMAP expression, neuronal differentiation markers, TIMAP overexpression, immunofluorescence for subcellular localization, nuclear interactome by mass spectrometry","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional overexpression with defined phenotype and direct localization experiment; single lab, two orthogonal methods","pmids":["38139189"],"is_preprint":false},{"year":2024,"finding":"BMP9 represses TIMAP expression via the SMAD1/5/8 pathway in endothelial cells; hypoxia and the prolyl hydroxylase inhibitor Roxadustat raise TIMAP expression by inhibiting SMAD1/5/8. VEGFA and IGF-I also elevate TIMAP partly by attenuating SMAD1/5/8. TIMAP deficiency in mice markedly reduces tumor growth and tumor angiogenesis in the E0771 breast cancer model.","method":"SMAD1/5/8 pathway activation assays, Roxadustat treatment, TIMAP KO mouse, E0771 breast cancer in vivo model, VEGF/IGF inhibitor experiments","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO model with tumor angiogenesis readout, and pathway inhibitor studies; single lab, multiple methods","pmids":["39344413"],"is_preprint":false}],"current_model":"PPP1R16B/TIMAP is a prenylated, CAAX box-dependent plasma membrane-anchored regulatory subunit of protein phosphatase 1β (PP1cβ) that is highly expressed in endothelial cells; it recruits PP1cβ to specific substrates including LAMR1, MLC2, ERM/moesin, merlin (phospho-Ser518), eEF1A1, and ECE-1 to control their dephosphorylation, while paradoxically inhibiting myosin phosphatase by competing with MYPT1 for PP1cβ and occluding the PP1cβ active site; TIMAP activity is tuned by PKA-primed GSK-3β phosphorylation at Ser333/Ser337 (which reduces inhibitory activity and promotes filopodia), by PKCα phosphorylation at Ser331 (which blocks ERM dephosphorylation), and by PLK4-mediated phosphorylation at Ser69 (which promotes membrane localization and migration); upstream, TGF-β represses TIMAP via HDAC3-associated Smad2/3 signaling, and BMP9/SMAD1/5/8 pathway suppresses TIMAP while hypoxia and angiogenic growth factors induce it, collectively making TIMAP a critical regulator of endothelial barrier integrity, Akt/PTEN-dependent angiogenesis, and tumor angiogenesis in vivo."},"narrative":{"mechanistic_narrative":"PPP1R16B/TIMAP is a prenylated, CAAX-box-dependent plasma-membrane-anchored regulatory subunit of protein phosphatase 1 that controls endothelial barrier integrity, migration, and angiogenesis by targeting and tuning PP1c activity toward specific membrane substrates [PMID:12055102, PMID:18586956, PMID:25007873]. Through its ankyrin-repeat cluster and PP1-interacting domain it preferentially binds the β-isoform of PP1c (PP1cβ) and acts as a substrate-targeting scaffold, recruiting the phosphatase to LAMR1, moesin/ERM proteins, MLC2, merlin (phospho-Ser518), eEF1A1, and ECE-1 to govern their dephosphorylation and membrane localization [PMID:16263087, PMID:18586956, PMID:23685145, PMID:26497934, PMID:26806547, PMID:27871951]. The complex exerts dual, substrate-dependent effects on PP1c: TIMAP-bound PP1cβ efficiently dephosphorylates MLC2 and ERM substrates, yet TIMAP also inhibits PP1c toward other substrates and, by competing with MYPT1 for PP1cβ and occluding the active site, antagonizes myosin phosphatase to paradoxically promote MLC2 phosphorylation [PMID:23685145, PMID:31315927]. TIMAP activity is set by a layered phospho-code: PKA-primed GSK-3β phosphorylation at Ser333/Ser337 relieves PP1c inhibition and drives filopodia and barrier protection, PKCα phosphorylation at Ser331 blocks ERM dephosphorylation and slows barrier recovery, and PLK4 phosphorylation at Ser69 promotes membrane enrichment and migration [PMID:17609201, PMID:21466834, PMID:27939168, PMID:34343028]. TIMAP positively regulates PTEN/Akt signaling required for endothelial proliferation, survival, and angiogenic sprouting [PMID:25007873], and its expression is repressed by TGF-β via HDAC3-associated Smad2/3 and by BMP9 via SMAD1/5/8 while induced by hypoxia and angiogenic growth factors, with TIMAP loss reducing tumor angiogenesis in vivo [PMID:27709267, PMID:39344413].","teleology":[{"year":2002,"claim":"Established TIMAP as a multidomain, membrane-targeted PP1 regulatory subunit and identified its transcriptional repression by TGF-β, defining the protein's basic architecture and a key regulatory input.","evidence":"GFP-fusion localization, domain analysis, and TGF-β1 treatment with inhibitors","pmids":["12055102"],"confidence":"Medium","gaps":["No direct PP1c interaction or substrate demonstrated","Mechanism of CAAX prenylation not biochemically resolved"]},{"year":2005,"claim":"Showed TIMAP functions as a substrate-targeting subunit by recruiting PP1 to LAMR1 via a specific ankyrin repeat, the first identified TIMAP-PP1 substrate.","evidence":"Yeast two-hybrid, co-IP, in vitro phosphatase assay, deletion mutagenesis","pmids":["16263087"],"confidence":"High","gaps":["PP1c isoform selectivity not yet addressed","LAMR1 phosphosites undefined at this stage"]},{"year":2007,"claim":"Defined the PKA-primed GSK-3β phospho-code at Ser333/Ser337 that controls TIMAP-PP1c activity, linking phosphoregulation to endothelial filopodia formation.","evidence":"32P metabolic labeling, cell-free kinase assays, mutagenesis, filopodia imaging","pmids":["17609201"],"confidence":"High","gaps":["Quantitative effect on PP1c binding not measured","Substrate underlying filopodia phenotype not identified"]},{"year":2008,"claim":"Demonstrated PP1cβ-isoform preference and a functional role in endothelial barrier protection through PKA-mediated moesin dephosphorylation.","evidence":"Isoform-specific pull-down, siRNA with TER measurements, co-IP, forskolin/PKA activation","pmids":["18586956"],"confidence":"High","gaps":["Direct demonstration that moesin is a TIMAP-PP1c substrate in vitro not shown here","Basis of β-isoform selectivity unresolved"]},{"year":2011,"claim":"Provided quantitative binding constants and showed double phosphorylation attenuates TIMAP's inhibitory effect on PP1c without abolishing binding, mechanistically explaining barrier protection by forskolin.","evidence":"Surface plasmon resonance, in vitro thiophosphorylation, phospho-moesin phosphatase assay, GSK3β inhibitor","pmids":["21466834"],"confidence":"High","gaps":["Structural basis of inhibition relief not resolved","In-cell phosphostoichiometry unmeasured"]},{"year":2013,"claim":"Resolved the dual substrate-specific behavior of the complex—dephosphorylating MLC2 while masking LAMR1 sites—and modeled how the ankyrin cluster engages the PP1c C-terminus while leaving the active site accessible.","evidence":"In vitro phosphatase assays on phosphorylase a/MLC2/LAMR1, structural modeling, mutagenesis, C-terminal truncations","pmids":["23685145"],"confidence":"High","gaps":["No experimental structure of the TIMAP-PP1c complex","Determinants of substrate selectivity incompletely defined"]},{"year":2014,"claim":"Placed TIMAP upstream of PTEN/Akt signaling, establishing its requirement for endothelial proliferation, survival, and angiogenic sprouting.","evidence":"siRNA depletion, proliferation/EdU/caspase assays, 3D sprouting, co-IP, PTEN inhibitor rescue","pmids":["25007873"],"confidence":"High","gaps":["Whether PTEN S370 is a direct TIMAP-PP1c substrate not shown","Kinase opposing this dephosphorylation unidentified"]},{"year":2015,"claim":"Identified eEF1A1 as a direct TIMAP interactor and ROCK-phosphorylated substrate whose membrane localization depends on TIMAP, expanding the substrate repertoire beyond cytoskeletal regulators.","evidence":"Pull-down, LC-MS/MS, Far-Western, co-IP, fractionation, siRNA, immunofluorescence","pmids":["26497934"],"confidence":"High","gaps":["Functional consequence of eEF1A1 dephosphorylation unresolved","Phosphosite on eEF1A1 not mapped"]},{"year":2016,"claim":"Defined multiple new substrates and inputs—HDAC3/Smad2/3-mediated transcriptional repression with MLC dephosphorylation in macrophages, PKCα-Ser331 phosphorylation blocking ERM dephosphorylation, ECE-1 control of endothelin-1 secretion, and merlin Ser518 dephosphorylation via FERM-domain binding.","evidence":"ChIP, in vitro PKC kinase assays, mutagenesis, co-IP, GST domain mapping, siRNA, ECIS, endothelin-1 secretion assays","pmids":["27709267","27939168","26806547","27871951"],"confidence":"High","gaps":["Integration of competing phospho-inputs (PKCα vs GSK3β) on the same complex unresolved","Single-lab substrate identifications lacking reciprocal in vivo validation"]},{"year":2019,"claim":"Demonstrated TIMAP antagonizes myosin phosphatase by competing with MYPT1 for PP1cβ and occluding the active site, explaining the paradoxical enhancement of MLC2 phosphorylation.","evidence":"Endogenous co-IP, reconstituted GST-TIMAP/His-MLC2 interaction, microcystin-LR affinity, KO mouse, siRNA, proteasome inhibition","pmids":["31315927"],"confidence":"High","gaps":["Stoichiometric balance of TIMAP vs MYPT1 in native cells unquantified","How active-site occlusion is reconciled with productive substrate dephosphorylation unresolved"]},{"year":2021,"claim":"Extended the model with PLK4-Ser69 phosphorylation driving membrane enrichment and migration, and defined a phospho-regulated LR-TIMAP/PP1c complex maintaining barrier integrity via VE-cadherin Ser665.","evidence":"Mutagenesis (S69D/A), fractionation, co-IP with PLK4, ECIS; AAV gene delivery, LC-MS/MS phosphosite mapping, PET imaging","pmids":["34343028","33639401"],"confidence":"Medium","gaps":["Whether PLK4 phosphorylation is regulated physiologically unclear","Mechanism linking LR phospho-state to VE-cadherin not fully resolved"]},{"year":2024,"claim":"Established BMP9/SMAD1/5/8 as a repressor and hypoxia/angiogenic factors as inducers of TIMAP, and demonstrated TIMAP is required for tumor angiogenesis in vivo.","evidence":"SMAD1/5/8 pathway assays, Roxadustat treatment, TIMAP KO mouse, E0771 breast cancer model, VEGF/IGF inhibitor studies","pmids":["39344413"],"confidence":"Medium","gaps":["Direct SMAD occupancy of the TIMAP promoter not shown in this study","Endothelial-autonomous vs systemic contribution to tumor angiogenesis not dissected"]},{"year":2023,"claim":"Revealed a non-endothelial role: TIMAP is downregulated during neuronal differentiation, inhibits it when overexpressed, and translocates to the nucleus where it engages a distinct interactome.","evidence":"qPCR/Western for expression and differentiation markers, overexpression, immunofluorescence, nuclear interactome by mass spectrometry","pmids":["38139189"],"confidence":"Medium","gaps":["Nuclear functions and direct partners undefined","Whether nuclear TIMAP retains PP1c-targeting activity unknown"]},{"year":null,"claim":"How the competing kinase inputs and substrate-specific dual activity are integrated into a unified structural and signaling logic, and what TIMAP does in the nucleus, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No experimental high-resolution structure of TIMAP-PP1c-substrate complexes","Nuclear TIMAP function uncharacterized","Quantitative model reconciling active-site occlusion with substrate dephosphorylation lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,5,7,11,12]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,4,5,12]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,7,11]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,3,9,14]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[15]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[6,8,16]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[6,16]}],"complexes":["TIMAP-PP1cβ holophosphatase","LR-TIMAP/PP1c membrane complex"],"partners":["PPP1CB","LAMR1/RPSA","MSN","NF2","EEF1A1","ECE1","MYPT1/PPP1R12A","PLK4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96T49","full_name":"Protein phosphatase 1 regulatory inhibitor subunit 16B","aliases":["Ankyrin repeat domain-containing protein 4","CAAX box protein TIMAP","TGF-beta-inhibited membrane-associated protein","hTIMAP"],"length_aa":567,"mass_kda":63.6,"function":"Regulator of protein phosphatase 1 (PP1) that acts as a positive regulator of pulmonary endothelial cell (EC) barrier function (PubMed:18586956). Involved in the regulation of the PI3K/AKT signaling pathway, angiogenesis and endothelial cell proliferation (PubMed:25007873). Regulates angiogenesis and endothelial cell proliferation through the control of ECE1 dephosphorylation, trafficking and activity (By similarity). Protects the endothelial barrier from lipopolysaccharide (LPS)-induced vascular leakage (By similarity). Involved in the regulation of endothelial cell filopodia extension (By similarity). May be a downstream target for TGF-beta1 signaling cascade in endothelial cells (PubMed:16263087, PubMed:18586956). Involved in PKA-mediated moesin dephosphorylation which is important in EC barrier protection against thrombin stimulation (PubMed:18586956). Promotes the interaction of PPP1CA with RPSA/LAMR1 and in turn facilitates the dephosphorylation of RPSA/LAMR1 (PubMed:16263087). Involved in the dephosphorylation of EEF1A1 (PubMed:26497934)","subcellular_location":"Cell membrane; Cell membrane; Nucleus; Cell projection","url":"https://www.uniprot.org/uniprotkb/Q96T49/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PPP1R16B","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":[],"url":"https://opencell.sf.czbiohub.org/search/PPP1R16B","total_profiled":1310},"omim":[{"mim_id":"613275","title":"PROTEIN PHOSPHATASE 1, REGULATORY SUBUNIT 16B; PPP1R16B","url":"https://www.omim.org/entry/613275"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Nuclear speckles","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":73.0},{"tissue":"lymphoid tissue","ntpm":39.3}],"url":"https://www.proteinatlas.org/search/PPP1R16B"},"hgnc":{"alias_symbol":["KIAA0823","TIMAP","ANKRD4"],"prev_symbol":[]},"alphafold":{"accession":"Q96T49","domains":[{"cath_id":"1.25.40.20","chopping":"69-189","consensus_level":"high","plddt":94.7987,"start":69,"end":189},{"cath_id":"1.25.40.20","chopping":"201-334","consensus_level":"high","plddt":93.1825,"start":201,"end":334},{"cath_id":"1.20.5","chopping":"7-52","consensus_level":"high","plddt":86.7583,"start":7,"end":52}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96T49","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96T49-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96T49-F1-predicted_aligned_error_v6.png","plddt_mean":70.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PPP1R16B","jax_strain_url":"https://www.jax.org/strain/search?query=PPP1R16B"},"sequence":{"accession":"Q96T49","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96T49.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96T49/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96T49"}},"corpus_meta":[{"pmid":"12055102","id":"PMC_12055102","title":"TIMAP, a novel CAAX box protein regulated by TGF-beta1 and expressed in endothelial cells.","date":"2002","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/12055102","citation_count":48,"is_preprint":false},{"pmid":"18586956","id":"PMC_18586956","title":"TIMAP is a positive regulator of pulmonary endothelial barrier function.","date":"2008","source":"American journal of physiology. Lung cellular and molecular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/18586956","citation_count":38,"is_preprint":false},{"pmid":"16263087","id":"PMC_16263087","title":"The protein phosphatase-1 targeting subunit TIMAP regulates LAMR1 phosphorylation.","date":"2005","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/16263087","citation_count":34,"is_preprint":false},{"pmid":"27709267","id":"PMC_27709267","title":"TIMAP repression by TGFβ and HDAC3-associated Smad signaling regulates macrophage M2 phenotypic phagocytosis.","date":"2016","source":"Journal of molecular medicine (Berlin, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/27709267","citation_count":34,"is_preprint":false},{"pmid":"17609201","id":"PMC_17609201","title":"Phosphorylation of TIMAP by glycogen synthase kinase-3beta activates its associated protein phosphatase 1.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17609201","citation_count":22,"is_preprint":false},{"pmid":"25007873","id":"PMC_25007873","title":"TIMAP promotes angiogenesis by suppressing PTEN-mediated Akt inhibition in human glomerular endothelial cells.","date":"2014","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/25007873","citation_count":20,"is_preprint":false},{"pmid":"29140585","id":"PMC_29140585","title":"TIMAP, the versatile protein phosphatase 1 regulator in endothelial cells.","date":"2017","source":"IUBMB life","url":"https://pubmed.ncbi.nlm.nih.gov/29140585","citation_count":17,"is_preprint":false},{"pmid":"27939168","id":"PMC_27939168","title":"PKC mediated phosphorylation of TIMAP regulates PP1c activity and endothelial barrier function.","date":"2016","source":"Biochimica et biophysica acta. Molecular cell research","url":"https://pubmed.ncbi.nlm.nih.gov/27939168","citation_count":16,"is_preprint":false},{"pmid":"21466834","id":"PMC_21466834","title":"Characterization of the effect of TIMAP phosphorylation on its interaction with protein phosphatase 1.","date":"2011","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/21466834","citation_count":16,"is_preprint":false},{"pmid":"33639401","id":"PMC_33639401","title":"The role of LR-TIMAP/PP1c complex in the occurrence and development of no-reflow.","date":"2021","source":"EBioMedicine","url":"https://pubmed.ncbi.nlm.nih.gov/33639401","citation_count":14,"is_preprint":false},{"pmid":"26497934","id":"PMC_26497934","title":"Elongation factor-1A1 is a novel substrate of the protein phosphatase 1-TIMAP complex.","date":"2015","source":"The international journal of biochemistry & cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/26497934","citation_count":14,"is_preprint":false},{"pmid":"23685145","id":"PMC_23685145","title":"Multi-directional function of the protein phosphatase 1 regulatory subunit TIMAP.","date":"2013","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/23685145","citation_count":14,"is_preprint":false},{"pmid":"21907835","id":"PMC_21907835","title":"TIMAP protects endothelial barrier from LPS-induced vascular leakage and is down-regulated by LPS.","date":"2011","source":"Respiratory physiology & neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/21907835","citation_count":11,"is_preprint":false},{"pmid":"16817016","id":"PMC_16817016","title":"Potential protein partners for the human TIMAP revealed by bacterial two-hybrid screening.","date":"2006","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/16817016","citation_count":11,"is_preprint":false},{"pmid":"27871951","id":"PMC_27871951","title":"Regulation of merlin by protein phosphatase 1-TIMAP and EBP50 in endothelial cells.","date":"2016","source":"The international journal of biochemistry & cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/27871951","citation_count":9,"is_preprint":false},{"pmid":"31315927","id":"PMC_31315927","title":"TIMAP inhibits endothelial myosin light chain phosphatase by competing with MYPT1 for the catalytic protein phosphatase 1 subunit PP1cβ.","date":"2019","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/31315927","citation_count":9,"is_preprint":false},{"pmid":"26806547","id":"PMC_26806547","title":"TIMAP-protein phosphatase 1-complex controls endothelin-1 production via ECE-1 dephosphorylation.","date":"2016","source":"The international journal of biochemistry & cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/26806547","citation_count":8,"is_preprint":false},{"pmid":"38139189","id":"PMC_38139189","title":"TIMAP, a Regulatory Subunit of Protein Phosphatase 1, Inhibits In Vitro Neuronal Differentiation.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/38139189","citation_count":4,"is_preprint":false},{"pmid":"34181349","id":"PMC_34181349","title":"TIMAP Upregulation Correlates Negatively with Survival in HER2- Negative Subtypes of Breast Cancer.","date":"2021","source":"Asian Pacific journal of cancer prevention : APJCP","url":"https://pubmed.ncbi.nlm.nih.gov/34181349","citation_count":4,"is_preprint":false},{"pmid":"39344413","id":"PMC_39344413","title":"Hypoxia-induced TIMAP upregulation in endothelial cells and TIMAP-dependent tumor angiogenesis.","date":"2024","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/39344413","citation_count":3,"is_preprint":false},{"pmid":"34343028","id":"PMC_34343028","title":"Ser69 phosphorylation of TIMAP affects endothelial cell migration.","date":"2021","source":"Experimental lung research","url":"https://pubmed.ncbi.nlm.nih.gov/34343028","citation_count":3,"is_preprint":false},{"pmid":"41170526","id":"PMC_41170526","title":"TIMAP downregulation in Burkitt's lymphoma reveals key molecules and signaling pathways in B-cell lymphomagenesis.","date":"2025","source":"Experimental biology and medicine (Maywood, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/41170526","citation_count":0,"is_preprint":false},{"pmid":"41968320","id":"PMC_41968320","title":"Loss of protein phosphatase 1 regulator TIMAP protein triggers EMT in A549 cells.","date":"2026","source":"Cell communication and signaling : CCS","url":"https://pubmed.ncbi.nlm.nih.gov/41968320","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15437,"output_tokens":5488,"usd":0.064315,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":14033,"output_tokens":4568,"usd":0.092183,"stage2_stop_reason":"end_turn"},"total_usd":0.156498,"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\": 2002,\n      \"finding\": \"TIMAP (PPP1R16B) contains five ankyrin repeats, a PP1-interacting domain, and a COOH-terminal CAAX box; a GFP-TIMAP fusion protein localizes to the plasma membrane in a CAAX box-dependent fashion, and TGF-β1 represses TIMAP through a protein synthesis- and histone deacetylase-dependent process.\",\n      \"method\": \"Immunofluorescence of GFP-fusion protein, Northern blot, domain analysis, TGF-β1 treatment with inhibitor studies\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment with CAAX-box dependency shown, and transcriptional repression mechanism characterized; single lab, two orthogonal methods\",\n      \"pmids\": [\"12055102\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"TIMAP interacts with the 37/67 kDa laminin receptor (LAMR1) via its fourth ankyrin repeat (amino acids 261–290); this interaction recruits PP1 to LAMR1 and targets PP1-mediated dephosphorylation of LAMR1, as full-length TIMAP (but not the ankyrin-deletion mutant) abolished LAMR1 phosphorylation in cells.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, co-localization at plasma membrane, domain-deletion mutagenesis, in vitro PP1 phosphatase assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (yeast two-hybrid, co-IP, in vitro phosphatase assay, deletion mutagenesis) in a single study establishing substrate/mechanism\",\n      \"pmids\": [\"16263087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TIMAP is phosphorylated by PKA at Ser333/Ser337 and, after PKA priming, by GSK-3β at the same sites; this phosphorylation controls TIMAP association with PP1c and PP1c activity. Abolishing PP1c binding (TIMAPV64A/F66A) causes hyper-phosphorylation of TIMAP, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Reduced PP1c activity (via TIMAP mutants with low PP1c binding) strongly stimulates endothelial cell filopodia formation.\",\n      \"method\": \"Metabolic 32P-labeling, cell-free kinase assays, site-directed mutagenesis (Ser→Ala, Val/Phe→Ala), phosphomimic substitutions, immunoprecipitation, endothelial cell filopodia imaging\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assays combined with mutagenesis and cellular functional readout; multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"17609201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Recombinant TIMAP preferentially binds the β-isoform of PP1c (PP1cβ) from pulmonary artery endothelial cells. TIMAP depletion by siRNA attenuates barrier-protective responses and enhances barrier disruption. TIMAP co-immunoprecipitates with moesin and is involved in PKA-mediated moesin dephosphorylation at the cell periphery, contributing to endothelial barrier protection.\",\n      \"method\": \"Pull-down assay with isoform-specific PP1c, siRNA depletion with transendothelial electrical resistance measurements, immunofluorescent co-localization, co-immunoprecipitation, forskolin/PKA activation experiments\",\n      \"journal\": \"American journal of physiology. Lung cellular and molecular physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, pull-down isoform selectivity, functional siRNA phenotype with mechanistic follow-up; multiple orthogonal methods, replicated aspects in subsequent papers\",\n      \"pmids\": [\"18586956\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Non-phosphorylated TIMAP binds PP1c with a binding constant Ka = 1.80 × 10⁶ M⁻¹ (surface plasmon resonance). PKA mono-phosphorylation slightly decreases the dissociation rate; double (PKA+GSK3β) phosphorylation of TIMAP does not substantially reduce PP1c binding but strongly attenuates TIMAP's inhibitory effect on PP1c activity toward phospho-moesin substrate (<10% inhibition vs ~60% for non-phosphorylated TIMAP). PKA activation followed by GSK3β activation is required for the barrier-protective effect of forskolin in thrombin-treated endothelial cells.\",\n      \"method\": \"Surface plasmon resonance binding assay, in vitro thiophosphorylation, phosphatase activity assay with phospho-moesin substrate, specific GSK3β inhibitor in bovine pulmonary endothelial cells\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative binding constants by SPR, in vitro kinase and phosphatase assays with mutagenesis-equivalent thiophosphorylation; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"21466834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TIMAP associates with all three PP1c isoforms in vitro but endogenous TIMAP in endothelial cells preferentially co-immunoprecipitates PP1cβ. Structural modeling predicts PP1c C-terminus is buried in the TIMAP ankyrin cluster while the active site remains accessible—consistent with experiments showing PP1c C-terminal phosphorylation by cdk2-cyclinA is masked by TIMAP. TIMAP inhibits PP1c activity toward phosphorylase a (IC₅₀ ~0.4–1.2 nM). TIMAP-bound PP1cβ effectively dephosphorylates MLC2 (direct substrate), while TIMAP inhibits PP1cβ activity toward LAMR1 by masking its phosphorylation sites. TIMAP C-terminal length and Ser333/Ser337 phosphomimic mutations modulate the inhibitory effect.\",\n      \"method\": \"Co-immunoprecipitation, in vitro phosphatase activity assays (phosphorylase a, MLC2, LAMR1), structural modeling, site-directed mutagenesis, C-terminal truncation variants\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro phosphatase reconstitution with substrate specificity profiling, mutagenesis, and structural modeling; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"23685145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TIMAP is necessary for Akt-dependent endothelial cell proliferation, survival, and angiogenic sprout formation. TIMAP and PTEN co-localize and co-immunoprecipitate in endothelial cells. TIMAP depletion reduces inhibitory phosphorylation of PTEN at S370 (but not S380/T382/T383), leading to reduced Akt phosphorylation; this effect is rescued by the PTEN inhibitor bpV(phen), placing TIMAP upstream of PTEN/Akt.\",\n      \"method\": \"siRNA depletion, impedance-based proliferation assay, EdU incorporation, caspase 3 activity, 3D angiogenic sprouting assay, co-immunoprecipitation, co-localization, pharmacological PTEN inhibition rescue\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis established by rescue with PTEN inhibitor, co-IP of endogenous proteins, multiple functional readouts; single lab with orthogonal methods\",\n      \"pmids\": [\"25007873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TIMAP directly interacts with eukaryotic elongation factor 1A1 (eEF1A1) via a TD-NEM-like motif in TIMAP. TIMAP regulates membrane localization of eEF1A1 (eEF1A1 disappears from the membrane in TIMAP-depleted cells). ROCK-phosphorylated eEF1A1 is a substrate for the TIMAP-PP1 complex.\",\n      \"method\": \"Pull-down, LC-MS/MS identification, Far-Western blot, co-immunoprecipitation, subcellular fractionation, siRNA depletion, immunofluorescence\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal binding assays (pull-down, Far-Western, co-IP) plus functional localization data and substrate identification; single lab\",\n      \"pmids\": [\"26497934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TGF-β transcriptionally downregulates TIMAP through HDAC3-associated Smad2/3 signaling; HDAC3 is recruited to the Smad binding element on the TIMAP promoter. TIMAP dephosphorylates myosin light chain (MLC) in macrophages, and TGF-β-induced macrophage migration and phagocytosis occur in a TIMAP- and MLC-phosphorylation-dependent manner.\",\n      \"method\": \"ChIP for HDAC3/Smad2/3 at TIMAP promoter, specific HDAC3 inhibition reversal experiments, TIMAP overexpression, MLC phosphorylation assays in macrophages, migration and phagocytosis assays\",\n      \"journal\": \"Journal of molecular medicine (Berlin, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and pharmacological rescue established the HDAC3/Smad mechanism; MLC substrate shown by phosphorylation assay; single lab\",\n      \"pmids\": [\"27709267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PKCα phosphorylates TIMAP at Ser331 in vitro and in endothelial cells. PKC-phosphorylated TIMAP is enriched at the plasma membrane, but phospho-Ser331 TIMAP shows severely reduced binding to ERM proteins, inhibiting TIMAP-PP1 activity toward phospho-ERM. Phosphomimic S331D TIMAP increases membrane phospho-ERM levels while phosphonull S331A lowers them, indicating Ser331 phosphorylation by PKC inhibits ERM dephosphorylation and slows endothelial barrier recovery.\",\n      \"method\": \"Co-immunoprecipitation of endogenous PKCα and TIMAP, in vitro PKC kinase assay on recombinant TIMAP, site-directed mutagenesis (S331D/S331A), subcellular fractionation, electric cell-substrate impedance sensing (ECIS)\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay plus mutagenesis with functional cellular readout; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"27939168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TIMAP-PP1c complex interacts with endothelin converting enzyme-1 (ECE-1); TIMAP depletion reduces TIMAP-PP1c activity toward ECE-1, causing increased ECE-1 at the plasma membrane and elevated endothelin-1 secretion. PKC-phosphorylated ECE-1 is identified as a TIMAP-PP1c substrate.\",\n      \"method\": \"Co-immunoprecipitation of TIMAP-PP1c with ECE-1, TIMAP siRNA depletion, measurement of ECE-1 membrane levels, endothelin-1 secretion assay, PKC activation experiments\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and functional depletion with defined molecular readout; single lab, two orthogonal methods\",\n      \"pmids\": [\"26806547\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PP1c-TIMAP complex dephosphorylates merlin at Ser518 in endothelial cells. TIMAP N-terminal half (aa 1–290) interacts with the FERM domain of merlin, and PP1c is present in TIMAP-merlin complexes. Without TIMAP, PP1c cannot bind merlin. TIMAP or PP1c depletion increases phospho-Ser518 merlin at the membrane. EBP50 binds unphosphorylated merlin in the nucleus and is required for merlin's nuclear localization.\",\n      \"method\": \"GST pull-down domain mapping, co-immunoprecipitation, siRNA depletion, subcellular fractionation, immunofluorescence, ECIS wound healing assay\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-mapped interaction, co-IP of endogenous proteins, functional phosphorylation readout with depletion; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"27871951\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TIMAP inhibits myosin phosphatase activity in endothelial cells by competing with MYPT1 for PP1cβ and blocking the PP1cβ active site. Excess TIMAP reduces MYPT1-PP1cβ association and leads to proteasomal MYPT1 degradation. TIMAP-associated PP1cβ does not interact with microcystin-LR (active-site inhibitor), indicating the active site is blocked when PP1cβ is bound to TIMAP. TIMAP overexpression paradoxically enhances MLC2 phosphorylation by displacing the more active myosin phosphatase MYPT1/PP1cβ.\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins, purified recombinant protein interaction assay (GST-TIMAP with His-MLC2), microcystin-LR affinity binding, TIMAP overexpression/knockout mouse, siRNA depletion, proteasome inhibitor experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reconstituted direct protein interaction, active-site probe (microcystin), endogenous co-IP, KO mouse, and cellular functional readout; multiple orthogonal methods\",\n      \"pmids\": [\"31315927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TIMAP-PP1c forms a complex with laminin receptor (LR) on the cell membrane (LR-TIMAP/PP1c complex). pTyr47-LR determines the stability of this complex. TIMAP/PP1c binding to LR activates PP1c phosphatase activity and regulates LR phosphorylation at Thr125; low phospho-LR stabilizes the complex and maintains endothelial barrier integrity via regulation of VE-cadherin Ser665 phosphorylation.\",\n      \"method\": \"Co-immunoprecipitation, in vivo gene delivery (AAV), LC-MS/MS phosphosite identification, Western blot for phospho-specific residues, PET perfusion imaging\",\n      \"journal\": \"EBioMedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP of endogenous complex and in vivo functional validation; single group, two orthogonal methods\",\n      \"pmids\": [\"33639401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Phosphorylation of TIMAP at Ser69 by polo-like kinase 4 (PLK4) promotes enrichment of TIMAP at the plasma membrane and increases membrane protrusions, leading to faster wound healing and endothelial cell migration. Ser69 phosphorylation does not affect binding of PP1c, ERM, or RACK1 to TIMAP. Phosphomimic S69D TIMAP showed enhanced membrane localization compared to wild-type.\",\n      \"method\": \"Site-directed mutagenesis (S69D/S69A), subcellular fractionation, immunofluorescence, pull-down, co-immunoprecipitation with PLK4, ECIS wound healing assay\",\n      \"journal\": \"Experimental lung research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with functional localization and migration readout; interaction with PLK4 shown by co-IP; single lab\",\n      \"pmids\": [\"34343028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TIMAP is expressed in SH-SY5Y neuroblastoma cells, is downregulated during neuronal differentiation, and its overexpression inhibits differentiation. During differentiation, TIMAP translocates from the plasma membrane to the nucleus, where its nuclear interactome comprises more than 50 proteins.\",\n      \"method\": \"qPCR and Western blot for TIMAP expression, neuronal differentiation markers, TIMAP overexpression, immunofluorescence for subcellular localization, nuclear interactome by mass spectrometry\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional overexpression with defined phenotype and direct localization experiment; single lab, two orthogonal methods\",\n      \"pmids\": [\"38139189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"BMP9 represses TIMAP expression via the SMAD1/5/8 pathway in endothelial cells; hypoxia and the prolyl hydroxylase inhibitor Roxadustat raise TIMAP expression by inhibiting SMAD1/5/8. VEGFA and IGF-I also elevate TIMAP partly by attenuating SMAD1/5/8. TIMAP deficiency in mice markedly reduces tumor growth and tumor angiogenesis in the E0771 breast cancer model.\",\n      \"method\": \"SMAD1/5/8 pathway activation assays, Roxadustat treatment, TIMAP KO mouse, E0771 breast cancer in vivo model, VEGF/IGF inhibitor experiments\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO model with tumor angiogenesis readout, and pathway inhibitor studies; single lab, multiple methods\",\n      \"pmids\": [\"39344413\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PPP1R16B/TIMAP is a prenylated, CAAX box-dependent plasma membrane-anchored regulatory subunit of protein phosphatase 1β (PP1cβ) that is highly expressed in endothelial cells; it recruits PP1cβ to specific substrates including LAMR1, MLC2, ERM/moesin, merlin (phospho-Ser518), eEF1A1, and ECE-1 to control their dephosphorylation, while paradoxically inhibiting myosin phosphatase by competing with MYPT1 for PP1cβ and occluding the PP1cβ active site; TIMAP activity is tuned by PKA-primed GSK-3β phosphorylation at Ser333/Ser337 (which reduces inhibitory activity and promotes filopodia), by PKCα phosphorylation at Ser331 (which blocks ERM dephosphorylation), and by PLK4-mediated phosphorylation at Ser69 (which promotes membrane localization and migration); upstream, TGF-β represses TIMAP via HDAC3-associated Smad2/3 signaling, and BMP9/SMAD1/5/8 pathway suppresses TIMAP while hypoxia and angiogenic growth factors induce it, collectively making TIMAP a critical regulator of endothelial barrier integrity, Akt/PTEN-dependent angiogenesis, and tumor angiogenesis in vivo.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PPP1R16B/TIMAP is a prenylated, CAAX-box-dependent plasma-membrane-anchored regulatory subunit of protein phosphatase 1 that controls endothelial barrier integrity, migration, and angiogenesis by targeting and tuning PP1c activity toward specific membrane substrates [#0, #3, #6]. Through its ankyrin-repeat cluster and PP1-interacting domain it preferentially binds the β-isoform of PP1c (PP1cβ) and acts as a substrate-targeting scaffold, recruiting the phosphatase to LAMR1, moesin/ERM proteins, MLC2, merlin (phospho-Ser518), eEF1A1, and ECE-1 to govern their dephosphorylation and membrane localization [#1, #3, #5, #7, #10, #11]. The complex exerts dual, substrate-dependent effects on PP1c: TIMAP-bound PP1cβ efficiently dephosphorylates MLC2 and ERM substrates, yet TIMAP also inhibits PP1c toward other substrates and, by competing with MYPT1 for PP1cβ and occluding the active site, antagonizes myosin phosphatase to paradoxically promote MLC2 phosphorylation [#5, #12]. TIMAP activity is set by a layered phospho-code: PKA-primed GSK-3β phosphorylation at Ser333/Ser337 relieves PP1c inhibition and drives filopodia and barrier protection, PKCα phosphorylation at Ser331 blocks ERM dephosphorylation and slows barrier recovery, and PLK4 phosphorylation at Ser69 promotes membrane enrichment and migration [#2, #4, #9, #14]. TIMAP positively regulates PTEN/Akt signaling required for endothelial proliferation, survival, and angiogenic sprouting [#6], and its expression is repressed by TGF-β via HDAC3-associated Smad2/3 and by BMP9 via SMAD1/5/8 while induced by hypoxia and angiogenic growth factors, with TIMAP loss reducing tumor angiogenesis in vivo [#8, #16].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established TIMAP as a multidomain, membrane-targeted PP1 regulatory subunit and identified its transcriptional repression by TGF-β, defining the protein's basic architecture and a key regulatory input.\",\n      \"evidence\": \"GFP-fusion localization, domain analysis, and TGF-β1 treatment with inhibitors\",\n      \"pmids\": [\"12055102\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct PP1c interaction or substrate demonstrated\", \"Mechanism of CAAX prenylation not biochemically resolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed TIMAP functions as a substrate-targeting subunit by recruiting PP1 to LAMR1 via a specific ankyrin repeat, the first identified TIMAP-PP1 substrate.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, in vitro phosphatase assay, deletion mutagenesis\",\n      \"pmids\": [\"16263087\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PP1c isoform selectivity not yet addressed\", \"LAMR1 phosphosites undefined at this stage\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined the PKA-primed GSK-3β phospho-code at Ser333/Ser337 that controls TIMAP-PP1c activity, linking phosphoregulation to endothelial filopodia formation.\",\n      \"evidence\": \"32P metabolic labeling, cell-free kinase assays, mutagenesis, filopodia imaging\",\n      \"pmids\": [\"17609201\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative effect on PP1c binding not measured\", \"Substrate underlying filopodia phenotype not identified\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated PP1cβ-isoform preference and a functional role in endothelial barrier protection through PKA-mediated moesin dephosphorylation.\",\n      \"evidence\": \"Isoform-specific pull-down, siRNA with TER measurements, co-IP, forskolin/PKA activation\",\n      \"pmids\": [\"18586956\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct demonstration that moesin is a TIMAP-PP1c substrate in vitro not shown here\", \"Basis of β-isoform selectivity unresolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Provided quantitative binding constants and showed double phosphorylation attenuates TIMAP's inhibitory effect on PP1c without abolishing binding, mechanistically explaining barrier protection by forskolin.\",\n      \"evidence\": \"Surface plasmon resonance, in vitro thiophosphorylation, phospho-moesin phosphatase assay, GSK3β inhibitor\",\n      \"pmids\": [\"21466834\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of inhibition relief not resolved\", \"In-cell phosphostoichiometry unmeasured\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved the dual substrate-specific behavior of the complex—dephosphorylating MLC2 while masking LAMR1 sites—and modeled how the ankyrin cluster engages the PP1c C-terminus while leaving the active site accessible.\",\n      \"evidence\": \"In vitro phosphatase assays on phosphorylase a/MLC2/LAMR1, structural modeling, mutagenesis, C-terminal truncations\",\n      \"pmids\": [\"23685145\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No experimental structure of the TIMAP-PP1c complex\", \"Determinants of substrate selectivity incompletely defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Placed TIMAP upstream of PTEN/Akt signaling, establishing its requirement for endothelial proliferation, survival, and angiogenic sprouting.\",\n      \"evidence\": \"siRNA depletion, proliferation/EdU/caspase assays, 3D sprouting, co-IP, PTEN inhibitor rescue\",\n      \"pmids\": [\"25007873\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PTEN S370 is a direct TIMAP-PP1c substrate not shown\", \"Kinase opposing this dephosphorylation unidentified\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified eEF1A1 as a direct TIMAP interactor and ROCK-phosphorylated substrate whose membrane localization depends on TIMAP, expanding the substrate repertoire beyond cytoskeletal regulators.\",\n      \"evidence\": \"Pull-down, LC-MS/MS, Far-Western, co-IP, fractionation, siRNA, immunofluorescence\",\n      \"pmids\": [\"26497934\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of eEF1A1 dephosphorylation unresolved\", \"Phosphosite on eEF1A1 not mapped\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined multiple new substrates and inputs—HDAC3/Smad2/3-mediated transcriptional repression with MLC dephosphorylation in macrophages, PKCα-Ser331 phosphorylation blocking ERM dephosphorylation, ECE-1 control of endothelin-1 secretion, and merlin Ser518 dephosphorylation via FERM-domain binding.\",\n      \"evidence\": \"ChIP, in vitro PKC kinase assays, mutagenesis, co-IP, GST domain mapping, siRNA, ECIS, endothelin-1 secretion assays\",\n      \"pmids\": [\"27709267\", \"27939168\", \"26806547\", \"27871951\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Integration of competing phospho-inputs (PKCα vs GSK3β) on the same complex unresolved\", \"Single-lab substrate identifications lacking reciprocal in vivo validation\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrated TIMAP antagonizes myosin phosphatase by competing with MYPT1 for PP1cβ and occluding the active site, explaining the paradoxical enhancement of MLC2 phosphorylation.\",\n      \"evidence\": \"Endogenous co-IP, reconstituted GST-TIMAP/His-MLC2 interaction, microcystin-LR affinity, KO mouse, siRNA, proteasome inhibition\",\n      \"pmids\": [\"31315927\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometric balance of TIMAP vs MYPT1 in native cells unquantified\", \"How active-site occlusion is reconciled with productive substrate dephosphorylation unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extended the model with PLK4-Ser69 phosphorylation driving membrane enrichment and migration, and defined a phospho-regulated LR-TIMAP/PP1c complex maintaining barrier integrity via VE-cadherin Ser665.\",\n      \"evidence\": \"Mutagenesis (S69D/A), fractionation, co-IP with PLK4, ECIS; AAV gene delivery, LC-MS/MS phosphosite mapping, PET imaging\",\n      \"pmids\": [\"34343028\", \"33639401\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PLK4 phosphorylation is regulated physiologically unclear\", \"Mechanism linking LR phospho-state to VE-cadherin not fully resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established BMP9/SMAD1/5/8 as a repressor and hypoxia/angiogenic factors as inducers of TIMAP, and demonstrated TIMAP is required for tumor angiogenesis in vivo.\",\n      \"evidence\": \"SMAD1/5/8 pathway assays, Roxadustat treatment, TIMAP KO mouse, E0771 breast cancer model, VEGF/IGF inhibitor studies\",\n      \"pmids\": [\"39344413\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct SMAD occupancy of the TIMAP promoter not shown in this study\", \"Endothelial-autonomous vs systemic contribution to tumor angiogenesis not dissected\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed a non-endothelial role: TIMAP is downregulated during neuronal differentiation, inhibits it when overexpressed, and translocates to the nucleus where it engages a distinct interactome.\",\n      \"evidence\": \"qPCR/Western for expression and differentiation markers, overexpression, immunofluorescence, nuclear interactome by mass spectrometry\",\n      \"pmids\": [\"38139189\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Nuclear functions and direct partners undefined\", \"Whether nuclear TIMAP retains PP1c-targeting activity unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the competing kinase inputs and substrate-specific dual activity are integrated into a unified structural and signaling logic, and what TIMAP does in the nucleus, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimental high-resolution structure of TIMAP-PP1c-substrate complexes\", \"Nuclear TIMAP function uncharacterized\", \"Quantitative model reconciling active-site occlusion with substrate dephosphorylation lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 5, 7, 11, 12]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 4, 5, 12]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 7, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 3, 9, 14]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [6, 8, 16]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [6, 16]}\n    ],\n    \"complexes\": [\"TIMAP-PP1cβ holophosphatase\", \"LR-TIMAP/PP1c membrane complex\"],\n    \"partners\": [\"PPP1CB\", \"LAMR1/RPSA\", \"MSN\", \"NF2\", \"EEF1A1\", \"ECE1\", \"MYPT1/PPP1R12A\", \"PLK4\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}