{"gene":"CACYBP","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":2002,"finding":"CacyBP/SIP binds multiple S100 family proteins (S100A1, S100A6, S100A12, S100B, S100P but not S100A4, calbindin D9k, parvalbumin, or calmodulin) in a Ca2+-dependent manner, and this interaction occurs via the C-terminal fragment of CacyBP/SIP (residues 155–229). Co-immunoprecipitation from brain and tumor cell extracts confirmed physiological relevance.","method":"CacyBP/SIP affinity chromatography, nitrocellulose overlay assay with 125I-CacyBP/SIP, Co-immunoprecipitation from tissue/cell extracts","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal biochemical methods (affinity chromatography, overlay assay, Co-IP) with domain mapping, replicated across multiple S100 proteins and tissue sources","pmids":["12042313"],"is_preprint":false},{"year":2001,"finding":"CacyBP/SIP (identified as SIP) interacts with Siah-1 and Skp1 and functions as a component of a ubiquitin ligase complex that promotes proteasomal degradation of β-catenin.","method":"Referenced in multiple corpus abstracts as the foundational finding (Matsuzawa and Reed, Mol Cell 2001); confirmed in downstream studies by Co-immunoprecipitation and ubiquitination assays","journal":"Molecular Cell (referenced; confirmed by multiple subsequent papers in corpus)","confidence":"High","confidence_rationale":"Tier 2 / Strong — independently replicated across multiple labs and cancer models using Co-IP and ubiquitination assays; founding result referenced consistently throughout corpus","pmids":["12042313","16340196","17400182","22295074","35184390"],"is_preprint":false},{"year":2009,"finding":"CacyBP/SIP directly binds ERK1/2 kinases; S100A6 competes for this interaction. A point mutant E217K of CacyBP/SIP does not bind ERK1/2 but retains S100A6 binding, implicating the C-terminal region (residues 189–219) in ERK1/2 binding. CacyBP/SIP–ERK1/2 interaction inhibits phosphorylation of the Elk-1 transcription factor in vitro and in the nuclear fraction of NB2a cells.","method":"Co-immunoprecipitation, site-directed mutagenesis (E217K), molecular modeling, in vitro kinase/phosphorylation assay, nuclear fractionation","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis, in vitro assay, and cellular fractionation in single lab; multiple orthogonal methods within one study","pmids":["19166809"],"is_preprint":false},{"year":2010,"finding":"CacyBP/SIP exhibits intrinsic phosphatase activity toward ERK1/2; the E217K mutant lacks this activity. Km and Vmax for p-NPP substrate and inhibition by okadaic acid (IC50 ~45 nM) are consistent with a PP2A-like phosphatase. Sequence analysis reveals similarity to phosphatase-like proteins and MAP kinase phosphatases.","method":"In vitro phosphatase activity assay (p-NPP substrate), okadaic acid inhibition, E217K mutagenesis, sequence analysis","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with mutagenesis and kinetic characterization; single lab but multiple orthogonal methods","pmids":["21110948"],"is_preprint":false},{"year":2010,"finding":"CacyBP/SIP directly binds actin, co-localizes with actin in NB2a cells, induces G-actin polymerization and formation of circular actin filament bundles. The N-terminal fragment (residues 1–179) is sufficient for this activity; the C-terminal fragment (residues 178–229) is not. CacyBP/SIP can simultaneously interact with both tubulin and actin, suggesting it may link the two cytoskeletal networks.","method":"Zero-length cross-linking, co-sedimentation assay, immunofluorescence co-localization, proximity ligation assay, stable cell line overexpression with adhesion/migration readout, N- and C-terminal fragment analysis","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — direct binding confirmed by cross-linking and co-sedimentation, domain mapping, multiple orthogonal methods in one study","pmids":["20637809"],"is_preprint":false},{"year":2012,"finding":"CacyBP/SIP binds directly to tropomyosin, and binding to the actin–tropomyosin complex causes conformational changes in tropomyosin (increased fluorescence). CacyBP/SIP destabilizes actin filaments in the absence of tropomyosin, but tropomyosin reverses this effect. CacyBP/SIP reduces actin-activated myosin S1 ATPase activity, and tropomyosin enhances this inhibitory effect.","method":"Fluorescence assay with AEDANS-labeled tropomyosin, direct binding assay, electron microscopy, actin-activated myosin S1 ATPase colorimetric assay","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — reconstituted filament assays, structural imaging, enzymatic readout; multiple orthogonal in vitro methods in single study","pmids":["23266554"],"is_preprint":false},{"year":2007,"finding":"CacyBP/SIP binds tubulin (established prior work cited by corpus), and in neurons its co-localization with tubulin and tau changes with age: in young rats CacyBP/SIP is present in cytoplasm and neuronal processes together with tubulin/tau; in aged rats it shifts to cytoplasm of neuronal somata, suggesting involvement in cytoskeletal physiology.","method":"Immunohistochemistry, immunofluorescence in cultured neurons and rat brain sections at multiple ages","journal":"Journal of neural transmission","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — localization study with functional inference; replicated across age groups but no direct functional rescue experiment","pmids":["18506390"],"is_preprint":false},{"year":2016,"finding":"Casein kinase II (CKII) phosphorylates CacyBP/SIP at threonine 184 in vitro; S100A6 inhibits this phosphorylation in a Ca2+-dependent manner. The phosphomimetic T184E mutant has lower phosphatase activity toward p-ERK1/2 compared to T184A or wild-type, indicating that CKII-mediated phosphorylation at T184 negatively regulates CacyBP/SIP's phosphatase activity.","method":"In vitro phosphorylation assay (CKII), site-directed mutagenesis (T184E, T184A), Western blot in NB2a cells, phosphatase activity assay toward p-ERK1/2","journal":"Journal of cellular biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay, phosphomimetic/non-phosphorylable mutagenesis with functional readout; single lab, multiple orthogonal methods","pmids":["26085436"],"is_preprint":false},{"year":2017,"finding":"CacyBP/SIP binds and dephosphorylates p38 MAP kinase; the middle CS domain is responsible for p38 dephosphorylation. In NB2a cells treated with hydrogen peroxide, CacyBP/SIP-mediated dephosphorylation of phospho-p38 is more effective than in untreated cells, implicating CacyBP/SIP in oxidative stress responses.","method":"Co-immunoprecipitation from NB2a cell lysate and with recombinant proteins, in vitro phosphatase assay using domain fragments, Western blot for phospho-p38 under H2O2 stress","journal":"Amino acids","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal interaction and domain-mapped phosphatase assay with recombinant proteins; single lab, multiple complementary methods","pmids":["28283909"],"is_preprint":false},{"year":2013,"finding":"CacyBP/SIP is sumoylated at lysine 16 in neuroblastoma NB2a cells; it binds the E2 SUMO ligase Ubc9 in cell extract. Uniquely, sumoylated CacyBP/SIP is found in the cytoplasmic rather than the nuclear fraction.","method":"Immunoprecipitation for SUMO-modified proteins, cell fractionation, site-directed identification of K16 as sumoylation site","journal":"Neurochemical research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — binding to E2 ligase shown by IP, sumoylation confirmed in cells with fractionation; single lab, multiple complementary methods","pmids":["24078263"],"is_preprint":false},{"year":2019,"finding":"Under normal conditions, CacyBP/SIP (SIP) inhibits ubiquitination and proteasomal degradation of BRUCE/Apollon, likely by blocking Nrdp1 binding to BRUCE. Upon starvation, SIP together with Rab8 enhances translocation of BRUCE into the recycling endosome and promotes autophagic destruction of BRUCE by optineurin-mediated autophagy, thereby relieving BRUCE-mediated proteasomal degradation of LC3-I and stimulating autophagy. Deletion of SIP in cells reduces autophagic degradation of damaged mitochondria and cytosolic protein aggregates.","method":"Co-immunoprecipitation, ubiquitination assay, SIP knockout cell lines, autophagy flux assay (LC3-I/II), mitophagy assay, live-cell imaging of BRUCE trafficking with Rab8","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ubiquitination assay, KO cells, trafficking assay) with mechanistic follow-up and clear phenotypic readouts; single lab but rigorous","pmids":["31213539"],"is_preprint":false},{"year":2019,"finding":"RNF41 binds CACYBP via its C-terminal substrate binding domain, ubiquitinates CACYBP, and promotes its degradation via both proteasome- and lysosome-dependent pathways. CACYBP overexpression stimulates Ser10, Thr157, and Thr198 phosphorylation of P27Kip1 and its cytoplasmic retention; RNF41 co-expression attenuates this. CACYBP depletion causes G1/S arrest and increased apoptosis in HCC cells.","method":"Co-immunoprecipitation (endogenous and exogenous), ubiquitination assay, Western blot for P27Kip1 phosphorylation, immunofluorescence for P27Kip1 localization, flow cytometry, xenograft model, P27Kip1-S10D/S10A reconstitution","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, ubiquitination assay, mutagenesis rescue, in vivo model; multiple orthogonal methods in single rigorous study","pmids":["31754404"],"is_preprint":false},{"year":2017,"finding":"CacyBP/SIP nuclear translocation induced by gastrin promotes proteasome-dependent degradation of p27Kip1 in colon cancer cells. CacyBP/SIP binds Skp1 (shown by Co-IP); a CacyBP/SIP truncation mutant lacking the Skp1 binding site fails to promote p27Kip1 degradation even after nuclear translocation. Proteasome inhibitor MG132 prevents gastrin-induced p27Kip1 reduction.","method":"Co-immunoprecipitation, truncation mutagenesis, confocal immunofluorescence, Western blot, MG132 proteasome inhibitor, siRNA knockdown","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP, mutagenesis of Skp1-binding domain with functional readout, pharmacological inhibition; multiple orthogonal methods, single lab","pmids":["28196083"],"is_preprint":false},{"year":2016,"finding":"CacyBP/SIP nuclear translocation in gastric cancer cells promotes proteasome-dependent p27Kip1 degradation; CacyBP/SIP binds Skp1 (Co-IP), and a Skp1-binding-deficient mutant of CacyBP/SIP fails to stimulate p27Kip1 degradation even when it translocates to the nucleus. G1-phase CacyBP/SIP is cytoplasmic; it translocates to nucleus at G2 where β-catenin and p-ERK1/2 are reduced.","method":"Co-immunoprecipitation, truncation mutagenesis, immunofluorescence, Western blot, MG132 proteasome inhibitor, cell cycle synchronization","journal":"World journal of gastroenterology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and mutagenesis with functional readout, single lab, partially overlapping with PMID 28196083","pmids":["27099442"],"is_preprint":false},{"year":2018,"finding":"Cell cycle-dependent nuclear translocation of CacyBP/SIP, Siah-1, and Skp1: these proteins are cytoplasmic in G1 and translocate to the nucleus in G2. In G2, CacyBP/SIP phosphorylation and binding capacity to Siah-1 and Skp1 increase, while β-catenin and p-ERK1/2 decrease. A CacyBP/SIP mutant lacking the S100-binding domain (CacyBP-ΔS100) shows increased nuclear translocation, further suppressing β-catenin and proliferation; this is reversed by β-catenin activator LiCl.","method":"Immunofluorescence after cell cycle synchronization, Co-immunoprecipitation, Western blot, phosphorylation assay, LiCl/XAV-939 pharmacological modulation","journal":"Anti-cancer drugs","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — cell cycle synchronization, Co-IP, domain mutant with pharmacological rescue; single lab","pmids":["29099417"],"is_preprint":false},{"year":2014,"finding":"Gastrin stimulation triggers nuclear translocation of CacyBP/SIP in gastric cancer cells; CacyBP/SIP nuclear translocation promotes cell cycle progression from G1 phase and enhances proliferation. siRNA-mediated prevention of nuclear translocation abolishes these proliferative effects.","method":"Immunofluorescence, subcellular fractionation + Western blot, MTT assay, colony formation assay, flow cytometry cell cycle analysis, siRNA knockdown","journal":"World journal of gastroenterology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct localization experiment with functional consequence; multiple readouts; single lab","pmids":["25110433"],"is_preprint":false},{"year":2018,"finding":"Ca2+-dependent binding of S100A6 to CacyBP/SIP is required for Ca2+-induced nuclear translocation of CacyBP/SIP in colon cancer SW480 cells: ionomycin-elevated [Ca2+]i triggers translocation, and knockdown of S100A6 abolishes this effect.","method":"Immunofluorescence, Co-immunoprecipitation under varying [Ca2+], siRNA knockdown of S100A6","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct localization experiment with S100A6 knockdown establishing mechanistic dependency; single lab, Co-IP plus imaging","pmids":["29534068"],"is_preprint":false},{"year":2017,"finding":"CacyBP/SIP promotes Siah1-mediated ubiquitination and degradation of cytoplasmic p27Kip1, inhibiting glioma cell migration and invasion. Silencing CacyBP/SIP reduces the Siah1–p27 interaction and attenuates p27 ubiquitination; overexpression increases the Siah1–p27 interaction and p27 degradation. Siah1 knockdown blocks p27 degradation.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA/overexpression of CacyBP/SIP and Siah1, migration/invasion assays (Transwell), Western blot","journal":"Cell biology international","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and ubiquitination assay establish mechanistic pathway; Siah1 epistasis confirmed by knockdown; single lab","pmids":["29024247"],"is_preprint":false},{"year":2020,"finding":"CacyBP/SIP interacts with Hsp90 and acts as a co-chaperone; overexpression of CacyBP/SIP in HEp-2 cells increases cellular resistance to stress-induced death. The Hsf1 transcription factor binds to the CacyBP/SIP gene promoter and up-regulates CacyBP/SIP expression under stress conditions (H2O2 and radicicol).","method":"Co-immunoprecipitation (CacyBP/SIP–Hsp90), ChIP assay (Hsf1 on CacyBP/SIP promoter), Western blot, cell viability assay, CacyBP/SIP overexpression","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP binding and ChIP for transcriptional regulation; functional cell viability assay; single lab","pmids":["29660399"],"is_preprint":false},{"year":2020,"finding":"CacyBP/SIP directly interacts with α-synuclein and protects it from aggregation at the initial phase of fibril formation. The fragment overlapping the N-terminal domain and CS domain of CacyBP/SIP is critical for this chaperone-like activity. In HEK293 cells overexpressing CacyBP/SIP, there are fewer α-synuclein inclusions and greater viability upon rotenone treatment.","method":"Thioflavin T fluorescence aggregation assay, high-speed ultracentrifugation + dot-blot, transmission electron microscopy, proximity ligation assay (PLA) in cells, in vitro assay with purified recombinant proteins, cell viability assay","journal":"Cells","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro reconstitution with purified proteins, multiple orthogonal methods (ThT, EM, ultracentrifugation, PLA, cellular assay), domain mapping; single lab","pmids":["33049998"],"is_preprint":false},{"year":2018,"finding":"CacyBP/SIP interacts with NPM1 (nucleophosmin) directly; their complex is found in the cell nucleus. CacyBP/SIP overexpression favors phosphorylation of NPM1 on S125 and increases binding of 28S and 18S rRNA to NPM1. Under oxidative stress, CacyBP/SIP overexpression alters NPM1 nuclear distribution and maintains nucleolar structure (assessed by fibrillarin staining); CacyBP/SIP is required for preserving nucleolar integrity under stress.","method":"Mass spectrometry identification, Co-immunoprecipitation, direct binding assay, RNA immunoprecipitation (RIP), immunofluorescence (fibrillarin), Western blot","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct binding plus RIP and functional imaging; single lab with multiple orthogonal methods","pmids":["29806702"],"is_preprint":false},{"year":2022,"finding":"SFRS8 splicing factor mediates alternative splicing of CACYBP pre-mRNA, reducing isoform 1 (NM_014412.3) and increasing isoform 2 (NM_001007214.1). Isoform switching alters ubiquitination and degradation of β-catenin, promoting myeloma progression. Exosomal siRNA targeting CACYBP isoform 2 inhibits tumor growth in PDX models.","method":"RNA immunoprecipitation sequencing (RIP-seq), RIP-qPCR, Co-IP, xenograft models, PDX model, exosome-mediated siRNA delivery","journal":"Clinical and translational medicine","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — RIP-seq identifies isoform regulation, functional in vivo rescue; single lab","pmids":["35184390"],"is_preprint":false},{"year":2023,"finding":"CacyBP binds MyD88 via its Toll/IL-1 receptor (TIR) domain and protects MyD88 from Siah-1-mediated proteasomal degradation by competitive binding. CacyBP–MyD88 signaling promotes HDAC1-mediated H3K9ac and H3K27ac modifications on the CX3CL1 promoter, increasing CX3CL1 transcription and secretion in HCC cells, which enhances tumor-associated macrophage infiltration. CacyBP inhibition reduces immunosuppressive microenvironment and sensitizes tumors to anti-PD-1 therapy.","method":"Co-immunoprecipitation, immunofluorescence, Co-IP competition assay, in vitro and in vivo macrophage recruitment assays, chromatin immunoprecipitation (H3K9ac/H3K27ac on CX3CL1 promoter), xenograft models","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP binding, ChIP for histone modification, in vivo model; single lab, multiple complementary methods","pmids":["37968706"],"is_preprint":false},{"year":2020,"finding":"CacyBP/SIP promotes degradation of mutant p53 by enhancing Mdm2 E3 ubiquitin ligase activity in U251 glioma cells. CacyBP/SIP interacts with both p53 and Mdm2, increases p53 ubiquitination in a dose-dependent manner in the presence of Mdm2, and Mdm2 inhibition reverses this. In wild-type p53 U87 cells, CacyBP/SIP has no effect on proliferation or p53 levels.","method":"Co-immunoprecipitation (CacyBP/SIP with p53 and Mdm2), ubiquitination assay (dose–response), Mdm2 inhibitor treatment, Western blot, shRNA knockdown","journal":"Neoplasma","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and ubiquitination assay establish the mechanistic pathway; dose-response ubiquitination; single lab","pmids":["32880469"],"is_preprint":false},{"year":2016,"finding":"CacyBP/SIP overexpression reduces p-ERK1/2 levels in undifferentiated NB2a cells, leading to decreased CREB phosphorylation and reduced BDNF mRNA; silencing has the opposite effect. In differentiated NB2a cells, CacyBP/SIP overexpression has a different effect on p-ERK1/2, which correlates with changes in CacyBP/SIP post-translational modifications and protein ligands.","method":"CacyBP/SIP overexpression and siRNA silencing, Western blot for p-ERK1/2, p-CREB, RT-qPCR for BDNF mRNA, 2D-PAGE analysis of CacyBP/SIP modification state","journal":"Neurochemistry international","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — bidirectional manipulation (OE/KD) with pathway readouts in two cell states; single lab","pmids":["27180052"],"is_preprint":false},{"year":2012,"finding":"CacyBP/SIP exhibits phosphatase activity toward ERK1/2 in the nuclear fraction of undifferentiated NB2a cells but not in HCT116 colon cancer cells. The phosphorylation state of CacyBP/SIP differs between these cell lines (assessed by 2D-electrophoresis), and nuclear β-catenin levels are inversely correlated: low in NB2a (with CacyBP/SIP overexpression) and high in HCT116.","method":"CacyBP/SIP overexpression, nuclear fractionation, phosphatase activity assay, 2D-electrophoresis, Western blot","journal":"Biochemistry and cell biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — cell-type-specific phosphatase activity demonstrated with fractionation; single lab, multiple methods","pmids":["22480271"],"is_preprint":false},{"year":2012,"finding":"S100A6 negatively regulates CacyBP/SIP-mediated inhibition of gastric cancer cell proliferation. A CacyBP/SIP truncation mutant lacking the S100-binding domain (CacyBP/SIPΔS100) shows reduced S100A6 co-immunoprecipitation and further reduces proliferation and tumorigenesis compared to wild-type, demonstrating that S100A6 binding attenuates CacyBP/SIP's antiproliferative effect through modulation of β-catenin expression and Tcf/LEF transcriptional activity.","method":"Co-immunoprecipitation, truncation mutagenesis, MTT assay, FACS, clonogenic assay, tumor xenograft","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — domain mutant Co-IP plus functional in vitro and in vivo readouts; single lab","pmids":["22295074"],"is_preprint":false},{"year":2017,"finding":"NFAT1 transcription factor directly binds to the CacyBP/SIP gene promoter and up-regulates CacyBP/SIP expression; inhibition or stimulation of NFAT transcriptional activity correspondingly decreases or increases CacyBP/SIP levels.","method":"Western blot, RT-PCR, luciferase reporter assay, EMSA, chromatin immunoprecipitation (ChIP)","journal":"Immunobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding demonstrated by EMSA and ChIP, functional reporter assay; single lab","pmids":["28526484"],"is_preprint":false},{"year":2022,"finding":"Heterozygous CACYBP/SIP p.E177Q variant is linked to paucity of interlobular bile ducts (PILBD) in a dominant family. CACYBP/SIP forms a ubiquitin ligase complex that degrades non-phosphorylated β-catenin; the variant may form a more active/stable complex, enhancing β-catenin degradation and delaying bile duct maturation.","method":"Next-generation sequencing (family), immunohistochemistry for CACYBP and β-catenin in patient liver","journal":"Journal of human genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — genetic variant identification with IHC; no in vitro reconstitution of enhanced activity; single family, mechanistic claim is partially inferred","pmids":["35087201"],"is_preprint":false},{"year":2024,"finding":"CacyBP interacts directly with OTUD5 deubiquitinase, enhances ubiquitination and proteasomal degradation of OTUD5, and thereby promotes lung adenocarcinoma tumorigenesis.","method":"Co-immunoprecipitation, ubiquitination assay, overexpression/knockdown functional assays (proliferation, migration, invasion), Western blot","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP and ubiquitination assay establish mechanism; functional phenotypic validation; single lab","pmids":["38558058"],"is_preprint":false},{"year":2025,"finding":"CacyBP/SIP interacts directly with ribosomal protein RPL6; the interaction was confirmed by multiple biochemical methods and in silico domain mapping. Cells with stably silenced CacyBP/SIP show reduced perinuclear nascent polypeptide labeling (OPP staining) and lower Hsp70 induction after heat shock, suggesting CacyBP/SIP influences ribosome function and protein synthesis efficiency.","method":"Mass spectrometry, Co-immunoprecipitation, direct binding assay, in silico domain mapping, OPP (O-propargyl-puromycin) labeling of nascent polypeptides, Western blot for Hsp70 under heat shock in CacyBP/SIP-silenced vs. control cells","journal":"Amino acids","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct binding and mass spectrometry; functional assay in silenced cells; single lab, single study","pmids":["40691326"],"is_preprint":false},{"year":2025,"finding":"CacyBP/SIP knockdown in cortical neurons of opossum (marsupial model) causes excessive branching and elongation of dendritic trees and axon arbors during upper cortical layer formation, without affecting proliferation or differentiation. This establishes a role for CacyBP/SIP in restraining neurite branching during cortical development.","method":"Lentiviral shRNA knockdown, primary cortical neuron cultures, morphometric analysis of dendritic and axonal arbors, cell proliferation and differentiation assays","journal":"Journal of neurochemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — loss-of-function with specific morphometric phenotypic readout; single lab, single model organism","pmids":["40476326"],"is_preprint":false},{"year":2025,"finding":"CacyBP/SIP stabilization by Cordycepin (via inhibiting its ubiquitin-proteasome degradation) suppresses NLRP3 inflammasome activation and enhances autophagosome-lysosome fusion, leading to autophagic degradation of α-synuclein in rotenone-treated SH-SY5Y cells. Blockade of CacyBP/SIP abrogates these neuroprotective effects.","method":"CacyBP/SIP protein stability assay with proteasome inhibitors, NLRP3 inflammasome activation assay, autophagosome-lysosome fusion assay, α-synuclein aggregation assay, siRNA knockdown of CacyBP/SIP, in vivo rotenone mouse model","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — pharmacological and genetic (siRNA) manipulation with mechanistic pathway readouts; single lab, in vitro plus in vivo","pmids":["40639630"],"is_preprint":false},{"year":2025,"finding":"CacyBP interacts directly with CDK1 (co-immunoprecipitation); CDK1 overexpression rescues the inhibitory effects of CacyBP knockdown on LUAD cell growth. CacyBP promotes LUAD progression at least in part through CDK1-mediated activation of the PI3K/AKT pathway (PI3K inhibitor LY294002 blocks CDK1-mediated growth).","method":"Co-immunoprecipitation (CacyBP–CDK1), CDK1 overexpression rescue, LY294002 PI3K inhibition, proliferation/apoptosis/migration assays, xenograft model","journal":"Biomolecules & biomedicine","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP binding, genetic rescue, and pharmacological epistasis; single lab","pmids":["40167359"],"is_preprint":false}],"current_model":"CacyBP/SIP is a multidomain scaffold protein that functions as: (1) a component of a Siah-1/Skp1-containing ubiquitin ligase complex that degrades β-catenin; (2) an intrinsic phosphatase for ERK1/2 and p38 MAP kinases, regulated by CK II-mediated phosphorylation at T184 and inhibited by S100A6 in a Ca2+-dependent manner; (3) a cytoskeletal regulator that directly binds actin (inducing polymerization via its N-terminal domain) and tropomyosin, links actin and tubulin networks, and regulates thin filament ATPase activity; (4) a promoter of autophagy by stabilizing BRUCE/Apollon and facilitating autophagosome formation; (5) an HSP90 co-chaperone that protects clients including α-synuclein from aggregation; (6) a regulator of nuclear functions including NPM1 phosphorylation, nucleolar integrity, and ribosome-associated translation efficiency via RPL6 interaction; with its subcellular localization (cytoplasmic vs. nuclear, regulated by Ca2+/S100A6 and cell cycle stage) being a key determinant of its functional output in proliferation, differentiation, stress response, and neurite morphogenesis."},"narrative":{"mechanistic_narrative":"CacyBP/SIP (CACYBP) is a multidomain, Ca2+-responsive scaffold whose subcellular distribution and partner repertoire determine which of several regulatory programs it executes — protein degradation, MAP kinase dephosphorylation, cytoskeletal organization, chaperoning, and autophagy [PMID:12042313, PMID:29099417]. Through its C-terminal region it binds multiple S100 proteins (S100A1, S100A6, S100A12, S100B, S100P) in a Ca2+-dependent manner [PMID:12042313], and S100A6 binding controls Ca2+-induced nuclear translocation that switches its activity [PMID:29534068]. As a component of a Siah-1/Skp1-containing ubiquitin ligase complex, CacyBP/SIP drives proteasomal degradation of β-catenin and of p27Kip1, with the Skp1-binding domain required for nuclear p27Kip1 turnover and antiproliferative output [PMID:12042313, PMID:16340196, PMID:17400182, PMID:22295074, PMID:35184390, PMID:28196083, PMID:29024247]; it likewise channels other substrates including mutant p53 (via Mdm2), OTUD5, and the MyD88 stability decision toward distinct cancer phenotypes [PMID:32880469, PMID:38558058, PMID:37968706]. Independently, CacyBP/SIP is an intrinsic PP2A-like phosphatase that binds and dephosphorylates ERK1/2 (C-terminal region; E217K abolishes binding and activity) and p38 (via its CS domain), an activity negatively regulated by CK II phosphorylation at T184 and competitively antagonized by S100A6 [PMID:19166809, PMID:21110948, PMID:26085436, PMID:28283909]. The protein also directly remodels the cytoskeleton: its N-terminal domain binds and polymerizes actin, it binds tropomyosin and modulates actin-activated myosin S1 ATPase, and it can bridge actin and tubulin networks [PMID:20637809, PMID:23266554]. CacyBP/SIP further acts as an HSP90 co-chaperone and protects α-synuclein from aggregation through its N-terminal/CS region, and promotes autophagy by stabilizing BRUCE/Apollon and facilitating autophagic flux [PMID:29660399, PMID:33049998, PMID:31213539]. A nuclear role encompasses NPM1 binding/phosphorylation and nucleolar maintenance under stress, plus RPL6 interaction that supports translation efficiency [PMID:29806702, PMID:40691326]. A heterozygous CACYBP p.E177Q variant segregates with paucity of interlobular bile ducts in a dominant family, consistent with altered β-catenin degradation during bile duct maturation [PMID:35087201].","teleology":[{"year":2001,"claim":"Established CacyBP/SIP's first defined molecular function — that it is not merely an S100-binding protein but a structural component of a ubiquitin ligase that targets β-catenin for degradation.","evidence":"Co-IP and ubiquitination assays placing SIP in a Siah-1/Skp1 complex, referenced and confirmed across multiple cancer models","pmids":["12042313","16340196","17400182","22295074","35184390"],"confidence":"High","gaps":["Stoichiometry and assembly order of the Siah-1/Skp1/CacyBP complex not resolved","Does not address how Ca2+/S100 status gates ligase assembly"]},{"year":2002,"claim":"Defined the calcium-sensing input by mapping Ca2+-dependent S100 binding to a discrete C-terminal segment, establishing CacyBP/SIP as a Ca2+/S100-regulated effector.","evidence":"Affinity chromatography, overlay assays, and Co-IP from brain/tumor extracts with domain mapping (residues 155–229)","pmids":["12042313"],"confidence":"High","gaps":["Functional consequence of each S100 partner not individually dissected","S100A6 selectivity over other S100s mechanistically unexplained"]},{"year":2010,"claim":"Revealed that CacyBP/SIP carries an intrinsic enzymatic activity — a PP2A-like phosphatase acting on ERK1/2 — defining a degradation-independent signaling function.","evidence":"In vitro phosphatase kinetics (p-NPP), okadaic acid inhibition, E217K mutagenesis, plus prior ERK1/2 direct-binding mapping","pmids":["21110948","19166809"],"confidence":"High","gaps":["Catalytic mechanism and active-site residues not structurally defined","Physiological substrate range beyond ERK1/2 not established"]},{"year":2010,"claim":"Showed CacyBP/SIP directly organizes the cytoskeleton, polymerizing actin via its N-terminus and potentially linking actin and tubulin networks.","evidence":"Zero-length cross-linking, co-sedimentation, immunofluorescence co-localization, and domain fragment analysis in NB2a cells","pmids":["20637809"],"confidence":"High","gaps":["In vivo relevance of actin/tubulin bridging not demonstrated","Regulation of cytoskeletal binding by Ca2+/S100 untested"]},{"year":2012,"claim":"Extended cytoskeletal function to thin-filament regulation, showing tropomyosin binding and modulation of actin-activated myosin ATPase.","evidence":"Fluorescence binding, electron microscopy, and myosin S1 ATPase assays with reconstituted actin/tropomyosin","pmids":["23266554"],"confidence":"High","gaps":["Physiological context (muscle vs non-muscle) of thin-filament regulation unclear","Connection to motility phenotypes not directly tested"]},{"year":2016,"claim":"Identified the regulatory switch governing phosphatase activity — CK II phosphorylation at T184 suppresses it, and S100A6 blocks that phosphorylation, integrating kinase and Ca2+ inputs.","evidence":"In vitro CKII kinase assay, T184E/T184A phosphomimetic mutagenesis, and phosphatase readout in NB2a cells","pmids":["26085436"],"confidence":"High","gaps":["Whether other phosphosites tune activity not addressed","In vivo CKII regulation in non-neuronal cells untested"]},{"year":2017,"claim":"Broadened phosphatase specificity to p38 and tied it to oxidative-stress responses, with the CS domain as the catalytic module.","evidence":"Reciprocal Co-IP, domain-mapped in vitro phosphatase assay, and phospho-p38 Westerns under H2O2 in NB2a cells","pmids":["28283909"],"confidence":"Medium","gaps":["Reconciliation of CS-domain p38 dephosphorylation with C-terminal ERK1/2 activity not made","Stress-dependent activation mechanism unresolved"]},{"year":2014,"claim":"Connected localization to function — gastrin-triggered nuclear translocation drives cell-cycle progression and proliferation, establishing localization as the key functional determinant.","evidence":"Fractionation, immunofluorescence, siRNA blockade of translocation, and proliferation/cell-cycle assays in gastric cancer cells","pmids":["25110433"],"confidence":"Medium","gaps":["Nuclear import machinery for CacyBP/SIP not identified","Direct nuclear targets after translocation only partly defined"]},{"year":2018,"claim":"Established the Ca2+/S100A6/cell-cycle logic of translocation — S100A6 binding is required for Ca2+-induced nuclear entry, and translocation in G2 increases Siah-1/Skp1 binding while lowering β-catenin and p-ERK1/2.","evidence":"Cell-cycle synchronization, Co-IP under varying Ca2+, S100A6 knockdown, and ΔS100 domain mutants with LiCl rescue across colon and gastric cancer cells","pmids":["29534068","29099417","27099442","28196083"],"confidence":"Medium","gaps":["Direct evidence linking phosphorylation state to nuclear retention incomplete","Mostly single-lab cancer-cell systems"]},{"year":2019,"claim":"Defined a proteostasis role in autophagy — CacyBP/SIP stabilizes BRUCE/Apollon and, with Rab8, redirects it for autophagic destruction to promote clearance of damaged mitochondria and aggregates.","evidence":"Co-IP, ubiquitination assays, KO cells, autophagy/mitophagy flux assays, and live-cell BRUCE trafficking","pmids":["31213539"],"confidence":"High","gaps":["How starvation switches CacyBP/SIP from protective to pro-degradative not fully resolved","Selectivity of cargo clearance untested"]},{"year":2020,"claim":"Defined CacyBP/SIP as an HSP90 co-chaperone and direct anti-aggregation factor for α-synuclein, expanding it from a degradation adaptor to a folding guardian.","evidence":"Hsp90 Co-IP, Hsf1 ChIP on the CACYBP promoter, ThT/EM/ultracentrifugation aggregation assays with purified proteins, PLA, and viability assays","pmids":["29660399","33049998"],"confidence":"High","gaps":["Client range of the Hsp90 co-chaperone activity not catalogued","Relationship between chaperone and phosphatase domains unclear"]},{"year":2018,"claim":"Uncovered nuclear/nucleolar functions through NPM1 binding, S125 phosphorylation, rRNA association, and maintenance of nucleolar integrity under stress.","evidence":"Mass spectrometry, Co-IP, RIP, and fibrillarin immunofluorescence under oxidative stress","pmids":["29806702"],"confidence":"Medium","gaps":["Kinase responsible for NPM1 S125 phosphorylation downstream of CacyBP/SIP not identified","Direct vs indirect role in rRNA binding unresolved"]},{"year":2022,"claim":"Expanded the substrate/partner network across cancers — mutant p53 (via Mdm2), OTUD5, MyD88 stabilization, and CACYBP isoform switching all feed proliferation, immune microenvironment, and β-catenin signaling.","evidence":"Co-IP, ubiquitination assays, ChIP for histone marks, RIP-seq, and xenograft/PDX models across glioma, LUAD, HCC, and myeloma","pmids":["32880469","38558058","37968706","35184390","29024247"],"confidence":"Medium","gaps":["Many partners shown in single cancer-type studies without cross-validation","Whether these reflect the same ligase complex or distinct activities unclear"]},{"year":2022,"claim":"Provided the first human disease link, associating a heterozygous CACYBP p.E177Q variant with paucity of interlobular bile ducts through altered β-catenin degradation.","evidence":"Family next-generation sequencing and IHC for CACYBP and β-catenin in patient liver","pmids":["35087201"],"confidence":"Low","gaps":["No in vitro reconstitution confirming enhanced ligase activity of the variant","Single family; causality not functionally proven"]},{"year":2025,"claim":"Connected CacyBP/SIP to translation and neurodevelopment — RPL6 binding supports protein-synthesis efficiency, and loss restrains neurite branching during cortical development.","evidence":"Mass spectrometry/Co-IP with OPP nascent-peptide labeling and Hsp70 induction; lentiviral shRNA knockdown with morphometry in opossum cortical neurons","pmids":["40691326","40476326"],"confidence":"Medium","gaps":["Mechanism by which RPL6 binding alters translation undefined","Neurodevelopmental phenotype shown only in marsupial model"]},{"year":null,"claim":"How a single protein arbitrates among ligase, phosphatase, cytoskeletal, chaperone, autophagy, and translational activities — and what structural/post-translational state selects each — remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated structural model linking domain organization to the competing activities","No unified accounting of how Ca2+/S100A6, CKII phosphorylation, SUMOylation, and cell-cycle stage jointly set output","Many cancer-specific partnerships lack independent replication"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,3,7,8]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[3,25]},{"term_id":"GO:0060090","term_label":"molecular adaptor 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Probably serves as a molecular bridge in ubiquitin E3 complexes. Participates in the ubiquitin-mediated degradation of beta-catenin (CTNNB1)","subcellular_location":"Nucleus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9HB71/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CACYBP","classification":"Not Classified","n_dependent_lines":136,"n_total_lines":1208,"dependency_fraction":0.11258278145695365},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"PTGES3","stoichiometry":4.0},{"gene":"CAPZB","stoichiometry":0.2},{"gene":"FKBP5","stoichiometry":0.2},{"gene":"FKBP8","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CACYBP","total_profiled":1310},"omim":[{"mim_id":"619294","title":"NIBAN APOPTOSIS REGULATOR 1; NIBAN1","url":"https://www.omim.org/entry/619294"},{"mim_id":"606186","title":"CALCYCLIN-BINDING PROTEIN; CACYBP","url":"https://www.omim.org/entry/606186"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Cytosol","reliability":"Enhanced"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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evaluation of CacyBP/SIP protein, β-catenin, and immunoproteasome subunit LMP7 in the heart of rats with hypertension of different etiology.","date":"2018","source":"Experimental biology and medicine (Maywood, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/30472885","citation_count":9,"is_preprint":false},{"pmid":"27099442","id":"PMC_27099442","title":"CacyBP/SIP nuclear translocation regulates p27Kip1 stability in gastric cancer cells.","date":"2016","source":"World journal of gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/27099442","citation_count":7,"is_preprint":false},{"pmid":"24078263","id":"PMC_24078263","title":"The CacyBP/SIP protein is sumoylated in neuroblastoma NB2a cells.","date":"2013","source":"Neurochemical research","url":"https://pubmed.ncbi.nlm.nih.gov/24078263","citation_count":7,"is_preprint":false},{"pmid":"34530574","id":"PMC_34530574","title":"[CacyBP promotes the proliferation and invasion of non-small cell lung 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sciences","url":"https://pubmed.ncbi.nlm.nih.gov/37373509","citation_count":6,"is_preprint":false},{"pmid":"32544715","id":"PMC_32544715","title":"CacyBP/SIP in the rat spinal cord in norm and after transection - Influence on the phosphorylation state of ERK1/2 and p38 kinases.","date":"2020","source":"Neurochemistry international","url":"https://pubmed.ncbi.nlm.nih.gov/32544715","citation_count":6,"is_preprint":false},{"pmid":"29099417","id":"PMC_29099417","title":"Cell cycle-dependent translocation and regulatory mechanism of CacyBP/SIP in gastric cancer cells.","date":"2018","source":"Anti-cancer drugs","url":"https://pubmed.ncbi.nlm.nih.gov/29099417","citation_count":5,"is_preprint":false},{"pmid":"36576589","id":"PMC_36576589","title":"CACYBP knockdown inhibits progression of prostate cancer via p53.","date":"2022","source":"Journal of cancer research and clinical 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physiology","url":"https://pubmed.ncbi.nlm.nih.gov/29806702","citation_count":3,"is_preprint":false},{"pmid":"40639630","id":"PMC_40639630","title":"Cordycepin promotes autophagic degradation of α-synuclein via CacyBP/SIP activation for ameliorating olfactory dysfunction against Parkinson's disease.","date":"2025","source":"Free radical biology & medicine","url":"https://pubmed.ncbi.nlm.nih.gov/40639630","citation_count":3,"is_preprint":false},{"pmid":"37156124","id":"PMC_37156124","title":"Involvement of CacyBP/SIP in differentiation and the immune response of HaCaT keratinocytes.","date":"2023","source":"Immunobiology","url":"https://pubmed.ncbi.nlm.nih.gov/37156124","citation_count":2,"is_preprint":false},{"pmid":"38558058","id":"PMC_38558058","title":"CacyBP promotes the development of lung adenocarcinoma by regulating OTUD5.","date":"2024","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/38558058","citation_count":1,"is_preprint":false},{"pmid":"38780511","id":"PMC_38780511","title":"Comparative assessment of CacyBP/SIP, β-catenin and cannabinoid receptors in the adrenals of hypertensive rats.","date":"2024","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/38780511","citation_count":1,"is_preprint":false},{"pmid":"40476326","id":"PMC_40476326","title":"CacyBP/SIP Protein Regulates the Length and Branching of Neuronal Processes During Cortical Development.","date":"2025","source":"Journal of neurochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40476326","citation_count":1,"is_preprint":false},{"pmid":"38203261","id":"PMC_38203261","title":"Evaluation of the Expression and Localization of the Multifunctional Protein CacyBP/SIP and Elements of the MAPK Signaling Pathway in the Adrenal Glands of Rats with Primary and Secondary Hypertension.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/38203261","citation_count":1,"is_preprint":false},{"pmid":"40691326","id":"PMC_40691326","title":"CacyBP/SIP - RPL6 interaction: potential influence on ribosome function.","date":"2025","source":"Amino acids","url":"https://pubmed.ncbi.nlm.nih.gov/40691326","citation_count":0,"is_preprint":false},{"pmid":"40167359","id":"PMC_40167359","title":"Silencing CACYBP suppresses lung adenocarcinoma growth via CDK1 inhibition.","date":"2025","source":"Biomolecules & biomedicine","url":"https://pubmed.ncbi.nlm.nih.gov/40167359","citation_count":0,"is_preprint":false},{"pmid":"41692428","id":"PMC_41692428","title":"Analysis of CacyBP/SIP, ERK1/2, and p38 Expression in Low- and High-Grade Papillary Urothelial Carcinoma.","date":"2026","source":"Cancer medicine","url":"https://pubmed.ncbi.nlm.nih.gov/41692428","citation_count":0,"is_preprint":false},{"pmid":"39644709","id":"PMC_39644709","title":"Evaluation of CacyBP/SIP expression and its relationship with ERK1/2 and p38 kinase in testicular seminoma.","date":"2024","source":"Pathology, research and practice","url":"https://pubmed.ncbi.nlm.nih.gov/39644709","citation_count":0,"is_preprint":false},{"pmid":"35087201","id":"PMC_35087201","title":"Heterozygous calcyclin-binding protein/Siah1-interacting protein (CACYBP/SIP) gene pathogenic variant linked to a dominant family with paucity of interlobular bile duct.","date":"2022","source":"Journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/35087201","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.09.17.676758","title":"Proteomic and Kinetic Characterization of Prion Seeding in Distinct Human CJD Strains Unveils Early Diagnostic Biomarkers","date":"2025-09-17","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.17.676758","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":40202,"output_tokens":9672,"usd":0.132843,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":19727,"output_tokens":5390,"usd":0.116693,"stage2_stop_reason":"end_turn"},"total_usd":0.249536,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"CacyBP/SIP binds multiple S100 family proteins (S100A1, S100A6, S100A12, S100B, S100P but not S100A4, calbindin D9k, parvalbumin, or calmodulin) in a Ca2+-dependent manner, and this interaction occurs via the C-terminal fragment of CacyBP/SIP (residues 155–229). Co-immunoprecipitation from brain and tumor cell extracts confirmed physiological relevance.\",\n      \"method\": \"CacyBP/SIP affinity chromatography, nitrocellulose overlay assay with 125I-CacyBP/SIP, Co-immunoprecipitation from tissue/cell extracts\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal biochemical methods (affinity chromatography, overlay assay, Co-IP) with domain mapping, replicated across multiple S100 proteins and tissue sources\",\n      \"pmids\": [\"12042313\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CacyBP/SIP (identified as SIP) interacts with Siah-1 and Skp1 and functions as a component of a ubiquitin ligase complex that promotes proteasomal degradation of β-catenin.\",\n      \"method\": \"Referenced in multiple corpus abstracts as the foundational finding (Matsuzawa and Reed, Mol Cell 2001); confirmed in downstream studies by Co-immunoprecipitation and ubiquitination assays\",\n      \"journal\": \"Molecular Cell (referenced; confirmed by multiple subsequent papers in corpus)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — independently replicated across multiple labs and cancer models using Co-IP and ubiquitination assays; founding result referenced consistently throughout corpus\",\n      \"pmids\": [\"12042313\", \"16340196\", \"17400182\", \"22295074\", \"35184390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CacyBP/SIP directly binds ERK1/2 kinases; S100A6 competes for this interaction. A point mutant E217K of CacyBP/SIP does not bind ERK1/2 but retains S100A6 binding, implicating the C-terminal region (residues 189–219) in ERK1/2 binding. CacyBP/SIP–ERK1/2 interaction inhibits phosphorylation of the Elk-1 transcription factor in vitro and in the nuclear fraction of NB2a cells.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (E217K), molecular modeling, in vitro kinase/phosphorylation assay, nuclear fractionation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis, in vitro assay, and cellular fractionation in single lab; multiple orthogonal methods within one study\",\n      \"pmids\": [\"19166809\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CacyBP/SIP exhibits intrinsic phosphatase activity toward ERK1/2; the E217K mutant lacks this activity. Km and Vmax for p-NPP substrate and inhibition by okadaic acid (IC50 ~45 nM) are consistent with a PP2A-like phosphatase. Sequence analysis reveals similarity to phosphatase-like proteins and MAP kinase phosphatases.\",\n      \"method\": \"In vitro phosphatase activity assay (p-NPP substrate), okadaic acid inhibition, E217K mutagenesis, sequence analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with mutagenesis and kinetic characterization; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"21110948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CacyBP/SIP directly binds actin, co-localizes with actin in NB2a cells, induces G-actin polymerization and formation of circular actin filament bundles. The N-terminal fragment (residues 1–179) is sufficient for this activity; the C-terminal fragment (residues 178–229) is not. CacyBP/SIP can simultaneously interact with both tubulin and actin, suggesting it may link the two cytoskeletal networks.\",\n      \"method\": \"Zero-length cross-linking, co-sedimentation assay, immunofluorescence co-localization, proximity ligation assay, stable cell line overexpression with adhesion/migration readout, N- and C-terminal fragment analysis\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct binding confirmed by cross-linking and co-sedimentation, domain mapping, multiple orthogonal methods in one study\",\n      \"pmids\": [\"20637809\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CacyBP/SIP binds directly to tropomyosin, and binding to the actin–tropomyosin complex causes conformational changes in tropomyosin (increased fluorescence). CacyBP/SIP destabilizes actin filaments in the absence of tropomyosin, but tropomyosin reverses this effect. CacyBP/SIP reduces actin-activated myosin S1 ATPase activity, and tropomyosin enhances this inhibitory effect.\",\n      \"method\": \"Fluorescence assay with AEDANS-labeled tropomyosin, direct binding assay, electron microscopy, actin-activated myosin S1 ATPase colorimetric assay\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — reconstituted filament assays, structural imaging, enzymatic readout; multiple orthogonal in vitro methods in single study\",\n      \"pmids\": [\"23266554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CacyBP/SIP binds tubulin (established prior work cited by corpus), and in neurons its co-localization with tubulin and tau changes with age: in young rats CacyBP/SIP is present in cytoplasm and neuronal processes together with tubulin/tau; in aged rats it shifts to cytoplasm of neuronal somata, suggesting involvement in cytoskeletal physiology.\",\n      \"method\": \"Immunohistochemistry, immunofluorescence in cultured neurons and rat brain sections at multiple ages\",\n      \"journal\": \"Journal of neural transmission\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — localization study with functional inference; replicated across age groups but no direct functional rescue experiment\",\n      \"pmids\": [\"18506390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Casein kinase II (CKII) phosphorylates CacyBP/SIP at threonine 184 in vitro; S100A6 inhibits this phosphorylation in a Ca2+-dependent manner. The phosphomimetic T184E mutant has lower phosphatase activity toward p-ERK1/2 compared to T184A or wild-type, indicating that CKII-mediated phosphorylation at T184 negatively regulates CacyBP/SIP's phosphatase activity.\",\n      \"method\": \"In vitro phosphorylation assay (CKII), site-directed mutagenesis (T184E, T184A), Western blot in NB2a cells, phosphatase activity assay toward p-ERK1/2\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay, phosphomimetic/non-phosphorylable mutagenesis with functional readout; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"26085436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CacyBP/SIP binds and dephosphorylates p38 MAP kinase; the middle CS domain is responsible for p38 dephosphorylation. In NB2a cells treated with hydrogen peroxide, CacyBP/SIP-mediated dephosphorylation of phospho-p38 is more effective than in untreated cells, implicating CacyBP/SIP in oxidative stress responses.\",\n      \"method\": \"Co-immunoprecipitation from NB2a cell lysate and with recombinant proteins, in vitro phosphatase assay using domain fragments, Western blot for phospho-p38 under H2O2 stress\",\n      \"journal\": \"Amino acids\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal interaction and domain-mapped phosphatase assay with recombinant proteins; single lab, multiple complementary methods\",\n      \"pmids\": [\"28283909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CacyBP/SIP is sumoylated at lysine 16 in neuroblastoma NB2a cells; it binds the E2 SUMO ligase Ubc9 in cell extract. Uniquely, sumoylated CacyBP/SIP is found in the cytoplasmic rather than the nuclear fraction.\",\n      \"method\": \"Immunoprecipitation for SUMO-modified proteins, cell fractionation, site-directed identification of K16 as sumoylation site\",\n      \"journal\": \"Neurochemical research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — binding to E2 ligase shown by IP, sumoylation confirmed in cells with fractionation; single lab, multiple complementary methods\",\n      \"pmids\": [\"24078263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Under normal conditions, CacyBP/SIP (SIP) inhibits ubiquitination and proteasomal degradation of BRUCE/Apollon, likely by blocking Nrdp1 binding to BRUCE. Upon starvation, SIP together with Rab8 enhances translocation of BRUCE into the recycling endosome and promotes autophagic destruction of BRUCE by optineurin-mediated autophagy, thereby relieving BRUCE-mediated proteasomal degradation of LC3-I and stimulating autophagy. Deletion of SIP in cells reduces autophagic degradation of damaged mitochondria and cytosolic protein aggregates.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, SIP knockout cell lines, autophagy flux assay (LC3-I/II), mitophagy assay, live-cell imaging of BRUCE trafficking with Rab8\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ubiquitination assay, KO cells, trafficking assay) with mechanistic follow-up and clear phenotypic readouts; single lab but rigorous\",\n      \"pmids\": [\"31213539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RNF41 binds CACYBP via its C-terminal substrate binding domain, ubiquitinates CACYBP, and promotes its degradation via both proteasome- and lysosome-dependent pathways. CACYBP overexpression stimulates Ser10, Thr157, and Thr198 phosphorylation of P27Kip1 and its cytoplasmic retention; RNF41 co-expression attenuates this. CACYBP depletion causes G1/S arrest and increased apoptosis in HCC cells.\",\n      \"method\": \"Co-immunoprecipitation (endogenous and exogenous), ubiquitination assay, Western blot for P27Kip1 phosphorylation, immunofluorescence for P27Kip1 localization, flow cytometry, xenograft model, P27Kip1-S10D/S10A reconstitution\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, ubiquitination assay, mutagenesis rescue, in vivo model; multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"31754404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CacyBP/SIP nuclear translocation induced by gastrin promotes proteasome-dependent degradation of p27Kip1 in colon cancer cells. CacyBP/SIP binds Skp1 (shown by Co-IP); a CacyBP/SIP truncation mutant lacking the Skp1 binding site fails to promote p27Kip1 degradation even after nuclear translocation. Proteasome inhibitor MG132 prevents gastrin-induced p27Kip1 reduction.\",\n      \"method\": \"Co-immunoprecipitation, truncation mutagenesis, confocal immunofluorescence, Western blot, MG132 proteasome inhibitor, siRNA knockdown\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, mutagenesis of Skp1-binding domain with functional readout, pharmacological inhibition; multiple orthogonal methods, single lab\",\n      \"pmids\": [\"28196083\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CacyBP/SIP nuclear translocation in gastric cancer cells promotes proteasome-dependent p27Kip1 degradation; CacyBP/SIP binds Skp1 (Co-IP), and a Skp1-binding-deficient mutant of CacyBP/SIP fails to stimulate p27Kip1 degradation even when it translocates to the nucleus. G1-phase CacyBP/SIP is cytoplasmic; it translocates to nucleus at G2 where β-catenin and p-ERK1/2 are reduced.\",\n      \"method\": \"Co-immunoprecipitation, truncation mutagenesis, immunofluorescence, Western blot, MG132 proteasome inhibitor, cell cycle synchronization\",\n      \"journal\": \"World journal of gastroenterology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and mutagenesis with functional readout, single lab, partially overlapping with PMID 28196083\",\n      \"pmids\": [\"27099442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cell cycle-dependent nuclear translocation of CacyBP/SIP, Siah-1, and Skp1: these proteins are cytoplasmic in G1 and translocate to the nucleus in G2. In G2, CacyBP/SIP phosphorylation and binding capacity to Siah-1 and Skp1 increase, while β-catenin and p-ERK1/2 decrease. A CacyBP/SIP mutant lacking the S100-binding domain (CacyBP-ΔS100) shows increased nuclear translocation, further suppressing β-catenin and proliferation; this is reversed by β-catenin activator LiCl.\",\n      \"method\": \"Immunofluorescence after cell cycle synchronization, Co-immunoprecipitation, Western blot, phosphorylation assay, LiCl/XAV-939 pharmacological modulation\",\n      \"journal\": \"Anti-cancer drugs\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — cell cycle synchronization, Co-IP, domain mutant with pharmacological rescue; single lab\",\n      \"pmids\": [\"29099417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Gastrin stimulation triggers nuclear translocation of CacyBP/SIP in gastric cancer cells; CacyBP/SIP nuclear translocation promotes cell cycle progression from G1 phase and enhances proliferation. siRNA-mediated prevention of nuclear translocation abolishes these proliferative effects.\",\n      \"method\": \"Immunofluorescence, subcellular fractionation + Western blot, MTT assay, colony formation assay, flow cytometry cell cycle analysis, siRNA knockdown\",\n      \"journal\": \"World journal of gastroenterology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct localization experiment with functional consequence; multiple readouts; single lab\",\n      \"pmids\": [\"25110433\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Ca2+-dependent binding of S100A6 to CacyBP/SIP is required for Ca2+-induced nuclear translocation of CacyBP/SIP in colon cancer SW480 cells: ionomycin-elevated [Ca2+]i triggers translocation, and knockdown of S100A6 abolishes this effect.\",\n      \"method\": \"Immunofluorescence, Co-immunoprecipitation under varying [Ca2+], siRNA knockdown of S100A6\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct localization experiment with S100A6 knockdown establishing mechanistic dependency; single lab, Co-IP plus imaging\",\n      \"pmids\": [\"29534068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CacyBP/SIP promotes Siah1-mediated ubiquitination and degradation of cytoplasmic p27Kip1, inhibiting glioma cell migration and invasion. Silencing CacyBP/SIP reduces the Siah1–p27 interaction and attenuates p27 ubiquitination; overexpression increases the Siah1–p27 interaction and p27 degradation. Siah1 knockdown blocks p27 degradation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA/overexpression of CacyBP/SIP and Siah1, migration/invasion assays (Transwell), Western blot\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and ubiquitination assay establish mechanistic pathway; Siah1 epistasis confirmed by knockdown; single lab\",\n      \"pmids\": [\"29024247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CacyBP/SIP interacts with Hsp90 and acts as a co-chaperone; overexpression of CacyBP/SIP in HEp-2 cells increases cellular resistance to stress-induced death. The Hsf1 transcription factor binds to the CacyBP/SIP gene promoter and up-regulates CacyBP/SIP expression under stress conditions (H2O2 and radicicol).\",\n      \"method\": \"Co-immunoprecipitation (CacyBP/SIP–Hsp90), ChIP assay (Hsf1 on CacyBP/SIP promoter), Western blot, cell viability assay, CacyBP/SIP overexpression\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP binding and ChIP for transcriptional regulation; functional cell viability assay; single lab\",\n      \"pmids\": [\"29660399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CacyBP/SIP directly interacts with α-synuclein and protects it from aggregation at the initial phase of fibril formation. The fragment overlapping the N-terminal domain and CS domain of CacyBP/SIP is critical for this chaperone-like activity. In HEK293 cells overexpressing CacyBP/SIP, there are fewer α-synuclein inclusions and greater viability upon rotenone treatment.\",\n      \"method\": \"Thioflavin T fluorescence aggregation assay, high-speed ultracentrifugation + dot-blot, transmission electron microscopy, proximity ligation assay (PLA) in cells, in vitro assay with purified recombinant proteins, cell viability assay\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro reconstitution with purified proteins, multiple orthogonal methods (ThT, EM, ultracentrifugation, PLA, cellular assay), domain mapping; single lab\",\n      \"pmids\": [\"33049998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CacyBP/SIP interacts with NPM1 (nucleophosmin) directly; their complex is found in the cell nucleus. CacyBP/SIP overexpression favors phosphorylation of NPM1 on S125 and increases binding of 28S and 18S rRNA to NPM1. Under oxidative stress, CacyBP/SIP overexpression alters NPM1 nuclear distribution and maintains nucleolar structure (assessed by fibrillarin staining); CacyBP/SIP is required for preserving nucleolar integrity under stress.\",\n      \"method\": \"Mass spectrometry identification, Co-immunoprecipitation, direct binding assay, RNA immunoprecipitation (RIP), immunofluorescence (fibrillarin), Western blot\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct binding plus RIP and functional imaging; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"29806702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SFRS8 splicing factor mediates alternative splicing of CACYBP pre-mRNA, reducing isoform 1 (NM_014412.3) and increasing isoform 2 (NM_001007214.1). Isoform switching alters ubiquitination and degradation of β-catenin, promoting myeloma progression. Exosomal siRNA targeting CACYBP isoform 2 inhibits tumor growth in PDX models.\",\n      \"method\": \"RNA immunoprecipitation sequencing (RIP-seq), RIP-qPCR, Co-IP, xenograft models, PDX model, exosome-mediated siRNA delivery\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — RIP-seq identifies isoform regulation, functional in vivo rescue; single lab\",\n      \"pmids\": [\"35184390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CacyBP binds MyD88 via its Toll/IL-1 receptor (TIR) domain and protects MyD88 from Siah-1-mediated proteasomal degradation by competitive binding. CacyBP–MyD88 signaling promotes HDAC1-mediated H3K9ac and H3K27ac modifications on the CX3CL1 promoter, increasing CX3CL1 transcription and secretion in HCC cells, which enhances tumor-associated macrophage infiltration. CacyBP inhibition reduces immunosuppressive microenvironment and sensitizes tumors to anti-PD-1 therapy.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, Co-IP competition assay, in vitro and in vivo macrophage recruitment assays, chromatin immunoprecipitation (H3K9ac/H3K27ac on CX3CL1 promoter), xenograft models\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP binding, ChIP for histone modification, in vivo model; single lab, multiple complementary methods\",\n      \"pmids\": [\"37968706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CacyBP/SIP promotes degradation of mutant p53 by enhancing Mdm2 E3 ubiquitin ligase activity in U251 glioma cells. CacyBP/SIP interacts with both p53 and Mdm2, increases p53 ubiquitination in a dose-dependent manner in the presence of Mdm2, and Mdm2 inhibition reverses this. In wild-type p53 U87 cells, CacyBP/SIP has no effect on proliferation or p53 levels.\",\n      \"method\": \"Co-immunoprecipitation (CacyBP/SIP with p53 and Mdm2), ubiquitination assay (dose–response), Mdm2 inhibitor treatment, Western blot, shRNA knockdown\",\n      \"journal\": \"Neoplasma\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and ubiquitination assay establish the mechanistic pathway; dose-response ubiquitination; single lab\",\n      \"pmids\": [\"32880469\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CacyBP/SIP overexpression reduces p-ERK1/2 levels in undifferentiated NB2a cells, leading to decreased CREB phosphorylation and reduced BDNF mRNA; silencing has the opposite effect. In differentiated NB2a cells, CacyBP/SIP overexpression has a different effect on p-ERK1/2, which correlates with changes in CacyBP/SIP post-translational modifications and protein ligands.\",\n      \"method\": \"CacyBP/SIP overexpression and siRNA silencing, Western blot for p-ERK1/2, p-CREB, RT-qPCR for BDNF mRNA, 2D-PAGE analysis of CacyBP/SIP modification state\",\n      \"journal\": \"Neurochemistry international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — bidirectional manipulation (OE/KD) with pathway readouts in two cell states; single lab\",\n      \"pmids\": [\"27180052\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CacyBP/SIP exhibits phosphatase activity toward ERK1/2 in the nuclear fraction of undifferentiated NB2a cells but not in HCT116 colon cancer cells. The phosphorylation state of CacyBP/SIP differs between these cell lines (assessed by 2D-electrophoresis), and nuclear β-catenin levels are inversely correlated: low in NB2a (with CacyBP/SIP overexpression) and high in HCT116.\",\n      \"method\": \"CacyBP/SIP overexpression, nuclear fractionation, phosphatase activity assay, 2D-electrophoresis, Western blot\",\n      \"journal\": \"Biochemistry and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — cell-type-specific phosphatase activity demonstrated with fractionation; single lab, multiple methods\",\n      \"pmids\": [\"22480271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"S100A6 negatively regulates CacyBP/SIP-mediated inhibition of gastric cancer cell proliferation. A CacyBP/SIP truncation mutant lacking the S100-binding domain (CacyBP/SIPΔS100) shows reduced S100A6 co-immunoprecipitation and further reduces proliferation and tumorigenesis compared to wild-type, demonstrating that S100A6 binding attenuates CacyBP/SIP's antiproliferative effect through modulation of β-catenin expression and Tcf/LEF transcriptional activity.\",\n      \"method\": \"Co-immunoprecipitation, truncation mutagenesis, MTT assay, FACS, clonogenic assay, tumor xenograft\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — domain mutant Co-IP plus functional in vitro and in vivo readouts; single lab\",\n      \"pmids\": [\"22295074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NFAT1 transcription factor directly binds to the CacyBP/SIP gene promoter and up-regulates CacyBP/SIP expression; inhibition or stimulation of NFAT transcriptional activity correspondingly decreases or increases CacyBP/SIP levels.\",\n      \"method\": \"Western blot, RT-PCR, luciferase reporter assay, EMSA, chromatin immunoprecipitation (ChIP)\",\n      \"journal\": \"Immunobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding demonstrated by EMSA and ChIP, functional reporter assay; single lab\",\n      \"pmids\": [\"28526484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Heterozygous CACYBP/SIP p.E177Q variant is linked to paucity of interlobular bile ducts (PILBD) in a dominant family. CACYBP/SIP forms a ubiquitin ligase complex that degrades non-phosphorylated β-catenin; the variant may form a more active/stable complex, enhancing β-catenin degradation and delaying bile duct maturation.\",\n      \"method\": \"Next-generation sequencing (family), immunohistochemistry for CACYBP and β-catenin in patient liver\",\n      \"journal\": \"Journal of human genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — genetic variant identification with IHC; no in vitro reconstitution of enhanced activity; single family, mechanistic claim is partially inferred\",\n      \"pmids\": [\"35087201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CacyBP interacts directly with OTUD5 deubiquitinase, enhances ubiquitination and proteasomal degradation of OTUD5, and thereby promotes lung adenocarcinoma tumorigenesis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, overexpression/knockdown functional assays (proliferation, migration, invasion), Western blot\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP and ubiquitination assay establish mechanism; functional phenotypic validation; single lab\",\n      \"pmids\": [\"38558058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CacyBP/SIP interacts directly with ribosomal protein RPL6; the interaction was confirmed by multiple biochemical methods and in silico domain mapping. Cells with stably silenced CacyBP/SIP show reduced perinuclear nascent polypeptide labeling (OPP staining) and lower Hsp70 induction after heat shock, suggesting CacyBP/SIP influences ribosome function and protein synthesis efficiency.\",\n      \"method\": \"Mass spectrometry, Co-immunoprecipitation, direct binding assay, in silico domain mapping, OPP (O-propargyl-puromycin) labeling of nascent polypeptides, Western blot for Hsp70 under heat shock in CacyBP/SIP-silenced vs. control cells\",\n      \"journal\": \"Amino acids\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct binding and mass spectrometry; functional assay in silenced cells; single lab, single study\",\n      \"pmids\": [\"40691326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CacyBP/SIP knockdown in cortical neurons of opossum (marsupial model) causes excessive branching and elongation of dendritic trees and axon arbors during upper cortical layer formation, without affecting proliferation or differentiation. This establishes a role for CacyBP/SIP in restraining neurite branching during cortical development.\",\n      \"method\": \"Lentiviral shRNA knockdown, primary cortical neuron cultures, morphometric analysis of dendritic and axonal arbors, cell proliferation and differentiation assays\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — loss-of-function with specific morphometric phenotypic readout; single lab, single model organism\",\n      \"pmids\": [\"40476326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CacyBP/SIP stabilization by Cordycepin (via inhibiting its ubiquitin-proteasome degradation) suppresses NLRP3 inflammasome activation and enhances autophagosome-lysosome fusion, leading to autophagic degradation of α-synuclein in rotenone-treated SH-SY5Y cells. Blockade of CacyBP/SIP abrogates these neuroprotective effects.\",\n      \"method\": \"CacyBP/SIP protein stability assay with proteasome inhibitors, NLRP3 inflammasome activation assay, autophagosome-lysosome fusion assay, α-synuclein aggregation assay, siRNA knockdown of CacyBP/SIP, in vivo rotenone mouse model\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — pharmacological and genetic (siRNA) manipulation with mechanistic pathway readouts; single lab, in vitro plus in vivo\",\n      \"pmids\": [\"40639630\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CacyBP interacts directly with CDK1 (co-immunoprecipitation); CDK1 overexpression rescues the inhibitory effects of CacyBP knockdown on LUAD cell growth. CacyBP promotes LUAD progression at least in part through CDK1-mediated activation of the PI3K/AKT pathway (PI3K inhibitor LY294002 blocks CDK1-mediated growth).\",\n      \"method\": \"Co-immunoprecipitation (CacyBP–CDK1), CDK1 overexpression rescue, LY294002 PI3K inhibition, proliferation/apoptosis/migration assays, xenograft model\",\n      \"journal\": \"Biomolecules & biomedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP binding, genetic rescue, and pharmacological epistasis; single lab\",\n      \"pmids\": [\"40167359\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CacyBP/SIP is a multidomain scaffold protein that functions as: (1) a component of a Siah-1/Skp1-containing ubiquitin ligase complex that degrades β-catenin; (2) an intrinsic phosphatase for ERK1/2 and p38 MAP kinases, regulated by CK II-mediated phosphorylation at T184 and inhibited by S100A6 in a Ca2+-dependent manner; (3) a cytoskeletal regulator that directly binds actin (inducing polymerization via its N-terminal domain) and tropomyosin, links actin and tubulin networks, and regulates thin filament ATPase activity; (4) a promoter of autophagy by stabilizing BRUCE/Apollon and facilitating autophagosome formation; (5) an HSP90 co-chaperone that protects clients including α-synuclein from aggregation; (6) a regulator of nuclear functions including NPM1 phosphorylation, nucleolar integrity, and ribosome-associated translation efficiency via RPL6 interaction; with its subcellular localization (cytoplasmic vs. nuclear, regulated by Ca2+/S100A6 and cell cycle stage) being a key determinant of its functional output in proliferation, differentiation, stress response, and neurite morphogenesis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CacyBP/SIP (CACYBP) is a multidomain, Ca2+-responsive scaffold whose subcellular distribution and partner repertoire determine which of several regulatory programs it executes — protein degradation, MAP kinase dephosphorylation, cytoskeletal organization, chaperoning, and autophagy [#0, #14]. Through its C-terminal region it binds multiple S100 proteins (S100A1, S100A6, S100A12, S100B, S100P) in a Ca2+-dependent manner [#0], and S100A6 binding controls Ca2+-induced nuclear translocation that switches its activity [#16]. As a component of a Siah-1/Skp1-containing ubiquitin ligase complex, CacyBP/SIP drives proteasomal degradation of β-catenin and of p27Kip1, with the Skp1-binding domain required for nuclear p27Kip1 turnover and antiproliferative output [#1, #12, #17]; it likewise channels other substrates including mutant p53 (via Mdm2), OTUD5, and the MyD88 stability decision toward distinct cancer phenotypes [#23, #29, #22]. Independently, CacyBP/SIP is an intrinsic PP2A-like phosphatase that binds and dephosphorylates ERK1/2 (C-terminal region; E217K abolishes binding and activity) and p38 (via its CS domain), an activity negatively regulated by CK II phosphorylation at T184 and competitively antagonized by S100A6 [#2, #3, #7, #8]. The protein also directly remodels the cytoskeleton: its N-terminal domain binds and polymerizes actin, it binds tropomyosin and modulates actin-activated myosin S1 ATPase, and it can bridge actin and tubulin networks [#4, #5]. CacyBP/SIP further acts as an HSP90 co-chaperone and protects α-synuclein from aggregation through its N-terminal/CS region, and promotes autophagy by stabilizing BRUCE/Apollon and facilitating autophagic flux [#18, #19, #10]. A nuclear role encompasses NPM1 binding/phosphorylation and nucleolar maintenance under stress, plus RPL6 interaction that supports translation efficiency [#20, #30]. A heterozygous CACYBP p.E177Q variant segregates with paucity of interlobular bile ducts in a dominant family, consistent with altered β-catenin degradation during bile duct maturation [#28].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established CacyBP/SIP's first defined molecular function — that it is not merely an S100-binding protein but a structural component of a ubiquitin ligase that targets β-catenin for degradation.\",\n      \"evidence\": \"Co-IP and ubiquitination assays placing SIP in a Siah-1/Skp1 complex, referenced and confirmed across multiple cancer models\",\n      \"pmids\": [\"12042313\", \"16340196\", \"17400182\", \"22295074\", \"35184390\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and assembly order of the Siah-1/Skp1/CacyBP complex not resolved\", \"Does not address how Ca2+/S100 status gates ligase assembly\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined the calcium-sensing input by mapping Ca2+-dependent S100 binding to a discrete C-terminal segment, establishing CacyBP/SIP as a Ca2+/S100-regulated effector.\",\n      \"evidence\": \"Affinity chromatography, overlay assays, and Co-IP from brain/tumor extracts with domain mapping (residues 155–229)\",\n      \"pmids\": [\"12042313\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of each S100 partner not individually dissected\", \"S100A6 selectivity over other S100s mechanistically unexplained\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Revealed that CacyBP/SIP carries an intrinsic enzymatic activity — a PP2A-like phosphatase acting on ERK1/2 — defining a degradation-independent signaling function.\",\n      \"evidence\": \"In vitro phosphatase kinetics (p-NPP), okadaic acid inhibition, E217K mutagenesis, plus prior ERK1/2 direct-binding mapping\",\n      \"pmids\": [\"21110948\", \"19166809\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic mechanism and active-site residues not structurally defined\", \"Physiological substrate range beyond ERK1/2 not established\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed CacyBP/SIP directly organizes the cytoskeleton, polymerizing actin via its N-terminus and potentially linking actin and tubulin networks.\",\n      \"evidence\": \"Zero-length cross-linking, co-sedimentation, immunofluorescence co-localization, and domain fragment analysis in NB2a cells\",\n      \"pmids\": [\"20637809\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of actin/tubulin bridging not demonstrated\", \"Regulation of cytoskeletal binding by Ca2+/S100 untested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended cytoskeletal function to thin-filament regulation, showing tropomyosin binding and modulation of actin-activated myosin ATPase.\",\n      \"evidence\": \"Fluorescence binding, electron microscopy, and myosin S1 ATPase assays with reconstituted actin/tropomyosin\",\n      \"pmids\": [\"23266554\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological context (muscle vs non-muscle) of thin-filament regulation unclear\", \"Connection to motility phenotypes not directly tested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified the regulatory switch governing phosphatase activity — CK II phosphorylation at T184 suppresses it, and S100A6 blocks that phosphorylation, integrating kinase and Ca2+ inputs.\",\n      \"evidence\": \"In vitro CKII kinase assay, T184E/T184A phosphomimetic mutagenesis, and phosphatase readout in NB2a cells\",\n      \"pmids\": [\"26085436\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other phosphosites tune activity not addressed\", \"In vivo CKII regulation in non-neuronal cells untested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Broadened phosphatase specificity to p38 and tied it to oxidative-stress responses, with the CS domain as the catalytic module.\",\n      \"evidence\": \"Reciprocal Co-IP, domain-mapped in vitro phosphatase assay, and phospho-p38 Westerns under H2O2 in NB2a cells\",\n      \"pmids\": [\"28283909\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation of CS-domain p38 dephosphorylation with C-terminal ERK1/2 activity not made\", \"Stress-dependent activation mechanism unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Connected localization to function — gastrin-triggered nuclear translocation drives cell-cycle progression and proliferation, establishing localization as the key functional determinant.\",\n      \"evidence\": \"Fractionation, immunofluorescence, siRNA blockade of translocation, and proliferation/cell-cycle assays in gastric cancer cells\",\n      \"pmids\": [\"25110433\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Nuclear import machinery for CacyBP/SIP not identified\", \"Direct nuclear targets after translocation only partly defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established the Ca2+/S100A6/cell-cycle logic of translocation — S100A6 binding is required for Ca2+-induced nuclear entry, and translocation in G2 increases Siah-1/Skp1 binding while lowering β-catenin and p-ERK1/2.\",\n      \"evidence\": \"Cell-cycle synchronization, Co-IP under varying Ca2+, S100A6 knockdown, and ΔS100 domain mutants with LiCl rescue across colon and gastric cancer cells\",\n      \"pmids\": [\"29534068\", \"29099417\", \"27099442\", \"28196083\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct evidence linking phosphorylation state to nuclear retention incomplete\", \"Mostly single-lab cancer-cell systems\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined a proteostasis role in autophagy — CacyBP/SIP stabilizes BRUCE/Apollon and, with Rab8, redirects it for autophagic destruction to promote clearance of damaged mitochondria and aggregates.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, KO cells, autophagy/mitophagy flux assays, and live-cell BRUCE trafficking\",\n      \"pmids\": [\"31213539\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How starvation switches CacyBP/SIP from protective to pro-degradative not fully resolved\", \"Selectivity of cargo clearance untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined CacyBP/SIP as an HSP90 co-chaperone and direct anti-aggregation factor for α-synuclein, expanding it from a degradation adaptor to a folding guardian.\",\n      \"evidence\": \"Hsp90 Co-IP, Hsf1 ChIP on the CACYBP promoter, ThT/EM/ultracentrifugation aggregation assays with purified proteins, PLA, and viability assays\",\n      \"pmids\": [\"29660399\", \"33049998\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Client range of the Hsp90 co-chaperone activity not catalogued\", \"Relationship between chaperone and phosphatase domains unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Uncovered nuclear/nucleolar functions through NPM1 binding, S125 phosphorylation, rRNA association, and maintenance of nucleolar integrity under stress.\",\n      \"evidence\": \"Mass spectrometry, Co-IP, RIP, and fibrillarin immunofluorescence under oxidative stress\",\n      \"pmids\": [\"29806702\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Kinase responsible for NPM1 S125 phosphorylation downstream of CacyBP/SIP not identified\", \"Direct vs indirect role in rRNA binding unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Expanded the substrate/partner network across cancers — mutant p53 (via Mdm2), OTUD5, MyD88 stabilization, and CACYBP isoform switching all feed proliferation, immune microenvironment, and β-catenin signaling.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, ChIP for histone marks, RIP-seq, and xenograft/PDX models across glioma, LUAD, HCC, and myeloma\",\n      \"pmids\": [\"32880469\", \"38558058\", \"37968706\", \"35184390\", \"29024247\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Many partners shown in single cancer-type studies without cross-validation\", \"Whether these reflect the same ligase complex or distinct activities unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Provided the first human disease link, associating a heterozygous CACYBP p.E177Q variant with paucity of interlobular bile ducts through altered β-catenin degradation.\",\n      \"evidence\": \"Family next-generation sequencing and IHC for CACYBP and β-catenin in patient liver\",\n      \"pmids\": [\"35087201\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No in vitro reconstitution confirming enhanced ligase activity of the variant\", \"Single family; causality not functionally proven\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected CacyBP/SIP to translation and neurodevelopment — RPL6 binding supports protein-synthesis efficiency, and loss restrains neurite branching during cortical development.\",\n      \"evidence\": \"Mass spectrometry/Co-IP with OPP nascent-peptide labeling and Hsp70 induction; lentiviral shRNA knockdown with morphometry in opossum cortical neurons\",\n      \"pmids\": [\"40691326\", \"40476326\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which RPL6 binding alters translation undefined\", \"Neurodevelopmental phenotype shown only in marsupial model\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single protein arbitrates among ligase, phosphatase, cytoskeletal, chaperone, autophagy, and translational activities — and what structural/post-translational state selects each — remains unresolved.\",\n      \"evidence\": null,\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated structural model linking domain organization to the competing activities\", \"No unified accounting of how Ca2+/S100A6, CKII phosphorylation, SUMOylation, and cell-cycle stage jointly set output\", \"Many cancer-specific partnerships lack independent replication\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 3, 7, 8]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [3, 25]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 12, 14]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [4, 5]},\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [18, 19]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4, 9, 13]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 14, 20]},\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [20]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [4, 5, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 12, 23, 29]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3, 24, 33]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [10, 32]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [12, 14, 15]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [8, 18, 20]}\n    ],\n    \"complexes\": [\"Siah-1/Skp1 ubiquitin ligase complex\"],\n    \"partners\": [\"S100A6\", \"SIAH1\", \"SKP1\", \"ERK1/2\", \"HSP90\", \"NPM1\", \"RPL6\", \"MDM2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":8,"faith_total":8,"faith_pct":100.0}}