{"gene":"MYL12A","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2011,"finding":"MYL12A (and MYL12B) associate with non-muscle myosin heavy chains MYH9 (NMHC IIA) and MYH10 (NMHC IIB), and the essential light chain MYL6, in NIH 3T3 fibroblasts. Knockdown of MYL12A/MYL12B causes striking changes in cell morphology and dynamics, and significantly reduces the protein levels of MYH9, MYH10, and MYL6, establishing that non-muscle RLCs are required to maintain the stability/integrity of myosin II.","method":"Proteomic co-immunoprecipitation (Co-IP) / proteomic association analysis; siRNA knockdown with morphological phenotyping and quantitative western blot in NIH 3T3 fibroblasts","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus RNAi loss-of-function with quantitative protein-level readouts; multiple orthogonal methods in a focused mechanistic study","pmids":["21126233"],"is_preprint":false},{"year":2024,"finding":"The Salmonella effector kinase SteC directly recruits host MYL12A (Myl12a) and phosphorylates its Ser19 and Thr20 residues, causing actin rearrangement and enhanced migration/invasion of macrophages. Crystal structure of SteC revealed an atypical dimerization-mediated catalytic mechanism, and SteC can use a wide range of NTPs (not only ATP) to phosphorylate MYL12A. This phosphorylation is required for Salmonella to breach the gut-vascular barrier in vivo.","method":"Biochemical kinase assay (phosphorylation at defined residues), crystal structure determination, in vivo infection model, macrophage migration/invasion assays, site-directed mutagenesis (implicit in residue mapping)","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with in vitro kinase assay mapping specific phospho-residues and in vivo functional validation in a single rigorous study","pmids":["38528181"],"is_preprint":false},{"year":2012,"finding":"MYL12A (as MRLC3) forms a cytoplasmic complex with the transcriptional regulator AATF. Upon genotoxic stress, MK2-mediated phosphorylation of AATF disrupts the cytoplasmic MRLC3:AATF complex, allowing AATF to translocate to the nucleus where it represses pro-apoptotic p53 target genes (PUMA, BAX, BAK). Thus MYL12A acts as a cytoplasmic retention factor for AATF in the DNA damage response.","method":"Co-immunoprecipitation, subcellular fractionation, phospho-mimetic mutant expression, xenograft experiments","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional phospho-mimetic mutant and in vivo xenograft; single lab but multiple orthogonal methods","pmids":["22909821"],"is_preprint":false},{"year":2012,"finding":"The cytoplasmic MRLC3 (MYL12A):AATF complex is disrupted by p38MAPK/MK2-mediated phosphorylation of AATF, enabling rapid nuclear AATF localization. This places MYL12A as a component of the p38/MK2/AATF signaling module that represses p53-driven apoptosis.","method":"Biochemical epistasis (kinase inhibitor and phospho-mimetic studies), co-immunoprecipitation, nuclear localization assays","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic follow-up/review by same lab reaffirming the MRLC3:AATF interaction with pathway placement; corroborates PMID:22909821","pmids":["22983126"],"is_preprint":false},{"year":2021,"finding":"MYL12A (MYL12) is phosphorylated by smooth muscle myosin light chain kinase (smMLCK) in cochlear hair cells. Purified MYL12B was phosphorylated by smMLCK in vitro, and inhibition of smMLCK by ML-7 reduced MYL12 phosphorylation in outer hair cells and was accompanied by expansion of cell area, establishing that MYL12 phosphorylation by smMLCK drives apical constriction-like cellular shape changes in hair cells.","method":"In vitro phosphorylation assay with purified protein; ddPCR expression analysis; immunofluorescence; pharmacological inhibition (ML-7) with morphometric readout","journal":"Journal of the Association for Research in Otolaryngology : JARO","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay plus pharmacological functional readout; single lab, two orthogonal methods","pmids":["33877471"],"is_preprint":false},{"year":2024,"finding":"Necdin interacts with non-muscle myosin regulatory light chain MYL12A/B (identified by yeast two-hybrid screening) and stabilizes MYL12A/B protein via the SGT1-HSP90 chaperone machinery. Loss of necdin leads to reduced MYL12A/B levels and cardiac contractility defects; cardiac-specific overexpression of MYL12A rescued heart dysfunction in necdin-deficient mice.","method":"Yeast two-hybrid screening; co-immunoprecipitation; zebrafish morpholino knockdown of MYL12.1; cardiac-specific overexpression rescue in mouse; biochemical chaperone pathway analysis","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus Co-IP plus two independent in vivo models (zebrafish and mouse) with rescue; single lab, multiple orthogonal methods","pmids":["38832028"],"is_preprint":false},{"year":2015,"finding":"MYL12A (MRLC3) is phosphorylated at Thr18/Ser19 in HeLa cells, consistent with myosin II stimulation. Among the three MRLC isoforms, overproduction of MRLC2 or MRLC3 (MYL12A), but not MRLC1, could rescue defective cell spreading caused by depletion of all MRLCs, indicating that MYL12A plays a dominant role in cell spreading.","method":"siRNA depletion of all MRLC isoforms; rescue by individual isoform overexpression; western blot for phospho-Thr18/Ser19; FRAP (turnover analysis); computational 3D protein modeling","journal":"Cytoskeleton (Hoboken, N.J.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic rescue experiments with quantitative morphological readout; single lab, two orthogonal methods (depletion + rescue + biochemistry)","pmids":["26663899"],"is_preprint":false},{"year":2024,"finding":"OTUD3, a deubiquitinase, directly deubiquitinates MYL12A, enhancing cell survival in diffuse large B-cell lymphoma (DLBCL). Inhibition by rupatadine reduces OTUD3-mediated deubiquitination of MYL12A, implicating ubiquitin-mediated proteasomal regulation of MYL12A protein stability.","method":"Deubiquitinase activity assay; co-immunoprecipitation; pharmacological inhibition with rupatadine; cell viability assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — enzymatic deubiquitination assay and Co-IP identifying a specific writer/eraser for MYL12A ubiquitination; single lab, two orthogonal methods","pmids":["39097608"],"is_preprint":false},{"year":2025,"finding":"Non-muscle myosin II (NM2A) and MYL12A are expressed in hair bundles of frog saccular hair cells (confirmed by immunostaining). Pharmacological inhibition of NM2 or MLCK reduces hair bundle stiffness, inhibits spontaneous oscillation, and increases resting open probability of transduction channels, demonstrating that MYL12A-containing actomyosin regulates the mechanical properties and physiological operating point of hair bundles.","method":"Immunostaining (localization); NM2/MLCK pharmacological inhibition; hair bundle stiffness measurements; spontaneous oscillation recording; transduction channel open-probability measurement; auditory brainstem response in mice","journal":"Journal of the Association for Research in Otolaryngology : JARO","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization tied to multiple functional mechanical readouts; single lab but orthogonal in vitro and in vivo methods","pmids":["40240732"],"is_preprint":false},{"year":2020,"finding":"MYL12A was identified as a novel interaction partner of cardiac syndecan-2 by affinity purification/mass spectrometry and verified by co-immunoprecipitation in HEK293 cells, placing MYL12A in a cytoskeletal remodeling network associated with the syndecan-2 interactome.","method":"Affinity purification-mass spectrometry (AP-MS); co-immunoprecipitation in HEK293 cells","journal":"Frontiers in cell and developmental biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP/AP-MS verification with no further mechanistic follow-up specific to MYL12A","pmids":["32984315"],"is_preprint":false}],"current_model":"MYL12A is a non-muscle myosin II regulatory light chain that binds MYH9 and MYH10 heavy chains to stabilize the myosin II holoenzyme and is required for cell morphology, spreading, and division; its activity is principally regulated by phosphorylation at Ser19/Thr18 (by MLCK or bacterial kinases such as SteC), which drives actomyosin contraction, actin rearrangement, and cellular shape changes; it is stabilized post-translationally via the SGT1-HSP90 chaperone system (regulated by necdin) and protected from proteasomal degradation by OTUD3-mediated deubiquitination; in unstressed cells it sequesters the transcription factor AATF in the cytoplasm, and genotoxic-stress-induced MK2 phosphorylation of AATF disrupts this complex to allow nuclear AATF translocation and repression of pro-apoptotic genes."},"narrative":{"mechanistic_narrative":"MYL12A is a non-muscle myosin II regulatory light chain that maintains the integrity of the actomyosin contractile machinery: it associates with the non-muscle myosin heavy chains MYH9 and MYH10 and the essential light chain MYL6, and its depletion both destabilizes these myosin II subunits and produces marked changes in cell morphology and dynamics [PMID:21126233]. Its contractile output is gated by phosphorylation at the conserved Thr18/Ser19 (Thr20/Ser19) regulatory site — by smooth muscle myosin light chain kinase and by the bacterial effector kinase SteC — which drives actin rearrangement and cell-shape changes, with MYL12A playing a dominant role among regulatory light chain isoforms in supporting cell spreading [PMID:38528181, PMID:33877471, PMID:26663899]. In the cochlea this phosphorylation-driven actomyosin activity tunes hair-bundle stiffness and the operating point of mechanotransduction [PMID:40240732]. MYL12A protein abundance is set post-translationally: it is stabilized by necdin acting through the SGT1–HSP90 chaperone system, a function required for cardiac contractility, and it is protected from proteasomal turnover by OTUD3-mediated deubiquitination [PMID:38832028, PMID:39097608]. Beyond its cytoskeletal role, MYL12A serves as a cytoplasmic retention factor for the transcriptional regulator AATF, with genotoxic-stress-induced MK2 phosphorylation of AATF dissolving the complex to release AATF for nuclear repression of pro-apoptotic genes [PMID:22909821, PMID:22983126].","teleology":[{"year":2011,"claim":"Established that non-muscle regulatory light chains are not merely accessory but are structurally required to hold the myosin II holoenzyme together, answering whether RLCs maintain heavy-chain stability.","evidence":"Reciprocal Co-IP plus siRNA knockdown with quantitative western blot and morphological phenotyping in NIH 3T3 fibroblasts","pmids":["21126233"],"confidence":"High","gaps":["Does not distinguish the non-redundant contributions of MYL12A versus MYL12B","Mechanism of heavy-chain destabilization upon RLC loss not resolved"]},{"year":2012,"claim":"Revealed a non-cytoskeletal moonlighting role: MYL12A sequesters the transcription factor AATF in the cytoplasm, and stress-dependent MK2 phosphorylation of AATF releases it to repress p53 pro-apoptotic targets, linking MYL12A to the DNA damage response.","evidence":"Co-IP, subcellular fractionation, phospho-mimetic mutants, kinase-inhibitor epistasis, and xenograft experiments","pmids":["22909821","22983126"],"confidence":"Medium","gaps":["Whether AATF binding is to phosphorylated or unphosphorylated MYL12A is unclear","Structural basis of the MYL12A:AATF interaction undefined","Relationship between AATF sequestration and myosin II assembly not addressed"]},{"year":2015,"claim":"Defined MYL12A as the dominant regulatory light chain isoform for cell spreading and confirmed Thr18/Ser19 phosphorylation, distinguishing functional roles among the three MRLC isoforms.","evidence":"siRNA depletion of all MRLCs with single-isoform rescue, phospho-Thr18/Ser19 western blot, and FRAP turnover analysis in HeLa cells","pmids":["26663899"],"confidence":"Medium","gaps":["Molecular basis for MYL12A dominance over MRLC1 not identified","Does not map which spreading pathways require MYL12A specifically"]},{"year":2021,"claim":"Showed that smMLCK-dependent MYL12 phosphorylation drives apical-constriction-like shape changes in cochlear hair cells, extending the phospho-regulatory mechanism to a sensory-cell context.","evidence":"In vitro kinase assay with purified protein, ddPCR expression, immunofluorescence, and ML-7 pharmacological inhibition with morphometry","pmids":["33877471"],"confidence":"Medium","gaps":["ML-7 is not MYL12A-specific, leaving other kinase targets possible","Direct contractile consequence in the hair cell not measured at single-molecule level"]},{"year":2024,"claim":"Identified two post-translational stability controls for MYL12A protein levels — chaperone-mediated stabilization via necdin/SGT1-HSP90 and protection from proteasomal degradation by OTUD3 deubiquitination — tying MYL12A abundance to cardiac contractility and to B-cell lymphoma survival.","evidence":"Yeast two-hybrid, Co-IP, zebrafish morpholino and mouse cardiac overexpression rescue (necdin); deubiquitinase assay, Co-IP, and rupatadine inhibition with viability assays (OTUD3)","pmids":["38832028","39097608"],"confidence":"Medium","gaps":["The E3 ligase that ubiquitinates MYL12A is not identified","How chaperone stabilization and deubiquitination intersect is unknown","Whether these controls regulate myosin II assembly directly is untested"]},{"year":2024,"claim":"Demonstrated that a bacterial effector kinase, SteC, directly hijacks MYL12A by phosphorylating Ser19/Thr20 to remodel host actin and breach the gut-vascular barrier, revealing the regulatory site as a target of pathogen subversion.","evidence":"In vitro kinase assays mapping phospho-residues, SteC crystal structure, macrophage migration/invasion assays, and in vivo Salmonella infection model","pmids":["38528181"],"confidence":"High","gaps":["Whether SteC phosphorylation mimics or exceeds physiological MLCK signaling is not quantified","Downstream actomyosin contractile changes not structurally resolved"]},{"year":2025,"claim":"Linked MYL12A-containing actomyosin in hair bundles directly to mechanotransduction physiology, showing it sets bundle stiffness and the resting operating point of transduction channels.","evidence":"Immunostaining localization, NM2/MLCK pharmacological inhibition, stiffness and spontaneous oscillation recordings, transduction channel open-probability measurement, and auditory brainstem response in mice","pmids":["40240732"],"confidence":"Medium","gaps":["Pharmacology cannot isolate MYL12A from other RLC isoforms","Direct phosphorylation state of bundle MYL12A during oscillation not measured"]},{"year":null,"claim":"How MYL12A's distinct roles — myosin II structural stabilization, cytoplasmic AATF sequestration, and chaperone/ubiquitin-regulated abundance — are coordinated within a single cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated model linking phosphorylation, stability control, and the AATF moonlighting function","No structure of MYL12A bound to either heavy chains or AATF in the timeline"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,6]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[2,3]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,3]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,8]}],"pathway":[{"term_id":"R-HSA-397014","term_label":"Muscle contraction","supporting_discovery_ids":[5,8]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[2,3]}],"complexes":["non-muscle myosin II"],"partners":["MYH9","MYH10","MYL6","AATF","STEC","OTUD3","NECDIN","SDC2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P19105","full_name":"Myosin regulatory light chain 12A","aliases":["Epididymis secretory protein Li 24","HEL-S-24","MLC-2B","Myosin RLC","Myosin regulatory light chain 2, nonsarcomeric","Myosin regulatory light chain MRLC3"],"length_aa":171,"mass_kda":19.8,"function":"Myosin regulatory subunit that plays an important role in regulation of both smooth muscle and nonmuscle cell contractile activity via its phosphorylation. Implicated in cytokinesis, receptor capping, and cell locomotion (By similarity)","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/P19105/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MYL12A","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000101608","cell_line_id":"CID001436","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"cytoskeleton","grade":3}],"interactors":[{"gene":"MYH9","stoichiometry":10.0},{"gene":"MYH10","stoichiometry":10.0},{"gene":"MYL12A;MYL9","stoichiometry":10.0},{"gene":"LRRFIP1","stoichiometry":0.2},{"gene":"MYH14","stoichiometry":0.2},{"gene":"FLII","stoichiometry":0.2},{"gene":"LRRFIP2","stoichiometry":0.2},{"gene":"IPO4","stoichiometry":0.2},{"gene":"KIF2C","stoichiometry":0.2},{"gene":"RSF1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001436","total_profiled":1310},"omim":[],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"heart muscle","ntpm":3965.3},{"tissue":"skeletal muscle","ntpm":3053.6},{"tissue":"tongue","ntpm":1943.8}],"url":"https://www.proteinatlas.org/search/MYL12A"},"hgnc":{"alias_symbol":["MLCB","MYL2B","MRLC3","MRCL3"],"prev_symbol":[]},"alphafold":{"accession":"P19105","domains":[{"cath_id":"1.10.238.10","chopping":"25-93","consensus_level":"high","plddt":90.1623,"start":25,"end":93},{"cath_id":"1.10.238.10","chopping":"98-163","consensus_level":"high","plddt":93.4153,"start":98,"end":163}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P19105","model_url":"https://alphafold.ebi.ac.uk/files/AF-P19105-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P19105-F1-predicted_aligned_error_v6.png","plddt_mean":83.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MYL12A","jax_strain_url":"https://www.jax.org/strain/search?query=MYL12A"},"sequence":{"accession":"P19105","fasta_url":"https://rest.uniprot.org/uniprotkb/P19105.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P19105/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P19105"}},"corpus_meta":[{"pmid":"21126233","id":"PMC_21126233","title":"Myosin 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Series A, Biological sciences and medical sciences","url":"https://pubmed.ncbi.nlm.nih.gov/38808484","citation_count":1,"is_preprint":false},{"pmid":"41412545","id":"PMC_41412545","title":"Analysis of the transcriptional mechanisms of yellowfin tuna (Thunnus albacares) juveniles in response to acute cold stress in brain and muscle tissues.","date":"2025","source":"Comparative biochemistry and physiology. Part A, Molecular & integrative physiology","url":"https://pubmed.ncbi.nlm.nih.gov/41412545","citation_count":1,"is_preprint":false},{"pmid":"40240732","id":"PMC_40240732","title":"Influence of Myosin Regulatory Light Chain and Myosin Light Chain Kinase on the Physiological Function of Inner Ear Hair Cells.","date":"2025","source":"Journal of the Association for Research in Otolaryngology : JARO","url":"https://pubmed.ncbi.nlm.nih.gov/40240732","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":21223,"output_tokens":3037,"usd":0.054612,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10299,"output_tokens":3060,"usd":0.063997,"stage2_stop_reason":"end_turn"},"total_usd":0.118609,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"MYL12A (and MYL12B) associate with non-muscle myosin heavy chains MYH9 (NMHC IIA) and MYH10 (NMHC IIB), and the essential light chain MYL6, in NIH 3T3 fibroblasts. Knockdown of MYL12A/MYL12B causes striking changes in cell morphology and dynamics, and significantly reduces the protein levels of MYH9, MYH10, and MYL6, establishing that non-muscle RLCs are required to maintain the stability/integrity of myosin II.\",\n      \"method\": \"Proteomic co-immunoprecipitation (Co-IP) / proteomic association analysis; siRNA knockdown with morphological phenotyping and quantitative western blot in NIH 3T3 fibroblasts\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus RNAi loss-of-function with quantitative protein-level readouts; multiple orthogonal methods in a focused mechanistic study\",\n      \"pmids\": [\"21126233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The Salmonella effector kinase SteC directly recruits host MYL12A (Myl12a) and phosphorylates its Ser19 and Thr20 residues, causing actin rearrangement and enhanced migration/invasion of macrophages. Crystal structure of SteC revealed an atypical dimerization-mediated catalytic mechanism, and SteC can use a wide range of NTPs (not only ATP) to phosphorylate MYL12A. This phosphorylation is required for Salmonella to breach the gut-vascular barrier in vivo.\",\n      \"method\": \"Biochemical kinase assay (phosphorylation at defined residues), crystal structure determination, in vivo infection model, macrophage migration/invasion assays, site-directed mutagenesis (implicit in residue mapping)\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with in vitro kinase assay mapping specific phospho-residues and in vivo functional validation in a single rigorous study\",\n      \"pmids\": [\"38528181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MYL12A (as MRLC3) forms a cytoplasmic complex with the transcriptional regulator AATF. Upon genotoxic stress, MK2-mediated phosphorylation of AATF disrupts the cytoplasmic MRLC3:AATF complex, allowing AATF to translocate to the nucleus where it represses pro-apoptotic p53 target genes (PUMA, BAX, BAK). Thus MYL12A acts as a cytoplasmic retention factor for AATF in the DNA damage response.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, phospho-mimetic mutant expression, xenograft experiments\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional phospho-mimetic mutant and in vivo xenograft; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"22909821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The cytoplasmic MRLC3 (MYL12A):AATF complex is disrupted by p38MAPK/MK2-mediated phosphorylation of AATF, enabling rapid nuclear AATF localization. This places MYL12A as a component of the p38/MK2/AATF signaling module that represses p53-driven apoptosis.\",\n      \"method\": \"Biochemical epistasis (kinase inhibitor and phospho-mimetic studies), co-immunoprecipitation, nuclear localization assays\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic follow-up/review by same lab reaffirming the MRLC3:AATF interaction with pathway placement; corroborates PMID:22909821\",\n      \"pmids\": [\"22983126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MYL12A (MYL12) is phosphorylated by smooth muscle myosin light chain kinase (smMLCK) in cochlear hair cells. Purified MYL12B was phosphorylated by smMLCK in vitro, and inhibition of smMLCK by ML-7 reduced MYL12 phosphorylation in outer hair cells and was accompanied by expansion of cell area, establishing that MYL12 phosphorylation by smMLCK drives apical constriction-like cellular shape changes in hair cells.\",\n      \"method\": \"In vitro phosphorylation assay with purified protein; ddPCR expression analysis; immunofluorescence; pharmacological inhibition (ML-7) with morphometric readout\",\n      \"journal\": \"Journal of the Association for Research in Otolaryngology : JARO\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay plus pharmacological functional readout; single lab, two orthogonal methods\",\n      \"pmids\": [\"33877471\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Necdin interacts with non-muscle myosin regulatory light chain MYL12A/B (identified by yeast two-hybrid screening) and stabilizes MYL12A/B protein via the SGT1-HSP90 chaperone machinery. Loss of necdin leads to reduced MYL12A/B levels and cardiac contractility defects; cardiac-specific overexpression of MYL12A rescued heart dysfunction in necdin-deficient mice.\",\n      \"method\": \"Yeast two-hybrid screening; co-immunoprecipitation; zebrafish morpholino knockdown of MYL12.1; cardiac-specific overexpression rescue in mouse; biochemical chaperone pathway analysis\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus Co-IP plus two independent in vivo models (zebrafish and mouse) with rescue; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"38832028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"MYL12A (MRLC3) is phosphorylated at Thr18/Ser19 in HeLa cells, consistent with myosin II stimulation. Among the three MRLC isoforms, overproduction of MRLC2 or MRLC3 (MYL12A), but not MRLC1, could rescue defective cell spreading caused by depletion of all MRLCs, indicating that MYL12A plays a dominant role in cell spreading.\",\n      \"method\": \"siRNA depletion of all MRLC isoforms; rescue by individual isoform overexpression; western blot for phospho-Thr18/Ser19; FRAP (turnover analysis); computational 3D protein modeling\",\n      \"journal\": \"Cytoskeleton (Hoboken, N.J.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic rescue experiments with quantitative morphological readout; single lab, two orthogonal methods (depletion + rescue + biochemistry)\",\n      \"pmids\": [\"26663899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OTUD3, a deubiquitinase, directly deubiquitinates MYL12A, enhancing cell survival in diffuse large B-cell lymphoma (DLBCL). Inhibition by rupatadine reduces OTUD3-mediated deubiquitination of MYL12A, implicating ubiquitin-mediated proteasomal regulation of MYL12A protein stability.\",\n      \"method\": \"Deubiquitinase activity assay; co-immunoprecipitation; pharmacological inhibition with rupatadine; cell viability assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — enzymatic deubiquitination assay and Co-IP identifying a specific writer/eraser for MYL12A ubiquitination; single lab, two orthogonal methods\",\n      \"pmids\": [\"39097608\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Non-muscle myosin II (NM2A) and MYL12A are expressed in hair bundles of frog saccular hair cells (confirmed by immunostaining). Pharmacological inhibition of NM2 or MLCK reduces hair bundle stiffness, inhibits spontaneous oscillation, and increases resting open probability of transduction channels, demonstrating that MYL12A-containing actomyosin regulates the mechanical properties and physiological operating point of hair bundles.\",\n      \"method\": \"Immunostaining (localization); NM2/MLCK pharmacological inhibition; hair bundle stiffness measurements; spontaneous oscillation recording; transduction channel open-probability measurement; auditory brainstem response in mice\",\n      \"journal\": \"Journal of the Association for Research in Otolaryngology : JARO\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization tied to multiple functional mechanical readouts; single lab but orthogonal in vitro and in vivo methods\",\n      \"pmids\": [\"40240732\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MYL12A was identified as a novel interaction partner of cardiac syndecan-2 by affinity purification/mass spectrometry and verified by co-immunoprecipitation in HEK293 cells, placing MYL12A in a cytoskeletal remodeling network associated with the syndecan-2 interactome.\",\n      \"method\": \"Affinity purification-mass spectrometry (AP-MS); co-immunoprecipitation in HEK293 cells\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP/AP-MS verification with no further mechanistic follow-up specific to MYL12A\",\n      \"pmids\": [\"32984315\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MYL12A is a non-muscle myosin II regulatory light chain that binds MYH9 and MYH10 heavy chains to stabilize the myosin II holoenzyme and is required for cell morphology, spreading, and division; its activity is principally regulated by phosphorylation at Ser19/Thr18 (by MLCK or bacterial kinases such as SteC), which drives actomyosin contraction, actin rearrangement, and cellular shape changes; it is stabilized post-translationally via the SGT1-HSP90 chaperone system (regulated by necdin) and protected from proteasomal degradation by OTUD3-mediated deubiquitination; in unstressed cells it sequesters the transcription factor AATF in the cytoplasm, and genotoxic-stress-induced MK2 phosphorylation of AATF disrupts this complex to allow nuclear AATF translocation and repression of pro-apoptotic genes.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MYL12A is a non-muscle myosin II regulatory light chain that maintains the integrity of the actomyosin contractile machinery: it associates with the non-muscle myosin heavy chains MYH9 and MYH10 and the essential light chain MYL6, and its depletion both destabilizes these myosin II subunits and produces marked changes in cell morphology and dynamics [#0]. Its contractile output is gated by phosphorylation at the conserved Thr18/Ser19 (Thr20/Ser19) regulatory site — by smooth muscle myosin light chain kinase and by the bacterial effector kinase SteC — which drives actin rearrangement and cell-shape changes, with MYL12A playing a dominant role among regulatory light chain isoforms in supporting cell spreading [#1, #4, #6]. In the cochlea this phosphorylation-driven actomyosin activity tunes hair-bundle stiffness and the operating point of mechanotransduction [#8]. MYL12A protein abundance is set post-translationally: it is stabilized by necdin acting through the SGT1–HSP90 chaperone system, a function required for cardiac contractility, and it is protected from proteasomal turnover by OTUD3-mediated deubiquitination [#5, #7]. Beyond its cytoskeletal role, MYL12A serves as a cytoplasmic retention factor for the transcriptional regulator AATF, with genotoxic-stress-induced MK2 phosphorylation of AATF dissolving the complex to release AATF for nuclear repression of pro-apoptotic genes [#2, #3].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Established that non-muscle regulatory light chains are not merely accessory but are structurally required to hold the myosin II holoenzyme together, answering whether RLCs maintain heavy-chain stability.\",\n      \"evidence\": \"Reciprocal Co-IP plus siRNA knockdown with quantitative western blot and morphological phenotyping in NIH 3T3 fibroblasts\",\n      \"pmids\": [\"21126233\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not distinguish the non-redundant contributions of MYL12A versus MYL12B\", \"Mechanism of heavy-chain destabilization upon RLC loss not resolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Revealed a non-cytoskeletal moonlighting role: MYL12A sequesters the transcription factor AATF in the cytoplasm, and stress-dependent MK2 phosphorylation of AATF releases it to repress p53 pro-apoptotic targets, linking MYL12A to the DNA damage response.\",\n      \"evidence\": \"Co-IP, subcellular fractionation, phospho-mimetic mutants, kinase-inhibitor epistasis, and xenograft experiments\",\n      \"pmids\": [\"22909821\", \"22983126\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether AATF binding is to phosphorylated or unphosphorylated MYL12A is unclear\", \"Structural basis of the MYL12A:AATF interaction undefined\", \"Relationship between AATF sequestration and myosin II assembly not addressed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined MYL12A as the dominant regulatory light chain isoform for cell spreading and confirmed Thr18/Ser19 phosphorylation, distinguishing functional roles among the three MRLC isoforms.\",\n      \"evidence\": \"siRNA depletion of all MRLCs with single-isoform rescue, phospho-Thr18/Ser19 western blot, and FRAP turnover analysis in HeLa cells\",\n      \"pmids\": [\"26663899\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis for MYL12A dominance over MRLC1 not identified\", \"Does not map which spreading pathways require MYL12A specifically\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed that smMLCK-dependent MYL12 phosphorylation drives apical-constriction-like shape changes in cochlear hair cells, extending the phospho-regulatory mechanism to a sensory-cell context.\",\n      \"evidence\": \"In vitro kinase assay with purified protein, ddPCR expression, immunofluorescence, and ML-7 pharmacological inhibition with morphometry\",\n      \"pmids\": [\"33877471\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"ML-7 is not MYL12A-specific, leaving other kinase targets possible\", \"Direct contractile consequence in the hair cell not measured at single-molecule level\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified two post-translational stability controls for MYL12A protein levels — chaperone-mediated stabilization via necdin/SGT1-HSP90 and protection from proteasomal degradation by OTUD3 deubiquitination — tying MYL12A abundance to cardiac contractility and to B-cell lymphoma survival.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP, zebrafish morpholino and mouse cardiac overexpression rescue (necdin); deubiquitinase assay, Co-IP, and rupatadine inhibition with viability assays (OTUD3)\",\n      \"pmids\": [\"38832028\", \"39097608\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The E3 ligase that ubiquitinates MYL12A is not identified\", \"How chaperone stabilization and deubiquitination intersect is unknown\", \"Whether these controls regulate myosin II assembly directly is untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated that a bacterial effector kinase, SteC, directly hijacks MYL12A by phosphorylating Ser19/Thr20 to remodel host actin and breach the gut-vascular barrier, revealing the regulatory site as a target of pathogen subversion.\",\n      \"evidence\": \"In vitro kinase assays mapping phospho-residues, SteC crystal structure, macrophage migration/invasion assays, and in vivo Salmonella infection model\",\n      \"pmids\": [\"38528181\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SteC phosphorylation mimics or exceeds physiological MLCK signaling is not quantified\", \"Downstream actomyosin contractile changes not structurally resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked MYL12A-containing actomyosin in hair bundles directly to mechanotransduction physiology, showing it sets bundle stiffness and the resting operating point of transduction channels.\",\n      \"evidence\": \"Immunostaining localization, NM2/MLCK pharmacological inhibition, stiffness and spontaneous oscillation recordings, transduction channel open-probability measurement, and auditory brainstem response in mice\",\n      \"pmids\": [\"40240732\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Pharmacology cannot isolate MYL12A from other RLC isoforms\", \"Direct phosphorylation state of bundle MYL12A during oscillation not measured\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MYL12A's distinct roles — myosin II structural stabilization, cytoplasmic AATF sequestration, and chaperone/ubiquitin-regulated abundance — are coordinated within a single cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated model linking phosphorylation, stability control, and the AATF moonlighting function\", \"No structure of MYL12A bound to either heavy chains or AATF in the timeline\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-397014\", \"supporting_discovery_ids\": [5, 8]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"complexes\": [\"non-muscle myosin II\"],\n    \"partners\": [\"MYH9\", \"MYH10\", \"MYL6\", \"AATF\", \"SteC\", \"OTUD3\", \"necdin\", \"SDC2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}