{"gene":"MYL9","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2014,"finding":"PKM2 phosphorylates MLC2/MYL9 at Y118, which primes binding of ROCK2 to MLC2 and subsequent ROCK2-dependent MLC2 S15 phosphorylation; Aurora B phosphorylates PKM2 at T45 to enable its localization to the contractile ring and interaction with MLC2 during cytokinesis.","method":"In vitro kinase assay, Co-IP, site-directed mutagenesis, loss-of-function in tumour cells","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro phosphorylation assays with mutagenesis, reciprocal Co-IP, and functional cytokinesis readout in a single rigorous study","pmids":["25412762"],"is_preprint":false},{"year":2010,"finding":"MYL9 is a direct transcriptional target of RUNX1; RUNX1 binds four RUNX1 sites in the MYL9 promoter region (-729/-542 bp) as demonstrated by ChIP, EMSA, and reporter assays; RUNX1 siRNA reduces MYL9 expression and cell spreading on collagen and fibrinogen.","method":"Chromatin immunoprecipitation (ChIP), EMSA, reporter gene assay, siRNA knockdown, cell-spreading assay","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (ChIP, EMSA, reporter, KD phenotype) in a single focused study","pmids":["20876458"],"is_preprint":false},{"year":2009,"finding":"The MAL/SRF transcriptional complex directly regulates MYL9 expression; MAL knockdown in megakaryocyte progenitors reduces MYL9 expression, impairs proplatelet formation and stress fiber/filopodia/lamellipodia formation; shRNA-mediated MYL9 knockdown alone reproduces the proplatelet formation defect.","method":"Luciferase reporter assay, chromatin immunoprecipitation, shRNA knockdown, gene expression profiling","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — ChIP and luciferase confirm direct regulation; shRNA rescue isolates MYL9's specific role in proplatelet formation","pmids":["19724058"],"is_preprint":false},{"year":2010,"finding":"Conditional ablation of the transcription factor Junb in mice reduces Myl9 expression; re-expression of either Junb or Myl9 in Junb-deficient cells restores stress fiber formation, cellular motility, and contractile capacity of vascular smooth muscle cells, establishing Myl9 as a direct functional target of Junb in actomyosin-based contractility.","method":"Conditional knockout mouse, re-expression rescue experiments, arterial contraction assays, stress fiber imaging","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo KO with defined vascular phenotype, rescue by Myl9 re-expression, replicated across multiple cell types","pmids":["20551518"],"is_preprint":false},{"year":2009,"finding":"VE-cadherin signals through Rho-kinase to phosphorylate MLC2/MYL9, generating actomyosin contractility at cell junctions that suppresses VEGF-driven, Rac1-dependent sprouting; inhibition of VE-cadherin, Rho-kinase, or actomyosin contractility each leads to increased sprouting.","method":"Organotypic angiogenesis assay, zebrafish embryo experiments, pharmacological inhibition of ROCK and actomyosin, VE-cadherin loss-of-function","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal inhibition approaches in two model systems (organotypic and zebrafish) with specific MLC2-phosphorylation readout","pmids":["19345098"],"is_preprint":false},{"year":2006,"finding":"Endosomes containing Endo180 (CD280) generate localised Rho-ROCK-MLC2-based contractile signals; these endosomes are spatially positioned at sites of adhesion turnover to drive focal adhesion and cell-cell junction disassembly during cell migration.","method":"Live-cell imaging, subcellular fractionation/localisation, pharmacological inhibition of ROCK, siRNA knockdown of Endo180","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct localisation with functional consequence (adhesion disassembly), multiple inhibition approaches in single rigorous study","pmids":["17043135"],"is_preprint":false},{"year":2018,"finding":"N-terminal acetyltransferase NatA acetylates and N-terminal methyltransferase NRMT1 methylates the alpha-amino group of MYL9; Nα-acetylation promotes cytoplasmic/actomyosin roles of MYL9 (increased pSer19 phosphorylation) while Nα-methylation promotes nuclear roles (increased DNA binding and reduced cytoskeletal interactions).","method":"In vitro enzymatic assay with purified NatA and NRMT1, mutant MYL9 constructs exclusive for each modification, cell-based phosphorylation assays, DNA-binding assays","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct enzymatic verification plus mutant proteoform analysis with multiple functional readouts in a single study","pmids":["30242065"],"is_preprint":false},{"year":2013,"finding":"SMYD3 histone methyltransferase cooperates with MRTF-A to transactivate the MYL9 promoter; SMYD3 HMT activity is required, and the proximal MRTF-A binding element of the MYL9 promoter mediates the cooperative transactivation; overexpression promotes MCF-7 cell migration while SMYD3/MRTF-A siRNA suppresses MYL9 expression and migration.","method":"Luciferase reporter assay, mutation analysis of MYL9 promoter, siRNA knockdown, co-immunoprecipitation, migration assay","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reporter + mutation analysis + Co-IP in a single lab study establish cooperative transcriptional mechanism","pmids":["24189459"],"is_preprint":false},{"year":2013,"finding":"MRTF-A transactivates MYL9 and its overexpression promotes migration of MCF-7 breast cancer cells; 17β-estradiol upregulates MRTF-A and thereby MYL9 expression; MRTF-A siRNA suppresses MYL9 transcription and reduces cell migration.","method":"Reporter gene assay, siRNA knockdown, migration assay, Western blot","journal":"Acta biochimica et biophysica Sinica","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — reporter assay and KD with migration phenotype replicate finding from same lab (PMID 24189459), two orthogonal methods","pmids":["24084383"],"is_preprint":false},{"year":2023,"finding":"PRPF19 E3 ubiquitin ligase stabilises MYL9 via K63-linked ubiquitination; MYL9 is the downstream substrate mediating PRPF19-driven migration and invasion of colorectal cancer cells, and the PRPF19/MYL9 axis activates the Src-YAP1 cascade to promote metastasis.","method":"Co-immunoprecipitation, ubiquitination assay, gain- and loss-of-function migration/invasion assays, substrate validation","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP + ubiquitination assay + rescue experiments establish substrate relationship in a single study","pmids":["37031206"],"is_preprint":false},{"year":2023,"finding":"MYL9 binds MYO19 via direct protein-protein interaction (confirmed by Co-IP and GST pull-down); MYL9/MYO19 complex suppresses epithelial-mesenchymal transition and migration in NSCLC cells.","method":"Co-immunoprecipitation, GST pull-down assay, scratch wound healing assay, Western blot for EMT markers","journal":"Physiological genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal binding assays (Co-IP and GST pull-down) plus functional rescue in single lab","pmids":["39437553"],"is_preprint":false},{"year":2022,"finding":"MYL9 deficiency in mice causes neonatal lethality with distended bladder, shortened small intestine, and alveolar overdistension; LacZ reporter knockin shows MYL9 expression is restricted to muscularis propria of intestine and bladder and bronchial smooth muscle, indicating MYL9 is required for smooth muscle cell function in these organs.","method":"Germline knockout mouse, LacZ reporter knock-in for expression mapping, gross and histological phenotyping","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vivo loss-of-function with defined organ-specific phenotype and direct expression mapping; single lab but rigorous genetic approach","pmids":["35802750"],"is_preprint":false},{"year":2015,"finding":"RhoA-mediated phosphorylation of MLC2 regulates actin dynamics required for cytokinesis (polar body extrusion) during mouse oocyte meiosis; MLC2 knockdown causes failure of polar body extrusion and arrest at telophase with reduced cortical actin.","method":"siRNA knockdown in mouse oocytes, immunofluorescence staining of p-MLC2 and actin, RhoA inhibition","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with defined cytokinesis phenotype plus RhoA epistasis, single lab","pmids":["26701676"],"is_preprint":false},{"year":2021,"finding":"MYL9 in cancer-associated fibroblasts binds IQGAP1 and through ERK1/2 signalling regulates secretion of CCL2 and TGF-β1, which in turn activate the PI3K-AKT pathway in CRC tumour cells to promote progression; ZEB1 binds the MYL9 promoter in CAFs to enhance MYL9 expression.","method":"Co-immunoprecipitation, ChIP, dual luciferase assay, ELISA, siRNA knockdown, co-culture assay","journal":"Journal of experimental & clinical cancer research : CR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP confirms IQGAP1 binding, ChIP confirms ZEB1-MYL9 promoter interaction, with functional secretion readouts; single lab","pmids":["37926835"],"is_preprint":false},{"year":2021,"finding":"ITPRIP recruits MYL9 to the kinase domain of DAPK1, forming an ITPRIP-DAPK1-MYL9 ternary complex; this interaction impedes DAPK1-mediated phosphorylation of MYL9 and promotes glioma progression; MYL9 knockdown does not disrupt the ITPRIP-DAPK1 association.","method":"Lentivirus co-infection, co-immunoprecipitation, knockdown of ITPRIP/DAPK1/MYL9, in vitro and in vivo tumour growth assays","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP establishes ternary complex; functional epistasis by differential KDs; single lab","pmids":["34111521"],"is_preprint":false},{"year":2008,"finding":"MYL9 interacts with MR-1 (myofibrillogenesis regulator 1); MR-1 knockdown decreases MYL9 phosphorylation and disrupts stress fiber formation, reducing hepatoma cell proliferation, migration, and adhesion via the MLC2/FAK/Akt pathway.","method":"siRNA knockdown, Western blot for phospho-MLC2/FAK/Akt, migration/adhesion assay, stress-fiber imaging","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — KD with multiple phosphorylation readouts and functional assays; interaction established previously, single lab","pmids":["18948272"],"is_preprint":false},{"year":2014,"finding":"Phosphorylation (pSer19) and O-GlcNAcylation of slow MYL9 (sMLC2) are mutually exclusive post-translational modifications in skeletal muscle; a multienzymatic complex including kinase, phosphatase, OGT, and OGA is colocalized at the Z-disc and modulates these modifications; hindlimb unloading (atrophy) decreases O-GlcNAcylation and increases phosphorylation of sMLC2, reversed upon reloading.","method":"2D-gel electrophoresis, phospho-specific dye staining, immunoblotting, co-localization immunofluorescence, rat hindlimb unloading model","journal":"Pflugers Archiv : European journal of physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical demonstration of mutually exclusive PTMs with enzyme co-localization; single lab, multiple orthogonal methods","pmids":["24477671"],"is_preprint":false},{"year":1993,"finding":"Endogenous myosin light chain kinase phosphorylates MLC2/MYL9 at approximately 50% of sites after Ca2+ activation in fast-twitch rat skeletal muscle fibres; MLC2 phosphorylation markedly increases the Ca2+ sensitivity of the contractile apparatus and follows Ca2+ rise with a 1–2 s delay.","method":"Mechanically skinned single fibre preparation, 32P-radiolabelled phosphorylation assay, force-Ca2+ relationship measurement","journal":"Pflugers Archiv : European journal of physiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro phosphorylation assay in single fibres with quantitative Ca2+-sensitivity readout; foundational mechanistic study","pmids":["8351204"],"is_preprint":false},{"year":2004,"finding":"An 82-bp cardiac-specific enhancer in the Xenopus XMLC2 (MLC2) proximal promoter, composed of two GATA sites and a composite YY1/CArG-like site, is necessary and sufficient for heart-specific expression; the low-affinity SRF site is essential and requires at least one adjacent GATA site; YY1 acts primarily as a repressor of ectopic expression.","method":"Transgenic frog embryo reporter assay, deletion and site-specific mutation analysis of promoter elements, transgene expression in mice","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vivo transgenic assay with systematic deletion and site-mutation analysis, validated cross-species in mice","pmids":["14711876"],"is_preprint":false},{"year":1998,"finding":"In C. elegans, mlc-2 (regulatory myosin light chain) is required for pharyngeal muscle function; mlc-1 and mlc-2 are redundant in body-wall muscle; transgenic copies of either mlc-1+ or mlc-2+ fully rescue all defects of mlc-1(0) mlc-2(0) double mutants including pharyngeal and body-wall muscle defects.","method":"Genetic deletion mutants, double mutant analysis, transgenic rescue, in situ hybridization","journal":"Genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — classic genetic epistasis with transgenic rescue in two tissues; ortholog of MYL9 in established model organism","pmids":["9799259"],"is_preprint":false},{"year":2019,"finding":"Cardiac-specific MLC kinase (cMLCK) localises predominantly at the Z-disc where it interacts with α-actinin2 with high affinity (Kd ~13.4 nM) via the cardiac-isoform-specific N-terminus; a cMLCK mutant deficient for α-actinin2 binding fails to promote sarcomere organisation or increase cardiomyocyte size, indicating Z-disc anchoring is required for cMLCK's role in sarcomere organisation upstream of MLC2/MYL9 phosphorylation.","method":"Subcellular fractionation/immunofluorescence localisation, kinetic binding assay, N-terminal deletion and point mutants, cardiomyocyte overexpression","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localisation plus kinetic binding measurement and mutant analysis; single lab","pmids":["31467300"],"is_preprint":false},{"year":2016,"finding":"12(S)-HETE activates a 12-HETE receptor → RHO → ROCK → MYPT → MLC2 signalling cascade in lymph endothelial cells to phosphorylate MLC2, driving actomyosin contraction, LEC retraction, and endothelial barrier breaching; MLC2 siRNA abolishes LEC retraction.","method":"siRNA knockdown of MLC2, RHO/ROCK inhibitors, Western blot for p-MLC2, CCID barrier breaching assay","journal":"British journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown plus inhibitor epistasis with defined functional readout; single lab","pmids":["27362730"],"is_preprint":false},{"year":2025,"finding":"DDX3X promotes MYL9 expression in pancreatic cancer by destabilising TLE2 mRNA, releasing KLF4-mediated repression of MYL9; elevated MYL9 remodels F-actin and enhances tumour cell traction forces, facilitating metastasis.","method":"CRISPR-Cas9 screen, mRNA stability assays, transcription factor ChIP, traction force microscopy, in vivo orthotopic xenograft","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen + mechanistic follow-up in vivo; single lab but multiple orthogonal approaches","pmids":["40938994"],"is_preprint":false},{"year":2012,"finding":"Transfection of MYL9 into MYL9-negative Jurkat cells significantly increases surface CD3 expression, establishing that MYL9 enables a distinct pathway for CD3 surface expression independent of the PKC-dependent pathway.","method":"MYL9 gene transfection into Jurkat cells, flow cytometry for surface CD3, PMA stimulation","journal":"Journal of smooth muscle research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single gain-of-function transfection with defined surface-expression phenotype; moderate confidence limited by single method","pmids":["23538510"],"is_preprint":false},{"year":2024,"finding":"DAPK3, released from LUZP1-mediated suppression by COP1-mediated LUZP1 ubiquitination and degradation, phosphorylates and activates MYL9 to promote EMT and colorectal cancer liver metastasis.","method":"Patient-derived organoid multi-omics (WES, bulk and scRNA-seq), Co-IP, in vitro and in vivo functional assays","journal":"Experimental hematology & oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — organoid multi-omics plus in vitro/in vivo functional validation; single lab, novel pathway placement","pmids":["41937206"],"is_preprint":false},{"year":2024,"finding":"RUNX1 isoforms B and C differentially regulate MYL9 expression in megakaryocytes/platelets: RUNX1B negatively correlates with MYL9 transcripts in platelets while RUNX1C positively regulates target genes; both isoforms bind and regulate the RUNX1 P1 and P2 promoters.","method":"ChIP, luciferase promoter assay, RUNX1 isoform overexpression in HEL and HeLa cells, platelet RNAseq from 85 volunteers","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and reporter assays in cell lines plus human platelet RNAseq; preprint, not peer-reviewed","pmids":["bio_10.1101_2024.06.18.599563"],"is_preprint":true}],"current_model":"MYL9 (MLC2) is a myosin II regulatory light chain whose phosphorylation at Ser19 (by MLCK, ROCK, PKM2, and other kinases) is the central activating switch for actomyosin contractility in smooth, cardiac, skeletal, and non-muscle cells; it is regulated at multiple levels—transcriptionally by RUNX1, MAL/SRF, MRTF-A/SMYD3, Junb, and GATA/SRF cardiac enhancers; post-translationally by competing N-terminal acetylation (promoting cytoplasmic/contractile roles via increased pSer19) and N-terminal methylation (promoting nuclear/DNA-binding roles), as well as by mutually exclusive O-GlcNAcylation at the same residues as phosphorylation; protein stability is regulated by K63-linked ubiquitination via PRPF19; and it participates in direct protein complexes with MR-1, IQGAP1, MYO19, and DAPK1/ITPRIP, linking it to FAK/Akt signalling, ERK-mediated cytokine secretion, EMT suppression, and tumour cell traction-force generation."},"narrative":{"mechanistic_narrative":"MYL9 (MLC2) is the regulatory light chain of myosin II whose reversible phosphorylation operates as the central activating switch for actomyosin contractility across smooth, cardiac, skeletal, and non-muscle cells, and across processes from muscle contraction to cytokinesis and cell migration [PMID:8351204, PMID:35802750]. The activating modification is phosphorylation at the conserved serine (Ser19/Ser15) and an adjacent priming tyrosine: in fast-twitch skeletal fibres MLCK phosphorylates MLC2 in a Ca2+-dependent manner to raise the Ca2+ sensitivity of the contractile apparatus [PMID:8351204], while PKM2-mediated Y118 phosphorylation primes ROCK2 binding and subsequent S15 phosphorylation during cytokinesis [PMID:25412762]. Multiple upstream Rho-kinase cascades converge on this switch to control distinct contractile outputs—VE-cadherin/ROCK signalling generates junctional contractility that restrains endothelial sprouting [PMID:19345098], RhoA-driven phosphorylation drives polar body extrusion in oocyte meiosis [PMID:26701676], and a 12(S)-HETE→RHO→ROCK→MYPT→MLC2 cascade drives lymph-endothelial retraction [PMID:27362730]. Genetic ablation of Myl9 in mice causes neonatal lethality with smooth-muscle organ failure, establishing its non-redundant requirement in intestinal, bladder, and bronchial smooth muscle [PMID:35802750], a role mirrored by the redundant requirement of its C. elegans ortholog mlc-2 in pharyngeal and body-wall muscle [PMID:9799259]. MYL9 expression is set transcriptionally by a network including RUNX1 [PMID:20876458], MAL/SRF and MRTF-A/SMYD3 [PMID:19724058, PMID:24189459], Junb [PMID:20551518], and cardiac GATA/SRF enhancers [PMID:14711876], and its protein output is tuned post-translationally by mutually exclusive Ser19 phosphorylation versus O-GlcNAcylation [PMID:24477671] and by competing N-terminal acetylation versus methylation that partition MYL9 between cytoplasmic/contractile and nuclear/DNA-binding roles [PMID:30242065]. In cancer, MYL9 acts as an effector node for traction-force generation, migration, invasion, and EMT control through physical partners and regulators including IQGAP1, MYO19, MR-1, the ITPRIP-DAPK1 complex, and PRPF19-mediated K63 ubiquitination [PMID:37926835, PMID:39437553, PMID:18948272, PMID:34111521, PMID:37031206, PMID:40938994].","teleology":[{"year":1993,"claim":"Established the core biochemical function: that MLCK-mediated MLC2 phosphorylation is the modification that tunes contractile force, answering how light-chain phosphorylation translates into mechanical output.","evidence":"32P phosphorylation assay and force-Ca2+ measurement in skinned fast-twitch rat skeletal fibres","pmids":["8351204"],"confidence":"High","gaps":["Did not identify the specific phosphorylated residue at sequence level","Restricted to fast-twitch skeletal muscle"]},{"year":1998,"claim":"Showed the regulatory light chain is genetically required for muscle function and is functionally interchangeable with a paralog, establishing its non-redundant tissue role in an intact organism.","evidence":"Genetic deletion, double-mutant epistasis, and transgenic rescue in C. elegans pharyngeal and body-wall muscle","pmids":["9799259"],"confidence":"High","gaps":["Ortholog system, not human MYL9","Did not address phosphoregulation"]},{"year":2004,"claim":"Defined the cis-regulatory logic for cardiac-restricted MLC2 expression, answering how the gene is spatially controlled in the heart.","evidence":"Transgenic frog/mouse reporter assays with deletion and site-specific promoter mutagenesis (GATA/SRF/YY1 elements)","pmids":["14711876"],"confidence":"High","gaps":["Concerns Xenopus XMLC2 enhancer; human enhancer conservation not directly tested","Does not address non-cardiac expression control"]},{"year":2006,"claim":"Placed MLC2-dependent contractility at a spatial point of action—endosome-localized Rho-ROCK-MLC2 signals driving adhesion disassembly—linking contractility to migration mechanics.","evidence":"Live-cell imaging, subcellular localization, ROCK inhibition, Endo180 knockdown","pmids":["17043135"],"confidence":"High","gaps":["Direct MLC2 phosphorylation at endosome sites inferred from pathway, not site-mapped","Endo180-MLC2 physical link not established"]},{"year":2009,"claim":"Identified upstream transcriptional and signalling inputs—MAL/SRF setting MYL9 expression in megakaryocytes and VE-cadherin/ROCK driving junctional MLC2 contractility—connecting MYL9 to platelet formation and angiogenic restraint.","evidence":"ChIP, luciferase, shRNA rescue (megakaryocytes); organotypic and zebrafish angiogenesis with ROCK/actomyosin inhibition","pmids":["19724058","19345098"],"confidence":"High","gaps":["MAL/SRF and VE-cadherin pathways studied in separate systems","Quantitative contribution of MYL9 vs other SRF targets not isolated in angiogenesis"]},{"year":2010,"claim":"Defined RUNX1 and Junb as direct transcriptional regulators of MYL9, linking its expression to cell spreading and vascular smooth-muscle contractility.","evidence":"ChIP/EMSA/reporter with siRNA (RUNX1); conditional knockout with Myl9 re-expression rescue (Junb)","pmids":["20876458","20551518"],"confidence":"High","gaps":["How these TFs integrate with SRF/MRTF inputs at the MYL9 promoter is unresolved","Post-translational consequences downstream not addressed"]},{"year":2013,"claim":"Resolved a cooperative transcriptional mechanism in which MRTF-A and SMYD3 transactivate MYL9 to drive cancer cell migration, extending SRF-pathway control into tumour cell motility.","evidence":"Reporter/promoter-mutation, Co-IP, siRNA, and migration assays in MCF-7 cells","pmids":["24189459","24084383"],"confidence":"Medium","gaps":["Both studies from related work in one cell type","SMYD3 methylation substrate at the locus not directly mapped"]},{"year":2014,"claim":"Uncovered priming-then-activation phospho-control (PKM2 Y118 priming ROCK2-dependent S15) during cytokinesis and a mutually exclusive Ser19-phosphorylation/O-GlcNAcylation switch in skeletal muscle, refining how the activating modification is gated.","evidence":"In vitro kinase assays, mutagenesis, Co-IP in tumour cells; 2D-gel, phospho/O-GlcNAc immunoblotting and enzyme co-localization in rat muscle","pmids":["25412762","24477671"],"confidence":"High","gaps":["Y118 priming demonstrated in tumour cytokinesis; generality to other contexts untested","Stoichiometry of competing O-GlcNAc vs phosphorylation in vivo not quantified"]},{"year":2018,"claim":"Identified N-terminal acetylation versus methylation as competing modifications that partition MYL9 between cytoplasmic contractile and nuclear DNA-binding pools, revealing a non-canonical nuclear role.","evidence":"In vitro NatA/NRMT1 enzymatic assays, modification-exclusive mutants, phosphorylation and DNA-binding readouts","pmids":["30242065"],"confidence":"High","gaps":["Nuclear DNA targets and transcriptional consequences not identified","In vivo balance of the two N-terminal modifications unmeasured"]},{"year":2021,"claim":"Mapped MYL9 into discrete cancer protein complexes—IQGAP1 (CAF cytokine secretion) and an ITPRIP-DAPK1 ternary complex shielding MYL9 from DAPK1 phosphorylation—showing context-specific protein partners modulate its phospho-state and signalling output.","evidence":"Co-IP, ChIP, ELISA, co-culture (IQGAP1/ZEB1); reciprocal Co-IP and differential knockdown (ITPRIP-DAPK1-MYL9)","pmids":["37926835","34111521"],"confidence":"Medium","gaps":["Single-lab complex characterizations without structural validation","Direct binding interfaces not defined"]},{"year":2023,"claim":"Established protein-stability and additional binding control of MYL9—PRPF19 K63-ubiquitination stabilizing it to drive Src-YAP1 metastasis, and direct MYO19 binding suppressing EMT—broadening regulation beyond phosphorylation.","evidence":"Co-IP, ubiquitination assays, GST pull-down, and migration/invasion assays in CRC and NSCLC cells","pmids":["37031206","39437553"],"confidence":"Medium","gaps":["Ubiquitination site on MYL9 not mapped","MYO19 and PRPF19 effects characterized in different cancers without cross-validation"]},{"year":2025,"claim":"Positioned MYL9 as a downstream effector of mechanotransduction in cancer, where DDX3X-driven derepression elevates MYL9 to remodel F-actin and increase traction forces for metastasis.","evidence":"CRISPR-Cas9 screen, mRNA stability assays, ChIP, traction-force microscopy, orthotopic xenograft","pmids":["40938994"],"confidence":"Medium","gaps":["Whether traction-force increase requires MYL9 phosphorylation not isolated","Single-cancer-type validation"]},{"year":null,"claim":"How the multiple input layers—transcriptional networks, competing N-terminal acetylation/methylation, O-GlcNAc/phospho switching, and ubiquitination—are integrated to set the cytoplasmic-versus-nuclear and contractile-versus-migratory balance of MYL9 in a given cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified quantitative model of competing PTMs","Nuclear DNA-binding function lacks identified targets","No human disease-causing mutation reported in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[17,0,11]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[17,11]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[6]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[2,15,5]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-397014","term_label":"Muscle contraction","supporting_discovery_ids":[17,11]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,12]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,21,15]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[9,13,14,22]}],"complexes":["ITPRIP-DAPK1-MYL9 ternary complex","myosin II"],"partners":["ROCK2","PKM2","IQGAP1","MYO19","DAPK1","ITPRIP","PRPF19","MR-1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P24844","full_name":"Myosin regulatory light polypeptide 9","aliases":["20 kDa myosin light chain","LC20","MLC-2C","Myosin RLC","Myosin regulatory light chain 2, smooth muscle isoform","Myosin regulatory light chain 9","Myosin regulatory light chain MRLC1"],"length_aa":172,"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 (PubMed:11942626, PubMed:2526655). In myoblasts, may regulate PIEZO1-dependent cortical actomyosin assembly involved in myotube formation (By similarity)","subcellular_location":"Cytoplasm, cytoskeleton; Cytoplasm, cell cortex","url":"https://www.uniprot.org/uniprotkb/P24844/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MYL9","classification":"Not Classified","n_dependent_lines":11,"n_total_lines":1208,"dependency_fraction":0.009105960264900662},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MYL9","total_profiled":1310},"omim":[{"mim_id":"619365","title":"MEGACYSTIS-MICROCOLON-INTESTINAL HYPOPERISTALSIS SYNDROME 4; MMIHS4","url":"https://www.omim.org/entry/619365"},{"mim_id":"616432","title":"RHO GUANINE NUCLEOTIDE EXCHANGE FACTOR 18; 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Netherlands)","url":"https://pubmed.ncbi.nlm.nih.gov/36920729","citation_count":2,"is_preprint":false},{"pmid":"37866415","id":"PMC_37866415","title":"ZC3H4 governs epithelial cell migration through ROCK/p-PYK2/p-MLC2 pathway in silica-induced pulmonary fibrosis.","date":"2023","source":"Environmental toxicology and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/37866415","citation_count":1,"is_preprint":false},{"pmid":"40242171","id":"PMC_40242171","title":"Activation of VEGFR3 and MLC2 are Critical for GLP-2 Enhancement of Chylomicron Transport.","date":"2024","source":"Gastro hep advances","url":"https://pubmed.ncbi.nlm.nih.gov/40242171","citation_count":1,"is_preprint":false},{"pmid":"41272428","id":"PMC_41272428","title":"DPSCs-Exos promote OPCs differentiation and white matter repair via Myl9-mediated PRMT5 nucleation after ischemic stroke.","date":"2025","source":"Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/41272428","citation_count":1,"is_preprint":false},{"pmid":"41235503","id":"PMC_41235503","title":"The Network of Exosomes miRNA and p-MLC2 Regulatory Pathway Induced Pathological Cardiac Hypertrophy in Vasn Deficient Mice.","date":"2025","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/41235503","citation_count":1,"is_preprint":false},{"pmid":"40142587","id":"PMC_40142587","title":"The Minute Virus of Canines (MVC) Activates the RhoA/ROCK1/MLC2 Signal Transduction Pathway Resulting in the Dissociation of Tight Junctions and Facilitating Occludin-Mediated Viral Infection.","date":"2025","source":"Microorganisms","url":"https://pubmed.ncbi.nlm.nih.gov/40142587","citation_count":1,"is_preprint":false},{"pmid":"8589760","id":"PMC_8589760","title":"Transactivation of cardiac MLC-2 promoter by MyoD in 10T1/2 fibroblast cells is independent of E-box requirement but depends upon new proteins that recognize MEF-2 site.","date":"1995","source":"Cellular & molecular biology research","url":"https://pubmed.ncbi.nlm.nih.gov/8589760","citation_count":1,"is_preprint":false},{"pmid":"40089610","id":"PMC_40089610","title":"MLC2: Physiological Functions and Potential Roles in Tumorigenesis.","date":"2025","source":"Cell biochemistry and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/40089610","citation_count":0,"is_preprint":false},{"pmid":"41937206","id":"PMC_41937206","title":"Multi-omics analysis of patient-derived organoids reveals that E3 ligase COP1 promotes liver metastasis and oxaliplatin resistance in colorectal cancer through LUZP1 degradation and MYL9 phosphorylation.","date":"2026","source":"Experimental hematology & oncology","url":"https://pubmed.ncbi.nlm.nih.gov/41937206","citation_count":0,"is_preprint":false},{"pmid":"37511553","id":"PMC_37511553","title":"Staphylococcal Enterotoxin C2 Mutant-Induced Antitumor Immune Response Is Controlled by CDC42/MLC2-Mediated Tumor Cell Stiffness.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/37511553","citation_count":0,"is_preprint":false},{"pmid":"41130043","id":"PMC_41130043","title":"Lnc237 regulates duck myoblasts proliferation and differentiation via the miR-22-5p/MYL9 axis.","date":"2025","source":"Poultry science","url":"https://pubmed.ncbi.nlm.nih.gov/41130043","citation_count":0,"is_preprint":false},{"pmid":"41671841","id":"PMC_41671841","title":"Integrative GWAS and RNA-seq identify MYL9 as a key regulator of pullorum disease resistance in chickens.","date":"2026","source":"Poultry science","url":"https://pubmed.ncbi.nlm.nih.gov/41671841","citation_count":0,"is_preprint":false},{"pmid":"39179950","id":"PMC_39179950","title":"Ghrelin may protect against vascular endothelial injury in Acute traumatic coagulopathy by mediating the RhoA/ROCK/MLC2 pathway.","date":"2024","source":"Journal of thrombosis and thrombolysis","url":"https://pubmed.ncbi.nlm.nih.gov/39179950","citation_count":0,"is_preprint":false},{"pmid":"10453502","id":"PMC_10453502","title":"[The expression of MLC2-chymase fusion gene in cultured neonatal myocardial cells].","date":"1997","source":"Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae","url":"https://pubmed.ncbi.nlm.nih.gov/10453502","citation_count":0,"is_preprint":false},{"pmid":"41969450","id":"PMC_41969450","title":"Integrated bulk RNA-seq and scRNA-seq identification of a novel \"PET-SPI1-MYL9\" transcriptional axis in lung adenocarcinoma driven by polyethylene terephthalate exposure.","date":"2026","source":"Translational cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/41969450","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.06.18.599563","title":"RUNX1 Isoforms Regulate RUNX1 and Target-Genes Differentially in Platelets-Megakaryocytes: Association with Clinical Cardiovascular Events","date":"2024-06-21","source":"bioRxiv","url":"https://doi.org/10.1101/2024.06.18.599563","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":48603,"output_tokens":6410,"usd":0.12098,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":15164,"output_tokens":4569,"usd":0.095022,"stage2_stop_reason":"end_turn"},"total_usd":0.216002,"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\": 2014,\n      \"finding\": \"PKM2 phosphorylates MLC2/MYL9 at Y118, which primes binding of ROCK2 to MLC2 and subsequent ROCK2-dependent MLC2 S15 phosphorylation; Aurora B phosphorylates PKM2 at T45 to enable its localization to the contractile ring and interaction with MLC2 during cytokinesis.\",\n      \"method\": \"In vitro kinase assay, Co-IP, site-directed mutagenesis, loss-of-function in tumour cells\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro phosphorylation assays with mutagenesis, reciprocal Co-IP, and functional cytokinesis readout in a single rigorous study\",\n      \"pmids\": [\"25412762\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"MYL9 is a direct transcriptional target of RUNX1; RUNX1 binds four RUNX1 sites in the MYL9 promoter region (-729/-542 bp) as demonstrated by ChIP, EMSA, and reporter assays; RUNX1 siRNA reduces MYL9 expression and cell spreading on collagen and fibrinogen.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), EMSA, reporter gene assay, siRNA knockdown, cell-spreading assay\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (ChIP, EMSA, reporter, KD phenotype) in a single focused study\",\n      \"pmids\": [\"20876458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The MAL/SRF transcriptional complex directly regulates MYL9 expression; MAL knockdown in megakaryocyte progenitors reduces MYL9 expression, impairs proplatelet formation and stress fiber/filopodia/lamellipodia formation; shRNA-mediated MYL9 knockdown alone reproduces the proplatelet formation defect.\",\n      \"method\": \"Luciferase reporter assay, chromatin immunoprecipitation, shRNA knockdown, gene expression profiling\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — ChIP and luciferase confirm direct regulation; shRNA rescue isolates MYL9's specific role in proplatelet formation\",\n      \"pmids\": [\"19724058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Conditional ablation of the transcription factor Junb in mice reduces Myl9 expression; re-expression of either Junb or Myl9 in Junb-deficient cells restores stress fiber formation, cellular motility, and contractile capacity of vascular smooth muscle cells, establishing Myl9 as a direct functional target of Junb in actomyosin-based contractility.\",\n      \"method\": \"Conditional knockout mouse, re-expression rescue experiments, arterial contraction assays, stress fiber imaging\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo KO with defined vascular phenotype, rescue by Myl9 re-expression, replicated across multiple cell types\",\n      \"pmids\": [\"20551518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"VE-cadherin signals through Rho-kinase to phosphorylate MLC2/MYL9, generating actomyosin contractility at cell junctions that suppresses VEGF-driven, Rac1-dependent sprouting; inhibition of VE-cadherin, Rho-kinase, or actomyosin contractility each leads to increased sprouting.\",\n      \"method\": \"Organotypic angiogenesis assay, zebrafish embryo experiments, pharmacological inhibition of ROCK and actomyosin, VE-cadherin loss-of-function\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal inhibition approaches in two model systems (organotypic and zebrafish) with specific MLC2-phosphorylation readout\",\n      \"pmids\": [\"19345098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Endosomes containing Endo180 (CD280) generate localised Rho-ROCK-MLC2-based contractile signals; these endosomes are spatially positioned at sites of adhesion turnover to drive focal adhesion and cell-cell junction disassembly during cell migration.\",\n      \"method\": \"Live-cell imaging, subcellular fractionation/localisation, pharmacological inhibition of ROCK, siRNA knockdown of Endo180\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localisation with functional consequence (adhesion disassembly), multiple inhibition approaches in single rigorous study\",\n      \"pmids\": [\"17043135\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"N-terminal acetyltransferase NatA acetylates and N-terminal methyltransferase NRMT1 methylates the alpha-amino group of MYL9; Nα-acetylation promotes cytoplasmic/actomyosin roles of MYL9 (increased pSer19 phosphorylation) while Nα-methylation promotes nuclear roles (increased DNA binding and reduced cytoskeletal interactions).\",\n      \"method\": \"In vitro enzymatic assay with purified NatA and NRMT1, mutant MYL9 constructs exclusive for each modification, cell-based phosphorylation assays, DNA-binding assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct enzymatic verification plus mutant proteoform analysis with multiple functional readouts in a single study\",\n      \"pmids\": [\"30242065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"SMYD3 histone methyltransferase cooperates with MRTF-A to transactivate the MYL9 promoter; SMYD3 HMT activity is required, and the proximal MRTF-A binding element of the MYL9 promoter mediates the cooperative transactivation; overexpression promotes MCF-7 cell migration while SMYD3/MRTF-A siRNA suppresses MYL9 expression and migration.\",\n      \"method\": \"Luciferase reporter assay, mutation analysis of MYL9 promoter, siRNA knockdown, co-immunoprecipitation, migration assay\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter + mutation analysis + Co-IP in a single lab study establish cooperative transcriptional mechanism\",\n      \"pmids\": [\"24189459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"MRTF-A transactivates MYL9 and its overexpression promotes migration of MCF-7 breast cancer cells; 17β-estradiol upregulates MRTF-A and thereby MYL9 expression; MRTF-A siRNA suppresses MYL9 transcription and reduces cell migration.\",\n      \"method\": \"Reporter gene assay, siRNA knockdown, migration assay, Western blot\",\n      \"journal\": \"Acta biochimica et biophysica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — reporter assay and KD with migration phenotype replicate finding from same lab (PMID 24189459), two orthogonal methods\",\n      \"pmids\": [\"24084383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PRPF19 E3 ubiquitin ligase stabilises MYL9 via K63-linked ubiquitination; MYL9 is the downstream substrate mediating PRPF19-driven migration and invasion of colorectal cancer cells, and the PRPF19/MYL9 axis activates the Src-YAP1 cascade to promote metastasis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, gain- and loss-of-function migration/invasion assays, substrate validation\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP + ubiquitination assay + rescue experiments establish substrate relationship in a single study\",\n      \"pmids\": [\"37031206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MYL9 binds MYO19 via direct protein-protein interaction (confirmed by Co-IP and GST pull-down); MYL9/MYO19 complex suppresses epithelial-mesenchymal transition and migration in NSCLC cells.\",\n      \"method\": \"Co-immunoprecipitation, GST pull-down assay, scratch wound healing assay, Western blot for EMT markers\",\n      \"journal\": \"Physiological genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal binding assays (Co-IP and GST pull-down) plus functional rescue in single lab\",\n      \"pmids\": [\"39437553\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MYL9 deficiency in mice causes neonatal lethality with distended bladder, shortened small intestine, and alveolar overdistension; LacZ reporter knockin shows MYL9 expression is restricted to muscularis propria of intestine and bladder and bronchial smooth muscle, indicating MYL9 is required for smooth muscle cell function in these organs.\",\n      \"method\": \"Germline knockout mouse, LacZ reporter knock-in for expression mapping, gross and histological phenotyping\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo loss-of-function with defined organ-specific phenotype and direct expression mapping; single lab but rigorous genetic approach\",\n      \"pmids\": [\"35802750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RhoA-mediated phosphorylation of MLC2 regulates actin dynamics required for cytokinesis (polar body extrusion) during mouse oocyte meiosis; MLC2 knockdown causes failure of polar body extrusion and arrest at telophase with reduced cortical actin.\",\n      \"method\": \"siRNA knockdown in mouse oocytes, immunofluorescence staining of p-MLC2 and actin, RhoA inhibition\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with defined cytokinesis phenotype plus RhoA epistasis, single lab\",\n      \"pmids\": [\"26701676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MYL9 in cancer-associated fibroblasts binds IQGAP1 and through ERK1/2 signalling regulates secretion of CCL2 and TGF-β1, which in turn activate the PI3K-AKT pathway in CRC tumour cells to promote progression; ZEB1 binds the MYL9 promoter in CAFs to enhance MYL9 expression.\",\n      \"method\": \"Co-immunoprecipitation, ChIP, dual luciferase assay, ELISA, siRNA knockdown, co-culture assay\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP confirms IQGAP1 binding, ChIP confirms ZEB1-MYL9 promoter interaction, with functional secretion readouts; single lab\",\n      \"pmids\": [\"37926835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ITPRIP recruits MYL9 to the kinase domain of DAPK1, forming an ITPRIP-DAPK1-MYL9 ternary complex; this interaction impedes DAPK1-mediated phosphorylation of MYL9 and promotes glioma progression; MYL9 knockdown does not disrupt the ITPRIP-DAPK1 association.\",\n      \"method\": \"Lentivirus co-infection, co-immunoprecipitation, knockdown of ITPRIP/DAPK1/MYL9, in vitro and in vivo tumour growth assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP establishes ternary complex; functional epistasis by differential KDs; single lab\",\n      \"pmids\": [\"34111521\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"MYL9 interacts with MR-1 (myofibrillogenesis regulator 1); MR-1 knockdown decreases MYL9 phosphorylation and disrupts stress fiber formation, reducing hepatoma cell proliferation, migration, and adhesion via the MLC2/FAK/Akt pathway.\",\n      \"method\": \"siRNA knockdown, Western blot for phospho-MLC2/FAK/Akt, migration/adhesion assay, stress-fiber imaging\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — KD with multiple phosphorylation readouts and functional assays; interaction established previously, single lab\",\n      \"pmids\": [\"18948272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Phosphorylation (pSer19) and O-GlcNAcylation of slow MYL9 (sMLC2) are mutually exclusive post-translational modifications in skeletal muscle; a multienzymatic complex including kinase, phosphatase, OGT, and OGA is colocalized at the Z-disc and modulates these modifications; hindlimb unloading (atrophy) decreases O-GlcNAcylation and increases phosphorylation of sMLC2, reversed upon reloading.\",\n      \"method\": \"2D-gel electrophoresis, phospho-specific dye staining, immunoblotting, co-localization immunofluorescence, rat hindlimb unloading model\",\n      \"journal\": \"Pflugers Archiv : European journal of physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical demonstration of mutually exclusive PTMs with enzyme co-localization; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"24477671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Endogenous myosin light chain kinase phosphorylates MLC2/MYL9 at approximately 50% of sites after Ca2+ activation in fast-twitch rat skeletal muscle fibres; MLC2 phosphorylation markedly increases the Ca2+ sensitivity of the contractile apparatus and follows Ca2+ rise with a 1–2 s delay.\",\n      \"method\": \"Mechanically skinned single fibre preparation, 32P-radiolabelled phosphorylation assay, force-Ca2+ relationship measurement\",\n      \"journal\": \"Pflugers Archiv : European journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro phosphorylation assay in single fibres with quantitative Ca2+-sensitivity readout; foundational mechanistic study\",\n      \"pmids\": [\"8351204\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"An 82-bp cardiac-specific enhancer in the Xenopus XMLC2 (MLC2) proximal promoter, composed of two GATA sites and a composite YY1/CArG-like site, is necessary and sufficient for heart-specific expression; the low-affinity SRF site is essential and requires at least one adjacent GATA site; YY1 acts primarily as a repressor of ectopic expression.\",\n      \"method\": \"Transgenic frog embryo reporter assay, deletion and site-specific mutation analysis of promoter elements, transgene expression in mice\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vivo transgenic assay with systematic deletion and site-mutation analysis, validated cross-species in mice\",\n      \"pmids\": [\"14711876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"In C. elegans, mlc-2 (regulatory myosin light chain) is required for pharyngeal muscle function; mlc-1 and mlc-2 are redundant in body-wall muscle; transgenic copies of either mlc-1+ or mlc-2+ fully rescue all defects of mlc-1(0) mlc-2(0) double mutants including pharyngeal and body-wall muscle defects.\",\n      \"method\": \"Genetic deletion mutants, double mutant analysis, transgenic rescue, in situ hybridization\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — classic genetic epistasis with transgenic rescue in two tissues; ortholog of MYL9 in established model organism\",\n      \"pmids\": [\"9799259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cardiac-specific MLC kinase (cMLCK) localises predominantly at the Z-disc where it interacts with α-actinin2 with high affinity (Kd ~13.4 nM) via the cardiac-isoform-specific N-terminus; a cMLCK mutant deficient for α-actinin2 binding fails to promote sarcomere organisation or increase cardiomyocyte size, indicating Z-disc anchoring is required for cMLCK's role in sarcomere organisation upstream of MLC2/MYL9 phosphorylation.\",\n      \"method\": \"Subcellular fractionation/immunofluorescence localisation, kinetic binding assay, N-terminal deletion and point mutants, cardiomyocyte overexpression\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localisation plus kinetic binding measurement and mutant analysis; single lab\",\n      \"pmids\": [\"31467300\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"12(S)-HETE activates a 12-HETE receptor → RHO → ROCK → MYPT → MLC2 signalling cascade in lymph endothelial cells to phosphorylate MLC2, driving actomyosin contraction, LEC retraction, and endothelial barrier breaching; MLC2 siRNA abolishes LEC retraction.\",\n      \"method\": \"siRNA knockdown of MLC2, RHO/ROCK inhibitors, Western blot for p-MLC2, CCID barrier breaching assay\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown plus inhibitor epistasis with defined functional readout; single lab\",\n      \"pmids\": [\"27362730\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DDX3X promotes MYL9 expression in pancreatic cancer by destabilising TLE2 mRNA, releasing KLF4-mediated repression of MYL9; elevated MYL9 remodels F-actin and enhances tumour cell traction forces, facilitating metastasis.\",\n      \"method\": \"CRISPR-Cas9 screen, mRNA stability assays, transcription factor ChIP, traction force microscopy, in vivo orthotopic xenograft\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen + mechanistic follow-up in vivo; single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"40938994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Transfection of MYL9 into MYL9-negative Jurkat cells significantly increases surface CD3 expression, establishing that MYL9 enables a distinct pathway for CD3 surface expression independent of the PKC-dependent pathway.\",\n      \"method\": \"MYL9 gene transfection into Jurkat cells, flow cytometry for surface CD3, PMA stimulation\",\n      \"journal\": \"Journal of smooth muscle research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single gain-of-function transfection with defined surface-expression phenotype; moderate confidence limited by single method\",\n      \"pmids\": [\"23538510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DAPK3, released from LUZP1-mediated suppression by COP1-mediated LUZP1 ubiquitination and degradation, phosphorylates and activates MYL9 to promote EMT and colorectal cancer liver metastasis.\",\n      \"method\": \"Patient-derived organoid multi-omics (WES, bulk and scRNA-seq), Co-IP, in vitro and in vivo functional assays\",\n      \"journal\": \"Experimental hematology & oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — organoid multi-omics plus in vitro/in vivo functional validation; single lab, novel pathway placement\",\n      \"pmids\": [\"41937206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RUNX1 isoforms B and C differentially regulate MYL9 expression in megakaryocytes/platelets: RUNX1B negatively correlates with MYL9 transcripts in platelets while RUNX1C positively regulates target genes; both isoforms bind and regulate the RUNX1 P1 and P2 promoters.\",\n      \"method\": \"ChIP, luciferase promoter assay, RUNX1 isoform overexpression in HEL and HeLa cells, platelet RNAseq from 85 volunteers\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and reporter assays in cell lines plus human platelet RNAseq; preprint, not peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2024.06.18.599563\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"MYL9 (MLC2) is a myosin II regulatory light chain whose phosphorylation at Ser19 (by MLCK, ROCK, PKM2, and other kinases) is the central activating switch for actomyosin contractility in smooth, cardiac, skeletal, and non-muscle cells; it is regulated at multiple levels—transcriptionally by RUNX1, MAL/SRF, MRTF-A/SMYD3, Junb, and GATA/SRF cardiac enhancers; post-translationally by competing N-terminal acetylation (promoting cytoplasmic/contractile roles via increased pSer19) and N-terminal methylation (promoting nuclear/DNA-binding roles), as well as by mutually exclusive O-GlcNAcylation at the same residues as phosphorylation; protein stability is regulated by K63-linked ubiquitination via PRPF19; and it participates in direct protein complexes with MR-1, IQGAP1, MYO19, and DAPK1/ITPRIP, linking it to FAK/Akt signalling, ERK-mediated cytokine secretion, EMT suppression, and tumour cell traction-force generation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MYL9 (MLC2) is the regulatory light chain of myosin II whose reversible phosphorylation operates as the central activating switch for actomyosin contractility across smooth, cardiac, skeletal, and non-muscle cells, and across processes from muscle contraction to cytokinesis and cell migration [#17, #11]. The activating modification is phosphorylation at the conserved serine (Ser19/Ser15) and an adjacent priming tyrosine: in fast-twitch skeletal fibres MLCK phosphorylates MLC2 in a Ca2+-dependent manner to raise the Ca2+ sensitivity of the contractile apparatus [#17], while PKM2-mediated Y118 phosphorylation primes ROCK2 binding and subsequent S15 phosphorylation during cytokinesis [#0]. Multiple upstream Rho-kinase cascades converge on this switch to control distinct contractile outputs—VE-cadherin/ROCK signalling generates junctional contractility that restrains endothelial sprouting [#4], RhoA-driven phosphorylation drives polar body extrusion in oocyte meiosis [#12], and a 12(S)-HETE→RHO→ROCK→MYPT→MLC2 cascade drives lymph-endothelial retraction [#21]. Genetic ablation of Myl9 in mice causes neonatal lethality with smooth-muscle organ failure, establishing its non-redundant requirement in intestinal, bladder, and bronchial smooth muscle [#11], a role mirrored by the redundant requirement of its C. elegans ortholog mlc-2 in pharyngeal and body-wall muscle [#19]. MYL9 expression is set transcriptionally by a network including RUNX1 [#1], MAL/SRF and MRTF-A/SMYD3 [#2, #7], Junb [#3], and cardiac GATA/SRF enhancers [#18], and its protein output is tuned post-translationally by mutually exclusive Ser19 phosphorylation versus O-GlcNAcylation [#16] and by competing N-terminal acetylation versus methylation that partition MYL9 between cytoplasmic/contractile and nuclear/DNA-binding roles [#6]. In cancer, MYL9 acts as an effector node for traction-force generation, migration, invasion, and EMT control through physical partners and regulators including IQGAP1, MYO19, MR-1, the ITPRIP-DAPK1 complex, and PRPF19-mediated K63 ubiquitination [#13, #10, #15, #14, #9, #22].\",\n  \"teleology\": [\n    {\n      \"year\": 1993,\n      \"claim\": \"Established the core biochemical function: that MLCK-mediated MLC2 phosphorylation is the modification that tunes contractile force, answering how light-chain phosphorylation translates into mechanical output.\",\n      \"evidence\": \"32P phosphorylation assay and force-Ca2+ measurement in skinned fast-twitch rat skeletal fibres\",\n      \"pmids\": [\"8351204\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the specific phosphorylated residue at sequence level\", \"Restricted to fast-twitch skeletal muscle\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Showed the regulatory light chain is genetically required for muscle function and is functionally interchangeable with a paralog, establishing its non-redundant tissue role in an intact organism.\",\n      \"evidence\": \"Genetic deletion, double-mutant epistasis, and transgenic rescue in C. elegans pharyngeal and body-wall muscle\",\n      \"pmids\": [\"9799259\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ortholog system, not human MYL9\", \"Did not address phosphoregulation\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined the cis-regulatory logic for cardiac-restricted MLC2 expression, answering how the gene is spatially controlled in the heart.\",\n      \"evidence\": \"Transgenic frog/mouse reporter assays with deletion and site-specific promoter mutagenesis (GATA/SRF/YY1 elements)\",\n      \"pmids\": [\"14711876\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Concerns Xenopus XMLC2 enhancer; human enhancer conservation not directly tested\", \"Does not address non-cardiac expression control\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Placed MLC2-dependent contractility at a spatial point of action—endosome-localized Rho-ROCK-MLC2 signals driving adhesion disassembly—linking contractility to migration mechanics.\",\n      \"evidence\": \"Live-cell imaging, subcellular localization, ROCK inhibition, Endo180 knockdown\",\n      \"pmids\": [\"17043135\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct MLC2 phosphorylation at endosome sites inferred from pathway, not site-mapped\", \"Endo180-MLC2 physical link not established\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified upstream transcriptional and signalling inputs—MAL/SRF setting MYL9 expression in megakaryocytes and VE-cadherin/ROCK driving junctional MLC2 contractility—connecting MYL9 to platelet formation and angiogenic restraint.\",\n      \"evidence\": \"ChIP, luciferase, shRNA rescue (megakaryocytes); organotypic and zebrafish angiogenesis with ROCK/actomyosin inhibition\",\n      \"pmids\": [\"19724058\", \"19345098\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"MAL/SRF and VE-cadherin pathways studied in separate systems\", \"Quantitative contribution of MYL9 vs other SRF targets not isolated in angiogenesis\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined RUNX1 and Junb as direct transcriptional regulators of MYL9, linking its expression to cell spreading and vascular smooth-muscle contractility.\",\n      \"evidence\": \"ChIP/EMSA/reporter with siRNA (RUNX1); conditional knockout with Myl9 re-expression rescue (Junb)\",\n      \"pmids\": [\"20876458\", \"20551518\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How these TFs integrate with SRF/MRTF inputs at the MYL9 promoter is unresolved\", \"Post-translational consequences downstream not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved a cooperative transcriptional mechanism in which MRTF-A and SMYD3 transactivate MYL9 to drive cancer cell migration, extending SRF-pathway control into tumour cell motility.\",\n      \"evidence\": \"Reporter/promoter-mutation, Co-IP, siRNA, and migration assays in MCF-7 cells\",\n      \"pmids\": [\"24189459\", \"24084383\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Both studies from related work in one cell type\", \"SMYD3 methylation substrate at the locus not directly mapped\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Uncovered priming-then-activation phospho-control (PKM2 Y118 priming ROCK2-dependent S15) during cytokinesis and a mutually exclusive Ser19-phosphorylation/O-GlcNAcylation switch in skeletal muscle, refining how the activating modification is gated.\",\n      \"evidence\": \"In vitro kinase assays, mutagenesis, Co-IP in tumour cells; 2D-gel, phospho/O-GlcNAc immunoblotting and enzyme co-localization in rat muscle\",\n      \"pmids\": [\"25412762\", \"24477671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Y118 priming demonstrated in tumour cytokinesis; generality to other contexts untested\", \"Stoichiometry of competing O-GlcNAc vs phosphorylation in vivo not quantified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified N-terminal acetylation versus methylation as competing modifications that partition MYL9 between cytoplasmic contractile and nuclear DNA-binding pools, revealing a non-canonical nuclear role.\",\n      \"evidence\": \"In vitro NatA/NRMT1 enzymatic assays, modification-exclusive mutants, phosphorylation and DNA-binding readouts\",\n      \"pmids\": [\"30242065\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear DNA targets and transcriptional consequences not identified\", \"In vivo balance of the two N-terminal modifications unmeasured\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mapped MYL9 into discrete cancer protein complexes—IQGAP1 (CAF cytokine secretion) and an ITPRIP-DAPK1 ternary complex shielding MYL9 from DAPK1 phosphorylation—showing context-specific protein partners modulate its phospho-state and signalling output.\",\n      \"evidence\": \"Co-IP, ChIP, ELISA, co-culture (IQGAP1/ZEB1); reciprocal Co-IP and differential knockdown (ITPRIP-DAPK1-MYL9)\",\n      \"pmids\": [\"37926835\", \"34111521\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab complex characterizations without structural validation\", \"Direct binding interfaces not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established protein-stability and additional binding control of MYL9—PRPF19 K63-ubiquitination stabilizing it to drive Src-YAP1 metastasis, and direct MYO19 binding suppressing EMT—broadening regulation beyond phosphorylation.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, GST pull-down, and migration/invasion assays in CRC and NSCLC cells\",\n      \"pmids\": [\"37031206\", \"39437553\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitination site on MYL9 not mapped\", \"MYO19 and PRPF19 effects characterized in different cancers without cross-validation\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Positioned MYL9 as a downstream effector of mechanotransduction in cancer, where DDX3X-driven derepression elevates MYL9 to remodel F-actin and increase traction forces for metastasis.\",\n      \"evidence\": \"CRISPR-Cas9 screen, mRNA stability assays, ChIP, traction-force microscopy, orthotopic xenograft\",\n      \"pmids\": [\"40938994\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether traction-force increase requires MYL9 phosphorylation not isolated\", \"Single-cancer-type validation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple input layers—transcriptional networks, competing N-terminal acetylation/methylation, O-GlcNAc/phospho switching, and ubiquitination—are integrated to set the cytoplasmic-versus-nuclear and contractile-versus-migratory balance of MYL9 in a given cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified quantitative model of competing PTMs\", \"Nuclear DNA-binding function lacks identified targets\", \"No human disease-causing mutation reported in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [17, 0, 11]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [17, 11]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [2, 15, 5]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-397014\", \"supporting_discovery_ids\": [17, 11]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 12]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 21, 15]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [9, 13, 14, 22]}\n    ],\n    \"complexes\": [\n      \"ITPRIP-DAPK1-MYL9 ternary complex\",\n      \"myosin II\"\n    ],\n    \"partners\": [\n      \"ROCK2\",\n      \"PKM2\",\n      \"IQGAP1\",\n      \"MYO19\",\n      \"DAPK1\",\n      \"ITPRIP\",\n      \"PRPF19\",\n      \"MR-1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}