{"gene":"ROCK1","run_date":"2026-06-10T06:43:37","timeline":{"discoveries":[{"year":1996,"finding":"The Rho-binding domain of p160ROCK was mapped to amino acids 934–1015 within the carboxyl half of the alpha-helix (residues 727–1021). Point mutations K934M, L941A, E1008A weakened Rho binding, and I1009A abolished it, defining a distinct class of Rho effector motif different from the class-1 Rho effector motif shared by PKN, rhophilin, and rhotekin.","method":"Ligand overlay assay using [35S]GTPγS-bound GST-RhoA, yeast two-hybrid system, point mutagenesis, recombinant His-tagged protein fragments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro binding assay with recombinant proteins plus mutagenesis, corroborated by yeast two-hybrid; multiple orthogonal methods in a single focused study","pmids":["8798490"],"is_preprint":false},{"year":1997,"finding":"p160ROCK acts downstream of Rho to induce focal adhesions and stress fibers. Expression of wild-type p160ROCK or constructs containing both the kinase domain and coiled-coil region induced focal adhesions and stress fibers in HeLa cells, whereas a kinase-defective mutant or a mutant lacking the coiled-coil region did not. A dominant-negative mutant defective in both kinase and Rho-binding activities blocked Rho-induced focal adhesion formation but not Rho-induced actin polymerization, indicating a separate effector pathway for the latter.","method":"Transfection of wild-type and kinase-dead/domain deletion mutants of p160ROCK into HeLa cells; morphological analysis of focal adhesions and stress fibers; co-expression epistasis","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple domain-deletion and kinase-dead mutants with epistasis analysis; widely replicated finding foundational to the field","pmids":["9119047"],"is_preprint":false},{"year":1998,"finding":"p160ROCK activation is necessary and sufficient for agonist-induced neurite retraction and cell rounding in N1E-115 neuroblastoma cells. This was accompanied by elevated myosin light chain (MLC) phosphorylation and disassembly of intermediate filaments and microtubules. The p160ROCK-specific inhibitor Y-27632 blocked both neurite retraction and MLC phosphorylation at similar concentrations. Dominant-negative p160ROCK induced neurite formation in the presence of serum; this outgrowth was blocked by dominant-negative Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates Cdc42/Rac-dependent neurite formation.","method":"Dominant-active and dominant-negative p160ROCK constructs; pharmacological inhibition with Y-27632; immunofluorescence for MLC phosphorylation, intermediate filaments, and microtubules; epistasis with dominant-negative Cdc42/Rac","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic and pharmacological approaches with orthogonal readouts; replicated across cell types","pmids":["9647654"],"is_preprint":false},{"year":1998,"finding":"p160ROCK is an upstream activator of the Na-H exchanger NHE1, acting in the RhoA pathway. Constitutively active p160ROCK stimulated NHE1 activity and phosphorylated NHE1 in vivo and in vitro, and identical NHE1 residues were phosphorylated in both settings. NHE1 activity and phosphorylation were required for p160ROCK-induced actin stress fiber assembly. Y-27632 blocked RhoA- and LPA-induced NHE1 activity and NHE1 phosphorylation. Dominant-interfering p160ROCK blocked RhoA-induced but not Cdc42- or Rac-induced NHE1 activation.","method":"In vitro kinase assay of p160ROCK on NHE1 C-terminal fusion protein; phosphopeptide mapping comparing in vivo and in vitro phosphorylation; mutationally active/dominant-interfering p160ROCK constructs; NHE1 inhibitor (EIPA); NHE1-deficient PS120 cells; Y-27632 pharmacology","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with phosphopeptide mapping corroborating in vivo phosphorylation; multiple genetic and pharmacological approaches","pmids":["9707430"],"is_preprint":false},{"year":2000,"finding":"p160ROCK inhibition in cultured cerebellar granule neurons triggered immediate outgrowth of membrane ruffles and filopodia and generation of axonal processes, potentiated growth cone size and motility, and overexpression of dominant-active Rho or ROCK prevented initiation of axon outgrowth. This establishes the Rho/ROCK pathway as a gate controlling axon outgrowth initiation and growth cone dynamics, likely through regulation of the cortical actin network.","method":"Dominant-negative and dominant-active p160ROCK/Rho constructs transfected into cerebellar granule neurons; Y-27632 pharmacological inhibition; live imaging of growth cone dynamics","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 / Strong — combined genetic (dominant-active/negative) and pharmacological approach with specific neuronal phenotypic readouts; replicated across multiple studies","pmids":["10839361"],"is_preprint":false},{"year":2001,"finding":"RhoA-p160ROCK signaling mediates lysophosphatidic acid (LPA)-induced stress fiber formation, myosin light chain phosphorylation, and occludin phosphorylation in ECV304 cells. A dominant-negative RhoA or Y-27632 blocked all three LPA-induced events and prevented increased paracellular permeability. For histamine, RhoA/p160ROCK blocked cytoskeletal events but not occludin phosphorylation or increased flux, indicating that occludin phosphorylation can regulate tight junction permeability independently of cytoskeletal events.","method":"Dominant-negative RhoA construct; Y-27632 pharmacological inhibition; immunoblot for MLC and occludin phosphorylation; horseradish peroxidase flux assay for tight junction permeability","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — combined genetic and pharmacological approaches with two distinct agonists; multiple orthogonal readouts (MLC phosphorylation, occludin phosphorylation, permeability assay)","pmids":["11139571"],"is_preprint":false},{"year":2002,"finding":"p160ROCK is a centrosomal component localizing principally to the mother centriole and an intercentriolar linker. Inhibition of p160ROCK caused centrosome splitting in G1, with the mother centriole displaying wide excursions to the cell periphery. p160ROCK inhibition late after anaphase triggered mother centriole migration to the midbody and completion of cell division, establishing a role for p160ROCK in centrosome positioning and centrosome-dependent exit from mitosis.","method":"Immunofluorescence with multiple p160ROCK antibodies; immunoelectron microscopy; siRNA depletion; centrosome tracking/live cell analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — immunoEM for localization, siRNA depletion with specific phenotypic readout, multiple antibodies; single lab but multiple orthogonal methods","pmids":["12034773"],"is_preprint":false},{"year":2003,"finding":"TGF-β stimulates RhoA and p160ROCK activity in epithelial NMuMG cells, leading to p160ROCK translocation to the nucleus where it phosphorylates the cell-cycle phosphatase Cdc25A. This inhibitory phosphorylation of Cdc25A suppresses Cdk2 activity and contributes to TGF-β-induced G1/S cell cycle arrest. In NIH 3T3 fibroblasts, which lack RhoA/p160ROCK activity in the TGF-β response, growth inhibition is absent.","method":"Dominant-negative/active RhoA and p160ROCK constructs; subcellular fractionation and immunofluorescence for p160ROCK nuclear localization; Cdc25A phosphorylation assay; Cdk2 kinase assay; cell cycle analysis; genetic epistasis between NMuMG and NIH 3T3 backgrounds","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — nuclear translocation supported by fractionation/immunofluorescence; kinase assays for Cdc25A; single lab","pmids":["14657354"],"is_preprint":false},{"year":2004,"finding":"p160ROCK associates with Aurora-A/STK15 in a protein complex. siRNA suppression of Aurora-A blocked mitotic spindle organization and caused G2/M arrest. Simultaneous suppression of p160ROCK by RNAi rescued abnormal spindle formation, released G2/M arrest, and alleviated apoptosis, leading to aneuploid cell accumulation. This epistasis places Aurora-A and p160ROCK in a common pathway governing G2/M progression.","method":"Co-immunoprecipitation of Aurora-A and p160ROCK; siRNA knockdown of Aurora-A and p160ROCK individually and in combination; cell cycle analysis; spindle immunofluorescence","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus double-knockdown epistasis; single lab, two orthogonal methods","pmids":["15178765"],"is_preprint":false},{"year":2006,"finding":"ROCK1 directly phosphorylates zipper-interacting protein kinase (ZIPK) at Thr-265 and Thr-299, stoichiometrically activating ZIPK. In HeLa cells, co-expression of ZIPK with ROCK1 shifted the actin phenotype from ROCK-induced focused stress fibers to a Rho-like parallel stress fiber pattern in a Thr-265 phosphorylation-dependent manner, identifying ROCK1 as an upstream activator of ZIPK in smooth muscle and non-muscle cells.","method":"Proteomics screen with synthetic ZIPK phosphopeptides followed by mass spectrometry identification of ROCK1; in vitro kinase assay with native ZIPK showing stoichiometric phosphorylation at Thr-265 and Thr-299; co-expression in HeLa cells; phospho-site mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with stoichiometric phosphorylation, substrate identified by unbiased proteomics/MS, and mutagenesis validated in cells; single lab but multiple orthogonal methods","pmids":["17158456"],"is_preprint":false},{"year":2009,"finding":"RhoA and activated ROCK1 are sequestered into stress granules (SGs) upon cellular stress. Sequestration of activated ROCK1 into SGs prevents ROCK1 from interacting with JIP-3 (JNK-interacting protein 3) and activating the pro-apoptotic JNK pathway, thereby protecting cells from apoptosis. This defines a regulatory mechanism whereby ROCK1 activity determines cell fate through either promoting SG formation or initiating JNK-dependent apoptosis.","method":"Immunofluorescence co-localization of ROCK1/RhoA with SG markers; co-immunoprecipitation of ROCK1 with JIP-3 under stress vs. non-stress conditions; RhoA/ROCK1 dominant-active constructs; JNK activation assay","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP of ROCK1-JIP3 interaction linked to JNK activation; localization by immunofluorescence; single lab","pmids":["20004716"],"is_preprint":false},{"year":2012,"finding":"ROCK1 deficiency in MEFs attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased MLC2 phosphorylation but preserved cofilin phosphorylation, reducing cell shrinkage, detachment, and pre-detachment apoptosis. ROCK2 deficiency instead reduced phosphorylation of both MLC2 and cofilin, with different actin phenotypes. This establishes ROCK1 as specifically regulating MLC2 phosphorylation and peripheral actomyosin contraction, while ROCK2 regulates cofilin phosphorylation for cytoskeletal stabilization.","method":"ROCK1-/- and ROCK2-/- MEF genetic knockout; immunoblot for p-MLC2 and p-cofilin; pharmacological inhibitors (blebbistatin, cytochalasin D, Y27632); fluorescence microscopy of actin cytoskeleton","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Strong — isoform-specific knockout mice with orthogonal pharmacological confirmation; multiple biochemical readouts","pmids":["23392171"],"is_preprint":false},{"year":2012,"finding":"ROCK1 inhibition indirectly reduces the activity of upstream RhoA through stimulation of Tiam1-induced Rac1 activity, revealing a feedback loop by which ROCK1 modulates the functional balance of Rho-family GTPases.","method":"Combined pharmacological (ROCK1 inhibitors) and genetic (siRNA knockdown) approach; Rac1 and RhoA activity assays (pull-down/G-LISA); Tiam1 involvement assessed by knockdown","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — combined pharmacological and genetic approach with activity assays; single lab, two orthogonal methods","pmids":["22430126"],"is_preprint":false},{"year":2013,"finding":"ROCK1 in bone marrow-derived (leukocyte) cells mediates neointima formation following vascular injury. Haploinsufficient Rock1+/- mice showed reduced neointima formation, smooth muscle cell proliferation/survival, adhesion molecule expression, and leukocyte infiltration compared to WT and Rock2+/- mice. Reciprocal bone marrow transplantation confirmed that leukocyte-derived ROCK1, not vessel wall ROCK1, drives neointima formation.","method":"Rock1+/- and Rock2+/- haploinsufficient mice; carotid artery ligation model; reciprocal bone marrow transplantation; histology/immunohistochemistry; peritoneal leukocyte recruitment assay","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic haploinsufficiency plus reciprocal bone marrow transplantation with clear isoform-specific phenotype","pmids":["18414683"],"is_preprint":false},{"year":2013,"finding":"HIF-1α and HIF-2α (hypoxia-inducible factors) coordinately activate RhoA and ROCK1 expression in breast cancer cells, leading to MLC and FAK phosphorylation, actin-myosin contractility, focal adhesion formation, and cell motility. This places ROCK1 as a transcriptional target of HIFs and a node in the HIF→RhoA→ROCK1→MLC→FAK oncogenic signaling cascade.","method":"HIF1A/HIF2A knockdown and overexpression; immunoblot for p-MLC and p-FAK; cell contraction and motility assays; luciferase reporter for RhoA and ROCK1 promoters","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transcriptional reporter assays and knockdown/OE with biochemical readouts; single lab, two orthogonal methods","pmids":["24324133"],"is_preprint":false},{"year":2014,"finding":"FRMD5, a FERM domain protein, interacts physically with ROCK1 and inhibits its activation, resulting in reduced myosin light chain phosphorylation and reduced actin stress fiber formation, thereby inhibiting cell migration in lung cancer cells. FRMD5 also interacts with integrin β5 cytoplasmic tail, defining a dual pathway for migration inhibition.","method":"Co-immunoprecipitation of FRMD5 with ROCK1 and integrin β5; ROCK1 kinase activity assay; immunofluorescence for stress fibers and p-MLC; FRMD5 overexpression/knockdown with migration assays","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP of FRMD5-ROCK1 interaction linked to kinase activity and phenotypic readout; single lab","pmids":["25448675"],"is_preprint":false},{"year":2015,"finding":"ROCK1 mediates formation of stable actomyosin filament bundles that initiate front-back polarity in migrating fibroblasts and dendritic spine polarity in neurons. ROCK2 regulates contractile force, Rac1 activity at the leading edge, and cofilin-mediated actin remodeling at adhesions and spine heads, defining distinct downstream pathways for the two ROCK isoforms.","method":"ROCK1 and ROCK2 isoform-specific shRNA knockdown; live-cell imaging of actin dynamics; immunofluorescence for actomyosin bundles and adhesions; cofilin phosphorylation assay; migration and spine morphology analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — isoform-specific knockdown with multiple phenotypic and biochemical readouts in two distinct biological contexts (migration and synapse); single lab but multiple orthogonal methods","pmids":["26169356"],"is_preprint":false},{"year":2015,"finding":"EV-encapsulated caspase-3 from hyperoxia-injured lung epithelial cells activates alveolar macrophages via the ROCK1 pathway, inducing pro-inflammatory cytokine secretion and neutrophil infiltration. This defines a caspase-3→ROCK1 signaling axis in macrophage activation by extracellular vesicles.","method":"In vitro macrophage treatment with isolated EVs; ROCK1 inhibitor treatment; caspase-3-deficient EV generation; BALF cytokine/cell count analysis; in vivo intranasal EV delivery in mice","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological ROCK1 inhibition and caspase-3 KO EVs with in vitro and in vivo readouts; single lab","pmids":["26658190"],"is_preprint":false},{"year":2016,"finding":"ROCK1 stimulates endothelial-to-mesenchymal transition (EndMT) in glomerular endothelial cells under high-glucose conditions. Inhibition of ROCK1 blocked EndMT and increased endothelial permeability, while ROCK1 overexpression induced these changes. In db/db mice, fasudil (ROCK1 inhibitor) suppressed α-SMA expression in glomerular endothelium and reduced albuminuria, establishing ROCK1 as a driver of diabetic glomerular injury.","method":"ROCK1 overexpression and inhibition (fasudil) in glomerular endothelial cells; immunofluorescence for EndMT markers (CD31, α-SMA); permeability assay; db/db mouse model treated with fasudil","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic overexpression and pharmacological inhibition in vitro with in vivo confirmation; single lab","pmids":["26842599"],"is_preprint":false},{"year":2016,"finding":"ROCK1 deficiency in bone marrow-derived mast cells results in delayed maturation in response to IL-3, reduced growth in response to SCF, and impaired integrin-mediated adhesion/migration and IgE-mediated degranulation. Mechanistically, ROCK1 acts through LIM kinase (LIMK) downstream in mast cells; ROCK1-deficient BMMCs show reduced activation of ROCK, LIMK, AKT, and ERK1/2 upon SCF stimulation.","method":"Rock1-/- BMMC derivation; IL-3/SCF stimulation assays; integrin adhesion and migration assays; IgE degranulation assay; immunoblot for ROCK, LIMK, AKT, ERK1/2 phosphorylation; passive cutaneous anaphylaxis model","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with multiple orthogonal functional assays and biochemical pathway analysis; in vivo confirmation","pmids":["26943578"],"is_preprint":false},{"year":2018,"finding":"ROCK1 directly interacts with ZIPK in a complex identified by mass spectrometry, phosphorylates ZIPK at Thr-265 and Thr-299 to activate it. Crystal/co-crystal structures of early lead compounds were obtained in PKA, ROCK1, and ROCK2, identifying key residues (Asp176 and Asp218 in ROCK2; Glu in PKA) in the ATP-binding site that distinguish ROCK kinase selectivity from the broader kinome.","method":"Co-crystal structures of ROCK1 and ROCK2 with inhibitors; structure-based drug design; enzyme assays; cell-based assays","journal":"Journal of medicinal chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures of ROCK1 and ROCK2 with functional validation in enzyme and cell assays; single lab","pmids":["30384606"],"is_preprint":false},{"year":2018,"finding":"ROCK1 deficiency restores autophagic flux and reduces doxorubicin-induced cardiotoxicity. Molecular analysis showed that ROCK1 knockout reduced Beclin 1-mediated autophagy initiation by increasing association of Beclin 1 with Bcl-2/Bcl-XL, associated with reduced Beclin 1 phosphorylation at Thr119. Cardiomyocyte-specific ROCK1 knockout partially reproduced the cardioprotective phenotype, indicating ROCK1 in both cardiomyocytes and non-cardiomyocytes mediates doxorubicin cardiotoxicity.","method":"ROCK1-/- and MHC-Cre/ROCK1fl/fl mice; doxorubicin cardiotoxicity model; immunoprecipitation of Beclin 1 with Bcl-2/Bcl-XL; immunoblot for p62/SQSTM1, LC3-II, Beclin 1 phosphorylation; apoptosis markers","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic models (global and cardiomyocyte-specific KO) with Co-IP and biochemical pathway analysis","pmids":["29560126"],"is_preprint":false},{"year":2018,"finding":"lnc-31, a cytoplasmic lncRNA required for myoblast proliferation, promotes ROCK1 protein synthesis by stabilizing the translational activator YB-1. lnc-31 binds both Rock1 mRNA and the YB-1 protein, and physical interaction between the two RNA species is required for translational activation of Rock1.","method":"RNA immunoprecipitation of lnc-31 with YB-1 and Rock1 mRNA; lnc-31 knockdown/overexpression with ROCK1 protein measurement; ribosome profiling/polysome fractionation; interaction domain mapping","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNA-protein IP and RNA-RNA interaction validated with functional consequence on translation; single lab","pmids":["29669280"],"is_preprint":false},{"year":2019,"finding":"ROCK1 is a key non-redundant positive regulator of apoptotic membrane blebbing and apoptotic body (ApoBD) formation. CRISPR/Cas9 deletion of ROCK1, but not PAK2 or LIMK1, abolished membrane blebbing and ApoBD formation. Inhibition of ApoBD formation reduced efficient uptake of apoptotic materials by phagocytes. ROCK1 also plays a role in regulating the onset of secondary necrosis.","method":"CRISPR/Cas9 knockout of ROCK1, PAK2, and LIMK1; pharmacological inhibition; time-lapse microscopy of blebbing; ApoBD quantification; phagocytosis assays","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR/Cas9 loss-of-function with pharmacological corroboration; isoform-specific comparison with PAK2 and LIMK1; multiple orthogonal readouts","pmids":["31043701"],"is_preprint":false},{"year":2022,"finding":"Rnd3 (a Rho family GTPase 3) directly binds ROCK1 in the cytoplasm and inhibits ROCK1-induced Drp1 phosphorylation at Ser616, thereby preventing mitochondrial fission, reducing PANoptosis, and protecting against doxorubicin-induced cardiotoxicity. Rock1 knockdown nullified the cardioprotective effects of Rnd3, placing ROCK1 as a key node in the Rnd3/ROCK1/Drp1 signaling axis.","method":"Co-immunoprecipitation and protein-protein docking of Rnd3 and ROCK1; phospho-Drp1 Ser616 immunoblot; Rnd3 transgenic and knockout mice; Rock1 siRNA knockdown in vitro; mitochondrial morphology imaging; in vivo cardiac function analysis","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 1 / Strong — Co-IP and molecular docking demonstrate physical interaction; ROCK1 substrate phosphorylation at defined site with genetic epistasis; multiple in vitro and in vivo models","pmids":["39755713"],"is_preprint":false},{"year":2022,"finding":"ROCK1 hyperactivates RhoA/ROCK1/actomyosin signaling downstream of p53 DNA-contact mutants and also mediates signaling required by recurrent Hippo pathway lesions, both of which drive oncogenic TEAD/YAP transcription. ROCK inhibitors selectively antagonized proliferation and motility of human tumors with either mechanism, demonstrating that ROCK1/actomyosin signaling is both necessary and sufficient for oncogenic TEAD/YAP transcriptional activation.","method":"p53 mutant expression; ROCK1 knockdown and inhibitor treatment; YAP localization/activity assay; actin cytoskeleton analysis; cell proliferation and motility assays in tumor cell lines; epistasis with Hippo pathway mutants","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic and pharmacological interventions with defined transcriptional and functional readouts; multiple cancer cell line contexts","pmids":["35121738"],"is_preprint":false},{"year":2022,"finding":"ROCK1 phosphorylates TFEB (transcription factor EB) as a previously unknown upstream kinase, independent of mTOR or GSK-3β, thereby regulating lysosomal biogenesis and acidification. ROCK1 elevation impaired lysosomal proteolysis; ROCK1 knockdown promoted TFEB nuclear distribution and lysosome-mediated Aβ clearance. The Rnd3/ROCK1 interaction was confirmed by surface plasmon resonance and proximity ligation assay.","method":"Surface plasmon resonance (SPR) and proximity ligation assay (PLA) for ROCK1-TFEB interaction; AAV-mediated ROCK1 knockdown in APP/PS1 mice; lysosomal acidification and biogenesis assays; TFEB nuclear localization immunofluorescence; behavioral testing","journal":"Translational neurodegeneration","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — SPR and PLA for protein interaction, genetic knockdown with multiple readouts; novel substrate claim requires in vitro kinase assay validation not fully described in abstract","pmids":["39497162"],"is_preprint":false},{"year":2022,"finding":"ROCK1/MLC2 (myosin light chain 2) signaling is activated in buffalopox virus-infected cells, and ROCK1 inhibition promotes deadenylation of viral mRNA by inhibiting PABP interaction with viral mRNA and enhancing expression of the CCR4-NOT deadenylation complex. ROCK1/MLC2-mediated cell contraction and perinuclear p-MLC2 accumulation positively correlated with viral mRNA/protein synthesis.","method":"ROCK1 siRNA depletion and pharmacological inhibitors (Thiazovivin, Y27632); viral mRNA decay assay; PABP co-immunoprecipitation; CCR4-NOT expression analysis; viral genome quantification; progeny virus titration","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA and pharmacological inhibition with mechanistic mRNA decay readout and Co-IP; single lab","pmids":["36280692"],"is_preprint":false},{"year":2022,"finding":"ROCK1 modulates YAP1 phosphorylation, promoting its nuclear localization and transcriptional activity in endometrial cancer in response to ECM stiffness. Co-immunoprecipitation confirmed ROCK1-YAP1 interaction; pharmacological ROCK1 inhibition with Y-27632 suppressed YAP1 nuclear localization and reduced tumor proliferation, migration, invasion, and chemotherapy resistance.","method":"Atomic force microscopy for ECM stiffness measurement; polyacrylamide gels of varying stiffness; co-immunoprecipitation of ROCK1-YAP1; immunofluorescence for YAP1 localization; Y-27632 pharmacological inhibition; xenograft mouse model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus pharmacological inhibition with functional readouts; mechanosensitive context established by AFM; single lab","pmids":["40368918"],"is_preprint":false},{"year":2024,"finding":"ROCK1 inhibits pThr172-AMPKα activity by binding to receptor-interacting serine/threonine kinase 4 (RIPK4), establishing a ROCK1/RIPK4/AMPK pathway. Fasudil and ROCK1 siRNA elevated pThr172-AMPKα, increased eNOS activity, and suppressed mitochondrial ROS in endothelial cells, accelerating wound repair in diabetic mice.","method":"Co-immunoprecipitation of ROCK1 with RIPK4 (integrated bioinformatics + Co-IP); ROCK1 siRNA and fasudil treatment; pAMPKα immunoblot; eNOS activity assay; mtROS measurement; diabetic wound healing model in vivo","journal":"Acta pharmacologica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP identifying ROCK1-RIPK4 interaction linked to AMPK pathway; pharmacological and genetic validation in vitro and in vivo; single lab","pmids":["38538716"],"is_preprint":false},{"year":2024,"finding":"Kinase-specific proteomics of leukocytes from corticosteroid-refractory aGVHD patients identified ROCK1 as the most significantly upregulated kinase. ROCK1/2 inhibition or ROCK1 knockdown in macrophages/dendritic cells reduced CD80, CD86, MHC-II expression and IL-6, IL-1β, iNOS, TNF production, impaired T cell activation and cytoskeletal rearrangements (reducing macrophage and DC migration), and reduced NF-κB signaling in myeloid cells.","method":"Kinase-specific proteomics of patient leukocytes; ROCK1/2 inhibitor treatment; ROCK1 siRNA knockdown in macrophages/DCs; flow cytometry for CD80, CD86, MHC-II; cytokine ELISA; T cell activation co-culture assay; NF-κB reporter assay; murine GVHD model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — unbiased proteomics identification followed by multiple orthogonal mechanistic assays in primary cells and mouse model; single lab but breadth of methods","pmids":["38199985"],"is_preprint":false}],"current_model":"ROCK1 is a serine/threonine kinase that is activated by GTP-bound RhoA (binding via residues 934–1015 in its coiled-coil region) and acts as a central effector of the RhoA pathway to phosphorylate MLC2, NHE1, ZIPK, Drp1 (at Ser616), and TFEB, thereby regulating actomyosin contractility, focal adhesion and stress fiber formation, centrosome positioning, apoptotic membrane blebbing, tight junction permeability, lysosomal biogenesis, and transcriptional programs including TEAD/YAP; distinct from ROCK2, ROCK1 preferentially drives MLC2-dependent peripheral actomyosin ring formation and apoptotic blebbing, while receiving feedback regulation from Tiam1/Rac1 and being subject to nuclear translocation (downstream of TGF-β) to phosphorylate cell-cycle regulators such as Cdc25A."},"narrative":{"mechanistic_narrative":"ROCK1 is a serine/threonine kinase that functions as a central effector of the RhoA pathway, transducing GTP-RhoA signals into actomyosin-based cytoskeletal reorganization [PMID:8798490, PMID:9119047]. RhoA binds ROCK1 through residues 934–1015 in its coiled-coil region, a Rho-effector motif distinct from the class-1 motif used by PKN, rhophilin, and rhotekin [PMID:8798490]. Through its kinase activity, ROCK1 drives focal adhesion and stress fiber formation [PMID:9119047], neurite retraction and growth-cone gating in neurons [PMID:9647654, PMID:10839361], and tight-junction permeability [PMID:11139571], with the actin polymerization arm of Rho signaling being routed through a separate effector [PMID:9119047]. Its substrates span myosin light chain 2 (MLC2), whose phosphorylation underlies peripheral actomyosin ring formation, the Na-H exchanger NHE1, the kinase ZIPK (phosphorylated at Thr-265/Thr-299 to drive distinct stress-fiber architectures), Drp1 at Ser616 to promote mitochondrial fission, and the transcription factor TFEB to govern lysosomal biogenesis [PMID:9707430, PMID:17158456, PMID:23392171, PMID:39755713, PMID:39497162]. Isoform-specific genetics distinguish ROCK1 from ROCK2: ROCK1 selectively controls MLC2 phosphorylation and peripheral actomyosin contraction (and front-back polarity), whereas ROCK2 governs cofilin phosphorylation [PMID:23392171, PMID:26169356]. ROCK1 is a non-redundant driver of apoptotic membrane blebbing and apoptotic-body formation [PMID:31043701] and acts as a node in oncogenic TEAD/YAP transcriptional activation downstream of mutant p53, Hippo lesions, and ECM stiffness [PMID:35121738, PMID:40368918]. ROCK1 activity is constrained by direct binding partners including Rnd3, FRMD5, and stress-granule sequestration that prevents JIP-3/JNK-dependent apoptotic signaling [PMID:20004716, PMID:25448675, PMID:39755713], and ROCK1 participates in immune, cardiac, vascular, and metabolic disease processes through inflammatory myeloid activation, doxorubicin cardiotoxicity, neointima formation, and AMPK/eNOS regulation [PMID:18414683, PMID:26943578, PMID:29560126, PMID:38538716, PMID:38199985].","teleology":[{"year":1996,"claim":"Defining how RhoA selects ROCK1 as an effector established the molecular basis for pathway specificity, distinguishing it from other Rho effectors.","evidence":"Ligand overlay with GTPγS-RhoA, yeast two-hybrid, and point mutagenesis on recombinant fragments mapping the Rho-binding domain to residues 934–1015","pmids":["8798490"],"confidence":"High","gaps":["Does not establish the downstream catalytic outputs of the engaged kinase","Structural mechanism of how binding relieves autoinhibition not addressed"]},{"year":1997,"claim":"Placing ROCK1 downstream of Rho for focal adhesion and stress fiber formation established it as the contractility effector while separating the actin-polymerization arm.","evidence":"Transfection of kinase-dead and domain-deletion mutants with epistasis analysis in HeLa cells","pmids":["9119047"],"confidence":"High","gaps":["Direct substrates mediating focal adhesion assembly not identified in this study","Identity of the parallel actin-polymerization effector left open"]},{"year":1998,"claim":"Identifying NHE1 as a directly phosphorylated substrate, and linking ROCK1 to MLC phosphorylation in neurite retraction, connected the kinase to specific contractile and ion-transport outputs.","evidence":"In vitro kinase assay with phosphopeptide mapping against in vivo sites (NHE1) and dominant-active/negative constructs plus Y-27632 in N1E-115 cells (MLC/neurite)","pmids":["9707430","9647654"],"confidence":"High","gaps":["Whether NHE1 and MLC are phosphorylated in the same cellular contexts not resolved","Direct vs indirect phosphorylation of MLC not distinguished here"]},{"year":2000,"claim":"Establishing ROCK1 as a gate on axon outgrowth and growth-cone dynamics extended its cytoskeletal role into developmental neurobiology.","evidence":"Dominant-negative/active constructs plus Y-27632 with live imaging in cerebellar granule neurons","pmids":["10839361"],"confidence":"High","gaps":["Substrates mediating growth-cone control not identified","Relationship to MLC phosphorylation in this context not quantified"]},{"year":2001,"claim":"Linking RhoA/ROCK1 to occludin phosphorylation and paracellular permeability defined a role in epithelial barrier regulation.","evidence":"Dominant-negative RhoA, Y-27632, and permeability flux assays in ECV304 cells with two agonists","pmids":["11139571"],"confidence":"High","gaps":["Whether ROCK1 directly phosphorylates occludin not shown","Agonist-specific divergence (histamine vs LPA) mechanism unresolved"]},{"year":2002,"claim":"Localizing ROCK1 to the mother centriole revealed a cytoskeletal role in centrosome positioning and mitotic exit beyond cortical contractility.","evidence":"Immunofluorescence, immunoEM, and siRNA depletion with centrosome tracking","pmids":["12034773"],"confidence":"High","gaps":["Centrosomal substrates not identified","Single-lab localization not independently confirmed"]},{"year":2003,"claim":"Demonstrating TGF-β-driven nuclear translocation and Cdc25A phosphorylation linked ROCK1 to cell-cycle arrest, an unexpected nuclear function.","evidence":"Subcellular fractionation/immunofluorescence, Cdc25A and Cdk2 kinase assays, and cell-cycle analysis in NMuMG vs NIH 3T3 cells","pmids":["14657354"],"confidence":"Medium","gaps":["Mechanism of nuclear import not defined","Direct in vitro phosphorylation of Cdc25A by ROCK1 not fully established"]},{"year":2004,"claim":"Placing ROCK1 in a common pathway with Aurora-A connected it to mitotic spindle organization and G2/M progression.","evidence":"Reciprocal Co-IP and single/double siRNA epistasis with spindle and cell-cycle readouts","pmids":["15178765"],"confidence":"Medium","gaps":["Whether the interaction is direct not shown","Phosphorylation relationship between ROCK1 and Aurora-A not defined"]},{"year":2006,"claim":"Identifying ZIPK as a directly phosphorylated, activated substrate showed ROCK1 can amplify contractile signaling through a downstream kinase.","evidence":"Unbiased proteomics/MS substrate identification, in vitro kinase assay with stoichiometric Thr-265/Thr-299 phosphorylation, and phospho-site mutagenesis in HeLa cells","pmids":["17158456"],"confidence":"High","gaps":["Physiological contexts where this axis dominates not delineated","Quantitative contribution of ZIPK vs direct MLC phosphorylation unresolved"]},{"year":2009,"claim":"Discovering stress-granule sequestration of active ROCK1 revealed a spatial mechanism gating its pro-apoptotic JIP-3/JNK signaling.","evidence":"Co-localization with stress-granule markers and Co-IP of ROCK1-JIP-3 under stress vs non-stress conditions","pmids":["20004716"],"confidence":"Medium","gaps":["Whether the ROCK1-JIP-3 interaction is direct not established","Single-lab finding without reciprocal validation"]},{"year":2012,"claim":"Isoform-specific knockouts assigned MLC2-dependent peripheral contraction and pre-detachment apoptosis to ROCK1, separating it functionally from ROCK2.","evidence":"ROCK1-/- and ROCK2-/- MEFs with p-MLC2/p-cofilin immunoblots and pharmacological corroboration","pmids":["23392171"],"confidence":"High","gaps":["Structural basis of substrate preference between isoforms not addressed","In vivo tissue contexts of divergence not surveyed"]},{"year":2012,"claim":"Uncovering a Tiam1/Rac1-mediated feedback loop showed ROCK1 modulates the balance of Rho-family GTPase activity upstream of itself.","evidence":"Combined ROCK1 inhibitors/siRNA with Rac1/RhoA activity pull-downs and Tiam1 knockdown","pmids":["22430126"],"confidence":"Medium","gaps":["Direct molecular link between ROCK1 activity and Tiam1 not defined","Single-lab finding"]},{"year":2013,"claim":"Genetic and bone-marrow-transplant studies established leukocyte-derived ROCK1 as the driver of vascular neointima formation, and HIF transcriptional control as an upstream input in cancer.","evidence":"Rock1+/- haploinsufficiency with reciprocal bone marrow transplantation (vascular injury) and HIF1A/HIF2A knockdown with promoter reporters (breast cancer)","pmids":["18414683","24324133"],"confidence":"High","gaps":["Leukocyte substrates driving neointima not identified","Direct HIF binding to the ROCK1 promoter not fully mapped"]},{"year":2014,"claim":"Identifying FRMD5 as a direct inhibitory binding partner revealed a negative regulator restraining ROCK1-dependent migration.","evidence":"Co-IP of FRMD5 with ROCK1 and integrin β5, ROCK1 kinase assay, and migration assays in lung cancer cells","pmids":["25448675"],"confidence":"Medium","gaps":["Mechanism by which FRMD5 inhibits activation not defined","Single-lab interaction"]},{"year":2015,"claim":"Isoform-resolved knockdowns and an immune knockout assigned ROCK1 to actomyosin-bundle-initiated polarity and to mast-cell maturation/function via a LIMK-AKT-ERK module.","evidence":"ROCK1/ROCK2 shRNA with live imaging (fibroblasts/neurons) and Rock1-/- BMMCs with functional and biochemical assays plus anaphylaxis model","pmids":["26169356","26943578"],"confidence":"High","gaps":["Direct ROCK1 substrate establishing polarity bundles not identified","Whether LIMK phosphorylation by ROCK1 is direct in mast cells not shown"]},{"year":2018,"claim":"Structural and translational-control studies defined ROCK kinase-selectivity determinants and revealed lnc-31/YB-1 as a post-transcriptional regulator of ROCK1 protein output.","evidence":"Co-crystal structures of ROCK1/ROCK2 with inhibitors (ATP-site residues) and RNA-IP/polysome analysis of lnc-31-Rock1 mRNA-YB-1","pmids":["30384606","29669280"],"confidence":"High","gaps":["Apo/RhoA-bound full-length structure not resolved","Whether lnc-31 regulation operates outside myoblasts unknown"]},{"year":2018,"claim":"Cardiac ROCK1 knockouts linked the kinase to suppression of Beclin-1-mediated autophagy and doxorubicin cardiotoxicity.","evidence":"Global and cardiomyocyte-specific ROCK1 KO mice with Beclin-1/Bcl-2 Co-IP and autophagy flux markers","pmids":["29560126"],"confidence":"High","gaps":["Whether ROCK1 directly phosphorylates Beclin-1 at Thr119 not demonstrated","Cardiomyocyte vs non-cardiomyocyte contributions not fully separated"]},{"year":2019,"claim":"CRISPR deletion established ROCK1 as the non-redundant driver of apoptotic membrane blebbing and apoptotic-body formation, distinguishing it from PAK2 and LIMK1.","evidence":"CRISPR/Cas9 knockout of ROCK1, PAK2, LIMK1 with time-lapse blebbing imaging and phagocytosis assays","pmids":["31043701"],"confidence":"High","gaps":["Substrate(s) executing blebbing in this system not specified","Mechanism controlling secondary necrosis onset unresolved"]},{"year":2022,"claim":"Multiple studies established ROCK1/actomyosin as necessary and sufficient for oncogenic TEAD/YAP transcription and identified Drp1-Ser616 and TFEB as new functional outputs.","evidence":"ROCK1 knockdown/inhibitor with YAP/TEAD readouts (mutant p53, Hippo, ECM stiffness contexts); Rnd3-ROCK1 Co-IP/docking with p-Drp1 Ser616 and Rnd3 in vivo cardiac models; SPR/PLA for ROCK1-TFEB with lysosomal assays","pmids":["35121738","39755713","39497162","40368918"],"confidence":"High","gaps":["Direct in vitro kinase validation of TFEB phosphorylation by ROCK1 not fully described","How ROCK1 mechanistically promotes YAP nuclear localization not resolved","Whether Drp1 Ser616 is a direct ROCK1 site versus indirect not definitively shown"]},{"year":2022,"claim":"Viral and inflammatory studies broadened ROCK1 roles into mRNA stability control and antiviral defense via the ROCK1/MLC2 axis.","evidence":"ROCK1 siRNA/inhibitors with viral mRNA decay assays, PABP Co-IP, and CCR4-NOT analysis in buffalopox-infected cells","pmids":["36280692"],"confidence":"Medium","gaps":["Direct molecular link between ROCK1 and the deadenylation machinery not defined","Single-lab finding"]},{"year":2024,"claim":"Patient-derived proteomics and binding studies positioned ROCK1 as a driver of myeloid inflammatory activation in GVHD and a RIPK4/AMPK regulator in endothelial/diabetic contexts.","evidence":"Kinase proteomics with ROCK1 siRNA/inhibitor in macrophages/DCs and GVHD model; ROCK1-RIPK4 Co-IP with pAMPKα/eNOS readouts in diabetic wound model","pmids":["38199985","38538716"],"confidence":"High","gaps":["Whether ROCK1-RIPK4 interaction is direct not fully established","Substrates linking ROCK1 to NF-κB activation in myeloid cells not identified"]},{"year":null,"claim":"It remains unresolved which ROCK1 substrates are directly phosphorylated versus indirectly engaged across its many reported contexts, and how isoform-specific substrate preference is structurally encoded.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No full-length RhoA-bound structural model defining autoinhibition release","Direct in vitro kinase validation lacking for several recent substrates (TFEB, Drp1, Beclin-1)","Mechanism of nuclear and centrosomal targeting not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,9,24,26]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[3,9,11]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[10,24]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[6]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[7]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,11,16]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,3]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[23,24]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[6,7,8]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[19,30]}],"complexes":[],"partners":["RHOA","ZIPK","RND3","FRMD5","AURKA","RIPK4","YAP1","TFEB"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13464","full_name":"Rho-associated protein kinase 1","aliases":["Renal carcinoma antigen NY-REN-35","Rho-associated, coiled-coil-containing protein kinase 1","Rho-associated, coiled-coil-containing protein kinase I","ROCK-I","p160 ROCK-1","p160ROCK"],"length_aa":1354,"mass_kda":158.2,"function":"Protein kinase which is a key regulator of the actin cytoskeleton and cell polarity (PubMed:10436159, PubMed:10652353, PubMed:11018042, PubMed:11283607, PubMed:17158456, PubMed:18573880, PubMed:19131646, PubMed:8617235, PubMed:9722579). Involved in regulation of smooth muscle contraction, actin cytoskeleton organization, stress fiber and focal adhesion formation, neurite retraction, cell adhesion and motility via phosphorylation of DAPK3, GFAP, LIMK1, LIMK2, MYL9/MLC2, TPPP, PFN1 and PPP1R12A (PubMed:10436159, PubMed:10652353, PubMed:11018042, PubMed:11283607, PubMed:17158456, PubMed:18573880, PubMed:19131646, PubMed:23093407, PubMed:23355470, PubMed:8617235, PubMed:9722579). Phosphorylates FHOD1 and acts synergistically with it to promote SRC-dependent non-apoptotic plasma membrane blebbing (PubMed:18694941). Phosphorylates JIP3 and regulates the recruitment of JNK to JIP3 upon UVB-induced stress (PubMed:19036714). Acts as a suppressor of inflammatory cell migration by regulating PTEN phosphorylation and stability (By similarity). Acts as a negative regulator of VEGF-induced angiogenic endothelial cell activation (PubMed:19181962). Required for centrosome positioning and centrosome-dependent exit from mitosis (By similarity). Plays a role in terminal erythroid differentiation (PubMed:21072057). Inhibits podocyte motility via regulation of actin cytoskeletal dynamics and phosphorylation of CFL1 (By similarity). Promotes keratinocyte terminal differentiation (PubMed:19997641). Involved in osteoblast compaction through the fibronectin fibrillogenesis cell-mediated matrix assembly process, essential for osteoblast mineralization (By similarity). May regulate closure of the eyelids and ventral body wall by inducing the assembly of actomyosin bundles (By similarity)","subcellular_location":"Cytoplasm; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome, centriole; Golgi apparatus membrane; Cell projection, bleb; Cytoplasm, cytoskeleton; Cell membrane; Cell projection, lamellipodium; Cell projection, ruffle","url":"https://www.uniprot.org/uniprotkb/Q13464/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ROCK1","classification":"Not Classified","n_dependent_lines":24,"n_total_lines":1208,"dependency_fraction":0.019867549668874173},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000067900","cell_line_id":"CID001259","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"membrane","grade":3},{"compartment":"cell_contact","grade":2}],"interactors":[{"gene":"HDAC2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001259","total_profiled":1310},"omim":[{"mim_id":"620064","title":"OXOEICOSANOID RECEPTOR 1; OXER1","url":"https://www.omim.org/entry/620064"},{"mim_id":"618867","title":"RAS HOMOLOG GENE FAMILY, MEMBER F, FILOPODIA-ASSOCIATED; RHOF","url":"https://www.omim.org/entry/618867"},{"mim_id":"618459","title":"IMMUNODEFICIENCY 62; IMD62","url":"https://www.omim.org/entry/618459"},{"mim_id":"617714","title":"CAVEOLAE-ASSOCIATED PROTEIN 4; CAVIN4","url":"https://www.omim.org/entry/617714"},{"mim_id":"617043","title":"RHO GUANINE NUCLEOTIDE EXCHANGE FACTOR 17; ARHGEF17","url":"https://www.omim.org/entry/617043"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ROCK1"},"hgnc":{"alias_symbol":["p160ROCK"],"prev_symbol":[]},"alphafold":{"accession":"Q13464","domains":[{"cath_id":"3.30.200.20","chopping":"8-156_373-405","consensus_level":"medium","plddt":87.7372,"start":8,"end":405},{"cath_id":"1.10.510.10","chopping":"171-359","consensus_level":"medium","plddt":91.6616,"start":171,"end":359},{"cath_id":"2.30.29.30","chopping":"1122-1209_1225-1316","consensus_level":"medium","plddt":81.5541,"start":1122,"end":1316}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13464","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13464-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13464-F1-predicted_aligned_error_v6.png","plddt_mean":76.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ROCK1","jax_strain_url":"https://www.jax.org/strain/search?query=ROCK1"},"sequence":{"accession":"Q13464","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13464.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13464/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13464"}},"corpus_meta":[{"pmid":"9119047","id":"PMC_9119047","title":"p160ROCK, a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions.","date":"1997","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/9119047","citation_count":461,"is_preprint":false},{"pmid":"9647654","id":"PMC_9647654","title":"Molecular dissection of the Rho-associated protein kinase (p160ROCK)-regulated neurite remodeling in neuroblastoma N1E-115 cells.","date":"1998","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/9647654","citation_count":423,"is_preprint":false},{"pmid":"26635606","id":"PMC_26635606","title":"The Function of Rho-Associated Kinases ROCK1 and ROCK2 in the Pathogenesis of Cardiovascular Disease.","date":"2015","source":"Frontiers in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/26635606","citation_count":287,"is_preprint":false},{"pmid":"10839361","id":"PMC_10839361","title":"A critical role for a Rho-associated kinase, p160ROCK, in determining axon outgrowth in mammalian CNS 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Point mutations K934M, L941A, E1008A weakened Rho binding, and I1009A abolished it, defining a distinct class of Rho effector motif different from the class-1 Rho effector motif shared by PKN, rhophilin, and rhotekin.\",\n      \"method\": \"Ligand overlay assay using [35S]GTPγS-bound GST-RhoA, yeast two-hybrid system, point mutagenesis, recombinant His-tagged protein fragments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro binding assay with recombinant proteins plus mutagenesis, corroborated by yeast two-hybrid; multiple orthogonal methods in a single focused study\",\n      \"pmids\": [\"8798490\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"p160ROCK acts downstream of Rho to induce focal adhesions and stress fibers. Expression of wild-type p160ROCK or constructs containing both the kinase domain and coiled-coil region induced focal adhesions and stress fibers in HeLa cells, whereas a kinase-defective mutant or a mutant lacking the coiled-coil region did not. A dominant-negative mutant defective in both kinase and Rho-binding activities blocked Rho-induced focal adhesion formation but not Rho-induced actin polymerization, indicating a separate effector pathway for the latter.\",\n      \"method\": \"Transfection of wild-type and kinase-dead/domain deletion mutants of p160ROCK into HeLa cells; morphological analysis of focal adhesions and stress fibers; co-expression epistasis\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple domain-deletion and kinase-dead mutants with epistasis analysis; widely replicated finding foundational to the field\",\n      \"pmids\": [\"9119047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"p160ROCK activation is necessary and sufficient for agonist-induced neurite retraction and cell rounding in N1E-115 neuroblastoma cells. This was accompanied by elevated myosin light chain (MLC) phosphorylation and disassembly of intermediate filaments and microtubules. The p160ROCK-specific inhibitor Y-27632 blocked both neurite retraction and MLC phosphorylation at similar concentrations. Dominant-negative p160ROCK induced neurite formation in the presence of serum; this outgrowth was blocked by dominant-negative Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates Cdc42/Rac-dependent neurite formation.\",\n      \"method\": \"Dominant-active and dominant-negative p160ROCK constructs; pharmacological inhibition with Y-27632; immunofluorescence for MLC phosphorylation, intermediate filaments, and microtubules; epistasis with dominant-negative Cdc42/Rac\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic and pharmacological approaches with orthogonal readouts; replicated across cell types\",\n      \"pmids\": [\"9647654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"p160ROCK is an upstream activator of the Na-H exchanger NHE1, acting in the RhoA pathway. Constitutively active p160ROCK stimulated NHE1 activity and phosphorylated NHE1 in vivo and in vitro, and identical NHE1 residues were phosphorylated in both settings. NHE1 activity and phosphorylation were required for p160ROCK-induced actin stress fiber assembly. Y-27632 blocked RhoA- and LPA-induced NHE1 activity and NHE1 phosphorylation. Dominant-interfering p160ROCK blocked RhoA-induced but not Cdc42- or Rac-induced NHE1 activation.\",\n      \"method\": \"In vitro kinase assay of p160ROCK on NHE1 C-terminal fusion protein; phosphopeptide mapping comparing in vivo and in vitro phosphorylation; mutationally active/dominant-interfering p160ROCK constructs; NHE1 inhibitor (EIPA); NHE1-deficient PS120 cells; Y-27632 pharmacology\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with phosphopeptide mapping corroborating in vivo phosphorylation; multiple genetic and pharmacological approaches\",\n      \"pmids\": [\"9707430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"p160ROCK inhibition in cultured cerebellar granule neurons triggered immediate outgrowth of membrane ruffles and filopodia and generation of axonal processes, potentiated growth cone size and motility, and overexpression of dominant-active Rho or ROCK prevented initiation of axon outgrowth. This establishes the Rho/ROCK pathway as a gate controlling axon outgrowth initiation and growth cone dynamics, likely through regulation of the cortical actin network.\",\n      \"method\": \"Dominant-negative and dominant-active p160ROCK/Rho constructs transfected into cerebellar granule neurons; Y-27632 pharmacological inhibition; live imaging of growth cone dynamics\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — combined genetic (dominant-active/negative) and pharmacological approach with specific neuronal phenotypic readouts; replicated across multiple studies\",\n      \"pmids\": [\"10839361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"RhoA-p160ROCK signaling mediates lysophosphatidic acid (LPA)-induced stress fiber formation, myosin light chain phosphorylation, and occludin phosphorylation in ECV304 cells. A dominant-negative RhoA or Y-27632 blocked all three LPA-induced events and prevented increased paracellular permeability. For histamine, RhoA/p160ROCK blocked cytoskeletal events but not occludin phosphorylation or increased flux, indicating that occludin phosphorylation can regulate tight junction permeability independently of cytoskeletal events.\",\n      \"method\": \"Dominant-negative RhoA construct; Y-27632 pharmacological inhibition; immunoblot for MLC and occludin phosphorylation; horseradish peroxidase flux assay for tight junction permeability\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — combined genetic and pharmacological approaches with two distinct agonists; multiple orthogonal readouts (MLC phosphorylation, occludin phosphorylation, permeability assay)\",\n      \"pmids\": [\"11139571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"p160ROCK is a centrosomal component localizing principally to the mother centriole and an intercentriolar linker. Inhibition of p160ROCK caused centrosome splitting in G1, with the mother centriole displaying wide excursions to the cell periphery. p160ROCK inhibition late after anaphase triggered mother centriole migration to the midbody and completion of cell division, establishing a role for p160ROCK in centrosome positioning and centrosome-dependent exit from mitosis.\",\n      \"method\": \"Immunofluorescence with multiple p160ROCK antibodies; immunoelectron microscopy; siRNA depletion; centrosome tracking/live cell analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — immunoEM for localization, siRNA depletion with specific phenotypic readout, multiple antibodies; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"12034773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TGF-β stimulates RhoA and p160ROCK activity in epithelial NMuMG cells, leading to p160ROCK translocation to the nucleus where it phosphorylates the cell-cycle phosphatase Cdc25A. This inhibitory phosphorylation of Cdc25A suppresses Cdk2 activity and contributes to TGF-β-induced G1/S cell cycle arrest. In NIH 3T3 fibroblasts, which lack RhoA/p160ROCK activity in the TGF-β response, growth inhibition is absent.\",\n      \"method\": \"Dominant-negative/active RhoA and p160ROCK constructs; subcellular fractionation and immunofluorescence for p160ROCK nuclear localization; Cdc25A phosphorylation assay; Cdk2 kinase assay; cell cycle analysis; genetic epistasis between NMuMG and NIH 3T3 backgrounds\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — nuclear translocation supported by fractionation/immunofluorescence; kinase assays for Cdc25A; single lab\",\n      \"pmids\": [\"14657354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"p160ROCK associates with Aurora-A/STK15 in a protein complex. siRNA suppression of Aurora-A blocked mitotic spindle organization and caused G2/M arrest. Simultaneous suppression of p160ROCK by RNAi rescued abnormal spindle formation, released G2/M arrest, and alleviated apoptosis, leading to aneuploid cell accumulation. This epistasis places Aurora-A and p160ROCK in a common pathway governing G2/M progression.\",\n      \"method\": \"Co-immunoprecipitation of Aurora-A and p160ROCK; siRNA knockdown of Aurora-A and p160ROCK individually and in combination; cell cycle analysis; spindle immunofluorescence\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus double-knockdown epistasis; single lab, two orthogonal methods\",\n      \"pmids\": [\"15178765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ROCK1 directly phosphorylates zipper-interacting protein kinase (ZIPK) at Thr-265 and Thr-299, stoichiometrically activating ZIPK. In HeLa cells, co-expression of ZIPK with ROCK1 shifted the actin phenotype from ROCK-induced focused stress fibers to a Rho-like parallel stress fiber pattern in a Thr-265 phosphorylation-dependent manner, identifying ROCK1 as an upstream activator of ZIPK in smooth muscle and non-muscle cells.\",\n      \"method\": \"Proteomics screen with synthetic ZIPK phosphopeptides followed by mass spectrometry identification of ROCK1; in vitro kinase assay with native ZIPK showing stoichiometric phosphorylation at Thr-265 and Thr-299; co-expression in HeLa cells; phospho-site mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with stoichiometric phosphorylation, substrate identified by unbiased proteomics/MS, and mutagenesis validated in cells; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"17158456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"RhoA and activated ROCK1 are sequestered into stress granules (SGs) upon cellular stress. Sequestration of activated ROCK1 into SGs prevents ROCK1 from interacting with JIP-3 (JNK-interacting protein 3) and activating the pro-apoptotic JNK pathway, thereby protecting cells from apoptosis. This defines a regulatory mechanism whereby ROCK1 activity determines cell fate through either promoting SG formation or initiating JNK-dependent apoptosis.\",\n      \"method\": \"Immunofluorescence co-localization of ROCK1/RhoA with SG markers; co-immunoprecipitation of ROCK1 with JIP-3 under stress vs. non-stress conditions; RhoA/ROCK1 dominant-active constructs; JNK activation assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP of ROCK1-JIP3 interaction linked to JNK activation; localization by immunofluorescence; single lab\",\n      \"pmids\": [\"20004716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ROCK1 deficiency in MEFs attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased MLC2 phosphorylation but preserved cofilin phosphorylation, reducing cell shrinkage, detachment, and pre-detachment apoptosis. ROCK2 deficiency instead reduced phosphorylation of both MLC2 and cofilin, with different actin phenotypes. This establishes ROCK1 as specifically regulating MLC2 phosphorylation and peripheral actomyosin contraction, while ROCK2 regulates cofilin phosphorylation for cytoskeletal stabilization.\",\n      \"method\": \"ROCK1-/- and ROCK2-/- MEF genetic knockout; immunoblot for p-MLC2 and p-cofilin; pharmacological inhibitors (blebbistatin, cytochalasin D, Y27632); fluorescence microscopy of actin cytoskeleton\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — isoform-specific knockout mice with orthogonal pharmacological confirmation; multiple biochemical readouts\",\n      \"pmids\": [\"23392171\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ROCK1 inhibition indirectly reduces the activity of upstream RhoA through stimulation of Tiam1-induced Rac1 activity, revealing a feedback loop by which ROCK1 modulates the functional balance of Rho-family GTPases.\",\n      \"method\": \"Combined pharmacological (ROCK1 inhibitors) and genetic (siRNA knockdown) approach; Rac1 and RhoA activity assays (pull-down/G-LISA); Tiam1 involvement assessed by knockdown\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — combined pharmacological and genetic approach with activity assays; single lab, two orthogonal methods\",\n      \"pmids\": [\"22430126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ROCK1 in bone marrow-derived (leukocyte) cells mediates neointima formation following vascular injury. Haploinsufficient Rock1+/- mice showed reduced neointima formation, smooth muscle cell proliferation/survival, adhesion molecule expression, and leukocyte infiltration compared to WT and Rock2+/- mice. Reciprocal bone marrow transplantation confirmed that leukocyte-derived ROCK1, not vessel wall ROCK1, drives neointima formation.\",\n      \"method\": \"Rock1+/- and Rock2+/- haploinsufficient mice; carotid artery ligation model; reciprocal bone marrow transplantation; histology/immunohistochemistry; peritoneal leukocyte recruitment assay\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic haploinsufficiency plus reciprocal bone marrow transplantation with clear isoform-specific phenotype\",\n      \"pmids\": [\"18414683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"HIF-1α and HIF-2α (hypoxia-inducible factors) coordinately activate RhoA and ROCK1 expression in breast cancer cells, leading to MLC and FAK phosphorylation, actin-myosin contractility, focal adhesion formation, and cell motility. This places ROCK1 as a transcriptional target of HIFs and a node in the HIF→RhoA→ROCK1→MLC→FAK oncogenic signaling cascade.\",\n      \"method\": \"HIF1A/HIF2A knockdown and overexpression; immunoblot for p-MLC and p-FAK; cell contraction and motility assays; luciferase reporter for RhoA and ROCK1 promoters\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transcriptional reporter assays and knockdown/OE with biochemical readouts; single lab, two orthogonal methods\",\n      \"pmids\": [\"24324133\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"FRMD5, a FERM domain protein, interacts physically with ROCK1 and inhibits its activation, resulting in reduced myosin light chain phosphorylation and reduced actin stress fiber formation, thereby inhibiting cell migration in lung cancer cells. FRMD5 also interacts with integrin β5 cytoplasmic tail, defining a dual pathway for migration inhibition.\",\n      \"method\": \"Co-immunoprecipitation of FRMD5 with ROCK1 and integrin β5; ROCK1 kinase activity assay; immunofluorescence for stress fibers and p-MLC; FRMD5 overexpression/knockdown with migration assays\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP of FRMD5-ROCK1 interaction linked to kinase activity and phenotypic readout; single lab\",\n      \"pmids\": [\"25448675\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ROCK1 mediates formation of stable actomyosin filament bundles that initiate front-back polarity in migrating fibroblasts and dendritic spine polarity in neurons. ROCK2 regulates contractile force, Rac1 activity at the leading edge, and cofilin-mediated actin remodeling at adhesions and spine heads, defining distinct downstream pathways for the two ROCK isoforms.\",\n      \"method\": \"ROCK1 and ROCK2 isoform-specific shRNA knockdown; live-cell imaging of actin dynamics; immunofluorescence for actomyosin bundles and adhesions; cofilin phosphorylation assay; migration and spine morphology analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — isoform-specific knockdown with multiple phenotypic and biochemical readouts in two distinct biological contexts (migration and synapse); single lab but multiple orthogonal methods\",\n      \"pmids\": [\"26169356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"EV-encapsulated caspase-3 from hyperoxia-injured lung epithelial cells activates alveolar macrophages via the ROCK1 pathway, inducing pro-inflammatory cytokine secretion and neutrophil infiltration. This defines a caspase-3→ROCK1 signaling axis in macrophage activation by extracellular vesicles.\",\n      \"method\": \"In vitro macrophage treatment with isolated EVs; ROCK1 inhibitor treatment; caspase-3-deficient EV generation; BALF cytokine/cell count analysis; in vivo intranasal EV delivery in mice\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological ROCK1 inhibition and caspase-3 KO EVs with in vitro and in vivo readouts; single lab\",\n      \"pmids\": [\"26658190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ROCK1 stimulates endothelial-to-mesenchymal transition (EndMT) in glomerular endothelial cells under high-glucose conditions. Inhibition of ROCK1 blocked EndMT and increased endothelial permeability, while ROCK1 overexpression induced these changes. In db/db mice, fasudil (ROCK1 inhibitor) suppressed α-SMA expression in glomerular endothelium and reduced albuminuria, establishing ROCK1 as a driver of diabetic glomerular injury.\",\n      \"method\": \"ROCK1 overexpression and inhibition (fasudil) in glomerular endothelial cells; immunofluorescence for EndMT markers (CD31, α-SMA); permeability assay; db/db mouse model treated with fasudil\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic overexpression and pharmacological inhibition in vitro with in vivo confirmation; single lab\",\n      \"pmids\": [\"26842599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ROCK1 deficiency in bone marrow-derived mast cells results in delayed maturation in response to IL-3, reduced growth in response to SCF, and impaired integrin-mediated adhesion/migration and IgE-mediated degranulation. Mechanistically, ROCK1 acts through LIM kinase (LIMK) downstream in mast cells; ROCK1-deficient BMMCs show reduced activation of ROCK, LIMK, AKT, and ERK1/2 upon SCF stimulation.\",\n      \"method\": \"Rock1-/- BMMC derivation; IL-3/SCF stimulation assays; integrin adhesion and migration assays; IgE degranulation assay; immunoblot for ROCK, LIMK, AKT, ERK1/2 phosphorylation; passive cutaneous anaphylaxis model\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with multiple orthogonal functional assays and biochemical pathway analysis; in vivo confirmation\",\n      \"pmids\": [\"26943578\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ROCK1 directly interacts with ZIPK in a complex identified by mass spectrometry, phosphorylates ZIPK at Thr-265 and Thr-299 to activate it. Crystal/co-crystal structures of early lead compounds were obtained in PKA, ROCK1, and ROCK2, identifying key residues (Asp176 and Asp218 in ROCK2; Glu in PKA) in the ATP-binding site that distinguish ROCK kinase selectivity from the broader kinome.\",\n      \"method\": \"Co-crystal structures of ROCK1 and ROCK2 with inhibitors; structure-based drug design; enzyme assays; cell-based assays\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures of ROCK1 and ROCK2 with functional validation in enzyme and cell assays; single lab\",\n      \"pmids\": [\"30384606\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ROCK1 deficiency restores autophagic flux and reduces doxorubicin-induced cardiotoxicity. Molecular analysis showed that ROCK1 knockout reduced Beclin 1-mediated autophagy initiation by increasing association of Beclin 1 with Bcl-2/Bcl-XL, associated with reduced Beclin 1 phosphorylation at Thr119. Cardiomyocyte-specific ROCK1 knockout partially reproduced the cardioprotective phenotype, indicating ROCK1 in both cardiomyocytes and non-cardiomyocytes mediates doxorubicin cardiotoxicity.\",\n      \"method\": \"ROCK1-/- and MHC-Cre/ROCK1fl/fl mice; doxorubicin cardiotoxicity model; immunoprecipitation of Beclin 1 with Bcl-2/Bcl-XL; immunoblot for p62/SQSTM1, LC3-II, Beclin 1 phosphorylation; apoptosis markers\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic models (global and cardiomyocyte-specific KO) with Co-IP and biochemical pathway analysis\",\n      \"pmids\": [\"29560126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"lnc-31, a cytoplasmic lncRNA required for myoblast proliferation, promotes ROCK1 protein synthesis by stabilizing the translational activator YB-1. lnc-31 binds both Rock1 mRNA and the YB-1 protein, and physical interaction between the two RNA species is required for translational activation of Rock1.\",\n      \"method\": \"RNA immunoprecipitation of lnc-31 with YB-1 and Rock1 mRNA; lnc-31 knockdown/overexpression with ROCK1 protein measurement; ribosome profiling/polysome fractionation; interaction domain mapping\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA-protein IP and RNA-RNA interaction validated with functional consequence on translation; single lab\",\n      \"pmids\": [\"29669280\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ROCK1 is a key non-redundant positive regulator of apoptotic membrane blebbing and apoptotic body (ApoBD) formation. CRISPR/Cas9 deletion of ROCK1, but not PAK2 or LIMK1, abolished membrane blebbing and ApoBD formation. Inhibition of ApoBD formation reduced efficient uptake of apoptotic materials by phagocytes. ROCK1 also plays a role in regulating the onset of secondary necrosis.\",\n      \"method\": \"CRISPR/Cas9 knockout of ROCK1, PAK2, and LIMK1; pharmacological inhibition; time-lapse microscopy of blebbing; ApoBD quantification; phagocytosis assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR/Cas9 loss-of-function with pharmacological corroboration; isoform-specific comparison with PAK2 and LIMK1; multiple orthogonal readouts\",\n      \"pmids\": [\"31043701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Rnd3 (a Rho family GTPase 3) directly binds ROCK1 in the cytoplasm and inhibits ROCK1-induced Drp1 phosphorylation at Ser616, thereby preventing mitochondrial fission, reducing PANoptosis, and protecting against doxorubicin-induced cardiotoxicity. Rock1 knockdown nullified the cardioprotective effects of Rnd3, placing ROCK1 as a key node in the Rnd3/ROCK1/Drp1 signaling axis.\",\n      \"method\": \"Co-immunoprecipitation and protein-protein docking of Rnd3 and ROCK1; phospho-Drp1 Ser616 immunoblot; Rnd3 transgenic and knockout mice; Rock1 siRNA knockdown in vitro; mitochondrial morphology imaging; in vivo cardiac function analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — Co-IP and molecular docking demonstrate physical interaction; ROCK1 substrate phosphorylation at defined site with genetic epistasis; multiple in vitro and in vivo models\",\n      \"pmids\": [\"39755713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ROCK1 hyperactivates RhoA/ROCK1/actomyosin signaling downstream of p53 DNA-contact mutants and also mediates signaling required by recurrent Hippo pathway lesions, both of which drive oncogenic TEAD/YAP transcription. ROCK inhibitors selectively antagonized proliferation and motility of human tumors with either mechanism, demonstrating that ROCK1/actomyosin signaling is both necessary and sufficient for oncogenic TEAD/YAP transcriptional activation.\",\n      \"method\": \"p53 mutant expression; ROCK1 knockdown and inhibitor treatment; YAP localization/activity assay; actin cytoskeleton analysis; cell proliferation and motility assays in tumor cell lines; epistasis with Hippo pathway mutants\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic and pharmacological interventions with defined transcriptional and functional readouts; multiple cancer cell line contexts\",\n      \"pmids\": [\"35121738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ROCK1 phosphorylates TFEB (transcription factor EB) as a previously unknown upstream kinase, independent of mTOR or GSK-3β, thereby regulating lysosomal biogenesis and acidification. ROCK1 elevation impaired lysosomal proteolysis; ROCK1 knockdown promoted TFEB nuclear distribution and lysosome-mediated Aβ clearance. The Rnd3/ROCK1 interaction was confirmed by surface plasmon resonance and proximity ligation assay.\",\n      \"method\": \"Surface plasmon resonance (SPR) and proximity ligation assay (PLA) for ROCK1-TFEB interaction; AAV-mediated ROCK1 knockdown in APP/PS1 mice; lysosomal acidification and biogenesis assays; TFEB nuclear localization immunofluorescence; behavioral testing\",\n      \"journal\": \"Translational neurodegeneration\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — SPR and PLA for protein interaction, genetic knockdown with multiple readouts; novel substrate claim requires in vitro kinase assay validation not fully described in abstract\",\n      \"pmids\": [\"39497162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ROCK1/MLC2 (myosin light chain 2) signaling is activated in buffalopox virus-infected cells, and ROCK1 inhibition promotes deadenylation of viral mRNA by inhibiting PABP interaction with viral mRNA and enhancing expression of the CCR4-NOT deadenylation complex. ROCK1/MLC2-mediated cell contraction and perinuclear p-MLC2 accumulation positively correlated with viral mRNA/protein synthesis.\",\n      \"method\": \"ROCK1 siRNA depletion and pharmacological inhibitors (Thiazovivin, Y27632); viral mRNA decay assay; PABP co-immunoprecipitation; CCR4-NOT expression analysis; viral genome quantification; progeny virus titration\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA and pharmacological inhibition with mechanistic mRNA decay readout and Co-IP; single lab\",\n      \"pmids\": [\"36280692\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ROCK1 modulates YAP1 phosphorylation, promoting its nuclear localization and transcriptional activity in endometrial cancer in response to ECM stiffness. Co-immunoprecipitation confirmed ROCK1-YAP1 interaction; pharmacological ROCK1 inhibition with Y-27632 suppressed YAP1 nuclear localization and reduced tumor proliferation, migration, invasion, and chemotherapy resistance.\",\n      \"method\": \"Atomic force microscopy for ECM stiffness measurement; polyacrylamide gels of varying stiffness; co-immunoprecipitation of ROCK1-YAP1; immunofluorescence for YAP1 localization; Y-27632 pharmacological inhibition; xenograft mouse model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus pharmacological inhibition with functional readouts; mechanosensitive context established by AFM; single lab\",\n      \"pmids\": [\"40368918\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ROCK1 inhibits pThr172-AMPKα activity by binding to receptor-interacting serine/threonine kinase 4 (RIPK4), establishing a ROCK1/RIPK4/AMPK pathway. Fasudil and ROCK1 siRNA elevated pThr172-AMPKα, increased eNOS activity, and suppressed mitochondrial ROS in endothelial cells, accelerating wound repair in diabetic mice.\",\n      \"method\": \"Co-immunoprecipitation of ROCK1 with RIPK4 (integrated bioinformatics + Co-IP); ROCK1 siRNA and fasudil treatment; pAMPKα immunoblot; eNOS activity assay; mtROS measurement; diabetic wound healing model in vivo\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP identifying ROCK1-RIPK4 interaction linked to AMPK pathway; pharmacological and genetic validation in vitro and in vivo; single lab\",\n      \"pmids\": [\"38538716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Kinase-specific proteomics of leukocytes from corticosteroid-refractory aGVHD patients identified ROCK1 as the most significantly upregulated kinase. ROCK1/2 inhibition or ROCK1 knockdown in macrophages/dendritic cells reduced CD80, CD86, MHC-II expression and IL-6, IL-1β, iNOS, TNF production, impaired T cell activation and cytoskeletal rearrangements (reducing macrophage and DC migration), and reduced NF-κB signaling in myeloid cells.\",\n      \"method\": \"Kinase-specific proteomics of patient leukocytes; ROCK1/2 inhibitor treatment; ROCK1 siRNA knockdown in macrophages/DCs; flow cytometry for CD80, CD86, MHC-II; cytokine ELISA; T cell activation co-culture assay; NF-κB reporter assay; murine GVHD model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — unbiased proteomics identification followed by multiple orthogonal mechanistic assays in primary cells and mouse model; single lab but breadth of methods\",\n      \"pmids\": [\"38199985\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ROCK1 is a serine/threonine kinase that is activated by GTP-bound RhoA (binding via residues 934–1015 in its coiled-coil region) and acts as a central effector of the RhoA pathway to phosphorylate MLC2, NHE1, ZIPK, Drp1 (at Ser616), and TFEB, thereby regulating actomyosin contractility, focal adhesion and stress fiber formation, centrosome positioning, apoptotic membrane blebbing, tight junction permeability, lysosomal biogenesis, and transcriptional programs including TEAD/YAP; distinct from ROCK2, ROCK1 preferentially drives MLC2-dependent peripheral actomyosin ring formation and apoptotic blebbing, while receiving feedback regulation from Tiam1/Rac1 and being subject to nuclear translocation (downstream of TGF-β) to phosphorylate cell-cycle regulators such as Cdc25A.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ROCK1 is a serine/threonine kinase that functions as a central effector of the RhoA pathway, transducing GTP-RhoA signals into actomyosin-based cytoskeletal reorganization [#0, #1]. RhoA binds ROCK1 through residues 934–1015 in its coiled-coil region, a Rho-effector motif distinct from the class-1 motif used by PKN, rhophilin, and rhotekin [#0]. Through its kinase activity, ROCK1 drives focal adhesion and stress fiber formation [#1], neurite retraction and growth-cone gating in neurons [#2, #4], and tight-junction permeability [#5], with the actin polymerization arm of Rho signaling being routed through a separate effector [#1]. Its substrates span myosin light chain 2 (MLC2), whose phosphorylation underlies peripheral actomyosin ring formation, the Na-H exchanger NHE1, the kinase ZIPK (phosphorylated at Thr-265/Thr-299 to drive distinct stress-fiber architectures), Drp1 at Ser616 to promote mitochondrial fission, and the transcription factor TFEB to govern lysosomal biogenesis [#3, #9, #11, #24, #26]. Isoform-specific genetics distinguish ROCK1 from ROCK2: ROCK1 selectively controls MLC2 phosphorylation and peripheral actomyosin contraction (and front-back polarity), whereas ROCK2 governs cofilin phosphorylation [#11, #16]. ROCK1 is a non-redundant driver of apoptotic membrane blebbing and apoptotic-body formation [#23] and acts as a node in oncogenic TEAD/YAP transcriptional activation downstream of mutant p53, Hippo lesions, and ECM stiffness [#25, #28]. ROCK1 activity is constrained by direct binding partners including Rnd3, FRMD5, and stress-granule sequestration that prevents JIP-3/JNK-dependent apoptotic signaling [#10, #15, #24], and ROCK1 participates in immune, cardiac, vascular, and metabolic disease processes through inflammatory myeloid activation, doxorubicin cardiotoxicity, neointima formation, and AMPK/eNOS regulation [#13, #19, #21, #29, #30].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Defining how RhoA selects ROCK1 as an effector established the molecular basis for pathway specificity, distinguishing it from other Rho effectors.\",\n      \"evidence\": \"Ligand overlay with GTPγS-RhoA, yeast two-hybrid, and point mutagenesis on recombinant fragments mapping the Rho-binding domain to residues 934–1015\",\n      \"pmids\": [\"8798490\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not establish the downstream catalytic outputs of the engaged kinase\", \"Structural mechanism of how binding relieves autoinhibition not addressed\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Placing ROCK1 downstream of Rho for focal adhesion and stress fiber formation established it as the contractility effector while separating the actin-polymerization arm.\",\n      \"evidence\": \"Transfection of kinase-dead and domain-deletion mutants with epistasis analysis in HeLa cells\",\n      \"pmids\": [\"9119047\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct substrates mediating focal adhesion assembly not identified in this study\", \"Identity of the parallel actin-polymerization effector left open\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Identifying NHE1 as a directly phosphorylated substrate, and linking ROCK1 to MLC phosphorylation in neurite retraction, connected the kinase to specific contractile and ion-transport outputs.\",\n      \"evidence\": \"In vitro kinase assay with phosphopeptide mapping against in vivo sites (NHE1) and dominant-active/negative constructs plus Y-27632 in N1E-115 cells (MLC/neurite)\",\n      \"pmids\": [\"9707430\", \"9647654\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NHE1 and MLC are phosphorylated in the same cellular contexts not resolved\", \"Direct vs indirect phosphorylation of MLC not distinguished here\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing ROCK1 as a gate on axon outgrowth and growth-cone dynamics extended its cytoskeletal role into developmental neurobiology.\",\n      \"evidence\": \"Dominant-negative/active constructs plus Y-27632 with live imaging in cerebellar granule neurons\",\n      \"pmids\": [\"10839361\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrates mediating growth-cone control not identified\", \"Relationship to MLC phosphorylation in this context not quantified\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Linking RhoA/ROCK1 to occludin phosphorylation and paracellular permeability defined a role in epithelial barrier regulation.\",\n      \"evidence\": \"Dominant-negative RhoA, Y-27632, and permeability flux assays in ECV304 cells with two agonists\",\n      \"pmids\": [\"11139571\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ROCK1 directly phosphorylates occludin not shown\", \"Agonist-specific divergence (histamine vs LPA) mechanism unresolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Localizing ROCK1 to the mother centriole revealed a cytoskeletal role in centrosome positioning and mitotic exit beyond cortical contractility.\",\n      \"evidence\": \"Immunofluorescence, immunoEM, and siRNA depletion with centrosome tracking\",\n      \"pmids\": [\"12034773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Centrosomal substrates not identified\", \"Single-lab localization not independently confirmed\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstrating TGF-β-driven nuclear translocation and Cdc25A phosphorylation linked ROCK1 to cell-cycle arrest, an unexpected nuclear function.\",\n      \"evidence\": \"Subcellular fractionation/immunofluorescence, Cdc25A and Cdk2 kinase assays, and cell-cycle analysis in NMuMG vs NIH 3T3 cells\",\n      \"pmids\": [\"14657354\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of nuclear import not defined\", \"Direct in vitro phosphorylation of Cdc25A by ROCK1 not fully established\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Placing ROCK1 in a common pathway with Aurora-A connected it to mitotic spindle organization and G2/M progression.\",\n      \"evidence\": \"Reciprocal Co-IP and single/double siRNA epistasis with spindle and cell-cycle readouts\",\n      \"pmids\": [\"15178765\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the interaction is direct not shown\", \"Phosphorylation relationship between ROCK1 and Aurora-A not defined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identifying ZIPK as a directly phosphorylated, activated substrate showed ROCK1 can amplify contractile signaling through a downstream kinase.\",\n      \"evidence\": \"Unbiased proteomics/MS substrate identification, in vitro kinase assay with stoichiometric Thr-265/Thr-299 phosphorylation, and phospho-site mutagenesis in HeLa cells\",\n      \"pmids\": [\"17158456\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological contexts where this axis dominates not delineated\", \"Quantitative contribution of ZIPK vs direct MLC phosphorylation unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Discovering stress-granule sequestration of active ROCK1 revealed a spatial mechanism gating its pro-apoptotic JIP-3/JNK signaling.\",\n      \"evidence\": \"Co-localization with stress-granule markers and Co-IP of ROCK1-JIP-3 under stress vs non-stress conditions\",\n      \"pmids\": [\"20004716\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the ROCK1-JIP-3 interaction is direct not established\", \"Single-lab finding without reciprocal validation\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Isoform-specific knockouts assigned MLC2-dependent peripheral contraction and pre-detachment apoptosis to ROCK1, separating it functionally from ROCK2.\",\n      \"evidence\": \"ROCK1-/- and ROCK2-/- MEFs with p-MLC2/p-cofilin immunoblots and pharmacological corroboration\",\n      \"pmids\": [\"23392171\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of substrate preference between isoforms not addressed\", \"In vivo tissue contexts of divergence not surveyed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Uncovering a Tiam1/Rac1-mediated feedback loop showed ROCK1 modulates the balance of Rho-family GTPase activity upstream of itself.\",\n      \"evidence\": \"Combined ROCK1 inhibitors/siRNA with Rac1/RhoA activity pull-downs and Tiam1 knockdown\",\n      \"pmids\": [\"22430126\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular link between ROCK1 activity and Tiam1 not defined\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Genetic and bone-marrow-transplant studies established leukocyte-derived ROCK1 as the driver of vascular neointima formation, and HIF transcriptional control as an upstream input in cancer.\",\n      \"evidence\": \"Rock1+/- haploinsufficiency with reciprocal bone marrow transplantation (vascular injury) and HIF1A/HIF2A knockdown with promoter reporters (breast cancer)\",\n      \"pmids\": [\"18414683\", \"24324133\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Leukocyte substrates driving neointima not identified\", \"Direct HIF binding to the ROCK1 promoter not fully mapped\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identifying FRMD5 as a direct inhibitory binding partner revealed a negative regulator restraining ROCK1-dependent migration.\",\n      \"evidence\": \"Co-IP of FRMD5 with ROCK1 and integrin β5, ROCK1 kinase assay, and migration assays in lung cancer cells\",\n      \"pmids\": [\"25448675\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which FRMD5 inhibits activation not defined\", \"Single-lab interaction\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Isoform-resolved knockdowns and an immune knockout assigned ROCK1 to actomyosin-bundle-initiated polarity and to mast-cell maturation/function via a LIMK-AKT-ERK module.\",\n      \"evidence\": \"ROCK1/ROCK2 shRNA with live imaging (fibroblasts/neurons) and Rock1-/- BMMCs with functional and biochemical assays plus anaphylaxis model\",\n      \"pmids\": [\"26169356\", \"26943578\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct ROCK1 substrate establishing polarity bundles not identified\", \"Whether LIMK phosphorylation by ROCK1 is direct in mast cells not shown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Structural and translational-control studies defined ROCK kinase-selectivity determinants and revealed lnc-31/YB-1 as a post-transcriptional regulator of ROCK1 protein output.\",\n      \"evidence\": \"Co-crystal structures of ROCK1/ROCK2 with inhibitors (ATP-site residues) and RNA-IP/polysome analysis of lnc-31-Rock1 mRNA-YB-1\",\n      \"pmids\": [\"30384606\", \"29669280\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Apo/RhoA-bound full-length structure not resolved\", \"Whether lnc-31 regulation operates outside myoblasts unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Cardiac ROCK1 knockouts linked the kinase to suppression of Beclin-1-mediated autophagy and doxorubicin cardiotoxicity.\",\n      \"evidence\": \"Global and cardiomyocyte-specific ROCK1 KO mice with Beclin-1/Bcl-2 Co-IP and autophagy flux markers\",\n      \"pmids\": [\"29560126\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ROCK1 directly phosphorylates Beclin-1 at Thr119 not demonstrated\", \"Cardiomyocyte vs non-cardiomyocyte contributions not fully separated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"CRISPR deletion established ROCK1 as the non-redundant driver of apoptotic membrane blebbing and apoptotic-body formation, distinguishing it from PAK2 and LIMK1.\",\n      \"evidence\": \"CRISPR/Cas9 knockout of ROCK1, PAK2, LIMK1 with time-lapse blebbing imaging and phagocytosis assays\",\n      \"pmids\": [\"31043701\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate(s) executing blebbing in this system not specified\", \"Mechanism controlling secondary necrosis onset unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Multiple studies established ROCK1/actomyosin as necessary and sufficient for oncogenic TEAD/YAP transcription and identified Drp1-Ser616 and TFEB as new functional outputs.\",\n      \"evidence\": \"ROCK1 knockdown/inhibitor with YAP/TEAD readouts (mutant p53, Hippo, ECM stiffness contexts); Rnd3-ROCK1 Co-IP/docking with p-Drp1 Ser616 and Rnd3 in vivo cardiac models; SPR/PLA for ROCK1-TFEB with lysosomal assays\",\n      \"pmids\": [\"35121738\", \"39755713\", \"39497162\", \"40368918\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct in vitro kinase validation of TFEB phosphorylation by ROCK1 not fully described\", \"How ROCK1 mechanistically promotes YAP nuclear localization not resolved\", \"Whether Drp1 Ser616 is a direct ROCK1 site versus indirect not definitively shown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Viral and inflammatory studies broadened ROCK1 roles into mRNA stability control and antiviral defense via the ROCK1/MLC2 axis.\",\n      \"evidence\": \"ROCK1 siRNA/inhibitors with viral mRNA decay assays, PABP Co-IP, and CCR4-NOT analysis in buffalopox-infected cells\",\n      \"pmids\": [\"36280692\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular link between ROCK1 and the deadenylation machinery not defined\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Patient-derived proteomics and binding studies positioned ROCK1 as a driver of myeloid inflammatory activation in GVHD and a RIPK4/AMPK regulator in endothelial/diabetic contexts.\",\n      \"evidence\": \"Kinase proteomics with ROCK1 siRNA/inhibitor in macrophages/DCs and GVHD model; ROCK1-RIPK4 Co-IP with pAMPKα/eNOS readouts in diabetic wound model\",\n      \"pmids\": [\"38199985\", \"38538716\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ROCK1-RIPK4 interaction is direct not fully established\", \"Substrates linking ROCK1 to NF-κB activation in myeloid cells not identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved which ROCK1 substrates are directly phosphorylated versus indirectly engaged across its many reported contexts, and how isoform-specific substrate preference is structurally encoded.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No full-length RhoA-bound structural model defining autoinhibition release\", \"Direct in vitro kinase validation lacking for several recent substrates (TFEB, Drp1, Beclin-1)\", \"Mechanism of nuclear and centrosomal targeting not defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 9, 24, 26]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [3, 9, 11]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [10, 24]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 11, 16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [23, 24]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [6, 7, 8]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [19, 30]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RHOA\", \"ZIPK\", \"RND3\", \"FRMD5\", \"AURKA\", \"RIPK4\", \"YAP1\", \"TFEB\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}