Peptide substrates for Rho-associated kinase 2 (Rho-kinase 2/ROCK2).
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ABSTRACT:
SUBMITTER: Kang JH
PROVIDER: S-EPMC3144920 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Onconeural antibodies are associated with cancer and paraneoplastic encephalitis. While their pathogenic role is still largely unknown, their high diagnostic value is undisputed. In this study we describe the discovery of a novel target of autoimmunity in an index case of paraneoplastic encephalitis associated with urogenital cancer.A 75-year-old man with a history of invasive bladder carcinoma 6 years ago with multiple recurrences and a newly discovered renal cell carcinoma presented with seizures and progressive cognitive decline followed by super-refractory status epilepticus. Clinical and ancillary findings including brain biopsy suggested paraneoplastic encephalitis. Immunohistochemistry of the brain biopsy was used to characterize the inflammatory response. Indirect immunofluorescence assay (IFA) was used for autoantibody screening. The autoantigen was identified by histo-immunoprecipitation and mass spectrometry and was validated by expressing the recombinant antigen in HEK293 cells and neutralization tests. Sera from 125 control patients were screened using IFA to test for the novel autoantibodies.IFA analysis of serum revealed a novel autoantibody against brain tissue. An intracellular enzyme, Rho-associated protein kinase 2 (ROCK2), was identified as target-antigen. ROCK2 was expressed in affected brain tissue and archival bladder tumor samples of this patient. Brain histopathology revealed appositions of cytotoxic CD8+ T cells on ROCK2-positive neurons. ROCK2 antibodies were not found in the sera of 20 patients with bladder cancer and 17 with renal cancer, both without neurological symptoms, 49 healthy controls, and 39 patients with other antineuronal autoantibodies. In conclusion, novel onconeural antibodies targeting ROCK2 are associated with paraneoplastic encephalitis and should be screened for when paraneoplastic neurological syndromes, especially in patients with urogenital cancers, occur.
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Project description:Mechanisms of the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK) inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H2O2 production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H2O2 production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.
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Project description:High-throughput cellular profiling has successfully stimulated early drug discovery pipelines by facilitating targeted as well as opportunistic lead finding, hit annotation and SAR analysis. While automation-friendly universal assay formats exist to address most established drug target classes like GPCRs, NHRs, ion channels or Tyr-kinases, no such cellular assay technology is currently enabling an equally broad and rapid interrogation of the Ser/Thr-kinase space. Here we present the foundation of an emerging cellular Ser/Thr-kinase platform that involves a) coexpression of targeted kinases with promiscuous peptide substrates and b) quantification of intracellular substrate phosphorylation by homogeneous TR-FRET. Proof-of-concept data is provided for cellular AKT, B-RAF and CamK2delta assays. Importantly, comparable activity profiles were found for well characterized B-Raf inhibitors in TR-FRET assays relying on either promiscuous peptide substrates or a MEK1(WT) protein substrate respectively. Moreover, IC(50)-values correlated strongly between cellular TR-FRET assays and a gold standard Ba/F3 proliferation assay for B-Raf activity. Finally, we expanded our initial assay panel by screening a kinase-focused cDNA library and identified starting points for >20 cellular Ser/Thr-kinase assays.
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Project description:BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.
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Project description:Medulloblastoma is one of the most common malignant brain tumor types in children, with an overall survival of 70%. Mortality is associated with metastatic relapsed tumors. Rho-associated kinases (ROCKs), important for epithelial-mesenchymal transition (EMT) and proper nervous system development, have previously been identified as a promising drug target to inhibit cancer growth and metastatic spread. Here, we show that ROCKs are expressed in medulloblastoma, with higher ROCK2 mRNA expression in metastatic compared to non-metastatic tumors. By evaluating three ROCK inhibitors in a panel of medulloblastoma cell lines we demonstrated that medulloblastoma cells were sensitive for pharmacological ROCK inhibition. The specific ROCK inhibitor RKI-1447 inhibited the tumorigenicity in medulloblastoma cells as well as impeded cell migration and invasion. Differential gene expression analysis suggested that ROCK inhibition was associated with the downregulation of signaling pathways important in proliferation and metastasis e.g., TNFα via NFκβ, TGFβ, and EMT. Expression of key proteins in these pathways such as RHOA, RHOB, JUN, and vimentin was downregulated in ROCK inhibited cells. Finally, we showed that ROCK inhibition by RKI-1447 suppressed medulloblastoma growth and proliferation in vivo. Collectively, our results suggest that ROCK inhibition presents a potential new therapeutic option in medulloblastoma, especially for children with metastatic disease.
| S-EPMC7016943 | biostudies-literature
Project description:Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. Here we show that neuroblastoma harbors frequent mutations of genes controlling the Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. RhoA is activated in tumors from neuroblastoma patients, and elevated expression of Rho-associated kinase (ROCK)2 is associated with poor patient survival. Pharmacological or genetic inhibition of ROCK1 and 2, key molecules in Rho signaling, resulted in neuroblastoma cell differentiation and inhibition of neuroblastoma cell growth, migration, and invasion. Molecularly, ROCK inhibition induced glycogen synthase kinase 3β-dependent phosphorylation and degradation of MYCN protein. Small-molecule inhibition of ROCK suppressed MYCN-driven neuroblastoma growth in TH-MYCN homozygous transgenic mice and MYCN gene-amplified neuroblastoma xenograft growth in nude mice. Interference with Rho/Rac signaling might offer therapeutic perspectives for high-risk neuroblastoma.
| S-EPMC5559038 | biostudies-literature
Project description:BackgroundAcupuncture has been proven effective for various types of pain, and peripheral molecular signals around acupuncture-treated areas have been suggested to contribute to the analgesic effects of acupuncture. However, the underlying mechanism from these peripheral molecular signals to central ones remains unclear. The purpose of this study was to investigate whether peripheral Rho-associated protein kinase (ROCK) activation induced by acupuncture treatment mediates acupuncture analgesia, and also to investigate the relationship between ROCK activation and extracellular signal-regulated kinase (ERK), which has previously been proven to mediate acupuncture analgesia and other related molecular changes during acupuncture.MethodsAcupuncture was treated at the bilateral GB34 acupoints of C57BL/6 mice, after which changes in ROCK activation and the location of its expression in the skin were analyzed. To verify the role of ROCK in acupuncture analgesia, we administrated ROCK inhibitor Y-27632 (0.3 μg/ul) into the skin before acupuncture treatment with formalin and complete Freund adjuvant (CFA) induced pain models, then the nociceptive responses were analyzed.ResultsAcupuncture treatment produced ROCK2 activation in the skin after 30 and 60 min, and the histological analyses revealed that ROCK2 was activated in the fibroblast of the dermis. The acupuncture-induced ROCK2 expression was significantly attenuated by the ERK inhibitor, whereas phospho-ERK expression was not inhibited by ROCK inhibitor. In both the formalin- and CFA-induced mouse pain models, acupuncture analgesia was blocked by ROCK inhibitor administration.ConclusionAcupuncture treatment-induced ROCK2 expression is a downstream effector of phospho-ERK in the skin and plays a crucial role in acupuncture analgesia.
| S-EPMC11364124 | biostudies-literature
Project description:Cardiovascular diseases are associated with chronic activation of Rho-associated kinase. Rho-associated kinase activity is significantly correlated with endothelial function and Framingham risk score. However, there is no information on the prognostic value of Rho-associated kinase activity. We evaluated Rho-associated kinase activity in peripheral leukocytes by Western blot analysis in 633 subjects who underwent health-screening examination at Hiroshima University Hospital. We assessed the associations between Rho-associated kinase activity and first major cardiovascular events (death from cardiovascular causes, myocardial infarction, and stroke), death from cardiovascular causes, acute myocardial infarction, stroke, revascularization (percutaneous coronary intervention, coronary artery bypass grafting), and hospitalization for heart failure. During a median period of 42.0 months (interquartile range, 24.4-56.6 months) of follow-up, 29 subjects died (10 from cardiovascular causes), 2 myocardial infarction, 20 revascularization, 15 stroke, and 17 hospitalization for heart failure. After adjustment for age, sex, cardiovascular risk factors, and other relevant variables, Rho-associated kinase activity remained a strong independent indicator of first major cardiovascular events (hazard ratio, 2.19; 95% confidence interval, 1.35-3.70; P=0.002), death from cardiovascular disease (hazard ratio, 2.57; 95% confidence interval, 1.18-6.60; P=0.002), stroke (hazard ratio, 2.14; 95% confidence interval, 1.24-3.86; P=0.006), and revascularization (hazard ratio, 2.68; 95% confidence interval, 1.60-4.66; P<0.001). Leukocyte Rho-associated kinase activity may be a new biomarker of cardiovascular events. These findings suggest that inhibition of Rho-associated kinase activity may be a therapeutic target for prevention of cardiovascular events.
| S-EPMC4101172 | biostudies-literature
Project description:BACKGROUND AND OBJECTIVES:Recent studies indicate that in response to vasoconstrictor stimuli, the small GTPase RhoA and its down-stream effector, Rho-associated kinase 2 (ROCK)/Rho-kinase, are associated with hypercontraction of the vascular smooth muscle of coronary arteries through augmentation of myosin light chain phosphorylation and Ca(2+) sensitization. Expression of ROCK/Rho-kinase mRNA was significantly increased and up-regulated in the spastic coronary artery in a porcine model, and a specific inhibitor of ROCK/Rho-kinase inhibited coronary artery spasm in humans. We therefore explored the role of ROCK2 polymorphisms in the pathogenesis of vasospastic angina (VA). SUBJECTS AND METHODS:We studied 106 patients with VA who exhibited spontaneous or provoked coronary spasm during coronary angiography and compared the prevalence of ROCK2 polymorphisms between this group of patients with VA and controls whose angiograms were normal, and in whom the ergonovine test did not cause spasm (n=107). Five single nucleotide polymorphisms (SNPs) of the ROCK2 gene were selected. SNPs were genotyped by high-resolution melting. Linkage disequilibrium and haplotype analyses were performed using the SHEsis program. RESULTS:The prevalence of genotypes of the 5 interesting SNPs in patients with VA was not different from that in the control group. In haplotype analysis, the haplotype G-T-C-T-G (in order of rs978906, rs2271621, rs2230774, rs1515210, and rs3771106) was significantly associated with a decreased risk of VA (p=0.007). CONCLUSION:The haplotype G-T-C-T-G in the ROCK2 gene had a protective effect against VA, suggesting the involvement of ROCK2 in VA pathogenesis.
| S-EPMC3390426 | biostudies-literature
Project description:Formaldehyde, a common indoor air pollutant, exacerbates asthma and synergizes with allergen to induce airway hyperresponsiveness (AHR) in animal models. The mechanisms mediating formaldehyde-induced AHR remain poorly understood. We posit that formaldehyde modulates agonist-induced contractile response of human airway smooth muscle (HASM) cells to elicit AHR. HASM cells were exposed to formaldehyde or vehicle and agonist-induced intracellular Ca2+ ([Ca2+]i) and myosin light-chain phosphatase (MYPT1) phosphorylation were determined. Air-liquid interface-differentiated human bronchial epithelial (HBE) cells were exposed to formaldehyde or vehicle and cocultured with HASM cells. Agonist-induced [Ca2+]i and MYPT1 phosphorylation were determined in the cocultured HASM cells. Precision-cut human lung slices were exposed to PBS or varying concentrations of formaldehyde, and then carbachol-induced airway narrowing was determined 24 hours after exposure. HASM cells were transfected with nontargeting or nuclear factor erythroid-derived 2, like 2 (Nrf-2)-targeting small interfering RNA and exposed to formaldehyde or vehicle, followed by determination of antioxidant response (quinone oxido-reductase 1 and thioredoxin 1) and basal and agonist-induced MYPT1 phosphorylation. Formaldehyde enhanced the basal Rho-kinase activity and MYPT1 phosphorylation with little effect on agonist-induced [Ca2+]i in HASM cells. Formaldehyde induced Nrf-2-dependent antioxidant response in HASM cells, although the MYPT1 phosphorylation was independent of Nrf-2 induction. Although HBE cells exposed to formaldehyde had little effect on agonist-induced [Ca2+]i or MYPT1 phosphorylation in cocultured HASM cells, formaldehyde enhanced carbachol-induced airway responsiveness in precision-cut human lung slices. In conclusion, formaldehyde induces phosphorylation of the regulatory subunit of MYPT1, independent of formaldehyde-induced Nrf-2 activation in HASM cells. The findings suggest that the Rho kinase-dependent Ca2+ sensitization pathway plays a role in formaldehyde-induced AHR.
| S-EPMC5070102 | biostudies-literature