Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphorylation of myosin regulatory light chain (RLC) at Ser19 (mono-phosphorylation) promotes filament assembly and enhances actin-activated ATPase activity of non-muscle myosin, while phosphorylation at both Ser19 and Thr18 (di-phosphorylation) further enhances the ATPase activity. However, it has not well been addressed which type of phosphorylation is important in regulating myosin during cytokinesis. Here, we investigated subcellular localization in sea urchin eggs of mono-phosphorylated and di-phosphorylated RLC by both quantitative biochemical and spatiotemporal cytological approaches. Mono-phosphorylated RLC was dominant in the equatorial cortex throughout the whole process of cytokinesis. Inhibition of myosin light chain kinase (MLCK) decreased mono-phosphorylated RLC both in the cortex and in the cleavage furrow, and blocked both formation and contraction of the contractile ring. Two different types of ROCK inhibitor gave inconsistent results: H1152 blocked both RLC mono-phosphorylation in the cleavage furrow and contraction of the contractile ring, while Y27632 affected neither the mono-phosphorylation nor cell division. These results suggest that there may be other targets of H1152 than ROCK, which is involved in the RLC phosphorylation in the cleavage furrow. Furthermore, it was revealed that localization of myosin heavy chain in the cleavage furrow, but not in the cortex, was perturbed by inhibition of RLC mono-phosphorylation. These results suggested that RLC mono-phosphorylation by more than two RLC kinases play a main role in regulation and localization of myosin in the dividing sea urchin eggs.
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PMID:In vivo phosphorylation of regulatory light chain of myosin II in sea urchin eggs and its role in controlling myosin localization and function during cytokinesis. 1796 85

Caldesmon (Cd) and calponin (Cp) are two actin/calmodulin-binding proteins involved in 'actin-linked' regulation of smooth muscle and non-muscle Mg(2+) actin-activated myosin II ATPase activity. However, in the brain, Cd and Cp are associated with the regulation of the neuronal cytoskeleton. In this study we investigated the subcellular distribution of Cd and Cp in the amygdala and their possible relationship to metabotropic glutamate (mGluR1 alpha and 5) and TrkB receptors which interact with inputs from the cortex and are involved in associative learning. Cd and Cp immunoreactivity (IR) was mainly found in dendritic spines, along dendritic microtubules, and in neuronal perikarya but never in axon terminals. Punctate labeling representing spine labeling was restricted to small patches in the lateral nucleus of amygdala, intercalated cell masses (ICM), and the lateral subdivision of central nucleus. This restricted distribution may reflect local afferent activation. In addition, Cd, Cp, mGluR1 alpha and cortical afferents are co-distributed in the ICM distributed in the lateral nucleus and lateral capsular division of the central nucleus, and the lateral division of the central nucleus itself. Consistent with our previous studies, TrkB IR in the central nucleus was associated with Cd and Cp-immunoreactive spines whereas mGluR1 alpha IR and mGluR5 IR were almost exclusively associated with the PSDs of asymmetric synapses, in most cases apposed by cortical terminals. mGluR1 alpha and TrkB immunoreactivities were invariably associated with each other. Overall, these findings suggest that caldesmon and calponin in the amygdala are closely associated with afferents and receptors that have been strongly implicated in associative learning.
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PMID:Co-localization of caldesmon and calponin with cortical afferents, metabotropic glutamate and neurotrophic receptors in the lateral and central nuclei of the amygdala. 1858 38

Long-chain fatty acids (FA) are the primary energy source utilized by the adult heart. However, during pathological cardiac hypertrophy the fetal gene program is reactivated and glucose becomes the major fuel source metabolized by the heart. Herein we describe the metabolic phenotype associated with caveolin-1(Cav1) gene ablation (Cav1ko) in cardiac fibroblasts. Cav1, the primary protein component of caveolae in non-muscle cells co-localizes with a number of proteins involved in substrate metabolism, including, FA translocase (CD36) and the insulin receptor. We demonstrate that Cav1ko hearts develop cardiac hypertrophy and contractile dysfunction at 5-6mos of age. Surprisingly, we observed an increase in the uptake of Intralipid triglyceride and albumin bound FA by 25% and 47%, respectively, in Cav1ko hearts. Isolated perfused heart studies revealed no significant difference in glucose oxidation and glycolysis, however, we observed a trend toward increased FA oxidation in Cav1ko hearts. Real-time PCR analysis revealed no significant changes in the expression of genes involved in FA and glucose metabolism. We also report myocardial triglyceride, fatty acid and cholesterol levels are significantly reduced in Cav1ko hearts. Microarray gene expression analysis revealed changes in genes that regulate calcium ion and lipid transport as well as a number of genes not previously linked to cardiac hypertrophy. We observed a 4-fold increase in tetraspanin-2 gene expression, a transmembrane protein implicated in regulating intracellular trafficking. Oxysterol binding protein related protein-3, which has been implicated in intracellular lipid synthesis and transport, was increased 3.6-fold. In addition, sarcoplasmic reticulum Ca(2+)-ATPase 3, and calcyclin gene transcripts were significantly increased in Cav1ko hearts. In summary, targeted loss of Cav1 produces a unique model of cardiac hypertrophy with normal substrate utilization and expression of genes involved in energy metabolism.
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PMID:Hearts lacking caveolin-1 develop hypertrophy with normal cardiac substrate metabolism. 1871 68

Tropomyosins, a family of actin-binding regulatory proteins, are present in muscle and non-muscle cells. Multiple tropomyosin (TM) isoforms differ in actin affinity and regulatory properties, but little is known about the molecular bases of these differences. The C-terminus of actin stabilizes contacts between actin subunits in the filament and interacts with myosin and regulatory proteins. The goal of this work was to reveal how structural changes in actin and differences between TM isoforms affect binding between these proteins and affect thin filament regulation. Actin proteolytically truncated by three C-terminal amino acids exhibited 1.2-1.5 fold reduced affinity for non-muscle and smooth muscle tropomyosin isoforms. The truncation increased the cooperativity of myosin S1-induced tropomyosin binding for short tropomyosins (TM5a and TM1b9a), but it was neutral for long isoforms (smTM and TM2). Actin modification affected regulation of actomyosin ATPase activity in the presence of all tropomyosins by shifting the filament into a more active state. We conclude that the integrity of the actin C-terminus is important for actin-tropomyosin interactions, however the increased affinity of tropomyosin binding in the S1-induced state of the filament appears not to be involved in the tropomyosin isoform-dependent mechanism of the actomyosin ATPase activation.
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PMID:Effect of actin C-terminal modification on tropomyosin isoforms binding and thin filament regulation. 1904 30

The actin-binding protein caldesmon (CaD) reversibly inhibits smooth muscle contraction. In non-muscle cells, a shorter CaD isoform co-exists with microfilaments in the stress fibers at the quiescent state, but the phosphorylated CaD is found at the leading edge of migrating cells where dynamic actin filament remodeling occurs. We have studied the effect of a C-terminal fragment of CaD (H32K) on the kinetics of the in vitro actin polymerization by monitoring the fluorescence of pyrene-labeled actin. Addition of H32K or its phosphorylated form either attenuated or accelerated the pyrene emission enhancement, depending on whether it was added at the early or the late phase of actin polymerization. However, the CaD fragment had no effect on the yield of sedimentable actin, nor did it affect the actin ATPase activity. Our findings can be explained by a model in which nascent actin filaments undergo a maturation process that involves at least two intermediate conformational states. If present at early stages of actin polymerization, CaD stabilizes one of the intermediate states and blocks the subsequent filament maturation. Addition of CaD at a later phase accelerates F-actin formation. The fact that CaD is capable of inhibiting actin filament maturation provides a novel function for CaD and suggests an active role in the dynamic reorganization of the actin cytoskeleton.
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PMID:Differential effects of caldesmon on the intermediate conformational states of polymerizing actin. 1988 35

Cells sense the rigidity of their environment and respond to it. Most studies have been focused on the role of adhesion complexes in rigidity sensing. In particular, it has been clearly shown that proteins of the adhesion complexes were stretch-sensitive, and could thus trigger mechano-chemical signaling in response to applied forces. In order to understand how this local mechano-sensitivity could be coordinated at the cell scale, we have recently carried out single cell traction force measurements on springs of varying stiffness. We found that contractility at the cell scale (force, speed of contraction, mechanical power) was indeed adapted to external stiffness, and reflected ATPase activity of non-muscle myosin II and acto-myosin response to load. Here we suggest a scenario of rigidity sensing where local adhesions sensitivity to force could be coordinated by adaptation of the acto-myosin dependent cortical tension at the global cell scale. Such a scenario could explain how spreading and migration are oriented by the rigidity of the cell environment.
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PMID:Acto-myosin based response to stiffness and rigidity sensing. 2081 54

Myosin II ATPase activity is enhanced by the phosphorylation of MRLC (myosin II regulatory light chain) in non-muscle cells. It is well known that pMRLC (phosphorylated MRLC) co-localizes with F-actin (filamentous actin) in the CR (contractile ring) of dividing cells. Recently, we reported that HeLa cells expressing non-phosphorylatable MRLC show a delay in the speed of furrow ingression, suggesting that pMRLC plays an important role in the control of furrow ingression. However, it is still unclear how pMRLC regulates myosin II and F-actin at the CR to control furrow ingression during cytokinesis. In the present study, to clarify the roles of pMRLC, we measured the turnover of myosin II and actin at the CR in dividing HeLa cells expressing fluorescent-tagged MRLCs and actin by FRAP (fluorescence recovery after photobleaching). A myosin II inhibitor, blebbistatin, caused an enhancement of the turnover of MRLC and actin at the CR, which induced a delay in furrow ingression. Furthermore, only non-phosphorylatable MRLC and a Rho-kinase inhibitor, Y-27632, accelerated the turnover of MRLC and actin at the CR. Interestingly, the effect of Y-27632 was cancelled in the cell expressing phosphomimic MRLCs. Taken together, these results reveal that pMRLC reduces the turnover of myosin II and also actin at the CR. In conclusion, we show that the enhancement of myosin II and actin turnover at the CR induced slower furrowing in dividing HeLa cells.
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PMID:Enhancement of myosin II/actin turnover at the contractile ring induces slower furrowing in dividing HeLa cells. 2123 14

We used FRET to examine the kinetics and thermodynamics of structural changes associated with ADP release in myosin V, which is thought to be a strain-sensitive step in many muscle and non-muscle myosins. We also explored essential dynamics using FIRST/FRODA starting with three different myosin V X-ray crystal structures to examine intrinsic flexibility and correlated motions. Our steady-state and time-resolved FRET analysis demonstrates a temperature-dependent reversible conformational change in the nucleotide-binding pocket (NBP). Our kinetic results demonstrate that the NBP goes from a closed to an open conformation prior to the release of ADP, while the actin-binding cleft remains closed. Interestingly, we find that the temperature dependence of the maximum actin-activated myosin V ATPase rate is similar to the pocket opening step, demonstrating that this is the rate-limiting structural transition in the ATPase cycle. Thermodynamic analysis demonstrates that the transition from the open to closed NBP conformation is unfavorable because of a decrease in entropy. The intrinsic flexibility analysis is consistent with conformational entropy playing a role in this transition as the MV.ADP structure is highly flexible compared to the MV.APO structure. Our experimental and modeling studies support the conclusion of a novel post-power-stroke actomyosin.ADP state in which the NBP and actin-binding cleft are closed. The novel state may be important for strain sensitivity as the transition from the closed to open NBP conformation may be altered by lever arm position.
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PMID:Kinetics and thermodynamics of the rate-limiting conformational change in the actomyosin V mechanochemical cycle. 2131 83

MYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations in the gene for the heavy chain of non-muscle myosin-IIA (NMMHC-IIA). Recent in vitro studies led to the hypothesis that thrombocytopenia of MYH9-RD derives from an ectopic platelet release by megakaryocytes in the osteoblastic areas of bone marrow (BM), which are enriched in type I collagen, rather than in vascular spaces. SDF-1-driven migration of megakaryocytes within BM to reach the vascular spaces is a key mechanism for platelet biogenesis. Since myosin-IIA is implicated in polarised migration of different cell types, we hypothesised that MYH9 mutations could interfere with this mechanism. We therefore investigated the SDF-1-driven migration of a megakaryoblastic cell line, Dami cells, on type I collagen or fibrinogen by a modified transwell assay. Inhibition of myosin-IIA ATPase activity suppressed the SDF-1-driven migration of Dami cells, while over-expression of NMMHC-IIA increased the efficiency of chemotaxis, indicating a role for NMMHC-IIA in this mechanism. Transfection of cells with three MYH9 mutations frequently responsible for MYH9-RD (p.R702C, p.D1424H, or p.R1933X) resulted in a defective SDF-1-driven migration with respect to the wild-type counterpart and in increased cell spreading onto collagen. Analysis of differential localisation of wild-type and mutant proteins suggested that mutant NMMHC-IIAs had an impaired cytoplasmic re-organisation in functional cytoskeletal structures after cell adhesion to collagen. These findings support the hypothesis that a defect of SDF-1-driven migration of megakaryocytes induced by MYH9 mutations contributes to ectopic platelet release in the BM osteoblastic areas, resulting in ineffective platelet production.
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PMID:Mutations responsible for MYH9-related thrombocytopenia impair SDF-1-driven migration of megakaryoblastic cells. 2183 45

Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various cell-division inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34(+) cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts.
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PMID:Enucleation of human erythroblasts involves non-muscle myosin IIB. 2228 91


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