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)

Most vital cellular functions are dependent on a fine-tuned regulation of intracellular ion homeostasis. Here we have demonstrated, using COS cells that were untransfected or transfected with wild-type rat ouabain-resistant Na(+)-K(+)-ATPase, that partial inhibition of Na(+)-K(+)-ATPase has a dramatic influence on cell attachment to fibronectin. Ouabain dose-dependently decreased attachment in untransfected cells and in cells expressing wild-type Na(+)-K(+)-ATPase, but not in cells expressing ouabain-insensitive Na(+)-K(+)-ATPase, whereas inhibition of Na(+)-K(+)-ATPase by lowering extracellular K(+) concentration decreased attachment in all three cell types. Thirty percent inhibition of Na(+)-K(+)-ATPase significantly attenuated attachment. Na(+)-K(+)-ATPase inhibition caused a sustained increase in the intracellular Ca(2+) concentration that obscured Ca(2+) transients observed in untreated cells during attachment. Inhibitors of Ca(2+) transporters significantly decreased attachment, but inhibition of Na(+)/H(+) exchanger did not. Ouabain reduced focal adhesion kinase autophosphorylation but had no effect on cell surface integrin expression. These results suggest that the level of Na(+)-K(+)-ATPase activity strongly influences cell attachment, possibly by an effect on intracellular Ca(2+).
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PMID:Changes in Na(+)-K(+)-ATPase activity influence cell attachment to fibronectin. 1178 41

Plasma membrane Ca(2+) -ATPase isoform 4b (PMCA4b) is phosphorylated on a tyrosine residue during platelet activation resulting in inhibition of its ATPase activity. We now report that tyrosine 1176 (Y(1176)) in the carboxyl (C-) terminal domain of PMCA4b is the phosphorylated residue. Two tyrosine residues located in the C-terminus of PMCA4b, Y(1122) and Y(1176) can be removed by calpain-dependent cleavage. This truncation removes all of the tyrosine phosphates added to PMCA during platelet activation. Sequence analysis indicates that Y(1176) is a likely substrate for focal adhesion kinase (FAK), while Y(1122) is not located in a tyrosine phosphorylation motif. This is the same residue we reported earlier to be phosphorylated by Src kinase in vitro. Thus we conclude that Y(1176) is the only tyrosine phosphorylated during platelet activation. Results of co-immunoprecipitation, treatment with tyrosine kinase inhibitors and integrin inhibition experiments suggest that FAK is responsible for PMCA4b tyrosine phosphorylation during platelet activation.
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PMID:Plasma membrane Ca2+-ATPase isoform 4b is phosphorylated on tyrosine 1176 in activated human platelets. 1254 Sep 62

Ca2+ sensitivity of smooth muscle and nonmuscle myosin II reflects the ratio of activities of myosin light-chain kinase (MLCK) to myosin light-chain phosphatase (MLCP) and is a major, regulated determinant of numerous cellular processes. We conclude that the majority of phenotypes attributed to the monomeric G protein RhoA and mediated by its effector, Rho-kinase (ROK), reflect Ca2+ sensitization: inhibition of myosin II dephosphorylation in the presence of basal (Ca2+ dependent or independent) or increased MLCK activity. We outline the pathway from receptors through trimeric G proteins (Galphaq, Galpha12, Galpha13) to activation, by guanine nucleotide exchange factors (GEFs), from GDP. RhoA. GDI to GTP. RhoA and hence to ROK through a mechanism involving association of GEF, RhoA, and ROK in multimolecular complexes at the lipid cell membrane. Specific domains of GEFs interact with trimeric G proteins, and some GEFs are activated by Tyr kinases whose inhibition can inhibit Rho signaling. Inhibition of MLCP, directly by ROK or by phosphorylation of the phosphatase inhibitor CPI-17, increases phosphorylation of the myosin II regulatory light chain and thus the activity of smooth muscle and nonmuscle actomyosin ATPase and motility. We summarize relevant effects of p21-activated kinase, LIM-kinase, and focal adhesion kinase. Mechanisms of Ca2+ desensitization are outlined with emphasis on the antagonism between cGMP-activated kinase and the RhoA/ROK pathway. We suggest that the RhoA/ROK pathway is constitutively active in a number of organs under physiological conditions; its aberrations play major roles in several disease states, particularly impacting on Ca2+ sensitization of smooth muscle in hypertension and possibly asthma and on cancer neoangiogenesis and cancer progression. It is a potentially important therapeutic target and a subject for translational research.
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PMID:Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. 1450 7

We have shown that the Na/K-ATPase and Src form a signaling receptor complex. Here we determined how alterations in the amount and properties of the Na/K-ATPase affect basal Src activity and ouabain-induced signal transduction. Several alpha1 subunit knockdown cell lines were generated by transfecting LLC-PK1 cells with a vector expressing alpha1-specific small interference RNA. Although the alpha1 knockdown resulted in significant decreases in Na/K-ATPase activity, it increased the basal Src activity and tyrosine phosphorylation of focal adhesion kinase, a Src effector. Concomitantly it also abolished ouabain-induced activation of Src and ERK1/2. When the knockdown cells were rescued by a rat alpha1, both Na/K-ATPase activity and the basal Src activity were restored. In addition, ouabain was able to stimulate Src and ERK1/2 in the rescued cells at a much higher concentration, consistent with the established differences in ouabain sensitivity between pig and rat alpha1. Finally both fluorescence resonance energy transfer analysis and co-immunoprecipitation assay indicated that the pumping-null rat alpha1 (D371E) mutant could also bind Src. Expression of this mutant restored the basal Src activity and focal adhesion kinase tyrosine phosphorylation. Taken together, the new findings suggest that LLC-PK1 cells contain a pool of Src-interacting Na/K-ATPase that not only regulates Src activity but also serves as a receptor for ouabain to activate protein kinases.
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PMID:Functional characterization of Src-interacting Na/K-ATPase using RNA interference assay. 1669 1

This review focuses on using the knowledge on volume-sensitive transport systems in Ehrlich ascites tumour cells and NIH-3T3 cells to elucidate osmotic regulation of salt transport in epithelia. Using the intestine of the European eel (Anguilla anguilla) (an absorptive epithelium of the type described in the renal cortex thick ascending limb (cTAL)) we have focused on the role of swelling-activated K+- and anion-conductive pathways in response to hypotonicity, and on the role of the apical (luminal) Na+-K+-2Cl- cotransporter (NKCC2) in the response to hypertonicity. The shrinkage-induced activation of NKCC2 involves an interaction between the cytoskeleton and protein phosphorylation events via PKC and myosin light chain kinase (MLCK). Killifish (Fundulus heteroclitus) opercular epithelium is a Cl(-)-secreting epithelium of the type described in exocrine glands, having a CFTR channel on the apical side and the Na+/K+ ATPase, NKCC1 and a K+ channel on the basolateral side. Osmotic control of Cl- secretion across the operculum epithelium includes: (i) hyperosmotic shrinkage activation of NKCC1 via PKC, MLCK, p38, OSR1 and SPAK; (ii) deactivation of NKCC by hypotonic cell swelling and a protein phosphatase, and (iii) a protein tyrosine kinase acting on the focal adhesion kinase (FAK) to set levels of NKCC activity.
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PMID:The role of volume-sensitive ion transport systems in regulation of epithelial transport. 1728 11

Differentiation of stromal cells into decidual cells, which is critical to successful pregnancy, represents a complex transformation requiring changes in cytoskeletal architecture. We demonstrate that in vitro differentiation of human uterine fibroblasts into decidual cells includes down-regulation of alpha-smooth muscle actin and beta-tubulin, phosphorylation of focal adhesion kinase, and redistribution of vinculin. This is accompanied by varied adhesion to fibronectin and a modified ability to migrate. Cytoskeletal organization is determined primarily by actin-myosin II interactions governed by the phosphorylation of myosin light chain (MLC20). Decidualization induced by cAMP [with estradiol-17beta (E) and medroxyprogesterone acetate (P)] results in a 40% decrease in MLC20 phosphorylation and a 55% decline in the long (214 kDa) form of myosin light-chain kinase (MLCK). Destabilization of the cytoskeleton by inhibitors of MLCK (ML-7) or myosin II ATPase (blebbistatin) accelerates decidualization induced by cAMP (with E and P) but inhibits decidualization induced by IL-1beta (with E and P). Adenoviral infection of human uterine fibroblast cells with a constitutively active form of MLCK followed by decidualization stimuli leads to a 30% increase in MLC20 phosphorylation and prevents decidualization. These data provide evidence that the regulation of cytoskeletal dynamics by MLC20 phosphorylation is critical for decidualization.
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PMID:Increased phosphorylation of myosin light chain prevents in vitro decidualization. 1741 15

During heart development, cells from the proepicardial organ spread over the naked heart tube to form the epicardium. From here, epicardium-derived cells (EPDCs) migrate into the myocardium. EPDCs proved to be indispensable for the formation of the ventricular compact zone and myocardial maturation, by largely unknown mechanisms. In this study we investigated in vitro how EPDCs affect cardiomyocyte proliferation, cellular alignment and contraction, as well as the expression and cellular distribution of proteins involved in myocardial maturation. Embryonic quail EPDCs induced proliferation of neonatal mouse cardiomyocytes. This required cell-cell interactions, as proliferation was not observed in transwell cocultures. Western blot analysis showed elevated levels of electrical and mechanical junctions (connexin43, N-cadherin), sarcomeric proteins (Troponin-I, alpha-actinin), extracellular matrix (collagen I and periostin) in cocultures of EPDCs and cardiomyocytes. Immunohistochemistry indicated more membrane-bound expression of Cx43, N-cadherin, the mechanotransduction molecule focal adhesion kinase, and higher expression of the sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a). Newly developed software for analysis of directionality in immunofluorescent stainings showed a quantitatively determined enhanced cellular alignment of cardiomyocytes. This was functionally related to increased contraction. The in vitro effects of EPDCs on cardiomyocytes were confirmed in three reciprocal in vivo models for EPDC-depletion (chicken and mice) in which downregulation of myocardial N-cadherin, Cx43, and FAK were observed. In conclusion, direct interaction of EPDCs with cardiomyocytes induced proliferation, correct mechanical and electrical coupling of cardiomyocytes, ECM-deposition and concurrent establishment of cellular array. These findings implicate that EPDCs are ideal candidates as adjuvant cells for cardiomyocyte integration during cardiac (stem) cell therapy.
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PMID:Epicardium-derived cells enhance proliferation, cellular maturation and alignment of cardiomyocytes. 2065 24

Current polyvinylpyrrolidone-modified polysulfone (PVP-PSU) membranes in haemodialysers do not facilitate the attachment and proliferation of renal proximal tubule cells (RPTCs). For bioartificial kidney (BAK) development expensive extracellular matrices are employed to ensure the PVP-PSU membranes can serve as a substrate for RPTCs. In this study we modified PSU using an acrylic monomer (am-PSU) and polymerization using ultraviolet irradiation. We demonstrated that on adjusting the PSU or acrylic content of the membranes the wettability and surface chemistry were altered, and this affected the amount of fibronectin (Fn) that was adsorbed onto the membranes. Using an integrin blocking assay we ascertained that Fn is an important extracellular matrix component that mediates RPTC attachment. The amount of Fn adsorbed also led to different bioresponses of RPTCs, which were evaluated using attachment and proliferation assays and qualitative quantification of vinculin, focal adhesion kinase, zonula occludens and Na(+)/K(+) ATPase. Our optimized membrane, am-PSU1 (21.4% C-O groups, 19.1% PVP-PSU; contact angle 71.5-80.80, PVP-PSU: 52.4-67.50), supports a confluent monolayer of RPTCs and prevents creatinine and inulin diffusion from the apical to the basal side, meeting the requirements for application in BAKs. However, further in vivo evaluation to assess the full functionality of RPTCs on am-PSU1 is required.
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PMID:Surface characteristics of acrylic modified polysulfone membranes improves renal proximal tubule cell adhesion and spreading. 2123 68

The cytoplasmic Ca(2+) clearance rate affects neuronal excitability, plasticity, and synaptic transmission. Here, we examined the modulation of the plasma membrane Ca(2+) ATPase (PMCA) by tyrosine kinases. In rat sensory neurons grown in culture, the PMCA was under tonic inhibition by a member of the Src family of tyrosine kinases (SFKs). Ca(2+) clearance accelerated in the presence of selective tyrosine kinase inhibitors. Tonic inhibition of the PMCA was attenuated in cells expressing a dominant-negative construct or shRNA directed to message for the SFKs Lck or Fyn, but not Src. SFKs did not appear to phosphorylate the PMCA directly but instead activated focal adhesion kinase (FAK). Expression of constitutively active FAK enhanced and dominant-negative or shRNA knockdown of FAK attenuated tonic inhibition. Antisense knockdown of PMCA isoform 4 removed tonic inhibition of Ca(2+) clearance, indicating that FAK acts on PMCA4. The hyaluronan receptor CD44 activates SFK-FAK signaling cascades and is expressed in sensory neurons. Treating neurons with a CD44-blocking antibody or short hyaluronan oligosaccharides, which are produced during injury and displace macromolecular hyaluronan from CD44, attenuated tonic PMCA inhibition. Ca(2+)-activated K(+) channels mediate a slow afterhyperpolarization in sensory neurons that was inhibited by tyrosine kinase inhibitors and enhanced by knockdown of PMCA4. Thus, we describe a novel kinase cascade in sensory neurons that enables the extracellular matrix to alter Ca(2+) signals by modulating PMCA-mediated Ca(2+) clearance. This signaling pathway may influence the excitability of sensory neurons following injury.
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PMID:Inhibition of the plasma membrane Ca2+ pump by CD44 receptor activation of tyrosine kinases increases the action potential afterhyperpolarization in sensory neurons. 2132 3

Tumor cell migration is supported in part by the cyclic formation and disassembly of focal adhesions (FAs); however, the mechanisms that regulate this process are not fully defined. The large guanosine 5'-triphosphatase dynamin (Dyn) plays an important role in FA dynamics and is activated by tyrosine phosphorylation. Using a novel antibody specific to phospho-dynamin (pDyn-Tyr-231), we found that Dyn2 is phosphorylated at FAs by Src kinase and is recruited to FAs by a direct interaction with the 4.1/ezrin/radizin/moesin domain of focal adhesion kinase (FAK), which functions as an adaptor between Src and Dyn2 to facilitate Dyn2 phosphorylation. This Src-FAK-Dyn2 trimeric complex is essential for FA turnover, as mutants disrupting the formation of this complex inhibit FA disassembly. Importantly, phosphoactivated Dyn2 promotes FA turnover by mediating the endocytosis of integrins in a clathrin-dependent manner. This study defines a novel mechanism of how Dyn2 functions as a downstream effector of FAK-Src signaling in turning over FAs.
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PMID:A direct interaction between the large GTPase dynamin-2 and FAK regulates focal adhesion dynamics in response to active Src. 2141 25


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