Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.17 (CaMKII)
 4,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Caldesmon is a major calmodulin- and actin-binding protein of smooth muscle which interacts with calmodulin in a Ca2+-dependent manner or with actin in a Ca2+-independent manner. Isolated caldesmon is capable of inhibiting the actin-activated Mg2+-ATPase of smooth-muscle myosin, suggesting a possible physiological role for caldesmon in regulating the contractile state of smooth-muscle. Caldesmon can be phosphorylated in vitro by a co-purifying Ca2+/calmodulin-dependent protein kinase and dephosphorylated by a protein phosphatase, both of which are present in smooth muscle. We investigated further the phosphorylation of caldesmon and the effects which phosphorylation has on the functional properties of the protein. The kinetics of caldesmon phosphorylation were similar whether the caldesmon substrate was free or bound to actin, actin/tropomyosin or thin filaments. Caldesmon containing endogenous kinase activity was rapidly phosphorylated (to approx. 1 mol of Pi/mol of caldesmon in 5 min) when reconstituted with actin, myosin, tropomyosin, calmodulin and myosin light-chain kinase in the presence of Ca2+ and MgATP2-. Under conditions in which unphosphorylated caldesmon showed substantial inhibition of the actin-activated myosin Mg2+-ATPase, no inhibition was observed with phosphorylated caldesmon. This was the case whether caldesmon was phosphorylated before addition to the actomyosin Mg2+-ATPase system, or phosphorylation was allowed to take place during the ATPase reaction. Binding studies revealed maximal binding of 1 mol of unphosphorylated caldesmon/9.5 mol of actin and 1 mol of phosphorylated caldesmon/11.7 mol of actin. All the bound phosphorylated caldesmon could be released by Ca2+/calmodulin, with half-maximal release at 0.11 microM-Ca2+, whereas only 62% of the bound unphosphorylated caldesmon could be removed, with half-maximal release at 0.16 microM-Ca2+. However, under conditions in which inhibition of actomyosin Mg2+-ATPase activity by non-phosphorylated but not by phosphorylated caldesmon was observed, both forms of caldesmon would remain bound to the thin filament. These observations suggest a possible mechanism whereby caldesmon phosphorylation may prevent its inhibitory action on the actomyosin Mg2+-ATPase.
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PMID:The effects of phosphorylation of smooth-muscle caldesmon. 282 3

Caldesmon, a major actin- and calmodulin-binding protein of smooth muscle, has been implicated in regulation of the contractile state of smooth muscle. The isolated protein can be phosphorylated by a co-purifying Ca2+/calmodulin-dependent protein kinase, and phosphorylation blocks inhibition of the actomyosin ATPase by caldesmon [Ngai & Walsh (1987) Biochem. J. 244, 417-425]. We have examined the phosphorylation of caldesmon in more detail. Several lines of evidence indicate that caldesmon itself is a kinase and the reaction is an intermolecular autophosphorylation: (1) caldesmon (141 kDa) and a 93 kDa proteolytic fragment of caldesmon can be separated by ion-exchange chromatography: both retain caldesmon kinase activity, which is Ca2+/calmodulin-dependent; (2) chymotryptic digestion of caldesmon generates a Ca2+/calmodulin-independent form of caldesmon kinase; (3) caldesmon purified to electrophoretic homogeneity retains caldesmon kinase activity, and elution of enzymic activity from a fast-performance-liquid-chromatography ion-exchange column correlates with caldesmon of Mr 141,000; (4) caldesmon is photoaffinity-labelled with 8-azido-[alpha-32P]ATP; labelling is inhibited by ATP, GTP and CTP, indicating a lack of nucleotide specificity; (5) caldesmon binds tightly to Affi-Gel Blue resin, which recognizes proteins having a dinucleotide fold. Autophosphorylation of caldesmon occurs predominantly on serine residues (83.3%), with some threonine (16.7%) and no tyrosine phosphorylation. Autophosphorylation is site-specific: 98% of the phosphate incorporated is recovered in a 26 kDa chymotryptic peptide. Complete tryptic/chymotryptic digestion of this phosphopeptide followed by h.p.l.c. indicates three major phosphorylation sites. Caldesmon exhibits a high degree of substrate specificity: apart from autophosphorylation, brain synapsin I is the only good substrate among many potential substrates examined. These observations indicate that caldesmon may regulate its own function (inhibition of the actomyosin ATPase) by Ca2+/calmodulin-dependent autophosphorylation. Furthermore, caldesmon may regulate other cellular processes, e.g. neurotransmitter release, through the Ca2+/calmodulin-dependent phosphorylation of other proteins such as synapsin I.
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PMID:Autophosphorylation of smooth-muscle caldesmon. 341 67

Caldesmon, a major calmodulin- and actin-binding protein of smooth muscle (Sobue, K., Muramoto, Y., Fujita, M., and Kakiuchi, S. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5652-5655), has been obtained in highly purified form from chicken gizzard by a modification of a previously published procedure (Ngai, P. K., Carruthers, C. A., and Walsh, M. P. (1984) Biochem. J. 218, 863-870) and was found to cause a significant inhibition of both superprecipitation and actin-activated myosin Mg2+-ATPase activity in a system reconstituted from the purified contractile and regulatory proteins without influencing the phosphorylation state of myosin. This inhibitory effect was seen both in the presence and absence of tropomyosin. A Ca2+-and calmodulin-dependent kinase which catalyzed phosphorylation of caldesmon was identified in chicken gizzard; this kinase is distinct from myosin light-chain kinase. Caldesmon prepared by calmodulin-Sepharose affinity chromatography was contaminated with caldesmon kinase activity and was unable to inhibit actomyosin ATPase activity or superprecipitation. Phosphatase activity capable of dephosphorylating caldesmon was also identified in smooth muscle. These results indicate that caldesmon can inhibit smooth muscle actomyosin ATPase activity in vitro, and this function may itself be subject to regulation by reversible phosphorylation of caldesmon.
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PMID:Inhibition of smooth muscle actin-activated myosin Mg2+-ATPase activity by caldesmon. 615 36

Sarcomplasmic reticulum from rabbit fast skeletal muscle contains intrinsic protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) and a substrate. The protein kinase activity was Mg2+ dependent and could also phosphorylate exogenous protein substrates. Autophosphorylation of sarcoplasmic reticulum vesicles was not stimulated by cyclic AMP, neither was it inhibited by the heat-stable protein kinase inhibitor protein. The phosphorylated membranes had the characteristics of a protein with a phosphoester bond. An average of 73 pmol Pi/mg protein were incorporated in 10 min at 30 degrees C. Addition of exogenous cyclic AMP-dependent protein kinase increased the endogenous level of phosphorylation by 25-100%. Sarcoplasmic reticulum membrane phosphorylation, mediated by either endogenous cyclic AMP-independent or exogenous cyclic AMP-dependent protein kinase, occurred on a 100 000 dalton protein and both enzyme activities resulted in enhanced calcium uptake and Ca2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3), in a manner similar to cardiac microsomal preparations. Regulation of Ca2+ transport in skeletal sarcoplasmic reticulum may be mediated by phosphorylation of a 100 000 dalton component of these membranes.
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PMID:Phosphorylation of a 100 000 dalton component and its relationship to calcium transport in sarcoplasmic reticulum from rabbit skeletal muscle. 624 11

Microsomal Ca(2+)-ATPase inhibitors such as thapsigargin (THG), cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-hydroquinone (DBHQ) have been shown to inhibit Ca2+ reuptake by the intracellular stores and increase cytosolic free Ca2+ ([Ca2+]i). DBHQ is a commercially available non-toxic synthetic compound chemically unrelated to THG and CPA. In this study, we tested the feasibility of utilizing DBHQ to improve Cl- secretion via the Ca(2+)-dependent pathway, in the cystic fibrosis (CF)-derived pancreatic epithelial cell line CFPAC-1. DBHQ stimulated 125I efflux and mobilized intracellular free Ca2+ in a dose-dependent manner. The maximal effects were seen at concentrations of 25-50 microM. DBHQ (25 microM) caused a short-term rise in [Ca2+]i in the absence of ambient Ca2+, and a sustained elevation of [Ca2+]i in cell monolayers bathed in the efflux solution (1.2 mM Ca2+), which was largely attenuated by Ni2+ (5 mM). Bath-application of DBHQ induced an outwardly-rectifying whole-cell Cl- current, which was abolished by pipette addition of BAPTA (5 mM) or CaMK [273-302] (20 microM), an inhibitory peptide of multifunctional Ca2+/calmodulin-dependent protein kinase (CaMKII). Pretreatment of monolayers of CFPAC-1 cells with DBHQ for 4-5 min significantly increased the Ca(2+)-independent or autonomous activity of CaMKII assayed in the cell homogenates. Thus, DBHQ appears to enhance Cl- channel activity via a Ca(2+)-dependent mechanism involving CaMKII. Pretreatment of CFPAC-1 cells with up to 50 microM DBHQ for 6 h did not cause any detectable change in cell viability and did not significantly affect the cell proliferation rate. These results suggest that appropriate selective microsomal Ca(2+)-ATPase inhibitors may be therapeutically useful in improving Cl- secretion in CF epithelial cells.
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PMID:Calcium- and CaMKII-dependent chloride secretion induced by the microsomal Ca(2+)-ATPase inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone in cystic fibrosis pancreatic epithelial cells. 756 71

Phospholamban is the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SR). It is phosphorylated by a Ca2+/calmodulin-dependent protein kinase (SRCaM kinase) which is closely associated with cardiac SR membrane preparations. We found that, upon renaturation of pig cardiac SR proteins, blotted onto PVDF membrane, two polypeptides of 54 and 52 kDa showed Ca2+/calmodulin-dependent autophosphorylation. In Western blots of SR proteins, the 54/52 kDa polypeptides were recognized by an antibody specific for the delta-CaM kinase isoforms, but not by an anti-alpha-CaM kinase. The two polypeptides were selectively immunoprecipitated from solubilized SR vesicles with the anti-delta-CaM kinase. The CaM kinase inhibitors KN-62 and peptide CaMK-(281-302) inhibited the activity of the SRCaM kinase with IC50 values in the same range with those obtained for the brain isozyme. In addition, initial autophosphorylation (Ca(2+)-dependent) produced a partially Ca(2+)-independent enzyme while further autophosphorylation (Ca(2+)-independent) made the enzyme completely Ca(2+)-independent. Based on these results we suggest that the SRCaM kinase is a distinct delta-CaM kinase isozyme.
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PMID:The cardiac sarcoplasmic reticulum phospholamban kinase is a distinct delta-CaM kinase isozyme. 758 37

A novel Ca2+/calmodulin-dependent protein kinase II (CaM Kinase II) inhibitor, KN-93 potently inhibits gastric acid secretion from parietal cells. As previously reported (1), treatment of parietal cells with a selective inhibitor of CaM kinase II, KN-62 resulted in the inhibition of cholinergic-stimulated rabbit parietal cell secretion, whereas it failed to inhibit the histamine and forskolin response. In contrast effects of carbachol, histamine and forskolin were significantly inhibited by KN-93 with an IC50 of 0.15, 0.3 and 1 microM, respectively; these effects occurred without any changes in intracellular cyclic AMP and Ca2+ levels. In the present study we investigated the mechanism by which KN-93 acts upon the acid-secreting machinery of gastric parietal cells. Neither redistribution of the proton pump activity nor the morphological transformation were affected by KN-93. The drug only weakly inhibited the H+, K(+)-ATPase activity but strongly dissipated the proton gradient formed in the gastric membrane vesicles and reduced the volume of luminal space. Thus KN-93 acts at pH gradient formation whereas KN-62 acts only at CaM Kinase II.
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PMID:Inhibition of acid secretion in gastric parietal cells by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-93. 769 May 57

Calponin has been implicated in the regulation of smooth muscle contraction as a result of its ability to inhibit the actin-activated Mg ATPase of smooth muscle myosin. This inhibitory effect is abolished by phosphorylation of calponin by Ca2+/calmodulin-dependent protein kinase II or protein kinase C, and restored following dephosphorylation by a type 2A protein phosphatase. Confocal immunofluorescent images of isolated smooth muscle cells colabeled with antibodies to calponin and actin or to calponin and tropomyosin indicate that calponin is present on thin filaments throughout the cell cytoplasm. Both calponin phosphorylation and myosin light chain phosphorylation increased in intact smooth muscle tissue strips when they contracted in response to carbachol or the phosphatase inhibitor okadaic acid. These results support the hypothesis that calponin phosphorylation-dephosphorylation plays a role in regulating smooth muscle contraction.
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PMID:Calponin and smooth muscle regulation. 776 87

In both cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum (SR) there are several systems involved in the regulation of Ca(2+)-ATPase function. These include substrate level regulation, covalent modification via phosphorylation-dephosphorylation of phospholamban by both cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase (CaM kinase) as well as direct CaM kinase phosphorylation of the Ca(2+)-ATPase. Studies comparing the effects of PKA and CaM kinase on cardiac Ca(2+)-ATPase function have yielded differing results; similar studies have not been performed in slow-twitch skeletal muscle. It has been suggested recently, however, that phospholamban is not tightly coupled to the Ca(2+)-ATPase in SR vesicles from slow-twitch skeletal muscle. Our results indicate that assay conditions strongly influence the extent of CaM kinase-dependent Ca(2+)-ATPase stimulation seen in both cardiac and slow-twitch skeletal muscle. Addition of calmodulin (0.2 microM) directly to the Ca2+ transport assay medium results in minimal (approximately 112-130% of control) stimulation of Ca2+ uptake activity when the Ca2+ uptake reaction is initiated by the addition or either ATP or Ca2+/EGTA. On the other hand, prephosphorylation of the SR by the endogenous CaM kinase and subsequent transfer of the membranes to the Ca2+ transport assay medium results in stimulation of Ca2+ uptake activity (202% of control). These effects are observable in both cardiac and slow-twitch skeletal muscle SR. PKA stimulates Ca2+ uptake markedly (215% of control) when the Ca2+ uptake reaction is initiated by the addition of prephosphorylated SR membranes or by Ca2+/EGTA but minimally (130% of control) when the Ca2+ uptake reaction is initiated by the addition of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparison of the effects of the membrane-associated Ca2+/calmodulin-dependent protein kinase on Ca(2+)-ATPase function in cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum. 777 65

In fetal rat brain neurons, activation of voltage-dependent Na+ channels induced their own internalization, probably triggered by an increase in intracellular Na+ level. To investigate the role of phosphorylation in internalization, neurons were exposed to either activators or inhibitors of cyclic AMP- and cyclic GMP-dependent protein kinases, protein kinase C, and tyrosine kinase. None of the tested compounds mimicked or inhibited the effect of Na+ channel activation. An increase in intracellular Ca2+ concentration induced either by thapsigargin, a Ca(2+)-ATPase blocker, or by A23187, a Ca2+ ionophore, was unable to provoke Na+ channel internalization. However, Ca2+ seems to be necessary because both neurotoxin- and amphotericin B-induced Na+ channel internalizations were partially inhibited by BAPTA-AM. The selective inhibitor of Ca2+/calmodulin-dependent protein kinase II, KN-62, caused a dose-dependent inhibition of neurotoxin-induced internalization due to a blockade of channel activity but did not prevent amphotericin B-induced internalization. The rate of increase in Na+ channel density at the neuronal cell surface was similar before and after channel internalization, suggesting that recycling of internalized Na+ channels back to the cell surface was almost negligible. Pretreatment of the cells with an acidotropic agent such as chloroquine prevented Na+ channel internalization, indicating that an acidic endosomal/lysosomal compartment is involved in Na+ channel internalization in neurons.
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PMID:Internalization of voltage-dependent sodium channels in fetal rat brain neurons: a study of the regulation of endocytosis. 779 Aug 86


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