EC:2.7.11.17 - FACTA Search

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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)

Glucocorticoid hormones (GC) have profound effects on the development and homeostasis of the immune system. In this communication we present evidence that GC regulate Ca(2+)-mediated pathways of T cell activation by a mechanism that involves abrogation of the autophosphorylation of the multifunctional Ca2+/calmodulin kinase (CaM kinase II) and induction of protein phosphatase activity. Primary human T cells were stimulated with the combination of ionomycin and phorbol ester in the presence or absence of dexamethasone (Dex) (10(-6)-10(-12) M). Stimulation of T cells resulted in a rapid activation of CaM kinase II and protein kinase C (PKC) activity as determined by the phosphorylation of synthetic peptide substrates recognized by these enzymes. Dex inhibited the activity of CaM kinase II but not PKC activity in a dose-dependent fashion (minimum effective dose 10(-10) M). Stimulation of 32P-labeled T cells induced a rapid increase in the phosphorylation level of CaM kinase II which was inhibited by Dex. The inhibitory effect of Dex on this enzyme was fully reversed in the presence of the phosphatase inhibitor okadaic acid (250 nM) or RU 486, a glucocorticoid antagonist. These results suggest that GC inhibit the activation of CaM kinase during T cell activation through a mechanism that involves both the GC receptor and protein phosphatases 2A and/or 1. Inhibition of protein phosphorylation through the induction of protein phosphatase activity may represent a novel mechanism for the diverse effects of GC on eukaryotic cells.
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PMID:Glucocorticoid-mediated regulation of protein phosphorylation in primary human T cells. Evidence for induction of phosphatase activity. 763 35

Inorganic lead inhibits neurite initiation in cultured rat hippocampal neurons at concentrations as low as 100 nM. Conflicting reports suggest that Pb2+ may stimulate or inhibit protein kinase C, adenylyl cyclase, phosphodiesterase, and calmodulin, or increase intracellular free Ca2+ concentrations. Therefore, Pb2+ may alter the activities of Ca2+/calmodulin-dependent protein kinase (CaM kinase) or protein kinases C or A. We cultured rat hippocampal neurons in 100 nM PbCI2 alone or in combination with kinase or calmodulin inhibitors. Inhibiting protein kinase C with calphostin C exacerbated the inhibition of neurite initiation caused by PbCI2, but inhibiting protein kinase A with KT5720, CaM kinase with KN62, or calmodulin with calmidazolium completely reversed the effects of PbCI2. These results indicate that Pb2+ may inhibit neurite initiation by inappropriately stimulating protein phosphorylation by CaM kinase or cyclic AMP-dependent protein kinase (PKA), possibly by stimulating calmodulin. This hypothesis is supported by findings that other treatments that should increase protein phosphorylation (okadaic acid, a protein phosphatase inhibitor, and Sp-cAMPS, a PKA activator) also reduced neurite initiation. Whole-cell intracellular free Ca2+ ion concentrations were not significantly altered by 100 nM PbCI2 at 4, 12, 24, or 48 hr. Therefore, the hypothesized stimulatory effects of Pb2+ exposure on calmodulin, CaM kinase, or PKA are probably not caused by increases in whole-cell intracellular free Ca2+, but may be attributable either to intracellular Pb2+ or to localized increases in [Ca2+]in that are not reflected in whole-cell measurements.
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PMID:Inorganic lead may inhibit neurite development in cultured rat hippocampal neurons through hyperphosphorylation. 767 45

We report that the C-terminal domain of skeletal muscle dystrophin expressed as a fusion protein with glutathione S-transferase (designated GST-CT-1) is a substrate for Ca2+/calmodulin-dependent phosphorylation and dephosphorylation. GST-CT-1 and GST-CT-1F (GST-CT-1 truncated by 20-25 residues) were phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). The stoichiometries of phosphorylation by CaM kinase II were 1.65 mol of Pi/mol of GST-CT-1 and 0.39 mol of Pi/mol of GST-CT-1F, respectively, suggesting that the principal site(s) of phosphorylation is (are) located in the C-terminal 20-25 residues that are missing from GST-CT-1F. The GST-CT-1 fusion protein was phosphorylated on both serine and threonine residues, whereas GST-CT-1F was phosphorylated only on serine. CaM kinase II-phosphorylated GST-CT-1 and GST-CT-1F were efficiently dephosphorylated by calcineurin, a Ca2+/calmodulin-dependent protein phosphatase (type 2B protein phosphatase). Importantly, calcineurin was found to be associated with a purified sarcolemmal membrane preparation enriched in dystrophin. Type 2A protein phosphatase isolated from smooth muscle (SMP-I) and its catalytic subunit (SMP-ic) also dephosphorylated GST-CT-1, but were less active toward these substrates than was calcineurin. Type 2C phosphatase (SMP-II) and type 1 protein phosphatases [SMP-III, SMP-IV, and myosin-associated phosphatase (PP1M) of smooth muscle and skeletal muscle protein phosphatase 1c] were ineffective in dephosphorylating the C-terminal region of dystrophin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the recombinant C-terminal domain of dystrophin: phosphorylation by calmodulin-dependent protein kinase II and dephosphorylation by type 2B protein phosphatase. 772 17

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

Proliferation of mammalian cells both in vivo and in vitro is dependent upon physiological concentrations of extracellular Ca2+. Growth factor stimulation of quiescent cells at the G0/G1 border usually results in a rapid mobilization of Ca2+ from both intra- and extracellular pools. However, Ca2+ influx is also required for later phases of cell cycle transition, especially in the late G1 phase for initiation of DNA synthesis. Available evidence indicates that calmodulin plays the major and essential roles in the Ca(2+)-dependent regulation of cell proliferation. Ca2+ and calmodulin act at multiple points in the cell cycle, including the initiation of the S phase and both initiation and completion of the M phase. Ca2+ and calmodulin stimulate the expression of genes involved in the cell cycle progression, leading to activation of cyclin-dependent kinases p33cdk2 and p34cdc2. Ca2+ and calmodulin are also involved in activation of enzymes participating in nucleotide metabolism and DNA replication, as well as nuclear envelope breakdown and cytokinesis. Ca2+/calmodulin-dependent protein kinase II and protein phosphatase calcineurin are both involved in the Ca2+ and calmodulin-mediated signalling of growth regulation. As compared to normal cells, growth of transformed cells is independent of extracellular Ca2+ and much less sensitive to calmodulin antagonists, suggesting the existence of derangements in the Ca2+ and calmodulin-mediated growth regulation mechanisms.
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PMID:Calcium, calmodulin and cell cycle progression. 779 90

Protein phosphatase 2A (PP2A) isolated from whole rat brain homogenate supernatants has been compared with that extracted from rat synaptosomal membranes. Both purified enzymes are comprised of the three known PP2A polypeptide chains of 65 (A subunit), 55 (B/B' subunit), and 38 (C subunit) kDa and have okadaic acid inhibition curves (Ki = 0.05 nM) nearly identical to that reported for skeletal muscle PP2A. The isolated 38-kDa subunit of rat brain PP2A appears to contain phosphotyrosine based on cross-reactivity with a specific monoclonal antibody (PY-20). Amino acid compositions and sequences of peptides isolated from the 65- and 38-kDa species correspond to regions of the cDNA-deduced sequences of the regulatory and catalytic subunits of protein phosphatase 2A from several sources. Studies reported here also demonstrate that autophosphorylated protein kinases, particularly Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), are excellent substrates for brain PP2A. Furthermore, Ca(2+)-dependent K(+)-depolarization of hippocampal synaptosomes was accompanied by a sequential increase, then decrease, in CaM kinase II phosphorylation level over a 45-s time course. The decrease was blocked by 1 nM okadaic acid. These data demonstrate that the type 2A protein phosphatase is present at the synapses of CNS neurons where its localization could alter the functions of phosphoproteins involved in synaptic plasticity.
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PMID:Rat brain protein phosphatase 2A: an enzyme that may regulate autophosphorylated protein kinases. 779 31

The cardiac calcium release channel (CRC) of sarcoplasmic reticulum vesicles was incorporated into planar lipid membranes to evaluate modulation of channel activity by phosphorylation and dephosphorylation. For this purpose a microsyringe application directly to the membrane was used to achieve sequential and multiple treatments of channels with highly purified kinases and phosphatases. Cyclic application of protein kinase A (PKA) or Ca2+/calmodulin-dependent protein kinase II (CalPK) and potato acid phosphatase or protein phosphatase 1 revealed a channel block by Mg2+ (-mM), that is referable to dephosphorylated states of the channel, and that the Mg2+ block could be removed by phosphorylation of the CRC by either PKA or CalPK. By contrast, activation of endogenous CalPK (end CalPK) led to channel closure which could be reversed by dephosphorylation using potato acid phosphatase or protein phosphatase 1. Calmodulin by itself (which activates end CalPK in the presence of MgATP) blocks the channel in the dephosphorylated state, which can be overcome by treatment with CalPK but not PKA. Our findings reveal important insights regarding channel regulation of the ryanodine receptor: 1) the calcium release channel must be phosphorylated to be in the active state at conditions approximating physiological Mg2+ concentrations (-mM); and 2) there are multiple sites of phosphorylation on the calcium release channel with different functional consequences, which may be relevant to the regulation of E-C coupling. Phosphorylation of the CRC may be involved in recruitment of active channels, and/or it may be directly involved in each Ca2+ contraction cycle of the heart. For example, Ca2+ release may require phosphorylation of the CRC by protein kinases at sites which overcome the block by Mg2+. Inactivation may involve CRC block by calmodulin and/or phosphorylation by endogenous CalPK at the junctional face membrane.
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PMID:Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from cardiac muscle. 783 35

The modulation of the calcium release channel (CRC) by protein kinases and phosphatases was studied. For this purpose, we have developed a microsyringe applicator to achieve sequential and multiple treatments with highly purified kinases and phosphatases applied directly at the bilayer surface. Terminal cisternae vesicles of sarcoplasmic reticulum from rabbit fast twitch skeletal muscle were fused to planar lipid bilayers, and single-channel currents were measured at zero holding potential, at 0.15 microM free Ca2+, +/- 0.5 mM ATP and +/- 2.6 mM free Mg2+. Sequential dephosphorylation and rephosphorylation rendered the CRC sensitive and insensitive to block by Mg2+, respectively. Channel recovery from Mg2+ block was obtained by exogenous protein kinase A (PKA) or by Ca2+/calmodulin-dependent protein kinase II (CalPK II). Somewhat different characteristics were observed with the two kinases, suggesting two different states of phosphorylation. Channel block by Mg2+ was restored by dephosphorylation using protein phosphatase 1 (PPT1). Before application of protein kinases or phosphatases, channels were found to be "dephosphorylated" (inactive) in 60% and "phosphorylated" (active) in 40% of 51 single-channel experiments based on the criterion of sensitivity to block by Mg2+. Thus, these two states were interconvertable by treatment with exogenously added protein kinases and phosphatases. Endogenous Ca2+/calmodulin-dependent protein kinase (end CalPK) had an opposite action to exogenous CalPK II. Previously, dephosphorylated channels using PPT (Mg2+ absent) were blocked in the closed state by action of endogenous CalPK. This block was removed to normal activity by the action of either PPT or by exogenous CalPK II. Our findings are consistent with a physiological role for phosphorylation/dephosphorylation in the modulation of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. A corollary of our studies is that only the phosphorylated channel is active under physiological conditions (mM Mg2+). Our studies suggest that phosphorylation can be at more than one site and, depending on the site, can have different functional consequences on the CRC.
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PMID:Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. 785 21

We investigated the role of Ca/calmodulin-dependent protein kinase (CaMKII) in relaxation and cytosolic free [Ca] ([Ca]i) decline during steady-state (SS) and postrest (PR) twitches in intact rat ventricular myocytes. Half-time of mechanical relaxation and time constant of [Ca]i decline (tau) were twofold greater during PR than with SS at 1 Hz. This difference was 1) abolished by inhibition of sarcoplasmic reticulum (SR) Ca accumulation by thapsigargin or caffeine; 2) greater at higher stimulation frequency and extracellular [Ca], which affected only SS tau; 3) abolished by the protein phosphatase inhibitor okadaic acid (10 microM, which selectively accelerated [Ca]i decline during PR); 4) still present during stimulation or inhibition of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) by 10 microM forskolin or 1 microM H-89, respectively (SS and PR tau values were abbreviated and prolonged, respectively); and 5) suppressed by 10 microM KN-62, a selective inhibitor of CaMKII, which selectively prolonged [Ca]i decline during SS twitches. Both protein kinase inhibitors were also shown to decrease the SR Ca-uptake rate in digitonin-permeabilized rat myocytes. We conclude that CaMKII plays a major role in modulation of relaxation in rat ventricular myocytes, enhancing SR Ca uptake in a activity-dependent fashion. Our results are also compatible with a background, activity-independent stimulation of SR Ca uptake by PKA in intact rat myocytes.
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PMID:CaMKII is responsible for activity-dependent acceleration of relaxation in rat ventricular myocytes. 786 97

Engagement of the T cell receptor for antigen activates phospholipase C resulting in an increase in intracellular free calcium concentration ([Ca2+]i) and activation of protein kinase C (PKC). Increased [Ca2+]i activates Ca2+/calmodulin-dependent kinases including the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM-K II), as well as calcineurin, a type 2B protein phosphatase. Recent studies have identified calcineurin as a key enzyme for interleukin (IL)-2 and IL-4 promoter activation. However, the role of CaM-K II remains unknown. We have used mutants of these kinases and phosphatases (gamma B*CaM-K and delta CaM-AI, respectively) to explore their relative role in cytokine gene transcription and their interactions with PKC-dependent signaling systems. gamma B*CaM-K and delta CaM-AI, known to exhibit constitutive Ca(2+)-independent activity, were cotransfected (alone or in combination) in Jurkat T cells with a plasmid containing the intact IL-2 promoter driving the expression of the chloramphenicol acetyltransferase reporter gene. Cotransfection of gamma B*CaM-K with the IL-2 promoter construct downregulated its transcription in response to stimulation with ionomycin and phorbol myristate acetate (PMA). The inhibitory effect of CaM-K II on IL-2 promoter was associated with decreased transcription of its AP-1 and NF-AT transactivating pathways. Under the same conditions, delta CaM-AI superinduced IL-2 promoter activity (approximately twofold increase). When both mutants were used in combination, gamma B*CaM-K inhibited the induction of the IL-2 promoter by delta CaM-AI. Similar results were obtained when a construct containing the IL-4 promoter also was used. gamma B*CaM-K also downregulated the activation of AP-1 in response to transfection with a constitutively active mutant of PKC or stimulation with PMA. These results suggest that CaM-K II may exert negative influences on cytokine gene transcription in human T cells, and provide preliminary evidence for negative cross-talk with the calcineurin- and PKC-dependent signaling systems.
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PMID:Calcium/calmodulin-dependent protein kinase II downregulates both calcineurin and protein kinase C-mediated pathways for cytokine gene transcription in human T cells. 786 38

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