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

To determine whether densities of calmodulin (CaM) and CaM-binding proteins are related to phasic and tonic behavior of smooth muscles, we quantified these proteins in the opossum esophageal body (EB) and lower esophageal sphincter (LES), which represent phasic and tonic smooth muscles, respectively. Gel electrophoresis, immunoprecipitation, Western blot, and hemagglutinin epitope-tagged CaM (HA-CaM) overlay assay with quantitative scanning densitometry and phosphorylation measurements were used. Total protein content in the two smooth muscles was similar (approximately 30 mg protein/g frozen tissue). Total tissue concentration of CaM was significantly (25%) higher in EB than in LES (P < 0.05). HA-CaM-binding proteins were qualitatively similar in LES and EB extracts. Myosin, myristoylated alanine-rich C kinase substrate protein, Ca(2+)/CaM kinase II, and calponin contents were also similar in the two muscles. However, content and total activity of myosin light chain kinase (MLCK) and content of caldesmon (CaD) were three- to fourfold higher in EB than in LES. Increased CaM and MLCK content may allow for a wide range of contractile force varying from complete relaxation in the basal state to a large-amplitude, high-velocity contraction in EB phasic muscle. Increased content of CaD, which provides a braking mechanism on contraction, may further contribute to the phasic contractile behavior. In contrast, low CaM, MLCK, and CaD content may be responsible for a small range of contractile force seen in tonic muscle of LES.
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PMID:Differences in calmodulin and calmodulin-binding proteins in phasic and tonic smooth muscles. 1174 2

Sepiapterin reductase (SPR) catalyzes the last step in the pathway of tetrahydrobiopterin biosynthesis in tissues. SPR is phosphorylated by Ca2+-dependent protein kinases, which indicates that Ca2+-activated protein kinases may play a role in the regulation of SPR in vivo. Phosphorylation sites of rat sepiapterin reductase (rSPR) by Ca2+/calmodulin-dependent protein kinase II were determined in the present study. Using specific monoclonal anti-phospho-Ser and -Thr antibodies, we found that only Ser residues of rSPR were phosphorylated. We constructed several point mutants of SPR by systematically replacing the three Ser residues by Ala ones. These mutants showed that all three Ser residues, i.e. S46, S196, and S214, of rSPR were phosphorylated. We also recognized that only Ser-213 of human SPR was phosphorylated. Each of these serine residues in SPR was found in the consensus sequence (Arg-X-X-Ser/Thr) of the phosphorylation site.
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PMID:Mutational analysis of sites in sepiapterin reductase phosphorylated by Ca2+/calmodulin-dependent protein kinase II. 1182 21

We demonstrated previously that 5-lipoxygenase (5-LO), a key enzyme in leukotriene biosynthesis, can be phosphorylated by p38 MAPK-regulated MAPKAP kinases (MKs). Here we show that mutation of Ser-271 to Ala in 5-LO abolished MK2 catalyzed phosphorylation and clearly reduced phosphorylation by kinases prepared from stimulated polymorphonuclear leukocytes and Mono Mac 6 cells. Compared with heat shock protein 27 (Hsp-27), 5-LO was a weak substrate for MK2. However, the addition of unsaturated fatty acids (i.e. arachidonate 1-50 microm) up-regulated phosphorylation of 5-LO, but not of Hsp-27, by active MK2 in vitro, resulting in a similar phosphorylation as for Hsp-27. 5-LO was phosphorylated also by other serine/threonine kinases recognizing the motif Arg-Xaa-Xaa-Ser (protein kinase A, Ca(2+)/calmodulin-dependent kinase II), but these activities were not increased by fatty acids. HeLa cells expressing wild type 5-LO or S271A-5-LO, showed prominent 5-LO activity when incubated with Ca(2+)-ionophore plus arachidonate. However, when stimulated with only exogenous arachidonic acid, activity for the S271A mutant was significantly lower as compared with wild type 5-LO. It appears that phosphorylation at Ser-271 is more important for 5-LO activity induced by a stimulus that does not prominently increase intracellular Ca(2+) and that arachidonic acid stimulates leukotriene biosynthesis also by promoting this MK2-catalyzed phosphorylation.
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PMID:Arachidonic acid promotes phosphorylation of 5-lipoxygenase at Ser-271 by MAPK-activated protein kinase 2 (MK2). 1184 97

Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons. In the brain, these neurons are especially vulnerable to effects of beta-amyloid (A beta) peptides. Choline acetyltransferase is a substrate for several protein kinases. In the present study, we demonstrate that short term exposure of IMR32 neuroblastoma cells expressing human choline acetyltransferase to A beta-(1-42) changes phosphorylation of the enzyme, resulting in increased activity and alterations in its interaction with other cellular proteins. Using mass spectrometry, we identified threonine 456 as a new phosphorylation site in choline acetyltransferase from A beta-(1-42)-treated cells and in purified recombinant ChAT phosphorylated in vitro by calcium/calmodulin-dependent protein kinase II (CaM kinase II). Whereas phosphorylation of choline acetyltransferase by protein kinase C alone caused a 2-fold increase in enzyme activity, phosphorylation by CaM kinase II alone did not alter enzyme activity. A 3-fold increase in choline acetyltransferase activity was found with coordinate phosphorylation of threonine 456 by CaM kinase II and phosphorylation of serine 440 by protein kinase C. This phosphorylation combination was observed in choline acetyltransferase from A beta-(1-42)-treated cells. Treatment of cells with A beta-(1-42) resulted in two phases of activation of choline acetyltransferase, the first within 30 min and associated with phosphorylation by protein kinase C and the second by 10 h and associated with phosphorylation by both CaM kinase II and protein kinase C. We also show that choline acetyltransferase from A beta-(1-42)-treated cells co-immunoprecipitates with valosin-containing protein, and mutation of threonine 456 to alanine abolished the A beta-(1-42)-induced effects. These studies demonstrate that A beta-(1-42) can acutely regulate the function of choline acetyltransferase, thus potentially altering cholinergic neurotransmission.
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PMID:Phosphorylation of 69-kDa choline acetyltransferase at threonine 456 in response to amyloid-beta peptide 1-42. 1248 17

Rim1, a brain-specific Rab3a-binding protein, localizes to the presynaptic cytomatrix and plays an important role in synaptic transmission and synaptic plasticity. Rim2, a homologous protein, is more ubiquitously expressed and is found in neuroendocrine cells as well as in brain. Both Rim1 and Rim2 contain multiple domains, including an N-terminal zinc finger, which in Rim1 strongly enhances secretion in chromaffin and PC12 cells. The yeast two-hybrid technique identified 14-3-3 proteins as ligands of the N-terminal domain. In vitro protein binding experiments confirmed a high-affinity interaction between the N terminus of Rim1 and 14-3-3. The N-terminal domain of Rim2 also bound 14-3-3. The binding domains were localized to a short segment just C-terminal to the zinc finger. 14-3-3 proteins bind to specific phosphoserine residues. Alkaline phosphatase treatment of N-terminal domains of Rim1 and Rim2 almost completely inhibited the binding of 14-3-3. Two serine residues in Rim1 (Ser-241 and Ser-287) and one serine residue in Rim2 (Ser-335) were required for 14-3-3 binding. Incubation with Ca2+/calmodulin-dependent protein kinase II greatly stimulated the interaction of recombinant N-terminal Rim but not the S241/287A mutant with 14-3-3, again indicating the importance of the phosphorylation of these residues for the binding. Rabphilin3, another Rab3a effector, also bound 14-3-3. Serine-to-alanine mutations identified Ser-274 as the likely phosphorylated residue to which 14-3-3 binds. Because the phosphorylation of this residue had been shown to be stimulated upon depolarization in brain slices, the interaction of 14-3-3 with Rabphilin3 may be important in the dynamic function of central nervous system neurons.
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PMID:Rim, a component of the presynaptic active zone and modulator of exocytosis, binds 14-3-3 through its N terminus. 1287 46

2-Pyrrolidinone, a metabolite of aniracetam, potentiated currents through alpha7 receptors expressed in Xenopus oocytes, in a bell-shaped dose-dependent manner at concentrations ranged from 1 nM to 10 microM, with a maximum at 100 nM (189% of original levels 60 min after 20-min treatment). The potentiation was inhibited by GF109203X, a selective inhibitor of protein kinase C (PKC), but not by KN-93, a selective inhibitor of CaMKII, or H-89, a selective inhibitor of protein kinase A (PKA). In the PKC assay using reversed-phase high-performance liquid chromatography, 2-pyrrolidinone enhanced activity of PKC-epsilon activated by linoleic acid to about 1.8-times greater than that in the absence of 2-pyrrolidinone, although it did not directly activate PKC-epsilon. In the Western immunoblot analysis, rat hippocampal slices treated with 2-pyrrolidinone (100 nM) was more reactive to an antibody against phosphorylated myristoylated alanine-rich C kinase substrate (MARCKS) than untreated slices. 2-Pyrrolidinone (100 nM) induced a long-lasting facilitation of hippocampal synaptic transmission in the CA1 region of rat hippocampal slices, and the facilitation was inhibited by GF109203X or alpha-bungarotoxin, an inhibitor of alpha7 receptors. The results of the present study suggest that 2-pyrrolidinone enhances activity of activated PKC, thereby potentiating alpha7 receptor responses, and then leading to a facilitation of hippocampal synaptic transmission.
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PMID:2-pyrrolidinone induces a long-lasting facilitation of hippocampal synaptic transmission by enhancing alpha7 ACh receptor responses via a PKC pathway. 1449 85

Increases in neuronal activity in response to tissue or nerve injury can lead to prolonged functional changes in the spinal cord resulting in an enhancement/sensitization of nociceptive processing. To assess the contribution of alpha-calcium-calmodulin kinase II (alpha-CaMKII) to injury-induced inflammation and pain, we evaluated nociceptive responses in mice that carry a point mutation in the alpha-CaMKII gene at position 286 (threonine to alanine). The mutated protein is unable to autophosphorylate and thus cannot function independently of calcium and calmodulin. Responses to acute noxious stimuli did not differ between alpha-CaMKII T286A mutant and wild type mice. However, the ongoing pain produced by formalin injury was significantly reduced in the mutant mice, as was formalin-evoked spinal Fos-immunoreactivity. In contrast, the decreased mechanical and thermal thresholds associated with nerve injury, Complete Freund's Adjuvant-induced inflammation or formalin-evoked tissue injury were manifest equally in wild-type and mutant mice. Double-labeling immunofluorescence studies revealed that in the mouse alpha-CaMKII is expressed in the superficial dorsal horn as well as in a population of small diameter primary afferent neurons. In summary, our results suggest that alpha-CaMKII, perhaps secondary to an N-methyl-D-aspartate-mediated calcium increase in postsynaptic dorsal horn nociresponsive neurons, is a critical contributor to the spontaneous/ongoing component of tissue-injury evoked persistent pain.
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PMID:The contribution of autophosphorylated alpha-calcium-calmodulin kinase II to injury-induced persistent pain. 1546 94

The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) is under the control of an SR protein named phospholamban (PLN). Dephosphorylated PLN inhibits SERCA2a, whereas phosphorylation of PLN at either the Ser16 site by PKA or the Thr17 site by CaMKII reverses this inhibition, thus increasing SERCA2a activity and the rate of Ca2+ uptake by the SR. This leads to an increase in the velocity of relaxation, SR Ca2+ load and myocardial contractility. In the intact heart, beta-adrenoceptor stimulation results in phosphorylation of PLN at both Ser16 and Thr17 residues. Phosphorylation of the Thr17 residue requires both stimulation of the CaMKII signaling pathways and inhibition of PP1, the major phosphatase that dephosphorylates PLN. These two prerequisites appear to be fulfilled by beta-adrenoceptor stimulation, which as a result of PKA activation, triggers the activation of CaMKII by increasing intracellular Ca2+, and inhibits PP1. Several pathological situations such as ischemia-reperfusion injury or hypercapnic acidosis provide the required conditions for the phosphorylation of the Thr17 residue of PLN, independently of the increase in PKA activity, i.e., increased intracellular Ca2+ and acidosis-induced phosphatase inhibition. Our results indicated that PLN was phosphorylated at Thr17 at the onset of reflow and immediately after hypercapnia was established, and that this phosphorylation contributes to the mechanical recovery after both the ischemic and acidic insults. Studies on transgenic mice with Thr17 mutated to Ala (PLN-T17A) are consistent with these results. Thus, phosphorylation of the Thr17 residue of PLN probably participates in a protective mechanism that favors Ca2+ handling and limits intracellular Ca2+ overload in pathological situations.
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PMID:The importance of the Thr17 residue of phospholamban as a phosphorylation site under physiological and pathological conditions. 1664 92

Class II histone deacetylases (HDACs) act as repressors of cardiac hypertrophy, an adaptative response of the heart characterized by a reprogramming of fetal cardiac genes. Prolonged hypertrophy often leads to dilated cardiomyopathy and heart failure. Upstream endogenous regulators of class II HDACs that regulate hypertrophic growth are just beginning to emerge. Here we demonstrate that the delta B isoform of calcium/calmodulin-dependent protein kinase II (CaMKIIdeltaB), known to promote cardiac hypertrophy, transmits signals specifically to HDAC4 but not other class II HDACs. CaMKIIdeltaB efficiently phosphorylates both a glutathione S-transferase (GST)-HDAC4 fragment spanning amino acids 207-311 and full-length FLAG-HDAC4 but not the equivalents in HDAC5. Although previous studies in skeletal muscle cells have shown that HDAC4 lacking serine 246 cannot be phosphorylated by CaMKI/IV, a similar mutant is still phosphorylated by CaMKIIdeltaB. Importantly, mutation of serine 210 to alanine totally abolishes phosphorylation of the GST fragment and significantly reduces phosphorylation of full-length HDAC by CaMKIIdeltaB. RNA interference knockdown of CaMKIIdeltaB prevents the effects of hypertrophic stimuli. Overexpression of CaMKIIdeltaB in primary neonatal cardiomyocytes increases the activity of the Mef2 transcription factor and completely rescues HDAC4-mediated repression of MEF2 but only partially rescues inhibition by HDAC5 or the HDAC4 S210A mutant. CaMKIIdeltaB strongly interacts with HDAC4 in cells but not with HDAC5. These results demonstrate that CaMKIIdeltaB preferentially targets HDAC4, and this involves serine 210. These findings identify HDAC4 as a specific downstream substrate of CaMKIIdeltaB in cardiac cells and have broad applications for the signaling pathways leading to cardiac hypertrophy and heart failure.
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PMID:Nuclear calcium/calmodulin-dependent protein kinase IIdelta preferentially transmits signals to histone deacetylase 4 in cardiac cells. 1717 59

Cytosolic phospholipase A(2) (cPLA(2)) is activated by phosphorylation at serine-505 (S505) by extracellular regulated kinase 1/2 (ERK1/2). However, rat brain calcium/calmodulin-dependent kinase II (CaMKII) phosphorylates recombinant cPLA(2) at serine-515 (S515) and increases its activity in vitro. We have studied the sites of cPLA(2) phosphorylation and their significance in arachidonic acid (AA) release in response to norepinephrine (NE) in vivo in rabbit vascular smooth muscle cells (VSMCs) using specific anti-phospho-S515- and -S505 cPLA(2) antibodies and by mutagenesis of S515 and S505 to alanine. NE increased the phosphorylation of cPLA(2) at S515, followed by phosphorylation of ERK1/2 and consequently phosphorylation of cPLA(2) at S505. The CaMKII inhibitor 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzene-sulfonyl)]amino-N-(4-chlorocinnamyl)-methylbenzylamine attenuated cPLA(2) at S515 and S505, whereas the ERK1/2 inhibitor U0126 reduced phosphorylation at S505 but not at S515. NE in cells transduced with adenovirus carrying enhanced cyan fluorescent protein cPLA(2) wild type caused phosphorylation at S515 and S505 and increased AA release. Expression of the S515A mutant in VSMCs reduced the phosphorylation of S505, ERK1/2, and AA release in response to NE. Transduction with a double mutant (S515A/S505A) blocked the phosphorylation of cPLA(2) and AA release. These data suggest that the NE-stimulated phosphorylation of cPLA(2) at S515 is required for the phosphorylation of S505 by ERK1/2 and that both sites of phosphorylation are important for AA release in VSMCs.
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PMID:cPLA2 phosphorylation at serine-515 and serine-505 is required for arachidonic acid release in vascular smooth muscle cells. 1818 3


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