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)

Ca2+/calmodulin- and cAMP-dependent protein kinase activities were characterized in two subcellular membrane samples. Membranes from rat lacrimal gland were isolated by differential and density gradient centrifugation into six density windows. The present study focused on membranes from density windows III and V which contain mixtures of apical, Golgi, endosomal, and endoplasmic reticulum membranes in different proportions. Phosphorylation of membrane proteins was measured by incubating the samples in [g-32P]ATP and separating the proteins by discontinuous SDS-PAGE followed by autoradiography. The amount of phosphate incorporated into specific peptide bands was quantified by densitometry. Ca2+/calmodulin-dependent protein kinase phosphorylated a 52,000 MW peptide in membranes from both density windows with a maximal increase from 0.3 to 66 microM free Ca2+. Trifluoperazine and promethazine, two inhibitors of Ca2+/calmodulin-dependent protein kinases, inhibited this phosphorylation. cAMP-dependent protein kinase phosphorylated a 22,000 MW peptide and a 91,000 MW peptide which were present in membranes from density window III only. We conclude that a Ca2+/calmodulin-dependent protein kinase activity is present in membranes from both density window III and V whereas a cAMP-dependent protein kinase activity is present only in membranes from density window III.
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PMID:Protein phosphorylation in Golgi, endosomal, and endoplasmic reticulum membrane fractions of lacrimal gland. 867 Jul 24

Retinal cytosolic Ca2+/calmodulin-dependent protein kinase II (CaM KII) was isolated from hatched 6-wk chicken retinae by ultracentrifugation and affinity chromatography using calmodulin (CaM) and anti-CaM KII-alpha columns. Samples from different fractions were examined with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining or immunoblotting. Comparisons were made between the final antibody affinity eluates from retina and forebrain. Silver-stained gels showed that multiple proteins were present in the antibody affinity eluates from retina, including major proteins of 178, 56, and 45 kDa and several minor proteins. Immunoblots revealed that CaM KII-alpha was present in eluates from the retina and forebrain. CaM KII-beta was present in the antibody eluate from forebrain but not retina. The latter subunit was present in the crude homogenates of the retina. Regarding the antibody eluate from retina, the possibility that the major 56 kDa protein was tubulin was ruled out, but protein tau (tau) and synapsin I were present. The presence of multiple proteins in the antibody affinity eluate indicates that these proteins were coisolated in a CaM KII-alpha-associated protein complex. The finding that protein tau and synapsin I are associated with retinal CaM KII provides further insight into the mechanisms underlying the function of the kinase in this tissue. The lack of cytosolic CaM KII-beta subunit in the antibody affinity eluate from retina is indicative of a brain region-specificity in subunit composition of the kinase.
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PMID:The Ca2+/calmodulin-dependent protein kinase II-associated protein complex isolated from chicken retina. 883 78

1. Phosphorylation of caldesmon was assayed in canine colonic circular smooth muscle strips labelled with 32P and stimulated with 10 microM acetylcholine. Caldesmon was isolated by two-dimensional non-equilibrium pH gel electrophoresis. Stimulation with acetylcholine increased caldesmon phosphorylation significantly from a basal level of 0.6 +/- 0.07 to 1.1 +/- 0.15 mol P1 (mol caldesmon)-1 after 2 min. 2. MAP kinase activities were measured in SDS extracts of muscle by a gel reconstitution method using myelin basic protein. Myelin basic protein kinase activities were observed at 38, 44, 50 and 57 kDa by the gel reconstitution method. Endogenous caldesmon kinase activities were also identified by the gel reconstitution method at 38, 44 and 50 kDa. The 38 and 44 kDa kinases comigrated with proteins labelled by anti-ERK1 MAP kinase antibodies on Western blots. Both 38 and 44 kDa MBP kinase activities increased significantly during contractions induced by 10 microM acetylcholine, 0.1 microM neurokinin A and 70 mM potassium. 3. Phorbol dibutyrate (0.1 microM) potentiated activation of MAP kinases and contraction of depolarized muscles while producing a decrease in fura-2 fluorescence ratio. This suggests that protein kinase C activation is coupled to MAP kinase activity in colonic smooth muscle. 4. MAP kinases isolated form muscle homogenates by Mono Q chromatography were assayed using the specific MAP kinase substrate peptide APRTPGGRR. Stimulation of muscles for 2 min with 10 microM acetylcholine activated both ERK1 and ERK2 MAP kinase activities 2-fold. 5. To determine the effects of caldesmon phosphorylation by MAP kinase on the cross-bridge cycle, actin sliding velocity was measured with an in vitro motility assay. Unphosphorylated turkey gizzard caldesmon (3 microM) significantly reduced mean sliding velocity. Phosphorylation of caldesmon with sea star ERK1 MAP kinase reversed the inhibitory effect of caldesmon on sliding velocity. The results are consistent with a protein kinase cascade being activated by contractile agonists in gastrointestinal smooth muscle which activates ERK MAP kinases leading to phosphorylation of caldesmon. Phosphorylation of caldesmon in vivo may reverse inhibitory influences of caldesmon on cross-bridge cycling.
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PMID:Activation of MAP kinases and phosphorylation of caldesmon in canine colonic smooth muscle. 888 69

A synthetic peptide corresponding to the autophosphorylation site of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (residues 281-289) was conjugated to paramagnetic particles, and phosphorylated by a constitutively active CaMKII fragment. Using this phosphopeptide conjugate as a substrate, a calyculin A-insensitive, Mn(2+)-dependent, and poly-L-lysine-stimulated protein phosphatase activity was detected in the crude extract of rat brain. The protein phosphatase (designated as CaMKII phosphatase) (CaMKIIPase) was purified to near homogeneity from rat brain. CaMKIIPase showed apparent molecular weights of 54,000 and 65,000, on SDS-polyacrylamide gel electrophoresis and gel-filtration analysis, respectively. It was not inhibited by 100 nM calyculin A or 10 microM okadaic acid. Mn2+, but not Mg2+, was absolutely required for activity. CaMKIIPase was potently activated by polycations. Autophosphorylated CaMKII was dephosphorylated by CaMKIIPase, whereas phosphorylase kinase, mixed histones, myelin basic protein, and alpha-casein (which had been phosphorylated by cAMP-dependent protein kinase) and phosphorylase a (phosphorylated by phosphorylase kinase) were not significantly dephosphorylated. No other proteins than CaMKII in rat brain extract which had been phosphorylated by CaMKII were dephosphorylated. The stimulated Ca(2+)-independent activity of autophosphorylated CaMKII was reversed by the action of CaMKIIPase. Thus, CaMKIIPase appears to be a specialized protein phosphatase for the regulation of CaMKII.
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PMID:A novel protein phosphatase that dephosphorylates and regulates Ca2+/calmodulin-dependent protein kinase II. 944 23

A number of guanine nucleotide exchange factors have been identified that activate Rho family GTPases, by promoting the binding of GTP to these proteins. We have recently demonstrated that lysophosphatidic acid and several other agonists stimulate phosphorylation of the Rac1-specific exchange factor Tiam1 in Swiss 3T3 fibroblasts, and that protein kinase C is involved in Tiam1 phosphorylation (Fleming, I. N., Elliott, C. M., Collard, J. G., and Exton, J. H. (1997) J. Biol. Chem. 272, 33105-33110). We now show, through manipulation of intracellular [Ca2+] and the use of protein kinase inhibitors, that both protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II are involved in the phosphorylation of Tiam1 in vivo. Furthermore, we show that Ca2+/calmodulin-dependent protein kinase II phosphorylates Tiam1 in vitro, producing an electrophoretic retardation on SDS-polyacrylamide gel electrophoresis. Significantly, phosphorylation of Tiam1 by Ca2+/calmodulin-dependent protein kinase II, but not by protein kinase C, enhanced its nucleotide exchange activity toward Rac1, by approximately 2-fold. Furthermore, Tiam1 was preferentially dephosphorylated by protein phosphatase 1 in vitro, and treatment with this phosphatase abolished the Ca2+/calmodulin-dependent protein kinase II activation of Tiam1. These data demonstrate that protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II phosphorylate Tiam1 in vivo, and that the latter kinase plays a key role in regulating the activity of this exchange factor in vitro.
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PMID:Ca2+/calmodulin-dependent protein kinase II regulates Tiam1 by reversible protein phosphorylation. 1021 59

A novel protein kinase (BjCCaBPk) from etiolated Brassica juncea seedlings has been purified and partially characterized. The purified enzyme migrated on SDS/PAGE as a single band with an apparent molecular mass of 43 kDa. The optimum pH for the kinase activity was 8.0. It was stimulated more than sixfold by the protozoa Entamoeba histolytica calcium binding protein EhCaBP (10.5 nM) but not by calmodulin (CaM) when used at equimolar concentration. Moreover the kinase also did not bind CaM-Sepharose. There was neither inhibition of the kinase activity in the presence of W-7 (a CaM antagonist), KN-62 (a specific calcium/CaM kinase inhibitor) and anti-CaM Ig, nor any effect on BjCCaBPk activity of staurosporine (a protein kinase C inhibitor). Furthermore a CaM-kinase specific substrate, syntide-2, proved to be a poor substrate for the BjCCaBPk compared with histone III-S. The phosphorylation of histone III-S involved serine residues. Southern and Northern blot analysis showed the presence of EhCaBP homologues in Brassica. The data suggest that BjCCaBPk may be a novel protein kinase with an affinity towards a calcium binding protein like EhCaBP.
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PMID:A novel protein kinase from Brassica juncea stimulated by a protozoan calcium binding protein. Purification and partial characterization. 1082 2

Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons and, in humans, may be produced in 82- and 69-kDa forms. In this study, recombinant choline acetyltransferase from baculovirus and bacterial expression systems was used to identify protein isoforms by two-dimensional SDS/PAGE and as substrate for protein kinases. Whereas hexa-histidine-tagged 82- and 69-kDa enzymes did not resolve as individual isoforms on two-dimensional gels, separation of wild-type choline acetyltransferase expressed in insect cells revealed at least nine isoforms for the 69-kDa enzyme and at least six isoforms for the 82-kDa enzyme. Non-phosphorylated wild-type choline acetyltransferase expressed in Escherichia coli yielded six (69 kDa) and four isoforms (82 kDa) respectively. Immunofluorescent labelling of insect cells expressing enzyme showed differential subcellular localization with the 69-kDa enzyme localized adjacent to plasma membrane and the 82-kDa enzyme being cytoplasmic at 24 h. By 64 h, the 69-kDa form was in cytoplasm and the 82-kDa form was only present in nucleus. Studies in vitro showed that recombinant 69-kDa enzyme was a substrate for protein kinase C (PKC), casein kinase II (CK2) and alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaM kinase), but not for cAMP-dependent protein kinase (PKA); phosphorylation by PKC and CK2 enhanced enzyme activity. The 82-kDa enzyme was a substrate for PKC and CK2 but not for PKA or alpha-CaM kinase, with only PKC yielding increased enzyme activity. Dephosphorylation of both forms of enzyme by alkaline phosphatase decreased enzymic activity. These studies are of functional significance as they report for the first time that phosphorylation enhances choline acetyltransferase catalytic activity.
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PMID:Expression, purification and characterization of recombinant human choline acetyltransferase: phosphorylation of the enzyme regulates catalytic activity. 1086 Dec 22

Intracellular calcium ions (Ca2+) regulate steroidogenesis in the placenta, adrenal gland, testis, and ovary. Earlier data indicate that Ca2+/calmodulin-dependent protein kinase (CamK) may mediate Ca(2+)-dependent up-regulation of CYP11A (cholesterol side-chain cleavage). To examine this notion further, we assessed the expression and actions of isotype-specific CamK on in vitro transcription of the swine CYP11A gene promoter in primary cultures of ovarian granulosa-luteal cells. RT-PCR and oligodeoxynucleotide sequencing identified gene transcripts encoding CamKII and IV in granulosa and theca cells and corpora lutea. DNA sequence homology with the cognate human and rat genes was 97 and 94% (CamKII) and 96 and 88% (CamKIV), respectively. SDS-PAGE and isoform-specific immunoblotting corroborated expression of CamKII (approximately 52 kDa) and CamKIV (approximately 60 kDa) proteins. To monitor transcriptional control, granulosa-luteal cells were transfected transiently with a putative 5'-upstream regulatory region of the homologous CYP11A gene -2320 to +23 bp from the transcriptional start site driving luciferase (CYP11A/luc). Coexpression of constitutively active CamKIV elevated basal transcription by 3.5 +/- 0.2-fold (P < 0.001), whereas inactive mutant CamKIV and native CamKII had no effect. Forskolin, an activator of adenylyl cyclase, stimulated expression of CYP11A/luciferase by 4.5 +/- 0.9-fold (P < 0.001) and did not enhance transcriptional drive by exogenous CamKIV. Preliminary promoter-deletional analyses showed that a proximal 5'-fragment -100 to +23 bp, but not -50/+23 bp, retained full responsiveness to CamKIV (4.5 +/- 0.4-fold; P < 0.001). Threefold cotransfection of -100/+23 bp CYP11A/luciferase, active CamKIV, and a dominant-negative mutant of the cAMP-responsive element binding protein (10, 100, and 250 ng) inhibited CamKIV-stimulated transcriptional activity by 17, 47, and 48% (pooled sem+/- 2%) [P < 0.01]. The dominant-negative mutant of the cAMP-responsive element binding protein also repressed forskolin's stimulation of -100/+23 CYP11A/luciferase by 12, 38, and 52% (P < 0.01). Based on these ensemble outcomes, we postulate that endogenous CamKIV may serve as a Ca(2+)-dependent effector mechanism to maintain basal CYP11A gene expression in ovarian granulosa-luteal cells.
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PMID:Up-regulation of basal transcriptional activity of the cytochrome P450 cholesterol side-chain cleavage (CYP11A) gene by isoform-specific calcium-calmodulin-dependent protein kinase in primary cultures of ovarian granulosa cells. 1531 55

We developed a method for the detection of phosphatase activity using fluorogenic substrates after polyacrylamide gel electrophoresis. When phosphatases such as Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP), protein phosphatase 2C (PP2C), protein phosphatase 5 (PP5), and alkaline phosphatase were resolved by polyacrylamide gel electrophoresis in the absence of SDS and the gel was incubated with a fluorogenic substrate such as 4-methylumbelliferyl phosphate (MUP), all of these phosphatase activities could be detected in situ. Although 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as well as MUP could be used as a fluorogenic substrate for an in-gel assay, MUP exhibited lower background fluorescence. Using this procedure, several fluorescent bands that correspond to endogenous phosphatases were observed after electrophoresis of various crude samples. The in-gel phosphatase assay could also be used to detect protein phosphatases resolved by SDS-polyacrylamide gel electrophoresis. In this case, however, the denaturation/renaturation process of resolved proteins was necessary for the detection of phosphatase activity. This procedure could be used for detection of renaturable protein phosphatases such as CaMKP and some other phosphatases expressed in cell extracts. The present fluorescent in-gel phosphatase assay is very useful, since no radioactive compounds or no special apparatus are required.
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PMID:In-gel protein phosphatase assay using fluorogenic substrates. 2004 70

Synapsins are synaptic vesicle-associated phosphoproteins that play a major role in the fine regulation of neurotransmitter release. In Drosophila, synapsins are required for complex behavior including learning and memory. Synapsin isoforms were immunoprecipitated from homogenates of wild-type Drosophila heads using monoclonal antibody 3C11. Synapsin null mutants (Syn(97)) served as negative controls. The eluted proteins were separated by SDS-PAGE and visualized by silver staining. Gel pieces picked from five bands specific for wild type were analyzed by nano-LC-ESI-MS/MS following multienzyme digestion (trypsin, chymotrypsin, AspN, subtilisin, pepsin, and proteinase K). The protein was unambiguously identified with high sequence coverage (90.83%). A number of sequence conflicts were observed and the N-terminal amino acid was identified as methionine rather than leucine expected from the cDNA sequence. Several peptides from the larger isoform demonstrated that the in-frame UAG stop codon at position 582 which separates two large open reading frames is read through by tRNAs for lysine. Seven novel phosphorylation sites in Drosophila synapsin were identified at Thr-86, Ser-87, Ser-464, Thr-466, Ser-538, Ser-961, and Tyr-982 and verified by phosphatase treatment. No phosphorylation was observed at the conserved PKA/CaM kinase-I/IV site (RRFS, edited to RGFS) in domain A or a potential PKA site near domain E.
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PMID:Mass spectrometric analysis of synapsins in Drosophila melanogaster and identification of novel phosphorylation sites. 2102 12


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