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)

Dihydropyridine-sensitive Ca2+ channels from skeletal muscle are multisubunit proteins and are regulated by protein phosphorylation. The purpose of this study was to determine: 1) which subunits are the preferential targets of various protein kinases when the channels are phosphorylated in vitro in their native membrane-bound state and 2) the consequences of these phosphorylations in functional assays. Using as substrates channels present in purified transverse (T) tubule membranes, cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a multifunctional Ca2+/calmodulin-dependent protein kinase (CaM protein kinase) preferentially phosphorylated the 165-kDa alpha 1 subunit to an extent that was 2-5-fold greater than the 52-kDa beta subunit. A protein kinase endogenous to the skeletal muscle membranes preferentially phosphorylated the beta peptide and showed little activity toward the alpha 1 subunit; however, the extent of phosphorylation was low. Reconstitution of partially purified channels into liposomes was used to determine the functional consequences of phosphorylation by these kinases. Phosphorylation of channels by PKA or PKC resulted in an activation of the channels that was observed as increases in both the rate and extent of Ca2+ influx. However, phosphorylation of channels by either the CaM protein kinase or the endogenous kinase in T-tubule membranes was without effect. Phosphorylation did not affect the sensitivities of the channels toward the dihydropyridines. Taken together, the results demonstrate that the alpha 1 subunit is the preferred substrate of PKA, PKC, and CaM protein kinase when the channels are phosphorylated in the membrane-bound state and that phosphorylation of the channels by PKA and PKC, but not by CaM protein kinase or an endogenous T-tubule membrane protein kinase, results in activation of the dihydropyridine-sensitive Ca2+ channels from skeletal muscle.
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PMID:Dihydropyridine-sensitive calcium channels from skeletal muscle. II. Functional effects of differential phosphorylation of channel subunits. 165 34

The phosphorylation and dephosphorylation of the dihydropyridine-sensitive Ca2+ channel was studied in transverse-tubule membranes isolated from rabbit skeletal muscle. Exposure of these membranes to either the cAMP-dependent protein kinase or a Ca2+/calmodulin-dependent protein kinase resulted in a rapid phosphorylation of a protein with properties similar to the major component of the skeletal muscle Ca2+ channel. The molecular mass of the phosphoprotein was 140 or 160 kDa, depending on the electrophoretic conditions. The stoichiometry of the phosphorylation was calculated to be 0.4-1.0 mol of phosphate per mol of protein. Neither the rate nor the extent of phosphorylation was affected by dihydropyridines. Limited proteolytic digestion of the protein that had been phosphorylated by either or both protein kinases yielded a single phosphopeptide of approximately equal to 5.4 kDa. The Ca2+-dependent phosphatase calcineurin dephosphorylated the membrane-bound Ca2+ channel that had been previously phosphorylated by either protein kinase. The results suggest that the major component of the dihydropyridine-sensitive Ca2+ channel from skeletal muscle can be effectively phosphorylated and dephosphorylated in its native state by cAMP- and Ca2+-dependent processes.
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PMID:Phosphorylation and dephosphorylation of dihydropyridine-sensitive voltage-dependent Ca2+ channel in skeletal muscle membranes by cAMP- and Ca2+-dependent processes. 242 10

Calmodulin kinase (CaM kinase) activity and immunoreactivity were measured in the cytosol and crude synaptic membranes of light- and dark-adapted rat retinas. Dark adaptation increased the calcium-independent CaM kinase activity 2.7 times and calcium-stimulated activity 3.9 times in membrane fractions. Dark adaptation also increased membrane-bound CaM kinase immunoreactivity 2.4 times. In the cytosol, dark adaptation increased calcium- and calmodulin-independent kinase activity 3.3-fold but did not enhance calcium- and calmodulin-dependent activity. Soluble CaM kinase immunoreactivity was decreased by 13% by dark exposure. These changes in enzyme activity and immunoreactivity are likely due to changes in the endogenous state of autophosphorylation and compartmental concentrations of CaM kinase and may represent translocation of CaM kinase from cytosol to membranes. CaM kinase may have an important role in modulating visual processes.
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PMID:Dark-induced changes in activity and compartmentalization of retinal calmodulin kinase in the rat. 255 Jan 12

Dihydropyridine-sensitive Ca2+ channels exist in many different types of cells and are believed to be regulated by various protein phosphorylation and dephosphorylation reactions. The present study concerns the phosphorylation of a putative component of dihydropyridine-sensitive Ca2+ channels by the calcium and phospholipid-dependent protein kinase, protein kinase C. A skeletal muscle peptide of 165 kDa, which is known to contain receptors for dihydropyridines, phenylalkylamines, and other Ca2+ channel effectors, was found to be an efficient substrate for protein kinase C when the peptide was phosphorylated in its membrane-bound state. Protein kinase C incorporated 1.5-2.0 mol of phosphate/mol of peptide within 2 min into the 165-kDa peptide in incubations carried out at 37 degrees C. In contrast to the membrane-bound peptide, the purified 165-kDa peptide in detergent solution was phosphorylated to a markedly less extent than its membrane-bound counterpart; less than 0.1 mol of phosphate/mol of peptide was incorporated. Preincubation of the membranes with several types of drugs known to be Ca2+ channel activators or inhibitors had no specific effects on the rate and/or extent of phosphorylation of the 165-kDa peptide by protein kinase C. The phosphorylation of the membrane-bound 165-kDa peptide by protein kinase C was compared to that catalyzed by cAMP-dependent protein kinase and was found to be not additive. Prior phosphorylation of the 165-kDa peptide by cAMP-dependent protein kinase prevented subsequent phosphorylation of the peptide by protein kinase C. Phosphoamino acid analysis indicated that protein kinase C phosphorylated the 165-kDa peptide at both serine and threonine residues. Phosphopeptide mapping experiments showed that protein kinase C phosphorylated one unique site in the 165-kDa peptide, and, in addition, other sites that were phosphorylated by either cAMP-dependent protein kinase or a multifunctional Ca2+/calmodulin-dependent protein kinase. The results suggest that the 165-kDa dihydropyridine/phenylalkylamine receptor could serve as a physiological substrate of protein kinase C in intact cells. It is therefore possible that the regulation of dihydropyridine-sensitive Ca2+ channels by activators of protein kinase C may occur at the level of this peptide.
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PMID:Phosphorylation of the 165-kDa dihydropyridine/phenylalkylamine receptor from skeletal muscle by protein kinase C. 284 62

Homogenates of the Aplysia nervous system contain protein kinase activities sensitive to cAMP, cGMP, and Ca2+/calmodulin. The cAMP- and cGMP-dependent activities are either soluble enzymes or are only loosely bound to membranes, since they can be detected only in crude but not in washed membrane fractions, and are present in 20,000 or 100,000 X g supernatants prepared from homogenates. In contrast there are both soluble and tightly membrane-bound Ca2+/calmodulin-dependent protein kinase activities. The three activities present in supernatant fractions can be separated by chromatography on DE-cellulose, indicating that they are due to distinct enzyme species. Substrates for these enzymes were analyzed by two-dimensional gel electrophoresis. Protein phosphorylation within the identified Aplysia neuron R15 in vivo was measured by the intracellular injection of [gamma-32P]ATP. cAMP stimulates the phosphorylation of nine proteins and decreases phosphorylation of two proteins in this cell. This in vivo pattern was compared with in vitro phosphorylation measured in homogenates of whole ganglion. Most of the phosphoproteins affected by cAMP in neuron R15 in vivo are also substrates for cAMP-dependent protein kinase in vitro. Thus, the in vitro system will be a useful tool for detailed biochemical analysis of phosphoproteins which have been identified as being physiologically relevant in vivo.
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PMID:Calcium- and cyclic nucleotide-dependent protein kinases and their substrates in the Aplysia nervous system. 298 Dec 96

1.) Application of serotonin to Aplysia sensory neurons can result in facilitated synaptic transmission, both short- and long-term. This facilitation is likely to be produced by a complex set of molecular mechanisms: serotonin activates adenylate cyclase, increasing cAMP and protein kinase (Cedar and Schwartz, 1972); serotonin also changes the subcellular distribution of the Ca2+/calmodulin-dependent protein kinase (Saitoh and Schwartz, 1983). Recently, phorbol esters also have been shown to produce facilitation. We have therefore investigated how protein kinase C (PKC) participates in serotonin-mediated synaptic facilitation. 2.) We found that the Aplysia genome encodes PKC, which is expressed in nervous tissue as at least two abundant transcripts (about 0.003% of the total message). Its inferred amino acid sequence is 85% homologous to that of enzymes from mammals and Drosophila, and over 95% homologous if compared to both. The specific activity of the Aplysia kinase is comparable to that found in rat brain, with similar reaction parameters and dependencies on phosphatidylserine (PS), Ca2+, diacylglycerol and phorbol esters. While PKC is found on neuronal membrane in the basal state, the PKC activators, Ca2+ and phorbol esters, further translocate the kinase to membrane in crude extracts of neuronal tissue. The amounts of membrane-bound PKC, as determined by 3H-phorbol-ester binding, are greatest in neuropil and nerve. 3.) Exposure of sensory neurons and their terminals in Aplysia pleural-pedal ganglia to facilitating doses of either phorbol ester or serotonin results in the translocation of PKC from cytosol to membrane, activating the enzyme. cAMP does not produce this translocation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Activation of protein kinase C by serotonin: biochemical evidence that it participates in the mechanisms underlying facilitation in Aplysia. 327 94

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca2+/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca2+/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca2+/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed alpha, beta, gamma and delta, only the gamma- and delta-subunits were phosphorylated by the cAMP-dependent protein kinase (+ cAMP), or by its purified catalytic subunits.
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PMID:cAMP, not Ca2+/calmodulin, regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax. 632 Aug 88

The cellular mechanisms controlling reabsorption of amino acids in the renal proximal tubule are unknown. Ca(2+)-dependent protein kinases modulate the activity of several ion channels and carriers in the kidney. The role of these enzymes in regulating tubular amino acid transport has not been established. We investigated the effect of Ca(2+)- and phospholipid-dependent protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMK II) on Na(+)- and Cl(-)-dependent proline transport across the rat renal brush-border membrane (BBM). Bioassays utilizing selective peptide substrates for Ca(2+)-dependent protein kinases demonstrated the presence of PKC and CaMK II in the BBM. Renal brush-border membrane vesicles (BBMV) were phosphorylated using the "hyposmotic shock" technique. Endogenous (membrane-bound) CaMK II and PKC, as well as exogenous, highly purified PKC inhibited NaCl-linked proline uptake by phosphorylated, lysed/resealed BBMV compared with control vesicles. The inhibitory effect of Ca2+ on proline transport, without the presence of other kinase activators, was mediated by activation of endogenous CaMK II. The CaMK II- and PKC-induced inhibition of proline uptake was reversed by the specific kinase inhibitor peptides CaMK II-(281-302) and PKC-(19-31), respectively. These data suggest that Ca(2+)-dependent protein kinase-mediated phosphorylation inhibits NaCl-dependent proline transport across the tubular luminal membrane.
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PMID:Ca(2+)-dependent protein kinases modulate proline transport across the renal brush-border membrane. 784 Feb 41

Activation of a calmodulin (CaM)-dependent protein kinase associated with rabbit skeletal-muscle sarcoplasmic reticulum (SR) results in the phosphorylation of polypeptides of 450, 360, 165, 105, 89, 60, 34 and 20 kDa. Radioligand-binding studies indicated that a membrane-bound 60 kDa polypeptide contained both CaM- and ATP-binding domains. Under renaturing conditions on nitrocellulose blots, the 60 kDa polypeptide of the membrane exhibited CaM-dependent autophosphorylation activity, suggesting that it was the CaM-dependent protein kinase of SR. Ca2+/CaM-independent autophosphorylation of polypeptides of 62 and 45 kDa was found to occur in the light SR, whereas the Ca2+/CaM-dependent autophosphorylation activity was enriched in the heavy SR. Both these kinase activities were absent from transverse tubules, although these membranes were enriched in CaM-binding polypeptides of 160, 100 and 80 kDa. In the absence of Ca2+, CaM bound to a 33 kDa polypeptide of the membrane. The purified ryanodine receptor was not phosphorylated by the purified CaM kinase, although it was a substrate for protein kinase C. Affinity-purified antibodies to brain CaM kinase II cross-reacted with the 60 kDa polypeptide in Western blots and immunoprecipitated the 60 kDa polypeptide, along with the 360, 105, 89, 34 and 20 kDa phosphoproteins, from Nonidet-P-40-solubilized SR membranes. Antibodies raised against the 60 kDa kinase polypeptide did not cross-react with the other phosphoproteins, suggesting that these polypeptides were distinct and unrelated. Subcellular distribution of the 60 kDa kinase indicated the specific association of the polypeptide with the junctional-face membrane of SR. The CaM-dependent incorporation of 32P into various membrane proteins was inhibited by the CaM kinase II fragment (290-309), with an IC50 value of 2 nM for the inhibition of incorporation into the 60 kDa kinase polypeptide. Recent studies [Wang and Best (1992) Nature (London) 359, 739-741] have shown that a CaM kinase activity intrinsic to the membrane can inactivate the Ca(2+)-release channel of skeletal muscle SR. Since our results demonstrate that the 60 kDa polypeptide of SR is a CaM-dependent protein kinase, we suggest that this kinase, through its associations, may be responsible for gating the Ca(2+)-release channel.
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PMID:A 60 kDa polypeptide of skeletal-muscle sarcoplasmic reticulum is a calmodulin-dependent protein kinase that associates with and phosphorylates several membrane proteins. 824 Mar 1

Changes in tubular reabsorption of amino acids and other solutes are characteristic of the immature renal tubule and of various hereditary nephropathies. The cellular mechanisms governing these aberrations in renal amino acid transport have not been established. Calcium (Ca2+)-dependent protein kinases are known to phosphorylate membrane-bound carrier proteins, thereby modulating transport of various solutes by the proximal tubule. The role of these enzymes in regulating renal tubular amino acid transport, particularly during kidney development, is unknown. We investigated: (1) the effect of Ca(2+)- and phospholipid-dependent protein kinase [protein kinase C (PKC)] and Ca2+/calmodulin-dependent protein kinase II (CaMKII) on sodium chloride (NaCl)-linked proline transport by renal brush border membrane vesicles (BBMV) from adult rats using the "hypoosmotic shock" technique (lysis of vesicles); (2) the activity, expression and subcellular distribution (cytosol, particulate, BBM) of Ca(2+)-dependent protein kinases in kidneys from 7-day-old and adult rats using MBP 4-14 and autocamtide II phosphorylation assays for PKC and CaMKII, respectively, endogenous protein phosphorylation (using gel electrophoresis and autoradiography) and Western immunoblot analysis to detect PKC and CaMKII. The studies showed: (1) endogenous (membrane-bound) CaMKII and PKC as well as exogenous, highly purified PKC inhibit proline uptake by phosphorylated, lyzed/resealed BBMV when compared with control vesicles; the voltage-clamped, nonelectrogenic component of proline transport was inhibited by PKC- but not CaMKII-mediated phosphorylation; (2) a Ca(2+)-dependent activity of both kinases was evident in all subcellular fractions tested in immature and adult kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The role of protein phosphorylation in renal amino acid transport. 825 36


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