Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Brief freezing as a means of transiently permeabilizing synaptosomes was explored. Rat brain synaptosomes frozen and thawed in the presence of 5% dimethyl sulfoxide, a cryoprotectant, were shown to release, in a calcium-dependent manner, previously accumulated [3H]norepinephrine and [14C]acetylcholine in response to elevated [K+]. In addition, synaptosomes subjected to freeze/thaw were shown to retain their ability to exhibit resting protein phosphorylation, as well as stimulated protein phosphorylation occurring in response to calcium influx. Brief freezing of synaptosomes in the presence of [gamma-32P]ATP and either the catalytic subunit of cyclic AMP-dependent protein kinase or calcium/calmodulin-dependent protein kinase II rendered the synaptosomal interior accessible to these agents, as reflected by the phosphorylation of substrate proteins, such as synapsin I, which reside within the nerve terminal. Inclusion of inhibitors of these protein kinases during freeze/thaw blocked synaptosomal protein phosphorylation, indicating that the inhibitors were also introduced. After freezing, the synaptosomes resealed rapidly and spontaneously, as shown by the inability of any of the agents to elicit an effect on phosphorylation when added at the end of the freezing period. The permeabilization procedure should contribute to an understanding of the functional roles of phosphoproteins, and of their associated protein kinases and protein phosphatases, in nerve terminals.
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PMID:Introduction of impermeant molecules into synaptosomes using freeze/thaw permeabilization. 253 75

ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 as determined by SDS/PAGE) is a major cytosolic substrate for cAMP-stimulated protein phosphorylation in dopamine-innervated regions of rat CNS (Walaas et al., 1983c). This acidic phosphoprotein has now been identified in bovine caudate nucleus cytosol and purified to homogeneity from this source. The purification procedure involved diethylaminoethyl-cellulose chromatography, ammonium sulfate fractionation, phenyl-Sepharose CL-4B chromatography, and fast protein liquid chromatography using Mono Q anion-exchange resin. Two isoforms of ARPP-21 (ARPP-21A and ARPP-21B) were obtained, which were present in approximately equal amounts in the starting material. ARPP-21A was purified 2610-fold with a final yield of 20% and ARPP-21B was purified 2940-fold with a final yield of 21%. The purified preparations of both isoforms were judged to be homogenous by SDS/PAGE. ARPP-21A and ARPP-21B yielded identical 2-dimensional thin-layer tryptic phosphopeptide maps, identical amino acid compositions and closely related, but distinct, reverse-phase high-pressure liquid chromatograms of tryptic digests. The amino acid composition of ARPP-21 showed a high content of glutamic acid/glutamine, and no methionine, tryptophan, tyrosine, phenylalanine, or histidine. ARPP-21 was stable to heat denaturation and to 50% (vol/vol) ethanol treatment and was partially soluble at pH 2. The Mr determined for ARPP-21 by SDS/PAGE was 21,000. The Stokes radius of ARPP-21 was 26.3 A, and the sedimentation coefficient of ARPP-21 was 1.3 S; these values yield a calculated molecular mass of 13,700 Da and a frictional ratio of 1.7, indicative of an elongated tertiary structure. ARPP-21 was an excellent substrate for cAMP-dependent protein kinase and was either not phosphorylated or only poorly phosphorylated by cGMP-dependent protein kinase, calcium/calmodulin-dependent protein kinase I, calcium/calmodulin-dependent protein kinase II, casein kinase II, or protein kinase C. The purified catalytic subunit of cAMP-dependent protein kinase catalyzed the incorporation of 1.2 mol phosphate/mol purified ARPP-21. Phosphorylation occurred exclusively on seryl residues. Phospho-ARPP-21 was dephosphorylated effectively by protein phosphatase-1 or -2A, but not by protein phosphatase-2B or -2C. Rabbit polyclonal and mouse monoclonal antibodies were prepared to purified ARPP-21. These antibodies specifically immunoprecipitated ARPP-21, which was found to be highly enriched in the caudate nucleus and putamen of monkey brain.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions. I. Purification and characterization of the protein from bovine caudate nucleus. 253 84

The phenomenon of long-term potentiation (LTP), a long lasting increase in the strength of synaptic transmission which is due to brief, repetitive activation of excitatory afferent fibres, is one of the most striking examples of synaptic plasticity in the mammalian brain. In the CA1 region of the hippocampus, the induction of LTP requires activation of NMDA (N-methyl-D-aspartate) receptors by synaptically released glutamate with concomitant postsynaptic membrane depolarization. This relieves the voltage-dependent magnesium block of the NMDA-receptor ion channel, allowing calcium to flow into the dendritic spine. Although calcium has been shown to be a necessary trigger for LTP (refs 11, 12), little is known about the immediate biochemical processes that are activated by calcium and are responsible for LTP. The most attractive candidates have been calcium/calmodulin-dependent protein kinase II (CaM-KII) (refs 13-16), protein kinase C (refs 17-19), and the calcium-dependent protease, calpain. Extracellular application of protein kinase inhibitors to the hippocampal slice preparation blocks the induction of LTP (refs 21-23) but it is unclear whether this is due to a pre- and/or postsynaptic action. We have found that intracellular injection into CA1 pyramidal cells of the protein kinase inhibitor H-7, or of the calmodulin antagonist calmidazolium, blocks LTP. Furthermore, LTP is blocked by the injection of synthetic peptides that are potent calmodulin antagonists and inhibit CaM-KII auto- and substrate phosphorylation. These findings demonstrate that in the postsynaptic cell both activation of calmodulin and kinase activity are required for the generation of LTP, and focus further attention on the potential role of CaM-KII in LTP.
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PMID:An essential role for postsynaptic calmodulin and protein kinase activity in long-term potentiation. 254 23

The phosphorylation of caldesmon was studied to determine if kinase activity reflected either an endogenous kinase or caldesmon itself. Titration of kinase activity with calmodulin yielded maximum activity at substoichiometric ratios of calmodulin/caldesmon. The sites of phosphorylation on caldesmon for calcium/calmodulin-dependent protein kinase II and endogenous kinase were the same, but distinct from protein kinase C sites. Phosphorylation in the presence of Ca2+ and calmodulin resulted in a subsequent increase of endogenous kinase activity in the absence of Ca2+. These results suggest that caldesmon is not a protein kinase and that kinase activity in caldesmon preparations is due to calcium/calmodulin-dependent protein kinase II.
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PMID:Phosphorylation of caldesmon. 255 23

Microtubule-associated protein 2 (MAP-2), a cytoskeletal protein of 280 kilodalton that is highly enriched in dendrites and neuronal perikarya, is subject to both cyclic AMP-, calcium/calmodulin-, and calcium/phospholipid-regulated phosphorylation when incubated with [gamma-32P]ATP in vitro. We have analyzed the different sites in MAP-2 phosphorylated by these three kinases in fresh or boiled cytosol from different regions of the rat brain, in particular the olfactory bulb, where only one form (MAP-2B) is present, and the cerebral cortex, where both forms (MAP-2A and MAP-2B) are equally enriched. Cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II phosphorylated four common phosphorylation sites, as well as a number of distinct sites that were unique to each enzyme. Calcium/phospholipid-dependent protein kinase phosphorylated a minimum of 15 sites, only one of which appeared to be shared with the other protein kinases. Only serine residues were phosphorylated by cyclic AMP-dependent and calcium/phospholipid-dependent protein kinases, while both serine and threonine residues were phosphorylated by calcium/calmodulin-dependent protein kinase II. No differences were observed in the peptide maps of phospho-MAP-2 prepared from different brain regions. These results emphasize the complexity of the phosphorylation systems that may regulate the function of MAP-2 in situ.
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PMID:Multisite phosphorylation of microtubule-associated protein 2 (MAP-2) in rat brain: peptide mapping distinguishes between cyclic AMP-, calcium/calmodulin-, and calcium/phospholipid-regulated phosphorylation mechanisms. 256 75

P1, a high mobility group-like nuclear protein, phosphorylated by casein kinase II on multiple sites in situ, has been found to be phosphorylated in vitro by protein kinase C, cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II on multiple and mostly distinct thermolytic peptides. All these enzymes phosphorylated predominantly serine residues, with casein kinase II and protein kinase C also labeling threonine residues. Both casein kinase II and second messenger-regulated protein kinases, particularly protein kinase C, might therefore be involved in the physiological regulation of multisite phosphorylation of P1.
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PMID:Phosphorylation of P1, a high mobility group-like protein, catalyzed by casein kinase II, protein kinase C, cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II. 259 27

The endogenous phosphorylation of serotonin binding protein (SBP), a soluble protein found in central and peripheral serotonergic neurons, inhibits the binding of 5-hydroxytryptamine (5-HT, serotonin). A protein kinase activity that copurifies with SBP (SBP-kinase) was partially characterized and compared with calcium/calmodulin-dependent protein kinase II (CAM-PK II). SBP itself is not the enzyme since heating destroyed the protein kinase activity without affecting the capacity of the protein to bind [3H]5-HT. SBP-kinase and CAM-PK II kinase shared the following characteristics: (1) size of the subunits; (2) autophosphorylation in a Ca2+-dependent manner; and (3) affinity for Ca2+. In addition, both forms of protein kinase phosphorylated microtubule-associated proteins well and did not phosphorylate myosin, phosphorylase b, and casein. Phorbol esters or diacylglycerol had no effect on either of the protein kinases. However, substantial differences between SBP-kinase and CAM-PK II were observed: (1) CAM enhanced CAM-PK II activity, but had no effect on SBP-kinase; (2) synapsin I was an excellent substrate for CAM-PK II, but not for SBP-kinase; (3) 5-HT inhibited both the autophosphorylation of SBP-kinase and the phosphorylation of SBP, but had no effect on CAM-PK II. These data indicate that SBP-kinase is different from CAM-PK II. Phosphopeptide maps of SBP and SBP-kinase generated by digestion with S. aureus V8 protease are consistent with the conclusion that these proteins are distinct molecular entities. It is suggested that phosphorylation of SBP may regulate the transport of 5-HT within neurons.
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PMID:A Ca2+-dependent protein kinase activity associated with serotonin binding protein. 304 Sep 4

Bovine brain cytosol is shown to contain two heat-resistant inhibitors of protein kinase C, with the following characteristics: 1. One protein kinase C inhibitor can be easily purified to homogeneity. Evidence is presented that this polypeptide of Mr 19,000 is calmodulin. It inhibits protein kinase C with an EC50 of about 2.5 microM and the inhibition is Ca2+-independent. It inhibits only intact protein kinase C. Removal of the regulatory domain of protein kinase C, by limited proteolysis with trypsin, abolishes the inhibition. 2. Another protein kinase C inhibitory activity has been partially purified. Its Mr is low (Mr 600-700, as estimated by gel chromatography). It is not digested by proteases, is hydrophilic, acid- and alkali-resistant, acts Ca2+-independently, and, in contrast to calmodulin, inhibits even the catalytic fragment of protein kinase C after removal of the regulatory domain by limited proteolysis. This inhibition is, at least partially, due to a competition with ATP. Besides protein kinase C, calcium/calmodulin-dependent protein kinase II is inhibited to a similar extent. cAMP-dependent protein kinase is not affected.
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PMID:Heat-resistant inhibitors of protein kinase C from bovine brain. 218 Jun 96

The binding of synapsin I, a synaptic vesicle-associated phosphoprotein, to small synaptic vesicles has been examined. For this study, synapsin I was purified under nondenaturing conditions from rat brain, using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and characterized. Small synaptic vesicles were purified from rat neocortex by controlled pore glass chromatography as the last purification step, and binding was characterized at an ionic strength equivalent to 40 mM NaCl. After removal of endogenous synapsin I, exogenous dephospho-synapsin I bound with high affinity (Kd, 10 +/- 6 nM) to synaptic vesicles. The binding saturated at 76 +/- 40 micrograms synapsin I/mg of vesicle protein, which corresponded to the amount found endogenously in purified vesicles. Synapsin I binding exhibited a broad pH optimum around pH 7. Other basic proteins, specifically myelin basic protein and histone H2b, did not compete with synapsin I for binding to vesicles. Other membranes purified from rat brain and membranes derived from human erythrocytes did not show the high affinity binding site for synapsin I found in vesicles. The binding of three different forms of phosphosynapsin I to vesicles was investigated. Synapsin I, phosphorylated at sites 2 and 3 by purified calcium/calmodulin-dependent protein kinase II, bound with a 5-fold lower affinity to the vesicles than did dephospho-synapsin I. In contrast, synapsin I, phosphorylated at site 1 by purified catalytic subunit of cAMP-dependent protein kinase, bound with an affinity close to that of dephospho-synapsin I. Synapsin I phosphorylated on all three sites bound to the vesicles with an affinity comparable to that of synapsin I phosphorylated on sites 2 and 3. Under conditions of higher ionic strength (150 mM NaCl equivalent), synapsin I bound with a 5-fold lower affinity to vesicles, and no effect of phosphorylation on binding was observed under these conditions.
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PMID:Characterization of synapsin I binding to small synaptic vesicles. 308 73

Synapsin I is a neuron-specific phosphoprotein localized to the cytoplasmic surface of synaptic vesicles. This phosphoprotein is a major substrate for cyclic AMP-dependent and calcium/calmodulin-dependent protein kinases. Its state of phosphorylation can be altered both in vivo and in vitro by a variety of physiological and pharmacological manipulations known to affect synaptic function. Recent direct evidence suggests that it may be involved in the regulation of neurotransmitter release from the nerve terminal. In the nerve terminal, synaptic vesicles are embedded in a cytoskeletal network, consisting in part of actin. We report here the ability of the dephospho-form of synapsin I to bundle F-actin. This bundling activity is reduced when synapsin I is phosphorylated by cAMP-dependent protein kinase and virtually abolished when it is phosphorylated by calcium/calmodulin-dependent protein kinase II or by both kinases. These results, demonstrating an interaction of synapsin I with actin in vitro, support the possibility that synapsin I is involved in clustering of synaptic vesicles at the presynaptic terminal and that the phosphorylation of synapsin I may be involved in regulating the translocation of synaptic vesicles to their sites of release.
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PMID:Synapsin I bundles F-actin in a phosphorylation-dependent manner. 310


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