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)

We report the purification and characterization of an active catalytic fragment of Ca2+/calmodulin-dependent protein kinase II, derived from autophosphorylation and subsequent limited chymotryptic digestion of the purified rat forebrain soluble kinase. The purified fragment was completely Ca2+/calmodulin-independent, existed as a monomer, and phosphorylated synapsin I at the same sites as does the native form of Ca2+/calmodulin-dependent protein kinase II. Kinetic studies with the purified fragment revealed a more than 10-fold increase in Vmax and a 50% decrease in Km for synthetic peptide substrates, compared with native Ca2+/calmodulin-dependent protein kinase II. No 32P-labeled autophosphorylated residues were detected in the purified active fragment, indicating that the autophosphorylation sites were not contained within this fragment. Comparative studies of this active fragment (30 kDa) and its inactive counterpart (32-kDa fragment) revealed certain structural details of both fragments. Calmodulin-overlay study, immunoblot analysis, and direct amino acid sequencing suggest that both fragments contain the entire NH2-terminal catalytic domain and were generated by distinct cleavage within the regulatory domain. The putative cleavage sites for both fragments are discussed.
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PMID:Active catalytic fragment of Ca2+/calmodulin-dependent protein kinase II. Purification, characterization, and structural analysis. 165 29

Endogenous phosphorylation of the crude membrane fraction of cultured 3Y1 fibroblast cells was enhanced by the addition of Ca2+/calmodulin. Both Ca2+/calmodulin-dependent protein kinase activity and its substrate were present in a cytoskeletal fraction, obtained as a pellet after washing of the membrane fraction with 2 mM EGTA, 0.6 M NaCl, and 1% Triton X-100. The phosphorylatable protein in the Triton X-insoluble fraction was identified by immunoblotting as vimentin. This endogenous phosphorylation induced by calmodulin was inhibited by the addition of KN-62, a specific Ca2+/calmodulin-dependent protein kinase II inhibitor, in a dose-dependent manner. However, phosphorylation of the 59 kDa protein (vimentin) in this fraction was not stimulated by adding both phosphatidyl serine and cAMP, thereby suggesting the absence of protein kinase C or of cAMP-dependent protein kinase in this fraction. The protein kinase associated with the Triton X-insoluble fraction phosphorylated the Ca2+/calmodulin-dependent protein kinase II-specific site of synapsin I from the bovine cortex. Two-dimensional phosphopeptide maps of vimentin indicated that a major phosphopeptide phosphorylated by the endogenous calmodulin-dependent kinase also appears to be the same as a major phosphopeptide phosphorylated by the exogenous Ca2+/calmodulin-dependent protein kinase II. Our results suggest that cytoskeleton-associated Ca2+/calmodulin-dependent protein kinase II regulates dynamic cellular functions through the phosphorylation of cytoskeletal elements in non-neural cells.
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PMID:Ca2+/calmodulin-dependent protein phosphorylation associated with the cytoskeleton of quiescent rat fibroblast (3Y1) cells. 166 12

1. Presynaptic or simultaneous pre- and postsynaptic voltage-clamp protocols were implemented in the squid giant synapse in order to determine the magnitude and time course of the presynaptic calcium current (ICa) and its relation to transmitter release before and after presynaptic injection of proteins. These included several forms of synapsin I, calcium-calmodulin-dependent protein kinase II (CaM kinase II) and avidin. 2. The quantities and location of these proteins were monitored by fluorescence video-enhanced microscopy during the electrophysiological measurements. 3. Presynaptic injection of dephosphorylated synapsin I inhibited synaptic transmission with a time course consistent with diffusion of the protein through the terminal and action at the active release zone. A mathematical model relating the diffusion of synapsin I into the terminal with transmitter release was developed to aid in the interpretation of these results. 4. Synapsin I inhibition of transmitter release was reversible. 5. The action of synapsin I was highly specific, as phosphorylation of the tail region only or head and tail regions prevented synapsin I from inhibiting release. 6. Injections of heat-treated synapsin I or of avidin, a protein with a size and isoelectric point similar to those of synapsin I, had no effect on transmitter release. 7. CaM kinase II injected presynaptically was found to facilitate transmitter release. This facilitation, which could be as large as 700% of the control response, was related to the level of penetration of the enzyme along the length of the preterminal A mathematical model of this facilitation indicates a reasonable fit between the distribution of CaM kinase II within the terminal and the degree of facilitation. 8. The overall shape of the postsynaptic response was not modified by either synapsin I or CaM kinase II injection. 9. The data suggest that, in addition to releasing transmitter, calcium also penetrates the presynaptic cytosol and activates CaM kinase II. When activated, CaM kinase II phosphorylates synapsin I, which reduces its binding to vesicles and/or cytoskeletal structures, enabling more vesicles to be released during a presynaptic depolarization. The amplitude of the postsynaptic response will then be both directly and indirectly regulated by depolarization induced Ca2+ influx. This model provides a molecular mechanism for synaptic potentiation.
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PMID:Regulation by synapsin I and Ca(2+)-calmodulin-dependent protein kinase II of the transmitter release in squid giant synapse. 167 19

A number of recent reports have suggested that norepinephrine (NE) produces a form of synaptic enhancement that resembles long-term potentiation (LTP). LTP, thought to be an electrophysiological correlate of memory, in part involves an augmentation of transmitter release. Although the effects of NE have not been unequivocally linked to LTP, it is clear that NE can produce increased transmitter release in the dentate gyrus of the hippocampus. The purpose of this study was to determine whether NE was capable of enhancing the phosphorylation of synapsin I and synapsin II, two homologous phosphoproteins thought to be involved in modulation of neurotransmitter release. NE (10 microM) and isoproterenol (250 nM) produced an increase in the phosphorylation of synapsin I and synapsin II in dentate slices from young rats. Phosphorylation site analysis of synapsin I, performed by limited proteolysis, indicated that NE and isoproterenol increased the phosphorylation of synapsin I at sites modified by Ca2+/calmodulin-dependent protein kinase II as well as cAMP-dependent protein kinase. These data demonstrate that NE stimulates the phosphorylation of synapsin I at its Ca2+/calmodulin-dependent protein kinase II site, which is a site that has been shown to regulate the effect of synapsin I on neurotransmitter release. We have also examined the effects of NE and isoproterenol on synapsin phosphorylation in dentate slices prepared from aged animals. Such animals have previously been shown to exhibit deficits in NE sensitivity as well as significant impairment in their ability to exhibit LTP. Neither NE nor isoproterenol stimulated synapsin phosphorylation in slices prepared from aged animals. Interestingly, the basal level of phosphorylation of the synapsin proteins was higher in slices prepared from aged animals. This higher basal level of phosphorylation may underlie the failure of aged animals to exhibit NE-stimulated increases in phosphorylation of the synapsin proteins. We hypothesize that the beta-adrenergic agonist-stimulated phosphorylation of synapsin I and synapsin II in young rats plays a role in the increase in transmitter release produced by NE in the dentate. Thus, the failure of the aged rats to show such phosphorylation may underlie, in part, their failure to exhibit normal responsiveness to NE. Moreover, these deficits in synapsin phosphorylation may also play some role in the deficits in plasticity seen in aged rats.
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PMID:Norepinephrine and isoproterenol increase the phosphorylation of synapsin I and synapsin II in dentate slices of young but not aged Fisher 344 rats. 190 Sep 42

The molecular events that control synaptic vesicle availability in chemical synaptic junctions have not been fully clarified. Among the protein molecules specifically located in presynaptic terminals, synapsin I and calcium/calmodulin-dependent protein kinase II (CaM kinase II) have been shown to modulate evoked transmitter release in the squid giant synapse. In the present study, analysis of synaptic noise in this chemical junction was used to determine whether these proteins also play a role in the control of spontaneous and enhanced spontaneous transmitter release. Injections of dephosphorylated synapsin I into the presynaptic terminal reduced the rate of spontaneous and enhanced quantal release, whereas injection of phosphorylated synapsin I did not modify such release. By contrast CaM kinase II injection increased enhanced miniature release without affecting spontaneous miniature frequency. These results support the view that dephosphorylated synapsin I "cages" synaptic vesicles while CaM kinase II, by phosphorylating synapsin I, "decages" these organelles and increases their availability for release without affecting the release mechanism itself.
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PMID:Effects of synapsin I and calcium/calmodulin-dependent protein kinase II on spontaneous neurotransmitter release in the squid giant synapse. 197 21

The granule cell-enriched Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) is a recently discovered neuron-specific enzyme. The kinase avidly phosphorylates synapsin I and contains a polyglutamate sequence, which suggests an association with chromatin as well. A possible role in synapsin I phosphorylation and in nuclear Ca2+ signaling was supported by immunochemical and ultrastructural examination of CaM kinase-Gr distribution. CaM kinase-Gr immunoreactivity was present in the molecular and granule cell layers of the rat cerebellum. This pattern corresponded to the occurrence of the enzyme in the granule cell axons and nuclei, respectively. Immunoblots confirmed these findings. Thus, CaM kinase-Gr may mediate and coordinate Ca2(+)-signaling within different subcellular compartments.
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PMID:Nuclear and axonal localization of Ca2+/calmodulin-dependent protein kinase type Gr in rat cerebellar cortex. 201 93

Synapsin I is a major nerve terminal-specific phosphoprotein. It consists of a hydrophobic head region containing one phosphorylation site for either cAMP-dependent protein kinase or Ca2+/calmodulin-dependent protein kinase I and of a basic and elongated tail region containing two phosphorylation sites for Ca2+/calmodulin-dependent protein kinase II. The steady-state emission spectrum of synapsin I was centered at 330 nm and was markedly red shifted upon denaturation, as expected for tryptophan residues segregated from the external aqueous environment in native conditions. Quenching studies showed a low accessibility of synapsin I tryptophans at low ionic strength which was further decreased by exposure to 200 mM NaCl but not significantly affected by phosphorylation. The intrinsic fluorescence of synapsin I was resolved into three major decay components with lifetimes of about 0.2, 3, and 7 ns. Upon phosphorylation of synapsin I on the tail sites, the spectra associated with the intermediate and long lifetimes were shifted to the red region, while the spectrum associated with the short lifetime was shifted to the blue region, in the absence of significant changes of the lifetimes. Phosphorylation of synapsin I on the head site was less effective. The anisotropy decay of synapsin I labeled with the long-living chromophore pyrene on Cys-223 was also analyzed. A shorter rotational correlation time was found for the tail phosphorylated form (corresponding to a Stokes radius of 41-42 A) than for the dephosphorylated or for the head phosphorylated form (corresponding to a Stokes radius of 60-63 A). The data suggest that phosphorylation of the tail sites induces changes in the conformation and hydrodynamic properties of synapsin I which may play a role in the regulation of the molecular interactions of synapsin I within the nerve terminal.
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PMID:Time-resolved fluorescence study of the neuron-specific phosphoprotein synapsin I. Evidence for phosphorylation-dependent conformational changes. 211 21

The relationship of the kinase which co-purifies with caldesmon to Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) was investigated by studying the phosphorylation of bovine brain synapsin I, as well-characterized substrate of CaM-kinase II. Synapsin I is a very good substrate (Km = 90 nM) of the co-purifying kinase, which phosphorylates two sites in synapsin I, both of which are distinct from the single site phosphorylated by cyclic-AMP-dependent protein kinase. Phosphorylation of synapsin I is Ca2(+)- and calmodulin-dependent: half-maximal activation occurs at 0.13 microM-Ca2+ and maximal activity at 0.4 microM-Ca2+. Phosphorylation of the co-purifying kinase slightly enhances the rate, but does not alter the stoichiometry, of subsequent synapsin I phosphorylation; it does, however, circumvent the requirement for Ca2+ and calmodulin. The properties of this kinase therefore closely resemble those of CaM-kinase II, and we conclude that it is probably a smooth-muscle isoenzyme of CaM-kinase II.
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PMID:Kinase activity associated with caldesmon is Ca2+/calmodulin-dependent kinase II. 216 10

There is a great deal known about the in vitro properties of CaM kinase II, both in terms of its substrate specificity and its regulation by calmodulin and autophosphorylation. Much of this characterization is based on experiments performed with the rat brain isozyme of CaM kinase II, although in the aspects examined to date isozymes of the kinase from other tissues appear to behave in a broadly similar manner in vitro. However, relatively little is known about the functions of the kinase in vivo. The proteins phosphorylated by the kinase (with the probable exception of synapsin I and tyrosine hydroxylase) and the role of kinase autophosphorylation in vivo remain largely unknown. Investigation of the physiological role of the kinase in brain and other tissues will be a particularly exciting area for future work. The current knowledge of the in vitro properties and the availability of cDNA clones will hopefully expedite this research.
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PMID:Calcium/calmodulin-dependent protein kinase II. 217 93

Ileal brush border membranes contain an endogenous Ca2+/calmodulin (CaM)-dependent protein kinase activity that modulates the activity of the apical membrane Na+/H+ exchanger. To further characterize this kinase, synapsin I, a substrate for Ca2+/CaM-dependent protein kinases, was added to preparations of ileal brush border membranes. In the presence of Ca2+/CaM, synapsin I was phosphorylated. Phosphopeptide mapping demonstrated that the addition of Ca2+/CaM to brush border membranes stimulated the phosphorylation of sites in synapsin I specific for Ca2+/CaM-dependent protein kinase II. Immunoblots containing brush border and microvillus membrane proteins were probed with an antibody that recognizes the 50-kDa subunit of rat brain Ca2+/CaM-dependent protein kinase II. This antibody labeled major and minor species of 50 and 53 kDa, respectively, with more labeling of the brush border than the microvillus membranes. Right-side-out ileal villus cell brush border vesicles were prepared containing CaM, ATP, and 350 nM free Ca2+. Na+/H+ exchange was inhibited by the presence of Ca2+/CaM/ATP within the vesicles. A 21-amino acid peptide inhibitor of CaM kinase II was enclosed within some vesicle preparations by freeze-thaw. The effect on Na+/H+ exchange of Ca2+/CaM/ATP was partially reversed by the inhibitor peptide. These studies demonstrate the presence of Ca2+/CaM-dependent protein kinase II in rabbit ileal villus cell brush border membranes. Based on the effect of a specific inhibitor peptide of Ca2+/CaM kinase II, it is concluded that this kinase inhibits brush border Na+/H+ exchange, which participates in the regulation of ileal Na+ absorption.
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PMID:Rabbit ileal villus cell brush border Na+/H+ exchange is regulated by Ca2+/calmodulin-dependent protein kinase II, a brush border membrane protein. 217 71


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