Calcium/Calmodulin-Dependent Protein Kinase II Is Associated with NR2A/B Subunits of NMDA Receptor in Postsynaptic Densities

Abstract: NMDA receptors and Ca²⁺/calmodulin-dependent kinase II (CaMKII) have been reported to be highly concentrated in the postsynaptic density (PSD). Although the possibility that CaMKII in PSD might be associated with specific proteins has been put forward, the protein or proteins determining the targeting of the kinase in PSD have not yet been identified. Here we report that CaMKII binds to NR2A and NR2B subunits of NMDA receptors in PSD isolated from cortex and hippocampus. The association of NMDA receptor subunits and CaMKII was assessed by immunoprecipitating PSD proteins with antibodies specific for NR2A/B and CaMKII: CaMKII coprecipitated with NR2A/B and NR1 but not with other glutamate ionotropic receptor subunits, such as GluR1 and GluR2-3. A direct association between CaMKII and NR2A/B subunits was further confirmed by overlay experiments using either ³²P-autophosphorylated CaMKII or ³²P-NR2A/B and by evaluating the formation of a CaMKII-NR2A/B complex by means of the cross-linker disuccimidyl suberate. These data demonstrate an association between the NMDA receptor complex and CaMKII in the postsynaptic compartment, suggesting that this colocalization may be relevant for synaptic plasticity.     

Decreased calcium/calmodulin-dependent protein kinase II and protein kinase C activities mediate impairment of hippocampal long-term potentiation in the olfactory bulbectomized mice

Olfactory bulbectomized (OBX) mice showed significant impairment of learning and memory-related behaviors 14 days after olfactory bulbectomy, as measured by passive avoidance and Y-maze tasks. We here observed a large impairment of hippocampal long-term potentiation (LTP) in the OBX mice. Concomitant with decreased acetylcholinesterase expression, protein kinase C (PKC)alpha autophosphorylation and NR1(Ser-896) phosphorylation significantly decreased in the hippocampal CA1 region of OBX mice. Both PKCalpha and NR1(Ser-896) phosphorylation significantly increased following LTP in the control mice, whereas increases were not observed in OBX mice. Like PKC activities, calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation significantly decreased in the hippocampal CA1 region of OBX mice as compared with that of control mice. In addition, increased CaMKII autophosphorylation following LTP was not observed in OBX mice. Finally, the impairment of CaMKII autophosphorylation was closely associated with reduced pGluR1(Ser-831) phosphorylation, without change in synapsin I (site 3) phosphorylation in the hippocampal CA1 region of OBX mice. Taken together, in OBX mice NMDA receptor hypofunction, possibly through decreased PKCalpha activity, underlies decreased CaMKII activity in the post-synaptic regions, thereby impairing LTP induction in the hippocampal CA1 region. Both decreased PKC and CaMKII activities with concomitant LTP impairment account for the learning disability observed in OBX mice.     

Dephosphorylation of r Factor by Protein Phosphatase 2A in Synaptosomal Cytosol Fractions, and Inhibition by Aluminum

When the synaptosomal cytosol fraction from rat brain was chromatographed on a DEAE-cellulose column and assayed for protein phosphatases for τ factor and histone H1, two peaks of activities, termed peak 1 (major) and peak 2 (minor), were separated. Each peak was in a single form on Sephacryl S-300 column chromatography. Both peaks 1 and 2 dephosphorylated τ factor phosphorylated by Ca²⁺/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The K_m values were in the range of 0.42–0.84 μM for τ factor. There were no differences in kinetic properties of dephosphorylation between the substrates phosphorylated by the two kinases. The phosphatase activities did not depend on Ca²⁺, Mn²⁺, and Mg²⁺. Immunoprecipitation and immunoblotting analysis using polyclonal antibodies to the catalytic subunit of brain protein phosphatase 2A revealed that both protein phosphatases are the holoenzymic forms of protein phosphatase 2A. Aluminum chloride inhibited the activities of both peaks 1 and 2 with IC₅₀ values of 40–60 μM. These results suggest that dephosphorylation of r factor in presynaptic nerve terminals is controlled mainly by protein phosphatase 2A and that the neurotoxic effect of aluminum seems to be related mostly to inhibition of dephosphorylation of τ factor     

Post-translational excision of the carboxyl-terminal segment of CaM kinase phosphatase N and its cytosolic occurrence in the brain

Ca2+/Calmodulin-dependent protein kinase (CaM kinase) phosphatase, occurring in the cytoplasm of all tissues, dephosphorylates and thereby deactivates multifunctional CaM kinases, such as CaM kinases I, II and IV. In contrast, CaM kinase phosphatase N has been reported to occur almost exclusively in the brain and to be localized in the nucleus in the transfected COS-7 cells, as examined immunocytochemically with antibodies against the carboxyl-terminal segment of the enzyme, indicating its involvement in the deactivation of CaM kinase IV. Here, we show that the majority of the naturally occurring CaM kinase phosphatase N in the brain exists not in the intact form of the enzyme (83.4 kDa) but in a form (61.1 kDa) in which the carboxyl-terminal segment containing nuclear localization signals is deleted, and that it is present mostly in the cytoplasm but a little in the nucleus throughout the central nervous system, although occurring mostly in the nucleus in some large neurons. Strong immunostaining of the enzyme was also observed at postsynaptic density. These findings suggest that CaM kinase phosphatase N is involved in the regulation of not only CaM kinase IV but also CaM kinases II and I.     

CaM kinase II and protein kinase C activations mediate enhancement of long-term potentiation by nefiracetam in the rat hippocampal CA1 region

Nefiracetam is a pyrrolidine-related nootropic drug exhibiting various pharmacological actions such as cognitive-enhancing effect. We previously showed that nefiracetam potentiates NMDA-induced currents in cultured rat cortical neurons. To address questions whether nefiracetam affects NMDA receptor-dependent synaptic plasticity in the hippocampus, we assessed effects of nefiracetam on NMDA receptor-dependent long-term potentiation (LTP) by electrophysiology and LTP-induced phosphorylation of synaptic proteins by immunoblotting analysis. Nefiracetam treatment at 1-1000 nM increased the slope of fEPSPs in a dose-dependent manner. The enhancement was associated with increased phosphorylation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) without affecting synapsin I phosphorylation. In addition, nefiracetam treatment increased PKCalpha activity in a bell-shaped dose-response curve which peaked at 10 nM, thereby increasing phosphorylation of myristoylated alanine-rich protein kinase C substrate and NMDA receptor. Nefiracetam treatment did not affect protein kinase A activity. Consistent with the bell-shaped PKCalpha activation, nefiracetam treatment enhanced LTP in the rat hippocampal CA1 region with the same bell-shaped dose-response curve. Furthermore, nefiracetam-induced LTP enhancement was closely associated with CaMKII and PKCalpha activation with concomitant increases in phosphorylation of their endogenous substrates except for synapsin I. These results suggest that nefiracetam potentiates AMPA receptor-mediated fEPSPs through CaMKII activation and enhances NMDA receptor-dependent LTP through potentiation of the post-synaptic CaMKII and protein kinase C activities. Together with potentiation of nicotinic acetylcholine receptor function, nefiracetam-enhanced AMPA and NMDA receptor functions likely contribute to improvement of cognitive function.     

Spinophilin is phosphorylated by Ca2+/calmodulin-dependent protein kinase II resulting in regulation of its binding to F-actin

Spinophilin is a protein phosphatase-1- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We have recently shown that the interaction of spinophilin with the actin cytoskeleton depends upon phosphorylation by protein kinase A. We have now found that spinophilin is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in neurons. Ca(2+)/calmodulin-dependent protein kinase II, located within the post-synaptic density of dendritic spines, is known to play a role in synaptic plasticity and is ideally positioned to regulate spinophilin. Using tryptic phosphopeptide mapping, site-directed mutagenesis and microsequencing analysis, we identified two sites of CaMKII phosphorylation (Ser-100 and Ser-116) within the actin-binding domain of spinophilin. Phosphorylation by CaMKII reduced the affinity of spinophilin for F-actin. In neurons, phosphorylation at Ser-100 by CaMKII was Ca(2+) dependent and was associated with an enrichment of spinophilin in the synaptic plasma membrane fraction. These results indicate that spinophilin is phosphorylated by multiple kinases in vivo and that differential phosphorylation may target spinophilin to specific locations within dendritic spines.     

Chronic lithium enhances hippocampal long-term potentiation,but not neurogenesis,in the aged rat dentate gyrus

We investigated the hippocampal long-term potentiation (LTP), neurogenesis, and the activation of signaling molecules in the 20-month-old aged rats following chronic lithium treatment. Chronic lithium treatment produced a significant 79% increase in the numbers of BrdU(+) cells after treatment completion in the dentate gyrus (DG). Both LTP obtained from slices perfused with artificial cerebrospinal fluid (ACSF-LTP), and LTP recorded in the presence of bicuculline (bicuculline-LTP) were significantly greater in the lithium group than in the saline controls. Our results show that as with young rats, chronic lithium can substantially increase LTP and the number of BrdU(+) cells in the aged rats. However, neurogenesis, assessed by colocalization of NeuN and BrdU, was not detected in the aged rat DG subjected to chronic lithium treatment. Therefore, it is concluded that the increase in LTP and the number of BrdU(+) cells might not be associated with increases in neurogenesis in the granule cell layer of the DG. Lithium might has a beneficial effects through other signaling pathways in the aged brain.     

Regional and Temporal Alterations in Ca2+/Calmodulin-Dependent Protein Kinase II and Calcineurin in the Hippocampus of Rat Brain After Transient Forebrain Ischemia

We have investigated regional and temporal alterations in Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and calcineurin (Ca2+/calmodulin-dependent protein phosphatase) after transient forebrain ischemia. Immunoreactivity and enzyme activity of CaM kinase II decreased in regions CA1 and CA3, and in the dentate gyrus, of the hippocampus early (6-12 h) after ischemia, but the decrease in immunoreactivity gradually recovered over time, except in the CA1 region. Furthermore, the increase in Ca2+/calmodulin-independent activity was detected up to 3 days after ischemia in all regions tested, suggesting that the concentration of intracellular Ca2+ increased. In contrast to CaM kinase II, as immunohistochemistry and regional immunoblot analysis revealed, calcineurin was preserved in the CA1 region until 1.5 days and then lost with the increase in morphological degeneration of neurons. Immunoblot analysis confirmed the findings of the immunohistochemistry. These results suggest that there is a difference between CaM kinase II and calcineurin in regional and temporal loss after ischemia and that imbalance of Ca2+/calmodulin-dependent protein phosphorylation-dephosphorylation may occur.     

Phosphorylation status of the NR2B subunit of NMDA receptor regulates its interaction with calcium/calmodulin-dependent protein kinase II

Ca²⁺ influx through NMDA-type glutamate receptor at excitatory synapses causes activation of post-synaptic Ca²⁺/calmodulin-dependent protein kinase type II (CaMKII) and its translocation to the NR2B subunit of NMDA receptor. The major binding site for CaMKII on NR2B undergoes phosphorylation at Ser1303, in vivo . Even though some regulatory effects of this phosphorylation are known, the mode of dephosphorylation of NR2B-Ser1303 is still unclear. We show that phosphorylation status at Ser1303 enables NR2B to distinguish between the Ca²⁺/calmodulin activated form and the autonomously active Thr286-autophosphorylated form of CaMKII. Green fluorescent protein–α-CaMKII co-expressed with NR2B sequence in human embryonic kidney 293 cells was used to study intracellular binding between the two proteins. Binding in vitro was studied by glutathione- S -transferase pull-down assay. Thr286-autophosphorylated α-CaMKII or the autophosphorylation mimicking mutant, T286D-α-CaMKII, binds NR2B sequence independent of Ca²⁺/calmodulin unlike native wild-type α-CaMKII. We show enhancement of this binding by Ca²⁺/calmodulin. Phosphorylation or a phosphorylation mimicking mutation on NR2B (NR2B-S1303D) abolishes the Ca²⁺/calmodulin-independent binding whereas it allows the Ca²⁺/calmodulin-dependent binding of α-CaMKII in vitro . Similarly, the autonomously active mutants, T286D-α-CaMKII and F293E/N294D-α-CaMKII, exhibited Ca²⁺-independent binding to non-phosphorylatable mutant of NR2B under intracellular conditions. We also show for the first time that phosphatases in the brain such as protein phosphatase 1 and protein phosphatase 2A dephosphorylate phospho-Ser1303 on NR2B.     

Ischemia-Induced Translocation of Ca2+/Calmodulin-Dependent Protein Kinase II: Potential Role in Neuronal Damage

The activities of Ca2+/calmodulin (CaM)-dependent, Ca2+/phospholipid-dependent, and cyclic AMP-dependent protein kinases (CaM-KII, PKC, and PKA, respectively) were determined in rat brains after global ischemia. Both CaM-KII and PKC activities were significantly depressed in both hippocampal and cerebral cortical regions of ischemic animals, whereas no change was detected in PKA activity. The loss of CaM-KII activity was more dramatic and more sustained than the loss of PKC activity and correlated with the duration of ischemia. These decreases in enzyme activity were found in both supernatant and pellet fractions from crude homogenates. When the supernatant and pellet were analyzed for the amount of CaM-KII 50-kDa protein, a significant decrease was detected in supernatant fractions that paralleled a gain in the amount of CaM-KII in the pellet. Thus, the loss of CaM-KII activity in the supernatant can be explained by translocation of the enzyme to the pellet. Whether inactivation of CaM-KII occurs during or after the enzyme translocates from the supernatant to the pellet is unknown. Our results indicate that loss in CaM-KII activity parallels neuronal damage associated with ischemia; down-regulation of CaM-KII activity coincided with translocation of the enzyme to the particulate fraction, and it is proposed that this may be, in fact, a mechanism for controlling excessive CaM-KII phosphorylation.     

Calcium/Calmodulin-Dependent Protein Kinase II in Squid Synaptosomes

The Ca2+/calmodulin (CaM)-dependent protein kinase II system in squid nervous tissue was investigated. The Ca2+/CaM-dependent protein kinase II was found to be very active in the synaptosome preparation from optic lobe, where it was associated with the high-speed particulate fraction. Incubation of the synaptosomal homogenate with calcium, calmodulin, magnesium, and ATP resulted in partial and reversible conversion of the Ca2+/CaM-dependent protein kinase II from its calcium-dependent form to a calcium-independent species. The magnitude of this conversion reaction could be increased by inclusion of the protein phosphatase inhibitor NaF or by substitution of adenosine 5'-O-(3-thiotriphosphate) for ATP. When [gamma-32P]ATP was used, proteins of 54 and 58 kilodaltons (kDa) as well as proteins greater than 100 kDa were rapidly 32P-labeled in a calcium-dependent manner. Major 125I-CaM binding proteins in the synaptosome membrane fraction were 38 and 54 kDa. The Ca2+/CaM-dependent protein kinase II was purified from the squid synaptosome and was shown to consist of 54- and 58-60-kDa subunits. The purified kinase, like Ca2+/CaM-dependent protein kinase II from rat brain, catalyzed autophosphorylation associated with formation of the calcium-independent form. These studies, characterizing the Ca2+/CaM-dependent protein kinase II in squid neural tissue, are supportive of the putative role of this kinase in regulating calcium-dependent synaptic functions.     

Glutamate-Induced Loss of Ca2+/Calmodulin-Dependent Protein Kinase II Activity in Cultured Rat Hippocampal Neurons

Abstract: The exposure of cultured rat hippocampal neurons to 500 µ M glutamate for 20 min induced a 55% decrease in the total Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) activity. The Ca²⁺-independent activity and autophosphorylation of CaM kinase II decreased to the same extent as the changes observed in total CaM kinase II activity, and these decreases in activities were prevented by pretreatment with MK-801, an N -methyl- d -aspartate (NMDA)-type receptor antagonist, and the removal of extracellular calcium but not by antagonists against other types of glutamate receptors and protease inhibitors. Similarly, the decrease in the CaM kinase II activity was induced by a Ca²⁺ ionophore, ionomycin. Immunoblot analysis with the anti-CaM kinase II antibody revealed a significant decrease in the amount of the enzyme in the soluble fraction, in contrast with the inverse increase in the insoluble fraction; thus, the translocation was probably induced during treatment of the cells with glutamate. These results suggest that glutamate released during brain ischemia induces a loss of CaM kinase II activity in hippocampal neurons, by stimulation of the NMDA receptor, and that inactivation of the enzyme may possibly be involved in the cascade of the glutamate neurotoxicity following brain ischemia.     

Dephosphorylation of Microtubule Proteins by Brain Protein Phosphatases 1 and 2A, and Its Effect on Microtubule Assembly

Protein phosphatase C was purified 140-fold from bovine brain with 8% yield using histone H1 phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase (cyclic AMP-kinase). Brain protein phosphatase C was considered to consist of 10 and 90%, respectively, of the catalytic subunits of protein phosphatases 1 and 2A on the basis of the effects of ATP and inhibitor-2. Protein phosphatase C dephosphorylated microtubule-associated protein 2 (MAP2), tau factor, and tubulin phosphorylated by a multifunctional Ca2+/calmodulin-dependent protein kinase (calmodulin-kinase) and the catalytic subunit of cyclic AMP-kinase. The properties of dephosphorylation of MAP2, tau factor, and tubulin were compared. The Km values were in the ranges of 1.6-2.7 microM for MAP2 and tau factor. The Km value for tubulin decreased from 25 to 10-12.5 microM in the presence of 1.0 mM Mn2+. No difference in kinetic properties of dephosphorylation was observed between the substrates phosphorylated by the two kinases. Protein phosphatase C did not dephosphorylate the native tubulin, but universally dephosphorylated tubulin phosphorylated by the two kinases. The holoenzyme of protein phosphatase 2A from porcine brain could also dephosphorylate MAP2, tau factor, and tubulin phosphorylated by the two kinases. The phosphorylation of MAP2 and tau factor by calmodulin-kinase separately induced the inhibition of microtubule assembly, and the dephosphorylation by protein phosphatase C removed its inhibitory effect. These data suggest that brain protein phosphatases 1 and 2A are involved in the switch-off mechanism of both Ca2+/calmodulin-dependent and cyclic AMP-dependent regulation of microtubule formation.     

Staurosporine: An Effective Inhibitor for Ca2+/Calmodulin-Dependent Protein Kinase II

We investigated the effect of staurosporine on Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) purified from rat brain. (a) Staurosporine (10-100 nM) inhibited the activity of CaM kinase II. The half-maximal and maximal inhibitory concentrations were 20 and 100 nM, respectively. (b) The inhibition with staurosporine was of the noncompetitive type with respect to ATP, calmodulin, and phosphate acceptor (beta-casein). (c) Staurosporine suppressed the auto-phosphorylation of alpha- and beta-subunits of CaM kinase II at concentrations similar to those at which the enzyme activity was inhibited. (d) Staurosporine also attenuated the Ca2+/calmodulin-independent activity of the autophosphorylated CaM kinase II. These results suggest that staurosporine inhibits CaM kinase II by interacting with the catalytic domain, distinct from the ATP-binding site or substrate-binding site, of the enzyme and that staurosporine is an effective inhibitor for CaM kinase II in the cell system.     

Inactivation and Reactivation of the Multifunctional Calmodulin-Dependent Protein Kinase from Brain by Autophosphorylation and Dephosphorylation: Involvement of Protein Phosphatases from Brain

The multifunctional calmodulin-dependent protein kinase (calmodulin-kinase) from rat brain was autophosphorylated in a Ca2+- and calmodulin-dependent manner. The activity of the autophosphorylated enzyme was independent of Ca2+ and calmodulin. Calmodulin-kinase was dephosphorylated by protein phosphatase C from bovine brain, which is the catalytic subunits of protein phosphatases 1 and 2A. The holoenzyme of protein phosphatase 2A was also involved in the dephosphorylation of the enzyme. The autophosphorylated sites of calmodulin-kinase were universally dephosphorylated by protein phosphatase C. Calmodulin-kinase was inactivated and reactivated by autophosphorylation and dephosphorylation, respectively. Furthermore, the regulation of calmodulin-kinase by autophosphorylation and dephosphorylation was observed using calmodulin-kinase from canine heart. These results suggest that the activity of calmodulin-kinase is regulated by autophosphorylation and dephosphorylation, and that the regulation is the universal phenomenon for many other calmodulin-kinases in various tissues.     

Ca2+/Calmodulin Kinase II Translocates in a Hippocampal Slice Model of Ischemia

Abstract: Rat hippocampal slices were exposed to conditions that simulate an ischemic insult, and the subcellular distribution and the enzymatic activity of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase) were monitored. Semiquantitative western blots using a monoclonal antibody to the 50-kDa α subunit showed that there was a significant redistribution of the enzyme from a supernatant to a pellet fraction after 10 min of an anoxic/aglycemic insult. No significant change in the total amount of CaM kinase enzyme was detected in the homogenates for up to 20 min of exposure to the insult. Ca²⁺/CaM-dependent enzyme activity did not significantly change in the pellet during the 20-min insult. Supernatant activity decreased throughout the insult. The persistence of Ca²⁺/CaM-dependent CaM kinase activity in the pellet fraction and the detected movement of enzyme from the supernatant to the pellet indicate that redistribution may be an important mechanism in regulating the cellular location of CaM kinase activity.     

Involvement of mitogen-activated protein kinase in hippocampal long-term potentiation

Mitogen-activated protein kinase (MAPK) cascade classically is thought to be involved in cellular transformation, including proliferation and differentiation. Recent behavioral studies suggest that MAPK may also have a role in learning and memory. Long-term potentiation (LTP), a candidate mechanism for learning and memory, has at least two distinct temporal phases: an early phase (E-LTP) which lasts for 1–2 h and a late phase (L-LTP) which can persist 3 h. Here, we report that PD 098059, a selective inhibitor of MAPK cascade, attenuates L-LTP induced by bath application of forskolin without affecting basal synaptic transmission. This effect was mimicked by direct injection of animals with MAPK antisense oligonucleotide into the hippocampal CA1 region. MAPK activity measured by using a synthetic peptide corresponding to the sequence surrounding the major site of phosphorylation of the myelin-basic protein by MAPK was enhanced by forskolin. The same antisense treatment also completely inhibited the increased MAPK activity. These results demonstrate an involvement of MAPK in the induction of L-LTP in the hippocampal CA1 neurons.     

Persistent Translocation of Ca2+/Calmodulin-Dependent Protein Kinase II to Synaptic Junctions in the Vulnerable Hippocampal CA1 Region Following Transient Ischemia

Abstract: The influence of brain ischemia on the subcellular distribution and activity of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) was studied in various cortical rat brain regions during and after cerebral ischemia. Total CaM kinase II immunoreactivity (IR) and calmodulin binding in the crude synaptosomal fraction of all regions studied increase but decrease in the microsomal and cytosolic fractions, indicative of a translocation of CaM kinase II to synaptosomes. The translocation of CaM kinase II to synaptic junctions occurs but not to synaptic vesicles. The translocation in neocortex and CA3/DG (dentate gyrus) is transient, whereas in the hippocampal CA1 region, it persists for at least 1 day of reperfusion. The Ca²⁺/calmodulin-dependent activity of CaM kinase II in the subsynaptosomal fractions of neocortex is persistently decreased by up to 85%, despite the increase in CaM kinase II IR. The decrease in activity is more pronounced than the decline in IR, suggesting that CaM kinase II is covalently modified in the postischemic phase. The persistent translocation of CaM kinase II in the vulnerable ischemic CA1 region indicates that a pathological process is sustained in the area after the reperfusion phase and this may be of significance for ischemic brain injury.