Calmodulin-dependent Protein Kinase IIalpha Research Articles

CaMKIIa Neurons of the Ventromedial Hypothalamus Mediate Wakefulness and Anxiety-like Behavior.

Insomnia and anxiety are two common and closely related clinical problems that pose a threat to individuals' physical and mental well-being. There is a possibility that some nuclei and neural circuits in the brain are shared by both insomnia and anxiety. In the present study, using a combination of chemogenetics, optogenetics, polysomnographic recordings and the classic tests of anxiety-like behaviors, we verified that the calmodulin-dependent protein kinase II alpha (CaMKIIa) neurons of the ventromedial hypothalamus (VMH) are involved in the regulation of both wakefulness and anxiety. Chemogenetic manipulation of the VMH CaMKIIa neurons elicited an apparent increase in wakefulness during activation, whereas inhibition decreased wakefulness mildly. It substantiated that the VMH CaMKIIa neurons contribute to wakefulness. Then in millisecond-scale control of neuronal activity, short-term and long-term optogenetic activation induced the initiation and maintenance of wakefulness, respectively. We also observed that mice reduced exploratory behaviors in classic anxiety tests while activating the VMH CaMKIIa neurons and were anxiolytic while inhibiting. Additionally, photostimulation of the VMH CaMKIIa axons in the paraventricular hypothalamus (PVH) mediated wakefulness and triggered anxiety-like behaviors as well. In conclusion, our results demonstrate that the VMH participates in the control of wakefulness and anxiety, and offer a neurological explanation for insomnia and anxiety, which may be valuable for therapeutic interventions such as medication and transcranial magnetic stimulation.

Asenapine as a Potential Lead Inhibitor against Central Ca2+/Calmodulin-Dependent Protein Kinase II: Investigation by Docking Simulation and Experimental Validation

Aim: The aim of this potential repurposing study is to investigate the potential inhibitory activity of asenapine against central nervous system CaMKII isozymes using docking experiments and enzymatic assay. Background: The Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional protein kinase ubiquitously expressed throughout the brain. Emerging biological data have indicated that inhibiting central nervous system CaMKII isoforms, namely, CaMKIIα and CaMKIIβ, may be a promising therapeutic strategy for the potential treatment of many neurological diseases including schizophrenia, depression, epilepsy, and learning deficit. Objective: 1- Study the possible attractive interactions of asenapine within the binding sites of the central CaMKII isozymes. 2- Evaluate the inhibitory activities of asenapine against central CaMKII isozymes. Methods: Docking experiments of asenapine and other known CaMKII inhibitors were performed. Docking settings were validated using ROC analysis. After that, the inhibitory activities of asenapine against central CaMKII alpha and beta were evaluated by enzymatic assay. Result: Docking and scoring experiments of asenapine showed several binding interactions anchoring asenapine within CaMKIIα and CaMKIIβ catalytic sites while enzymatic assay results revealed that asenapine can inhibit CaMKIIα and CaMKIIβ in the micromolar range. Conclusion: Our study provides evidence that asenapine can serve as a promising lead for the development of new CaMKIIα and CaMKIIβ inhibitors. Moreover, this study reinforces how the investment in drug repurposing could boost the drug discovery process.

The memory modulatory effect of agmatine in passive avoidance task coincides with alterations of hippocampal CaMKII-α and ERK signaling in mice

Agmatine is a polyamine suggested to act as a supposed neurotransmitter in the brain. Evidence has indicated that acute agmatine administration might modulate memory. The present study aimed to investigate the effect of repeated agmatine treatment on passive avoidance memory, hippocampal calcium–calmodulin-dependent protein kinase II-alpha (CaMKII-α), and Extracellular Signal-Regulated Kinase (ERK) signaling pathways in naive mice. Adult male NMRI mice were treated with agmatine (10, 20, 30, 40, and 80 mg/kg/ip) or saline for 11 days. Acquisition and retention tests of passive avoidance memory were performed on days 10 and 11, respectively. Following the memory retention test, the hippocampi were assessed for the levels of CaMKII-α and ERK using the western blotting technique. The results revealed the dose-dependent effect of agmatine on the passive avoidance memory. Accordingly, the memory was impaired in lower doses, but was improved in higher ones. Agmatine in none of the doses affected the nociception of the mice in tail-flick test. Furthermore, agmatine increased the phosphorylation of CaMKII-α and ERK in the hippocampus at memory enhancing doses, while ERK phosphorylation decreased following the impairing doses of agmatine. Thus, the dose-dependent effect of agmatine on memory might be related to its modulatory effect on CaMKII-α and ERK signal transduction, eventually regulating the memory process.

Gastrodin relieved complete Freund's adjuvant-induced spontaneous pain by inhibiting inflammatory response

The analgesic effects of gastrodin (GAS), an active component derived from the Chinese herb Tian ma (Gastrodia elata Blume), on chronic inflammatory pain of mice and the involved molecular mechanisms were investigated. GAS significantly attenuated mice chronic inflammatory pain induced by hindpaw injection of complete Freund's adjuvant (CFA) and the accompanying anxiety-like behaviors. GAS administration reduced CFA-induced up-regulation of GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, GluN2A- and GluN2B-containing N-methyl-d-aspartate (NMDA) receptors, and Ca2+/calmodulin-dependent protein kinase II-alpha (CaMKII-α) in the anterior cingulate cortex (ACC). The GluN2A and GluN2B subunits of NMDA receptors, the GluR1 type of AMPA receptor, and CaMKII-α are key molecules responsible for neuroplasticity involved in chronic pain and the accompanying anxiety. Moreover, GAS administration reduced the activation of astrocyte and microglia and the induction of TNF-α and IL-6 in the ACC of the CFA-injected mice. Therefore, GAS administration relieved chronic pain, exerted anxiolytic effects by regulating neuroplasticity molecules, and attenuated the inflammatory response by reducing the induction of TNF-α and IL-6 in the ACC of the CFA-injected mice.

Effects of subchronic perfluorooctane sulfonate exposure of rats on calcium-dependent signaling molecules in the brain tissue

Perfluorooctane sulfonate (PFOS) is a persistent and bio-accumulative pollutant ubiquitous in wildlife and humans, which receives many concerns on the fate, transport, distribution, and toxicity. Studies have shown that PFOS-induced neurotoxicity in experimental animals; however, little is known about the potential mechanism of PFOS exposure on the central nervous system (CNS). Ca(2+)/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha), cAMP-response element binding protein (CREB), c-fos, and c-jun, which are important down-stream molecules of calcium signaling in describing neuron cells structure and function in the CNS, were examined in the paper with the purpose to evaluate the effect of PFOS exposure on brain and approach the molecular mechanisms involved in the neurotoxicity induced by PFOS. Adult male Sprague-Dawley rats were administered with PFOS at dosages of 1.7, 5.0, and 15.0 mg/L in drinking water for 91 consecutive days. LC/MS was used for PFOS analysis in brain tissues, and western blot was employed to determine the expression of CaMKIIalpha and pCREB in the isolated cortex and hippocampus. The expression of c-fos and c-jun was detected by real-time reverse transcription polymerase chain reaction. The results showed that the expression of CaMKIIalpha and pCREB exhibits a significant increase in cortex and hippocampus after treatment with PFOS, compared with the control. The transcription factor c-fos was up-regulated in hippocampus, and c-jun was elevated both in cortex and hippocampus in PFOS-treated groups. These results indicated that, at least in part, the neurotoxic effect induced by PFOS is mediated by the Ca(2+)-dependent molecules in calcium signaling.

Positive allosteric activation of GABAA receptors bi-directionally modulates hippocampal glutamate plasticity and behaviour

Long-term BZ (benzodiazepine) anxiolytic therapy increases the risk of physical dependence manifested as withdrawal anxiety. BZ-induced potentiation of GABA(A)R (gamma-aminobutyric acid type-A receptor) function by 1-week oral administration of FZP (flurazepam) bi-directionally modulates excitatory glutamatergic synaptic transmission in hippocampal CA1 neurons during drug withdrawal. Previous electrophysiological studies on acutely isolated and intact CA1 neurons, as well as immunofluorescence and post-embedding immunogold electron microscopy studies, suggest increased synaptic insertion of GluR (glutamate receptor) 2-lacking AMPARs (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors) in 2-day FZP-withdrawn rats. Preliminary studies indicated a similar increase in GluR1, then phospho-Ser(831)-GluR1, as well as CaMKIIalpha (Ca(2+)/calmodulin-dependent protein kinase IIalpha), but not phospho-Thr(286)-CaMKII levels at the same time point. In our studies, whole-cell recordings in hippocampal slices revealed that AMPAR mEPSC [miniature EPSC (excitatory postsynaptic current)] amplitude was increased in 1-day FZP-withdrawn rats followed by an increase in estimated single-channel conductance in 2-day-FZP-withdrawn rats. Enhanced conductance was no longer observed in slices pre-incubated for 2 h in the CaMKII inhibitor KN-93, but not the inactive analogue KN-92. To evaluate whether CaMKII-mediated AMPA potentiation could occlude LTP (long-term potentiation), LTP was induced by TBS (theta burst stimulation) and recorded using whole-cell and extracellular techniques. LTP was induced in both groups, but only maintained for <15 min in 2-day FZP-withdrawn rats. LTP was fully restored after 7-day withdrawal. Despite the lack of LTP maintenance, impairment of object recognition, place and context was not observed in 2-day-FZP-withdrawn rats. Since L-VGCC (L-type voltage-gated calcium channel) current density was doubled on drug withdrawal and up to 2 days, Ca(2+) entry through L-VGCCs and perhaps subsequently through Ca(2+)-permeable AMPARs are proposed to be responsible for enhanced CaMKIIalpha levels and AMPAR potentiation. Mechanisms associated with several different models of activity-dependent plasticity may underlie BZ physical dependence.

Cold‐induced exodus of postsynaptic proteins from dendritic spines

Dendritic spines are small protrusions on neuronal dendrites and the major target of the excitatory inputs in mammalian brains. Cultured neurons and brain slices are important tools in studying the biochemical and cellular properties of dendritic spines. During the processes of immunocytochemical studies of neurons and the preparation of brain slices, neurons were often kept at temperatures lower than 37 degrees C for varied lengths of time. This study sought to investigate whether and how cold treatment would affect the protein composition of dendritic spines. The results indicated that upon cold treatment four postsynaptic proteins, namely, alpha,beta-tubulins, calcium, calmodulin-dependent protein kinase IIalpha, and cytoplasmic dynein heavy chain and microtubule-associated protein 2, but not PSD-95 or AMPA receptors, exited from the majority of dendritic spines of cultured rat hippocampal neurons in a Gd(3+)-sensitive manner. The cold-induced exit of tubulins from dendritic spines was further found to be an energy-dependent process involving the activation of Gd(3+)-sensitive calcium channels and ryanodine receptors. The results thus indicate that changes in temperature, calcium concentration, and energy supply of the medium surrounding neurons would affect the protein composition of the dendritic spines and conceivably the protein composition of the subcellular organizations, such as the postsynaptic density, in the cytoplasm of dendritic spines.

The fine-structural distribution of G-protein receptor kinase 3, β-arrestin-2, Ca2+/calmodulin-dependent protein kinase II and phosphodiesterase PDE1C2, and a Cl−-cotransporter in rodent olfactory epithelia

The sequentially activated molecules of olfactory signal-onset are mostly concentrated in the long, thin distal parts of olfactory epithelial receptor cell cilia. Is this also true for molecules of olfactory signal-termination and -regulation? G-protein receptor kinase 3 (GRK3) supposedly aids in signal desensitization at the level of odor receptors, whereas beta-arrestin-2, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and phosphodiesterase (PDE) PDE1C2 are thought to do so at the level of the adenylyl cyclase, ACIII. The Na+, K(+)-2Cl(-)-cotransporter NKCC1 regulates Cl(-)-channel activity. In an attempt to localize the subcellular sites olfactory signal-termination and -regulation we used four antibodies to GRK3, two to beta-arrestin-2, five to CaMKII (one to both the alpha and beta form, and two each specific to CaMKII alpha and beta), two to PDE1C2, and three to Cl(-)-cotransporters. Only antibodies to Cl(-)-cotransporters labeled cytoplasmic compartments of, especially, supporting cells but also those of receptor cells. For all other antibodies, immunoreactivity was mostly restricted to the olfactory epithelial luminal border, confirming light microscopic studies that had shown that antibodies to GRK3, beta- arrestin-2, CaMKII, and PDE1C2 labeled this region. Labeling did indeed include receptor cell cilia but occurred in microvilli of neighboring supporting cells as well. Apical parts of microvillous cells that are distinct from supporting cells, and also of ciliated respiratory cells, immunoreacted slightly with most antibodies. When peptides were available, antibody preabsorption with an excess of peptide reduced labeling intensities. Though some of the antibodies did label apices and microvilli of vomeronasal (VNO) supporting cells, none immunoreacted with VNO sensory structures.

Calmodulin and Calmodulin-dependent Kinase IIα Regulate Osteoblast Differentiation by Controlling c-fos Expression

Ca(2+)/calmodulin-dependent protein kinase IIalpha (alpha-CaMKII) was once thought to be exclusively expressed in neuronal tissue, but it is becoming increasingly evident that CaMKII is also expressed in various extraneural cells. CaMKII plays a critical role in regulating various signaling pathways leading to modulation of several aspects of cellular functions, including proliferation, differentiation, cytoskeletal structure, and gene expression. The purpose of this study was to examine the expression of CaMKII in osteoblast-like cells (MC4) and to elucidate its role in osteoblast differentiation. We demonstrated that CaMKII, specifically the alpha isoform, is expressed in osteoblasts both in vitro and in vivo. Inhibition of CaMKII by the calmodulin antagonist trifluoperazine or the CaMKII antagonist KN93 reduces alkaline phosphatase activity and mineralization, as well as causes 85 and 56% decreases in alkaline phosphatase and osteocalcin gene expression, respectively. CaM and CaMKII antagonists, using the newborn mouse calvaria in vivo model, cause a 50% decrease in osteoblast number (N.Ob-BS) and a 32% decrease in mineralization (BV/TV). Pharmacologic and genetic inhibition of alpha-CaMKII by using trifluoperazine, KN93, and alpha-CaMKII small interfering RNA decreases the phosphorylation of ERK and of cAMP-response element-binding protein, leading to a significant decrease in the transactivation of serum response element and cAMP-response element. Inhibition of alpha-CaMKII decreases the expression of c-fos, AP-1 transactivation, and AP-1 DNA binding activity. Our findings demonstrated that alpha-CaMKII is expressed in osteoblasts and is involved in c-fos expression via regulation of serum response element and cAMP-response element. Inhibition of alpha-CaMKII results in a decrease in c-fos expression and AP-1 activation, leading to inhibition of osteoblast differentiation.

Stereological analysis of Ca2+/calmodulin‐dependent protein kinase IIα‐containing dorsal root ganglion neurons in the rat: Colocalization with isolectin Griffonia simplicifolia, calcitonin gene‐related peptide, or vanilloid receptor 1

The enzyme Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is widely distributed in the nervous system. A previous report describes immunostaining for CaMKII alpha in dorsal root ganglion (DRG) neurons. In this study, CaMKII alpha is colocalized in the rat with three putative markers of nociceptive DRG neurons, isolectin Griffonia simplicifolia (I-B4), identifying small-diameter, "peptide-poor" neurons; calcitonin gene-related peptide (CGRP), identifying " peptide-rich" neurons; or the vanilloid receptor 1 (VR1), identifying neurons activated by heat, acid, and capsaicin. Lumbar 4 and 5 DRG sections were labeled using immunofluorescence or lectin binding histochemistry, and percentages of single and double-labeled CaMKIIalpha neurons were determined. Stereological estimates of total neuron number in the L4 DRG were 13,815 +/- 2,798 and in the L5 DRG were 14,111 +/- 4,043. Percentages of single-labeled L4 DRG neurons were 41% +/- 2% CaMKII alpha, 38% +/- 3% I-B4, 44% +/- 3% CGRP, and 32% +/- 6% VR1. Percentages of single-labeled L5 DRG neurons were 44% +/- 5% CaMKII alpha, 48% +/- 2% I-B4, 41% +/- 7% CGRP, and 39% +/- 14% VR1. For L4 and L5, respectively, estimates of double-labeled CaMKII alpha neurons showed 34% +/- 2% and 38% +/- 17% labeled for I-B4, 25% +/- 14% and 19% +/- 10% labeled for CGRP, and 37% +/- 7% and 38% +/- 5% labeled for VR1. Conversely, for L4 and L5, respectively, 39% +/- 14% and 38% +/- 7% I-B4 binding neurons, 24% +/- 12% and 23% +/- 10% CGRP neurons, and 42% +/- 7% and 35% +/- 7% VR1 neurons labeled for CaMKIIalpha. The mean diameter of CaMKII alpha - labeled neurons was approximately 27 microm, confirming that this enzyme was preferentially localized in small DRG neurons. The results indicate that subpopulations of DRG neurons containing CaMKII alpha are likely to be involved in the processing of nociceptive information. Thus, this enzyme may play a critical role in the modulation of nociceptor activity and plasticity of primary sensory neurons.

Three-dimensional Reconstructions of Calcium/Calmodulin-dependent (CaM) Kinase IIα and Truncated CaM Kinase IIα Reveal a Unique Organization for Its Structural Core and Functional Domains

Studies of the structural organization of calcium/ calmodulin-dependent protein kinase IIalpha (CaM KIIalpha) and truncated CaM KIIalpha by three-dimensional electron microscopy and protein engineering show that the structures consist of 12 subunits that are organized in two stacked hexameric rings with 622 symmetry. The body of CaM KIIalpha is gear-shaped, consisting of six slanted flanges, and has six foot-like processes attached by narrow appendages to both ends of the flanges. Truncated CaM KIIalpha that lacks functional domains has a structure that is very similar to the body of CaM KIIalpha. Thus, the functional domains reside in the foot-like processes, and the association domain comprises the gear-shaped core. The ribbon diagram of the bilobate structure of CaM KI fits nicely in the envelope of the foot-like component and indicates that the crevice between the two lobes comprising the functional domains is near the middle portion of the foot. The clustering of the functional domains provides a favorable arrangement for the autophosphorylation reaction, and the unusual arrangement of the catalytic domain on extended tethers appears to be significant for the remarkable functional diversity of CaM KIIalpha in cellular regulation.

Comparative study of the distribution of calmodulin kinase II and calcineurin in the mouse brain

The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and the phosphatase calcineurin (CaN) are Ca(2+)/calmodulin-binding proteins that are very abundant in the central nervous system. In the mammalian brain, CaMKII is composed by the association of several similar subunits at different ratios produced by four different genes. CaN is composed of two different subunits produced by two genes. We selected the most abundant subunits of each enzyme in the rodent brain, CaMKII alpha and CaN A, and compared their pattern of expression in the mouse brain by using in situ hybridization histochemistry and immunohistochemistry. We found that CaMKII and CaN were mainly expressed in cerebral cortex, hippocampus, and striatum and that low levels of expression were observed in midbrain and brainstem. CaN was also expressed in cerebellum. In the cell, the kinase and the phosphatase were detected in the perikarya, the neuronal processes, and the nucleus. The present study shows that all the regions of the mouse brain in which CaMKII is expressed also show CaN expression. This fact is consistent with the presence of common substrates for both enzymes or with a regulatory action of one versus the other. The lack of correspondence in the cerebellum could be explained by the fact that the major subunit of the kinase in this brain region is CaMKII beta.

In vivo and in vitro characterization of the sequence requirement for oligomer formation of Ca2+/calmodulin-dependent protein kinase IIalpha.

Ca2+/calmodulin-dependent protein kinase II (CaMK II) is a multifunctional serine/threonine protein kinase that regulates ion channels, metabolic enzymes, cytoskeletal proteins, and possibly transcription factors. CaMK II holoenzymes have been shown to be large oligomers built from eight or more subunits. The oligomeric structure of CaMK II is required for a critical trans-autophosphorylation step by which each subunit traps bound calmodulin and renders the enzyme partially active in the absence of Ca2+/calmodulin. Here we define a minimal C-terminal domain of alphaCaMK II that is necessary and sufficient for oligomerization. The oligomeric structure of alphaCaMK II deletion mutants was investigated in vitro by gel filtration chromatography and in living cells by measuring nuclear exclusion and diffusion coefficients of green fluorescent protein-tagged mutants. These studies showed that a C-terminal region of alphaCaMK II of 135 amino acids is sufficient for oligomer formation. Oligomer formation was abolished by further C-terminal and N-terminal deletions, indicating that the same region is not only sufficient but also necessary for oligomerization. Thus, an oligomerization domain of only 15 kDa is sufficient to build the circular structure of CaMK II holoenzyme.

CDNA cloning and sequencing of Ca2+/calmodulin-dependent protein kinase IIalpha subunit and its mRNA expression in diisopropyl phosphorofluoridate (DFP)-treated hen central nervous system.

Diisopropyl phosphorofluoridate (DFP) produces delayed neurotoxicity, known as organophosphorus ester-induced delayed neurotoxicity (OPIDN), in hen, human, and other sensitive species. A single dose of DFP (1.7 mg/kg, se.) produces first mild ataxia followed by paralysis in 7-14 days in hens. DFP treatment also increases in vitro autophosphorylation of Ca2+ calmodulin-dependent protein kinase II (CaM kinase II) and the phosphorylation of several cytoskeletal proteins in the hen brain. To investigate whether increase in CaM kinase II activity is associated with increased expression of its mRNA, we cloned and sequenced CaM kinase II alpha subunit cDNA, and used it to study CaM kinase II expression in brain regions and spinal cord. Hen CaM kinase II alpha subunit differs in 7 amino acids from that of rat CaM kinase II. Its mRNA occurs predominantly as a 6.7 kb message, which is very close to that of human CaM kinase II alpha subunit. Northern blot analysis showed a transient increase in CaM kinase II alpha subunit mRNA in the cerebellum and spinal cord of DFP-treated chickens. The increase in CaM kinase II mRNA expression is consistent with the previously reported increase in its activity in brain and spinal cord, and its increased expression only in cerebellum and spinal cord, which are sensitive to the Wallerian-type degeneration characteristic of OPIDN, suggests the probable role of this enzyme in delayed neurotoxicity.

Neuronal nitric oxide synthase and calmodulin-dependent protein kinase IIalpha undergo neurotoxin-induced proteolysis.

Calpain (calcium-activated neutral protease) has been implicated as playing a role of neuronal injury in cerebral ischemia and excitotoxicity. Here we report that, in addition to extreme excitotoxic conditions [N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate challenges], other neurotoxins such as maitotoxin, A23187, and okadaic acid also induce calpain activation, as detected by m-calpain autolytic fragmentation and nonerythroid alpha-spectrin breakdown. Under the same conditions, calmodulin-dependent protein kinase II-alpha (CaMPK-IIalpha) and neuronal nitric oxide synthase (nNOS) are both proteolytically cleaved by calpain. Such fragmentation can be reduced by calpain inhibitors (acetyl-Leu-Leu-Nle-CHO and PD151746). In vitro digestion of protein extract from cortical cultures with purified mu- and m-calpain produced fragmentation patterns for CaMPK-IIalpha and nNOS similar to those produced in situ. Also, several other calpain-sensitive calmodulin-binding proteins (plasma membrane calcium pump, microtubule-associated protein 2, and calcineurin A) and protein kinase C-alpha are also degraded in neurotoxin-treated cultures. Lastly, in a rat pup model of acute excitotoxicity, intrastriatal injection of NMDA resulted in breakdown of CaMPK-IIalpha and nNOS. The degradation of CaMPK-IIalpha, nNOS, and other endogenous calpain substrates may contribute to the neuronal injury associated with various neurotoxins.

Perinuclear membrane localization of alphaKAP, a protein produced from a gene within the gene of calmodulin-dependent protein kinase IIalpha.

AlphaKAP is a protein produced from a gene within the gene of the a isoform of calmodulin-dependent protein kinase II (CaM-kinase IIa). It consists of the association domain of CaM-kinase IIa and a highly hydrophobic amino-terminal stretch consisting of 25 amino acids which is absent from CaM-kinase IIalpha. We previously demonstrated that alphaKAP is an integral membrane protein by subcellular fractionation analysis [Sugai, R., Takeuchi, M., Okuno, S., and Fujisawa, H. (1996) J. Biochem. 120, 773-779], but the exact subcellular localization of alphaKAP was not well understood. Here we demonstrate that alphaKAP is localized on the nuclear membrane of COS-7 cells transiently expressing alphaKAP. The nuclear membrane and perinuclear small vesicles were immunostained with an antibody against a synthetic peptide corresponding to the carboxyl-terminal 15 amino acids of alphaKAP. In contrast to the intact alphaKAP, the mutant alphaKAP, from which the hydrophobic amino-terminal segment had been deleted, accumulated within nuclei. Thus, alphaKAP may function as an anchoring protein for CaM-kinase II and/or other proteins in the perinuclear membrane.

Computation of the binding of fully flexible peptides to proteins with flexible side chains.

Docking algorithms play an important role in the process of rational drug design and in understanding the mechanism of molecular recognition. An important determinant for successful docking is the extent to which the configurational space (including conformational changes) of the ligand/receptor system is searched. Here we describe a new, combinatorial method for flexible docking of peptides to proteins that allows full rotation around all single bonds of the peptide ligand and around those of a large set of receptor side chains. We have simulated the binding of several viral peptides to murine major histocompatibility complex class I H-2Kb. In addition, we have explored the limits of our method by simulating a complex between calmodulin and an 18-residue long helical peptide from calmodulin-dependent protein kinase IIalpha. The calculated peptide conformations generally matched well with the X-ray structures. Essential information about local flexibility and about residues that are responsible for strong binding was obtained. We have frequently observed considerable side-chain flexibility during the simulations, showing the need for a flexible treatment of the receptor. Our method may also be useful whenever the receptor side-chain conformation is not available or uncertain, as illustrated by the docking of an H-2Kb binding nonapeptide to the receptor structure taken from an octapeptide/H-2Kb complex.

Interaction of Autophosphorylated Ca2+/Calmodulin-dependent Protein Kinase II with Neuronal Cytoskeletal Proteins: CHARACTERIZATION OF BINDING TO A 190-kDa POSTSYNAPTIC DENSITY PROTEIN

Subcellular localization of Ca2+/calmodulin-dependent protein kinase II (CaMKII) by interaction with specific anchoring proteins may be an important mechanism contributing to the regulation of CaMKII. Proteins capable of binding CaMKII were identified by the use of a gel overlay assay with recombinant mouse CaMKII alpha (mCaMKII alpha) or Xenopus CaMKII beta (xCaMKII beta) 32P-autophosphorylated at Thr286/287 as a probe. Numerous [32P]CaMKII-binding proteins were identified in various whole rat tissue extracts, but binding was most prominent to forebrain proteins of 190 kDa (p190) and 140 kDa (p140). Fractionation of forebrain extracts localized p190 and p140 to a crude particulate/cytoskeletal fraction and isolated postsynaptic densities. [32P]m-CaMKII alpha-bound to p190 with an apparent Kd of 609 nM (subunit concentration) and a Bmax of 7.0 pmol of mCaMKII alpha subunit bound per mg of P2 protein, as measured using the overlay assay. Binding of 100 nM [32P]m-CaMKII alpha to p190 was competed by nonradioactive mCaMKII alpha autophosphorylated on Thr286 (EC50% = 200 nM), but to a much lesser extent by nonradioactive mCaMKII alpha autophosphorylated on Thr306 (EC50% > 2000 nM). In addition, nonphosphorylated mCaMKII alpha was a poor competitor for [32P]mCaMKII alpha binding to p190. The competition data indicate that Ca2+/CaM-dependent autophosphorylation at Thr286 promotes binding to p190, whereas, Ca2+/CaM-independent autophosphorylation at Thr306 does not enhance binding. Therefore, CaMKII may become localized to postsynaptic densities by p190 following its activation by an increase of dendritic Ca2+ concentration.

Rapid translocation of cytosolic Ca2+/calmodulin-dependent protein kinase II into postsynaptic density after decapitation.

The postsynaptic density (PSD) fraction prepared from rat forebrains frozen with liquid nitrogen immediately after dissection (within 30 s after decapitation) contained major postsynaptic density protein (mPSDp), alpha subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKII) at a level of merely 2.7% of the total protein. The content of the protein in the fraction was increased to approximately 10% by placing the forebrains on ice for a few minutes. Accumulation, but to a lesser extent, of the protein after placement was also observed in the particulate, synaptosome, and synaptic plasma membrane fractions with its concomitant decrease in the cytosolic fraction. The distribution change may be translocation of the protein, because the amounts of the losses of the protein in the cytosolic fraction were balanced by the gains in the particulate fractions. By translocation, CaMKII became Triton X-100 insoluble and partially inactivated. The amount of CaMKII transferred from the cytosol to particulate fractions at 0 degrees C was about the same as that contained in the conventional PSD fraction. Furthermore, the thickness of the PSD was increased by the treatment of the forebrains at 37 degrees C, by which the content of CaMKII alpha in the PSD fraction was increased to twofold. These results suggest that most of the CaMKII alpha subunit associated with the PSD fraction (mPSDp) is translocated from cytosol after decapitation. We also showed similar translocation of CaMKII beta/beta'.