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ABSTRACT: How information encoded in glutamate release rates at individual synapses is converted into biochemical activation patterns of postsynaptic enzymes remains unexplored. To address this, we developed a dual fluorescence resonance energy transfer (FRET) imaging platform and recorded CaMKIIα and calcineurin activities in hippocampal neurons while varying glutamate uncaging frequencies. With little spine morphological change, 5 Hz spine glutamate uncaging strongly stimulated calcineurin, but not CaMKIIα. In contrast, 20 Hz spine glutamate uncaging, which induced spine growth, activated both CaMKIIα and calcineurin with distinct spatiotemporal kinetics. Higher temporal resolution recording in the soma revealed that CaMKIIα activity summed supralinearly and sensed both higher frequency and input number, thus acting as an input frequency/number decoder. In contrast, calcineurin activity summated sublinearly with increasing input number and showed little frequency dependence, thus functioning as an input number counter. These results provide evidence that CaMKIIα and calcineurin are fine-tuned to unique bandwidths and compute input variables in an asymmetric manner.
Cell reports. 04/2013;
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Hiroyuki Okuno,
Kaori Akashi,
Yuichiro Ishii,
Nan Yagishita-Kyo,
Kanzo Suzuki,
Mio Nonaka,
Takashi Kawashima,
Hajime Fujii,
Sayaka Takemoto-Kimura,
Manabu Abe,
Rie Natsume,
Shoaib Chowdhury,
Kenji Sakimura,
Paul F Worley, Haruhiko Bito
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ABSTRACT: The Arc/Arg3.1 gene product is rapidly upregulated by strong synaptic activity and critically contributes to weakening synapses by promoting AMPA-R endocytosis. However, how activity-induced Arc is redistributed and determines the synapses to be weakened remains unclear. Here, we show targeting of Arc to inactive synapses via a high-affinity interaction with CaMKIIβ that is not bound to calmodulin. Synaptic Arc accumulates in inactive synapses that previously experienced strong activation and correlates with removal of surface GluA1 from individual synapses. A lack of CaMKIIβ either in vitro or in vivo resulted in loss of Arc upregulation in the silenced synapses. The discovery of Arc's role in "inverse" synaptic tagging that is specific for weaker synapses and prevents undesired enhancement of weak synapses in potentiated neurons reconciles essential roles of Arc both for the late phase of long-term plasticity and for reduction of surface AMPA-Rs in stimulated neurons.
Cell 05/2012; 149(4):886-98. · 32.40 Impact Factor
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ABSTRACT: An increasing prevalence of mental health problems has been partly ascribed to abnormal brain development that is induced upon exposure to environmental chemicals. However, it has been extremely difficult to detect and assess such causality particularly at low exposure levels. To address this question, we here investigated higher brain function in mice exposed to dioxin in utero and via lactation by using our recently developed automated behavioral flexibility test and immunohistochemistry of neuronal activation markers Arc, at the 14 brain areas. Pregnant C57BL/6 mice were given orally a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at a dose of either 0, 0.6 or 3.0 µg/kg on gestation day 12.5. When the pups reached adulthood, they were group-housed in IntelliCage to assess their behavior. As a result, the offspring born to dams exposed to 0.6 µg TCDD/kg were shown to have behavioral inflexibility, compulsive repetitive behavior, and dramatically lowered competitive dominance. In these mice, immunohistochemistry of Arc exhibited the signs of hypoactivation of the medial prefrontal cortex (mPFC) and hyperactivation of the amygdala. Intriguingly, mice exposed to 3.0 µg/kg were hardly affected in both the behavioral and neuronal activation indices, indicating that the robust, non-monotonic dose-response relationship. In conclusion, this study showed for the first time that perinatal exposure to a low dose of TCDD in mice develops executive function deficits and social behavioral abnormality accompanied with the signs of imbalanced mPFC-amygdala activation.
PLoS ONE 01/2012; 7(12):e50741. · 4.09 Impact Factor
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ABSTRACT: When new learning occurs against the background of established prior knowledge, relevant new information can be assimilated into a schema and thereby expand the knowledge base. An animal model of this important component of memory consolidation reveals that systems memory consolidation can be very fast. In experiments with rats, we found that the hippocampal-dependent learning of new paired associates is associated with a striking up-regulation of immediate early genes in the prelimbic region of the medial prefrontal cortex, and that pharmacological interventions targeted at that area can prevent both new learning and the recall of remotely and even recently consolidated information. These findings challenge the concept of distinct fast (hippocampal) and slow (cortical) learning systems, and shed new light on the neural mechanisms of memory assimilation into schemas.
Science 08/2011; 333(6044):891-5. · 31.20 Impact Factor
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ABSTRACT: We present a fluorescence activation-coupled protein labeling (FAPL) method, which employs small-molecular probes that exhibit almost no basal fluorescence but acquire strong fluorescence upon covalent binding to tag-proteins. This method enables real-time imaging of protein labeling without any washout process and is uniquely suitable for real-time imaging of protein dynamics on the cell surface. We applied this method to address the spatiotemporal dynamics of the EGF receptor during cell migration.
Journal of the American Chemical Society 05/2011; 133(17):6745-51. · 9.91 Impact Factor
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ABSTRACT: Most of the currently available drugs for osteoporosis inhibit osteoclastic bone resorption; only a few drugs promote osteoblastic bone formation. It is thus becoming increasingly necessary to identify the factors that regulate bone formation. We found that osteoclasts express semaphorin 4D (Sema4D), previously shown to be an axon guidance molecule, which potently inhibits bone formation. The binding of Sema4D to its receptor Plexin-B1 on osteoblasts resulted in the activation of the small GTPase RhoA, which inhibits bone formation by suppressing insulin-like growth factor-1 (IGF-1) signaling and by modulating osteoblast motility. Sema4d-/- mice, Plxnb1-/- mice and mice expressing a dominant-negative RhoA specifically in osteoblasts showed an osteosclerotic phenotype due to augmented bone formation. Notably, Sema4D-specific antibody treatment markedly prevented bone loss in a model of postmenopausal osteoporosis. Thus, Sema4D has emerged as a new therapeutic target for the discovery and development of bone-increasing drugs.
Nature medicine 01/2011; 17(11):1473-80. · 27.14 Impact Factor
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ABSTRACT: Formation of a new memory requires plasticity at the synaptic level. However, it has also been shown that the consolidation and the maintenance of such a new memory involve processes that necessitate active mRNA at the nucleus of the cell. How can robust changes in synaptic efficacy specifically drive new transcription and translation of new gene transcripts, and thus transform an otherwise transient plasticity into a long-lasting and stable one? In this article, we highlight the conceptual advance that was gained by the discovery of a potent Synaptic Activity-Responsive Element (SARE) found ∼7 kb upstream of the transcription initiation site of the neuronal immediate early gene Arc. The unique genomic structure of SARE, which contained adjacent and cooperative binding sites for three major activity-dependent transcription factors within a 100-bp locus, was associated with an unusual responsiveness to neuronal stimuli. Taken together, these findings shed light on a new class of transcriptional sensor with enhanced sensitivity to synaptic activity.
Communicative & integrative biology 09/2010; 3(5):443-6.
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Haruhiko Bito
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ABSTRACT: Excitatory synapses are located in confined chemical spaces called the dendritic spines. These are atypical femtoliter-order microdomains where the behavior of even single molecules may have important biological consequences. Powerful chemical biological techniques have now been developed to decipher the dynamic stability of the synapses and to further interrogate the complex properties of neuronal circuits.
Nature Chemical Biology 08/2010; 6(8):560-3. · 14.69 Impact Factor
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ABSTRACT: Ca(2+) -regulated reorganization of actin cytoskeleton is one of the key cell biological events that critically regulate neuronal morphogenesis during circuit formation, spinogenesis during synapse development, and activity-dependent structural plasticity at mature synapses. However, it remains unclear as to what extent the underlying Ca(2+) signaling processes are shared or segregated. Here, we present evidence from the literature that collectively begins to suggest that distinct calmodulin-dependent protein kinase (CaMK) isoforms are differentially expressed in time and in subcellular space, and thus may be selectively activated and engaged by distinct upstream stimuli; each CaMK isoform, in turn, couples to related, but separate, cytoskeletal and transcriptional regulatory pathways, dependent on its abundance or physical proximity with either the upstream or downstream signaling complexes. These signal transduction characteristics provide the basis for better understanding the role of excitation-morphogenesis coupling via multiple CaMKs during neuronal circuit and synapse formation.
European Journal of Neuroscience 07/2010; 32(2):224-30. · 3.63 Impact Factor
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ABSTRACT: Weakly tetanized synapses in area CA1 of the hippocampus that ordinarily display long-term potentiation lasting approximately 3 h (called early-LTP) will maintain a longer-lasting change in efficacy (late-LTP) if the weak tetanization occurs shortly before or after strong tetanization of an independent, but convergent, set of synapses in CA1. The synaptic tagging and capture hypothesis explains this heterosynaptic influence on persistence in terms of a distinction between local mechanisms of synaptic tagging and cell-wide mechanisms responsible for the synthesis, distribution, and capture of plasticity-related proteins (PRPs). We now present evidence that distinct CaM kinase (CaMK) pathways serve a dissociable role in these mechanisms. Using a hippocampal brain-slice preparation that permits stable long-term recordings in vitro for >10 h and using hippocampal cultures to validate the differential drug effects on distinct CaMK pathways, we show that tag setting is blocked by the CaMK inhibitor KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) that, at low concentration, is more selective for CaMKII. In contrast, the CaMK kinase inhibitor STO-609 [7H-benzimidazo(2,1-a)benz(de)isoquinoline-7-one-3-carboxylic acid] specifically limits the synthesis and/or availability of PRPs. Analytically powerful three-pathway protocols using sequential strong and weak tetanization in varying orders and test stimulation over long periods of time after LTP induction enable a pharmacological dissociation of these distinct roles of the CaMK pathways in late-LTP and so provide a novel framework for the molecular mechanisms by which synaptic potentiation, and possibly memories, become stabilized.
Journal of Neuroscience 04/2010; 30(14):4981-9. · 7.11 Impact Factor
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Natsumi Ageta-Ishihara,
Sayaka Takemoto-Kimura,
Mio Nonaka,
Aki Adachi-Morishima,
Kanzo Suzuki,
Satoshi Kamijo,
Hajime Fujii,
Tatsuo Mano,
Frank Blaeser,
Talal A Chatila,
Hidenobu Mizuno,
Tomoo Hirano,
Yoshiaki Tagawa,
Hiroyuki Okuno, Haruhiko Bito
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ABSTRACT: Ca(2+) signaling plays important roles during both axonal and dendritic growth. Yet whether and how Ca(2+) rises may trigger and contribute to the development of long-range cortical connections remains mostly unknown. Here, we demonstrate that two separate limbs of the Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK)-CaMKI cascades, CaMKK-CaMKIalpha and CaMKK-CaMKIgamma, critically coordinate axonal and dendritic morphogenesis of cortical neurons, respectively. The axon-specific morphological phenotype required a diffuse cytoplasmic localization and a strikingly alpha-isoform-specific kinase activity of CaMKI. Unexpectedly, treatment with muscimol, a GABA(A) receptor agonist, selectively stimulated elongation of axons but not of dendrites, and the CaMKK-CaMKIalpha cascade critically mediated this axonogenic effect. Consistent with these findings, during early brain development, in vivo knockdown of CaMKIalpha significantly impaired the terminal axonal extension and thereby perturbed the refinement of the interhemispheric callosal projections into the contralateral cortices. Our findings thus indicate a novel role for the GABA-driven CaMKK-CaMKIalpha cascade as a mechanism critical for accurate cortical axon pathfinding, an essential process that may contribute to fine-tuning the formation of interhemispheric connectivity during the perinatal development of the CNS.
Journal of Neuroscience 10/2009; 29(43):13720-9. · 7.11 Impact Factor
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ABSTRACT: The neuronal immediate early gene Arc/Arg-3.1 is widely used as one of the most reliable molecular markers for intense synaptic activity in vivo. However, the cis-acting elements responsible for such stringent activity dependence have not been firmly identified. Here we combined luciferase reporter assays in cultured cortical neurons and comparative genome mapping to identify the critical synaptic activity-responsive elements (SARE) of the Arc/Arg-3.1 gene. A major SARE was found as a unique approximately 100-bp element located at >5 kb upstream of the Arc/Arg-3.1 transcription initiation site in the mouse genome. This single element, when positioned immediately upstream of a minimal promoter, was necessary and sufficient to replicate crucial properties of endogenous Arc/Arg-3.1's transcriptional regulation, including rapid onset of transcription triggered by synaptic activity and low basal expression during synaptic inactivity. We identified the major determinants of SARE as a unique cluster of neuronal activity-dependent cis-regulatory elements consisting of closely localized binding sites for CREB, MEF2, and SRF. Consistently, a SARE reporter could readily trace and mark an ensemble of cells that have experienced intense activity in the recent past in vivo. Taken together, our work uncovers a novel transcriptional mechanism by which a critical 100-bp element, SARE, mediates a predominant component of the synapse-to-nucleus signaling in ensembles of Arc/Arg-3.1-positive activated neurons.
Proceedings of the National Academy of Sciences 01/2009; 106(1):316-21. · 9.68 Impact Factor
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Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 03/2008; 53(4 Suppl):418-23.
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Sayaka Takemoto-Kimura,
Natsumi Ageta-Ishihara,
Mio Nonaka,
Aki Adachi-Morishima,
Tatsuo Mano,
Michiko Okamura,
Hajime Fujii,
Toshimitsu Fuse,
Mikio Hoshino,
Shingo Suzuki,
Masami Kojima,
Masayoshi Mishina,
Hiroyuki Okuno, Haruhiko Bito
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ABSTRACT: Ca(2+) signaling plays a central role in activity-dependent regulation of dendritic arborization, but key molecular mechanisms downstream of calcium elevation remain poorly understood. Here we show that the C-terminal region of the Ca(2+)/calmodulin-dependent protein kinase CLICK-III (CL3)/CaMKIgamma, a membrane-anchored CaMK, was uniquely modified by two sequential lipidification steps: prenylation followed by a kinase-activity-regulated palmitoylation. These modifications were essential for CL3 membrane anchoring and targeting into detergent-resistant lipid microdomains (or rafts) in the dendrites. We found that CL3 critically contributed to BDNF-stimulated dendritic growth. Raft insertion of CL3 specifically promoted dendritogenesis of cortical neurons by acting upstream of RacGEF STEF and Rac, both present in lipid rafts. Thus, CL3 may represent a key element in the Ca(2+)-dependent and lipid-raft-delineated switch that turns on extrinsic activity-regulated dendrite formation in developing cortical neurons.
Neuron 07/2007; 54(5):755-70. · 14.74 Impact Factor
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Shigeki Kiyonaka,
Minoru Wakamori,
Takafumi Miki,
Yoshitsugu Uriu,
Mio Nonaka, Haruhiko Bito,
Aaron M Beedle,
Emiko Mori,
Yuji Hara,
Michel De Waard,
Motoi Kanagawa,
Makoto Itakura,
Masami Takahashi,
Kevin P Campbell,
Yasuo Mori
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ABSTRACT: The molecular organization of presynaptic active zones is important for the neurotransmitter release that is triggered by depolarization-induced Ca2+ influx. Here, we demonstrate a previously unknown interaction between two components of the presynaptic active zone, RIM1 and voltage-dependent Ca2+ channels (VDCCs), that controls neurotransmitter release in mammalian neurons. RIM1 associated with VDCC beta-subunits via its C terminus to markedly suppress voltage-dependent inactivation among different neuronal VDCCs. Consistently, in pheochromocytoma neuroendocrine PC12 cells, acetylcholine release was significantly potentiated by the full-length and C-terminal RIM1 constructs, but membrane docking of vesicles was enhanced only by the full-length RIM1. The beta construct beta-AID dominant negative, which disrupts the RIM1-beta association, accelerated the inactivation of native VDCC currents, suppressed vesicle docking and acetylcholine release in PC12 cells, and inhibited glutamate release in cultured cerebellar neurons. Thus, RIM1 association with beta in the presynaptic active zone supports release via two distinct mechanisms: sustaining Ca2+ influx through inhibition of channel inactivation, and anchoring neurotransmitter-containing vesicles in the vicinity of VDCCs.
Nature Neuroscience 07/2007; 10(6):691-701. · 15.53 Impact Factor
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Haruhiko Bito
Seikagaku. The Journal of Japanese Biochemical Society 02/2007; 79(1):5-15. · 0.04 Impact Factor
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Kojiro Sato,
Ayako Suematsu,
Tomoki Nakashima,
Sayaka Takemoto-Kimura,
Kazuhiro Aoki,
Yasuyuki Morishita,
Hiroshi Asahara,
Keiichi Ohya,
Akira Yamaguchi,
Toshiyuki Takai,
Tatsuhiko Kodama,
Talal A Chatila, Haruhiko Bito,
Hiroshi Takayanagi
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ABSTRACT: Calcium (Ca(2+)) signaling is essential for a variety of cellular responses and higher biological functions. Ca(2+)/calmodulin-dependent kinases (CaMKs) and the phosphatase calcineurin activate distinct downstream pathways that are mediated by the transcription factors cAMP response element (CRE)-binding protein (CREB) and nuclear factor of activated T cells (NFAT), respectively. The importance of the calcineurin-NFAT pathway in bone metabolism has been demonstrated in osteoclasts, osteoblasts and chondrocytes. However, the contribution of the CaMK-CREB pathway is poorly understood, partly because of the difficulty of dissecting the functions of homologous family members. Here we show that the CaMKIV-CREB pathway is crucial for osteoclast differentiation and function. Pharmacological inhibition of CaMKs as well as the genetic ablation of Camk4 reduced CREB phosphorylation and downregulated the expression of c-Fos, which is required for the induction of NFATc1 (the master transcription factor for osteoclastogenesis) that is activated by receptor activator of NF-kappaB ligand (RANKL). Furthermore, CREB together with NFATc1 induced the expression of specific genes expressed by differentiated osteoclasts. Thus, the CaMK-CREB pathway biphasically functions to regulate the transcriptional program of osteoclastic bone resorption, by not only enhancing induction of NFATc1 but also facilitating NFATc1-dependent gene regulation once its expression is induced. This provides a molecular basis for a new therapeutic strategy for bone diseases.
Nature Medicine 01/2007; 12(12):1410-6. · 22.46 Impact Factor
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Norikazu Yamana,
Yoshiki Arakawa,
Tomohiro Nishino,
Kazuo Kurokawa,
Masahiro Tanji,
Reina E Itoh,
James Monypenny,
Toshimasa Ishizaki, Haruhiko Bito,
Kazuhiko Nozaki,
Nobuo Hashimoto,
Michiyuki Matsuda,
Shuh Narumiya
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ABSTRACT: Directed cell migration requires cell polarization and adhesion turnover, in which the actin cytoskeleton and microtubules work critically. The Rho GTPases induce specific types of actin cytoskeleton and regulate microtubule dynamics. In migrating cells, Cdc42 regulates cell polarity and Rac works in membrane protrusion. However, the role of Rho in migration is little known. Rho acts on two major effectors, ROCK and mDia1, among which mDia1 produces straight actin filaments and aligns microtubules. Here we depleted mDia1 by RNA interference and found that mDia1 depletion impaired directed migration of rat C6 glioma cells by inhibiting both cell polarization and adhesion turnover. Apc and active Cdc42, which work together for cell polarization, localized in the front of migrating cells, while active c-Src, which regulates adhesion turnover, localized in focal adhesions. mDia1 depletion impaired localization of these molecules at their respective sites. Conversely, expression of active mDia1 facilitated microtubule-dependent accumulation of Apc and active Cdc42 in the polar ends of the cells and actin-dependent recruitment of c-Src in adhesions. Thus, the Rho-mDia1 pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and delivering Apc/Cdc42 and c-Src to their respective sites of action.
Molecular and Cellular Biology 10/2006; 26(18):6844-58. · 5.53 Impact Factor
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ABSTRACT: Organization and dynamic remodeling of postsynaptic density (PSD) are thought to be critical in postsynaptic signal transduction, but the underlying molecular mechanisms are not well understood. We show here that four major scaffolding molecules, PSD-95, GKAP, Shank, and PSD-Zip45, show distinct instability in total molecular content per synapse. Fluorescence recovery after photobleaching also confirmed their distinct turnover rates. Among the PSD molecules examined, PSD-95 was most stable, but its elimination did not influence the dynamics of its direct binding partner GKAP. Multiple interactions of scaffolding molecules with the actin cytoskeleton have suggested their importance in both maintenance and remodeling of the PSD. Indeed, acute pharmacological disruption of F-actin rapidly eliminated the dynamic fraction of GKAP, Shank, and PSD-Zip45, without changing synaptic localization of PSD-95. GKAP content in synapses increased after pharmacological enhancement of neuronal activity, whereas Shank and PSD-Zip45 content showed reduction. Inhibition of F-actin dynamics prevented activity-dependent redistribution of all three scaffolds. We also assessed involvement of glutamate receptors in the regulation of PSD dynamics. Genetic manipulations eliminating either NMDA receptors or metabotropic glutamate receptors did not primarily influence mobility of their binding scaffolds. These results collectively indicate a critical role of filamentous actin in determining the extent of dynamic reorganization in PSD molecular composition.
Journal of Neuroscience 08/2006; 26(29):7693-706. · 7.11 Impact Factor
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Shogo Ohmae,
Sayaka Takemoto-Kimura,
Michiko Okamura,
Aki Adachi-Morishima,
Mio Nonaka,
Toshimitsu Fuse,
Satoshi Kida,
Masahiro Tanji,
Tomoyuki Furuyashiki,
Yoshiki Arakawa,
Shuh Narumiya,
Hiroyuki Okuno, Haruhiko Bito
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ABSTRACT: Despite the critical importance of Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) II signaling in neuroplasticity, only a limited amount of work has so far been available regarding the presence and significance of another predominant CaMK subfamily, the CaMKI/CaMKIV family, in the central nervous system. We here searched for kinases with a core catalytic structure similar to CaMKI and CaMKIV. We isolated full-length cDNAs encoding three mouse CaMKI/CaMKIV-related kinases, CLICK-I (CL1)/doublecortin and CaM kinase-Like (DCAMKL)1, CLICK-II (CL2)/DCAMKL2, and CLICK-I,II-related (CLr)/DCAMKL3, the kinase domains of which had an intermediate homology not only to CaMKI/CaMKIV but also to CaMKII. Furthermore, CL1, CL2, and CLr were highly expressed in the central nervous system, in a neuron-specific fashion. CL1alpha and CL1beta were shorter isoforms of DCAMKL1, which lacked the doublecortin-like domain (Dx). In contrast, CL2alpha and CL2beta contained a full N-terminal Dx, whereas CLr only possessed a partial and dysfunctional Dx. Interestingly, despite a large similarity in the kinase domain, CL1/CL2/CLr had an impact on CRE-dependent gene expression distinct from that of the related CaMKI/CaMKIV and CaMKII. Although these were previously shown to activate Ca(2+)/cAMP-response element-binding protein (CREB)-dependent transcription, we here show that CL1 and CL2 were unable to significantly phosphorylate CREB Ser-133 and rather inhibited CRE-dependent gene expression by a dominant mechanism that bypassed CREB and was mediated by phosphorylated TORC2.
Journal of Biological Chemistry 08/2006; 281(29):20427-39. · 4.77 Impact Factor
