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J. Biol. Chem., Vol. 281, Issue 29, 20427-20439, July 21, 2006
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1
111¶¶**2
From the
Department of Pharmacology, Kyoto University Faculty of Medicine, Yoshida-Konoecho, Sakyo-ku, Kyoto 606-8315, the Department of Neurochemistry and the ¶Center for Integrated Brain Medical Science, The University of Tokyo Graduate School of Medicine, Hongo, Bunkyo-ku, Tokyo 113-0033, the ||Department of Bioscience, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-8502, and the **Solution-Oriented Research in Science and Technology (SORST)-Japan Science and Technology Agency (JST), Kawaguchi 332-0012, Japan
Received for publication, December 12, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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and CL1
were shorter isoforms of DCAMKL1, which lacked the doublecortin-like domain (Dx). In contrast, CL2 and CL2 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 Ca2+/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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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As part of the large list of serine/threonine and tyrosine kinase families, the Ca2+/calmodulin (CaM)3-dependent protein kinase (CaMK) group stands out by the large number of its constituent kinases (1-3). Despite its nomenclature, however, only the classic CaMK subgroups such as CaMKII family, or the CaMKI/CaMKIV family, are genuinely catalytically Ca2+/CaM-dependent. Most of the kinases of the CaMK group actually lack the characteristic Ca2+/CaM-sensitive regulatory domain. They nonetheless belong to the CaMK group, because they share in common a significant homology in the primary structure of their kinase domains. Several classic CaMKs such as CaMKII and CaMKIV are highly expressed in the central nervous system and have been convincingly shown to play a critical role in long term synaptic plasticity and in several forms of long term memory (4-15). Thus, elucidation of the biological function of classic CaMKs is one of the key issues in neuroscience.
Despite these insights, not many investigations have specifically addressed how many CaMKs or related kinases are expressed in the central nervous system, and how many of them actually might play a role in regulation of brain circuitry formation, maturation, or plasticity. For example, it is only recently that the diversity of the CaMKI subfamily consisting of four separate genes has been appreciated (1, 2, 16-19) and that several intriguing novel functions have been proposed for neuronal CaMKI (20-22). To better understand the complexity of neuronal CaMK profile, we here have carried out a degenerate PCR-based search for kinases with a core catalytic structure similar to CaMKI and CaMKIV. Consistent with our goal, we identified three mouse CaMKI/CaMKIV-related kinases, CLICK-I (CL1)/DCAMKL1, CLICK-II (CL2)/DCAMKL2, and CLICK-I,II-related (CLr)/DCAMKL3, the kinase domains of which were structurally related not only to CaMKIV and CaMKI but that also had comparable homology to CaMKII. Furthermore, CL1, CL2, and CLr were highly expressed throughout the central nervous system, in a neuron-specific fashion, from embryonic stages until adulthood. We identified several distinct open reading frames for CL1, CL2, and CLr. Whereas CL1 and CL1 are shorter isoforms of DCAMKL1 that lack the doublecortin-like domain (23-30), CL2 and CL2 contain an N-terminal doublecortin-like domain (Dx), which, via tight association with the microtubules, allowed specific dendritic localization in mature hippocampal neurons. In contrast, CLr only contained an incomplete Dx-like homology and was unable to localize with microtubules. Interestingly, despite a large structural similarity in the kinase domain, the functional impact of kinase activity of CL1/CL2/CLr kinases appeared distinct from the related CaMKI/CaMKIV and CaMKII. Indeed, although both CaMKI/CaMKIV and CaMKII branches of the CaMK family are established Ca2+/cAMP-response element-binding protein (CREB) phosphorylating kinases (11, 31-37), we show here, using transcriptional readout assays, that CL1/CL2/CLr kinases were unlikely to target CREB but rather inhibited CRE-dependent gene expression by a dominant mechanism that bypassed CREB and was mediated by phosphorylated transducer of regulated CREB activity (TORC) 2.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Molecular Cloning of Full-length CLICK-I, CLICK-II, and CLICK-I,II-related cDNAs—To screen for the presence of yet uncharacterized CaMK-like kinases, degenerate oligonucleotide primers were constructed as follows: CaMK5'-1, 5'-GTICAYMGIGAYMTCAARCC-3' (Y = T/C, M = A/C, R = A/G); CaMK5'-2, 5'-GTICAYMGIGAYMTGAARCC-3' (Y = T/C, M = A/C, R = A/G); CaMK5'-3, 5'-GTICAYMGIGAYMTAAARCC-3' (Y = T/C, M = A/C, R = A/G); and CaMK3', 5'-CCYGGRGTYCCACAIRCYGT-3' (Y = T/C, R = A/G).
100 pmol of a 1:1:1 mixture of CaMK5'-1/CaMK5'-2/CaMK5'-3 and 100 pmol of CaMK3' were included with 
100 ng of Sprague-Dawley rat adult hippocampal cDNA in a standard 50-µl PCR carried out for 35 cycles at 30-s denaturation at 94 °C, 1-min annealing at 48 °C, 1-min extension at 72 °C, using native Taq polymerase (GeneTaq, Nippon Gene). After confirming the presence of an expected 140-bp band, this PCR fragment was co-digested with BspHI, BstXI, BclI in NEB buffer 2 (50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 10 mM Tris-HCl, pH 7.9, New England Biolabs), because these three enzymes were expected to digest the PCR fragments amplified from known CaMKI/CaMKIV cDNAs. A portion of the 140-bp fragment consistently remained resistant to enzyme digestion. To identify this fragment, this resistant PCR band was gelpurified from a 3% NuSieve GTG-agarose gel, and subcloned into T7Blue-2 (Novagen) by TA cloning. Isolation of independent colonies and sequence analysis revealed the presence of two related inserts that were homologous to, but had only limited identity with, known members of the CaMKI/CaMKIV family. These fragments were tentatively considered as CaMK-like CREB regulatory kinase candidates (CLICKs), and named CLICK-I (CL1) and CLICK-II (CL2), respectively. EST search revealed the presence of mouse orthologs for both CL1 and CL2, but none represented fulllength cDNAs. Full-length 5'-ends and 3'-ends for mouse CL1 and CL2 were identified by rapid amplification of 5'- and 3'-cDNA end (5'- and 3'-RACE) procedures using a Smart RACE cDNA amplification kit (Clontech). We obtained multiple 3'-RACE fragments of different sizes for both CL1 and CL2, and sequencing confirmed the presence of at least two C-terminal splice variants in each case. As for the 5'-end, the most remote ATG codon that was found in-frame was considered as the putative initiation site of the open reading frame. Finally, to isolate genuine full-length open reading frames (ORFs), PCR was carried out between the putative initiation ATG and the two alternate stop codons using Advantage High Fidelity Polymerase (Clontech) and ICR mouse adult hippocampal cDNA as a template. A CL1- and CL2-related mouse FANTOM clone C730036H08 was obtained from Dnaform (Tokyo, Japan). Although this clone was deposited as a partial cDNA, full sequencing of its 5'-end revealed a C insertion at position 566 of the originally deposited sequence. Correction of this frameshift unmasked a previously unrecognized cryptic N terminus with a functional ATG initiation codon starting from position 154. In total, six distinct full-length cDNAs were obtained in this study and designated as CLICK-I (GenBankTM accession number AY968047
[GenBank]
), CLICK-I (AY968048
[GenBank]
), CLICK-II (AY968049
[GenBank]
), CLICK-II1 (AY968050
[GenBank]
), CLICK-II2 (AY968051
[GenBank]
), and CLICK-I,II-related (DQ286388
[GenBank]
).
Genomic Mapping—Kinase domain sequences of CLICK-I (CL1), CLICK-II (CL2), and CLICK-I,II-related (CLr) were mapped on the Human and Mouse Genome Database, as made available by NCBI and Celera. BAC clones mapped to 3D and 3F1 were obtained from the BACPAC Resource Center (Children's Hospital Oakland Research Institute, Oakland, CA) to ascertain that both CL1/Dcamkl1 and CL2/Dcamkl2 loci were located indeed in relative vicinity on the mouse chromosomes 3D and 3F1, respectively.
Kinase Characterization—All expression vectors for wildtype and mutant CL1, CL2, and CLr were constructed in either pDEST26 or pEGFP-C1 vector backbones using PCR, TOPO cloning or GATEWAY technology (Invitrogen), with addition of either N-terminal Myc tag, HA tag, or EGFP tag, as indicated. Domain deletion and introduction of point mutations were carried out on CL1 and CL22 backbones, unless stated otherwise, using PCR and by use of QuikChange Mutagenesis kit (Stratagene). The presence of proper inserts and mutagenesis was confirmed by DNA sequencing. The plasmids used in this report were as follows: CL1 (amino acid (aa) position 1-421), CL1KD (aa 1-421 with Lys at position 111 mutated to Ala), CL1
C (aa 1-353), CL2 (aa 1-771), CL2 (aa 1-714 of CL22), Dx-CL2 (aa 377-714 of CL22), Dx-CL2KD (aa 377-714 with Lys at position 437 mutated to Ala), Dx-CL2C (aa 377-679 of CL22), and CL2-Dx (aa 1-431 of CL2). These constructs were transfected in COS-7 cells, and crude lysates were prepared in a Ca2+-free buffer containing (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40, 1mM dithiothreitol, 25 mM NaF, 10 mM -glycerophosphate, 5 mM sodium pyrophosphate, 0.1 mM sodium vanadate, 1 mM EGTA, 0.1 µM calyculin A, and 1 x EDTA-free Complete protease inhibitors (Roche Applied Science)). Immunoprecipitates were obtained using an anti-Myc monoclonal antibody (Santa Cruz Biotechnology) and were subjected to kinase assays as described using 5 µg of myelin basic protein as a substrate (17), except that Ca2+ and CaM were removed from the kinase reaction buffer. Integrity of expressed proteins was verified by silver staining or by immunoblot using a mouse anti-Myc monoclonal antibody (Santa Cruz Biotechnology). The 32P incorporation was visualized using BioMax x-ray films (Eastman Kodak Co.). Pull-down assays using CaM beads were done as described previously (17).
Transcript Analyses—Northern blot analyses were carried out using pre-made poly(A)+ RNA blots (2 µg per lane) obtained from Clontech. Blots were hybridized using a 32P-labeled DNA probe corresponding to the nucleotide position 58-1116 of CL1 (AY968047
[GenBank]
), 3-2039 of CL2 (AY968049
[GenBank]
), and 514-2373 of CLr (DQ286388
[GenBank]
) (for position see Fig. 1A), according to Ref. 17. In situ hybridization using DIG technology (Roche Diagnostics) was performed essentially as described (38) with some modifications. In brief, anesthetized mice were perfusion-fixed with Tissue Fixative (GenoStaff, Inc.), and dissected tissues were sectioned after paraffin embedding. For generation of antisense and sense cRNA probes, 294-, 255-, and 1860-bp fragments corresponding to the nucleotide position 898-1191 of CL1, 860-1114 of CL2, and 514-2373 of CLr were subcloned into pBluescript II KS+ vector (Stratagene). Digoxigenin-labeled cRNA probes were prepared with DIG RNA labeling Mix (Roche Applied Science). Coloring reactions were performed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate, an alkaline phosphatase color substrate, and tissue sections were counterstained with Kernechtrot stain solution (Muto Pure Chemicals Co., Ltd.). After mounting, 24-bit color images were acquired by scanning the sections using an Epson GT-8400UF digital scanner at 3200-dpi resolution (Fig. 3C and supplemental Fig. S1). DIG signals were isolated by uniformly subtracting the counterstaining color component using Photoshop version 7.0.1. (Adobe) and displayed in 8-bit grayscale without further correction (Fig. 3, B and D).
Immunocytochemical Analysis of CL1 and CL2 Expressed in HeLa Cells and in Central Neurons—Cerebelli and hippocampi were obtained from postnatal day 0-1 ICR mice, and cerebellar granule neurons and hippocampal neurons were cultured as described before (39, 40). Plasmid transfection was carried out using Lipofectamine 2000 (Invitrogen), at 0 day in vitro for cerebellar granule cells or at 9 days in vitro for hippocampal neurons, essentially as described (17, 41). About 48 h after transfection, HeLa cells and neurons were fixed in 4% paraformaldehyde/4% sucrose/Ca2+/Mg2+-free phosphate-buffered saline (PBS(-)) at 37 °C for 15 min, washed with 0.1 M glycine/PBS(-) at room temperature. Blocking and permeabilization was carried out as described (11). Primary and secondary antibody reactions were performed overnight at 4 °C and 1 h at room temperature, respectively. Antibodies used were: rabbit anti-Myc polyclonal (Santa Cruz Biotechnology), mouse anti--tubulin monoclonal (Sigma), and Alexa488- or Alexa555-conjugated anti-rabbit or anti-mouse antibodies (Molecular Probes). Immunofluorescent images were acquired using an Olympus DP-70 charge-coupled device camera (Fig. 4) or a Zeiss LSM510META confocal microscope (Supplemental Fig. S2). For the latter, maximal projection images were obtained from z-stacks of 10-15 confocal sections covering the entire depth of cells within the field of view.
Luciferase Assay—Dual luciferase assay was carried out in COS-7 cells essentially as described (17), except that 24-well plates were used for assays and luciferase activities were monitored using a Fluoroskan Ascent FL plate reader (ThermoLab-systems), with EF1-Rluc as a control vector (42). CRE-luc, Gal4-CREB, UAS-luc, and PKAcat constructs were from Stratagene. An SRE-luc (3D.Aluc) vector was obtained from Richard Treisman (Imperial Cancer Research Foundation). A V14Rho expression vector was used as described (39). A lacZ expression vector was created as a mock control in the pDEST26 backbone, based on SinRep/LacZ (Invitrogen). CaMK4C and CaMK2C were constructed in pIRES-EGFP vectors (Clontech) as described (43), based on CaMK4DA and CaMK2DA vectors (33) kindly provided by Richard Maurer (Oregon Health and Sciences University). A full-length CBP cDNA (44) was obtained from Akiyoshi Fukamizu (Tsukuba University), with permission from Marc Montminy (Salk Institute) and subcloned inframe immediately downstream of Gal4-(1-147) in a mammalian Matchmaker two-hybrid vector pM (Clontech) to yield a Gal4-CBP vector. To construct TORC2-GFP, an open reading frame of human TORC2 (OriGene clone TC107319) was subcloned by PCR into the EcoRI/SalI sites of pEGFP-C1 (Clontech). All point mutants were constructed using PCR and the QuikChange mutagenesis kit (Stratagene). Gal4-CREB-DA (pRc/RSV-GAL4-CREBDIEDML) and Gal4-CREB-WT (pRc/RSV-GAL4-CREB) (45) were kind gifts of Richard Goodman (Vollum Institute), and EF1-Rluc was kindly provided by Akiko Tabuchi and Masaaki Tsuda (Toyama Medical and Pharmaceutical University).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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140 bp was detected, consistent with the expected size of the amplified product from cDNAs of either CaMKI,-,-
, or CaMKIV. Based on available GenBankTM information, these predicted PCR products would be cleaved by BspHI, BstXI, or BclI. A diagnostic restriction enzyme reaction with these three enzymes, however, revealed that a previously unknown species, resistant to all three enzyme digestions, was present. To identify this molecular species, we gel-purified this digestion-resistant 140-bp fragment and subcloned it into the T7Blue-2 vector. Random sequencing of 78 independent clones revealed that 73 clones encoded a kinase or a kinaselike gene, and among them, more than 60 clones represented kinases belonging to the large CaMK group (1), indicating that the kinase screen was carried out properly. Three major species was isolated more than 10 times (49 times in total), and among them, two were yet uncharacterized, homologous to each other and were relatively related to the kinase domains of CaMKI and CaMKIV. These fragments were considered as partial clones from CaMK-like CREB regulatory kinase candidates (CLICKs), which were designated CLICK-I (CL1) and CLICK-II (CL2), respectively (Fig. 1A, thick lines). Using EST search and sequential RACE techniques, full-length mouse cDNAs were then obtained for both CL1 and CL2. Two distinct ORFs were isolated for CL1 (CL1, 421 aa and CL1, 433 aa (Fig. 1A)) and three distinct ORFs were isolated for CL2 (CL2, 771 aa; CL21 715 aa; and CL22, 714 aa (Fig. 1, A and B)). CL1 and CL21 contained an insertion of Glu at position 68 and of Gln at position 368, respectively, likely reflecting insertion and deletion polymorphisms (indels). Examination of genomic exon-intron boundaries revealed that CL21 and CL22 were borne out of use of distinct splice acceptor sites at the 5'-terminal end of exon 7 and resulted in an addition of one extra amino acid in CL21 (Fig. 1B).
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and CL1 were found to encode mouse orthologs for KIAA0369-BS/CaMK-VI/DCLK-short-B/DCK-1 (24, 28, 48, 49) and cpg-16/KIAA0369-BL/DCLK-short-A/DLK-2 (24-26, 48, 49), respectively. A longer splice variant possessing an N-terminal doublecortin-like domain (Dx), DCLK/DCAMKL1 (23, 24, 27, 29, 30), was further reported to be highly present in the embryonic brain (50-52). However, the precise biological function for DCAMKL1 and its numerous splice variants has not been established. In contrast to CL1/DCAMKL1, for which many cloning efforts, including ours, revealed the presence of shorter isoforms in the adult brain, all CL2 transcripts isolated from adult mouse hippocampal cDNAs in this study contained the Dx-like domain in their open reading frames, and no shorter splice variants were found. This indicated that CL2 may be a predominant Dx-containing CL/DCAMKL isoform in the mature central nervous system. Interestingly, genomic mapping revealed that the genomic loci for CL1 and CL2 genes were both located on mouse chromosome 3 (3D and 3F1, respectively) within close proximity and in an opposite tandem direction, suggesting that these two genes originated from gene duplication during evolution (Fig. 1C). In human, both genes were separated on chromosomes 13 (13q13) and 4 (4q31.23), respectively (Fig. 1C). Resequencing of a CL1/2-related cDNA, originally reported as a partial cDNA (FANTOM clone C730036H08), revealed a yet unreported nucleotide insertion within its putative open reading frame. The correction of this frameshift revealed the presence of a previously unrecognized N-terminal end with an in-frame ATG initiation codon. The accuracy of the newly obtained cDNA sequence, designated as CLICK-I,II-related (CLr), was confirmed by a gapless sequence alignment to existing mouse genome sequences. Furthermore, a GFP fusion protein constructed by a single round of PCR subcloning yielded a recombinant protein of the expected size as measured by SDS-PAGE and by Western blot (data not shown). Exon-intron structures were, however, distinct from CL1/Dcamkl1 and CL2/Dcamkl2 genes (Fig. 1B), and the mouse CLr/Dcamkl3 gene was mapped to chromosome 9 (9F3, Fig. 1C).
CL1/DCAMKL1, CL2/DCAMKL2, and CLr/DCAMKL3 Form a Novel Kinase Subfamily with Intermediate Homology with CaMKI/CaMKIV and CaMKII—Comparison of kinase domain sequence identity revealed that CLr/DCAMKL3 was indeed the closest kinase to CL1 and CL2 (56 and 54% identity, respectively), followed by CaMKIV (44 and 42%) and CaMKII (43 and 42%) (Fig. 2). Furthermore, using ClustalW, the CL1/CL2/CLr/DCAMKL kinase subfamily was confirmed to actually lie between the CaMKII gene family and the CaMKI/CaMKIV gene family. This raised the possibility that the kinase activity of CL1, CL2, and CLr may be regulated in a manner similar to CaMKI/CaMKIV and CaMKII. C-terminal deletion mutants for both CL1 (CL1C) and CL2 (Dx-CL2C) had a significant amount of Ca2+/CaM-independent kinase activity (data not shown), in keeping with prior findings obtained for CaMKII and CaMKIV (35-37). Surprisingly, however, intact forms of both CL1 and CL2 also showed notable amounts of kinase activities, even in the absence of calcium, and lacked significant CaM binding (data not shown), consistent with prior reports (26, 48, 53). Taken together, CL1/DCAMKL1, CL2/DCAML2, and CLr/DCAMKL3 are likely to form a novel subfamily of kinases with intermediate homology with CaMKI/CaMKIV and CaMKII, but with significantly reduced Ca2+/CaM affinity and dependence.
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), a kinase-dead full-length CL2 (CL2KD), and the doublecortin-like domain only of CL2 (CL2-Dx) all co-localized with microtubule structures, whereas Dx removal (Dx-CL2) resulted in a diffuse cytosolic distribution (Fig. 4A). Heavy bundling of microtubules was observed in a significant proportion of cells expressing CL2 constructs containing Dx such as CL2, CL2KD, and CL2-Dx but was absent in cells transfected with a CL2 mutant lacking Dx (Dx-CL2) (Fig. 4B). Together, the presence of Dx in CL2 appeared to be necessary and sufficient for CL2 co-localization with microtubules in HeLa cells (Fig. 4A), and the critical importance of Dx was also shown in cultured postmitotic neurons such as hippocampal pyramidal and cerebellar granules neurons (Ref. 53 and supplemental Fig. S2, A and B). Interestingly, CLr localized diffusely in the cytoplasm and in the nucleus with no particular association with the microtubule structures in HeLa cells, indicating that the weakly homologous Dx-like domain in its N terminus (Fig. 1A) may not be sufficient to provide efficient microtubule binding (Fig. 4C). In dividing HeLa cells, overexpression of Dx-containing CL2 constructs induced severe cytokinesis phenotypes such as aberrant multinucleate cells (>10% of transfected cells versus a few percent in mock controls). This phenotype was completely abolished by the removal of Dx and was attenuated in the absence of kinase activity in Dx-containing CL2 mutants (Fig. 4, D and E). This indicated that the intrinsic activity of an overexpressed Dx domain, or alternatively, sequestration of binding sites for endogenous Dx domains severely interfered with dynamics of endogenous microtubule structures and suggested the possibility that CL2 kinase activity may modulate to some extent the interaction of Dx with the microtubules.
The Kinase Activity of CL1/CL2/CLr Targets Components of CREB-dependent Gene Expression in a Manner Distinct from That of CaMKI, CaMKIV, and CaMKII—Finally, we addressed whether the kinase activity of CL1/CL2/CLr had an impact to gene transcription that was similar or distinct from that of CaMKI/CaMKIV and CaMKII. Previous work, including ours, has established that activated forms of CaMKI, CaMKIV, and CaMKII all have significant effects on CRE-mediated gene expression (11, 17, 31-37, 46, 47). Kinase domain-only constructs (CL1 and Dx-CL2,-WT, and-KD) were transfected into COS-7 cells along with CRE-luc and a control Renilla-luc vector driven by the EF1 promoter as a control, and the Luc/Renilla ratio was calculated as an index for CRE-dependent gene expression. Expression of an increasing amount of wildtype kinase constructs gradually reduced CRE-dependent gene expression in forskolin-stimulated COS-7 cells, whereas kinase-dead constructs showed no decrease at all (Fig. 5A). This clearly indicated that the kinase activity in CL1 and CL2 was sufficient for suppressing forskolin-stimulated CRE-dependent gene expression. These results were replicated using C-terminal-deleted kinases (CL1C and Dx-CL2C) as well (Fig. 5B and data not shown), and this CL1/CL2-mediated repression of CRE-dependent gene expression was rather specific, because Rho-stimulated SRE-dependent gene expression (55) was completely unaffected (Fig. 5B). Such specificity would be easily accounted for if CL1 and CL2 were able to phosphorylate and modulate specific regulators involved in CRE-dependent transcription. One obvious candidate is CREB, which has repeatedly been demonstrated to be phosphorylated at its Ser-133 residue by activated CaMKI, CaMKIV, and CaMKII (11, 17, 31-37, 46, 47). However, despite the structural similarity in the kinase domain with CaMKI, CaMKIV, and CaMKII, CL1 and CL2 strongly repressed both CaMKIV-induced (Fig. 5C, left panel, CaMK4C) or PKA-induced CREB activation as tested using a Gal4-CREB/UAS-Luc system (Fig. 5C, right panel, PKAcat). Thus, in contrast to CaMKI and CaMKIV, CL1 and CL2 clearly were not able to stimulate CREB. Rather, CL1 and CL2 tended to inhibit CREB-dependent transcription. Intriguingly, this CL phenotype could be qualitatively replicated by expression of a constitutively active form of CaMKII (CaMK2C) (Fig. 5C).
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C) (Fig. 5C). To directly test whether Ser-142 had any role in the inhibitory effect mediated by CL1/CL2, we introduced a Ser-142 
Ala mutation into the Gal4-CREB construct and examined its consequence on CL-mediated CREB inhibition. Again, however, both CL1C and Dx-CL2C were still able to significantly inhibit forskolin-stimulated CREB-dependent transcription, which argued that Ser-142 phosphorylation was unlikely to be the major mechanism of this repression (Fig. 6B, upper and lower panels). This is in sharp contrast to the case of CaMKII, which was previously shown to activate, rather than inhibit, Gal4-CREBS142A-dependent transcription (33, 46).
An additional possibility would be that CL1 and CL2 may alter the function of the CREB target CBP, as has been suggested for CaMKIV (56-58). We tested this by using a Gal4-CBP construct. Consistent with prior reports, both CaMKIV and PKA were able to significantly augment CBP-mediated transcription (Fig. 6C). However, CL1C and Dx-CL2C revealed no such activity, and no consistent inhibitory activity was shown either (Fig. 6C). Thus, unlike CaMKIV, neither CL1 nor CL2 appeared to modulate CBP significantly.
Recent evidence suggested that a novel type of CREB coactivator, transducer of regulated CREB activity (TORC), may mediate a significant part of CREB-dependent gene expression (59, 60). In particular, a critical phosphorylation site was found at serine-171 of TORC2, one of the TORC isoforms, and control of its phosphorylation state by either a serum-inducible kinase or an adenosine-monophosphate-dependent protein kinase was shown to regulate nucleocytoplasmic shuttling of TORC. Thus, increase in phospho-Ser-171-TORC2 prevented nuclear import of TORC, a CREB co-activator, and removal of TORC from the nucleus strongly diminished CREB-dependent gene expression (61-63). To ask whether CL1/CL2-mediated CREB repression may be mediated by phosphorylated TORC2, we first examined nucleocytoplasmic shuttling of TORC2 in the absence or presence of CL2 activity. Co-expression of active CL2 with TORC2-GFP resulted in an increased retention of TORC2-GFP in the cytoplasm (Fig. 7A), accompanied by an increase in the ratio of total cytoplasmic versus total nuclear GFP fluorescence (Fig. 7B). Consistent with a role for Ser-171 phosphorylation in CL2-mediated retention of TORC2 in the cytoplasm, TORC2-dependent potentiation of CRE-dependent transcription was strongly diminished in the presence of active CL2, however, an alanine substitution of Ser-171 completely abolished such CL2-dependent inhibition (Fig. 7C). Similar results were obtained with CL1 and CLr (data not shown). In contrast, CaMKII-dependent inhibition of TORC2-potentiated CREB activity still persisted even with a S171A mutation of TORC2, suggesting that CaMKII did not mediate CREB suppression in a phosphor-Ser-171-TORC2-dependent manner (Fig. 7C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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and CL1 were mouse orthologs of shorter isoforms of DCAMKL1 that did not contain a doublecortin-like domain (Dx). All isolated isoforms of CL2 (CL2, CL21, and CL22), however, included an intact microtubule-binding Dx at its N terminus, whereas CLr only contained a partial and dysfunctional Dx. Interestingly, the mouse Dcamkl1 and Dcamkl2 genes were located within relative proximity on an identical chromosome 3 in the mouse genome, in opposite direction, whereas the third related gene, Dcamkl3, has been mapped to mouse chromosome 9. Surprisingly, a dendrogram analysis, examining a putative phylogenic relationship based on the primary structure of the kinase domain, revealed that, in fact, the kinase domains of all three DCAMKL genes formed a novel kinase group that had an intermediate homology with both the CaMKI/CaMKIV and CaMKII subfamilies of CaMK. Given that CL1/DCAMKL1, CL2/DCAMKL2, and CLr/DCAMKL3 also share similarity with CaMKI/CaMKII/CaMKIV in their neuronspecific expression in the adult brain, it is likely that the DCAMKL genes, CaMKI/CaMKIV subfamily, and CaMKII subfamily may have co-evolved from a common ancestral CaMK during evolution. Recently, the role of doublecortin in formation of forebrain layer structures has been extensively characterized (e.g. Ref. 54). It is now believed that doublecortin largely functions as a microtubule-associated protein via its privileged interaction with assembled microtubules (64-66). The high homology (>70% identity, data not shown) between doublecortin and the doublecortin-like domain (Dx) of either CL1/DCAMKL1 or CL2/DCAMKL2 strongly indicates that these also possess microtubule-associated protein-like activity. In contrast, a Dx-like motif in CLr/DCAMKL3 had only a marginal homology (<40% identity in the most homologous stretch, data not shown), suggesting that CLr may not fully share such activity.
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One salient feature of the classic CaMKs, such as CaMKI/CaMKIV and CaMKII, i.e. the striking Ca2+/calmodulin dependence of their kinase activity, is largely absent in CL1 and CL2 (26, 48, 53). Consistently, both wild-type CL1 and CL2 demonstrated intact myelin basic protein as well as autophosphorylating activities in the absence of calcium (data not shown). In this respect, it is worth mentioning that one major member of the CaMK family, CaMKK, is shown to possess a degree of Ca2+/CaM-independent activity (19, 47). It thus remains to be seen in future studies whether a limited amount of Ca2+/CaM dependence could conversely be reacquired by CL1/CL2/CLr under certain conditions.
Furthermore, another common characteristic previously found for activated CaMKI/CaMKIV and CaMKII, namely their ability to functionally stimulate CREB via phosphorylation of Ser-133 (31-37, 46, 47), was missing in CL1/CL2/CL2/CLr (Figs. 5, 6, 7 and data not shown). In a previous report, Silverman et al. (26) noted that the rat ortholog for CL1, CPG16, is able to diminish CRE-dependent gene expression, but the mechanism underlying this phenomenon was not examined further. The present study clearly demonstrates: 1) that the kinase activity in CL1/CL2/CLr is responsible for suppression of stimulated CREB-dependent transcription, 2) that the kinase effect on transcription is unlikely to be mediated by either inhibition of Ser-133 phosphorylation, stimulation of phospho-Ser-133 dephosphorylation, or phosphorylation of an inhibitory Ser-142 site, and finally 3) that the kinase effect bypasses CREB and is likely mediated by phosphor-Ser-171-TORC2. Previous work suggests that TORC2 phosphorylation may occur in the cytoplasm thus favoring its nuclear exclusion and inhibition of CREB activity (61, 62). Whether this is indeed the case in the neuronal context still remains to be elucidated.
These lines of evidence also highlight the fact that, despite the structural homology found in the catalytic and activation domains between the DCAMKL subfamily and the CaMKI/CaMKIV and CaMKII subfamilies, the functional consequence of kinase activation in intact cells was dramatically opposite at the level of CREB regulation. Such a dichotomy could result, in principle, either from a distinct kinase substrate specificity or a completely different mode of kinase regulation (e.g. involvement of a completely distinct regulatory molecule, such as an upstream kinase or an auxiliary subunit), or both. Previous studies using synthetic substrate peptides and in vitro phosphorylation assays have reported mostly overlapping peptide selectivity or substrate recognition motifs between CaMKI/CaMKIV and DCAMKL1/2 (48, 49, 53). These studies thereby previously favored the view that DCAMKL might actually possess largely similar substrate specificities with CaMKI/CaMKIV. Our results using transcriptional reporters as functional cellular readout here shed novel light on the potential selectivity present between distinct branches within the CAMK family, because the CL1/CL2/CLr/DCAMKL class of kinases appears to regulate components of the CREB-dependent transcriptional machinery in a manner clearly distinct from that of the CaMKI/CaMKIV- and CaMKII-like classes of CAMK. Further work is needed to fully resolve the similarity and the distinction between the various branches of CAMK family genes in the context of living neurons.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to H. B., H. O., M. O., and S. T.-K.), from Japan Science and Technology Agency (to H. B.), and from Human Frontier Science Program (to H. B. and H. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.
1 These authors contributed equally to this work.
2 To whom correspondence should be addressed. Tel.: 81-3-5841-3559; Fax: 81-3-3814-8154; E-mail: hbito{at}m.u-tokyo.ac.jp.
3 The abbreviations used are: CaM, calmodulin; CaMK, Ca2+/CaM-dependent protein kinase; CLICK, CaMK-like CREB-regulatory kinase candidate; DCAMKL, doublecortin and CaM kinase-Like; CREB, Ca2+/cAMP-response element-binding protein; RACE, rapid amplification of cDNA end; ORF, open reading frame; PBS(-), Ca2+/Mg2+-free phosphate-buffered saline; CRE, Ca2+/cAMP-response element; PKA, protein kinase A; SRE, serum-response element; IRES, internal ribosome entry sequence; EGFP, enhanced green fluorescent protein; CBP, CREB-binding protein; DA, dominant active; W T, wild-type; EST, expressed sequence tag; indel, insertion and deletion polymorphism; DCK, doublecortin-like kinase; DCLK, doublecortin-like kinase; cpg, candidate plasticity-related gene; Dx, doublecortin-like domain; KID, kinase-inducible domain; TORC, transducer of regulated CREB activity; ANOVA, analysis of variance; aa, amino acid(s).
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