Kainate-Elicited Seizures Induce mRNA Encoding a CaMK-Related Peptide: A Putative Modulator of Kinase Activity in Rat Hippocampus

Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands.
Journal of Neurobiology (Impact Factor: 3.84). 05/1999; 39(1):41-50. DOI: 10.1002/(SICI)1097-4695(199904)39:13.0.CO;2-X
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By means of differential display techniques, we have previously identified an mRNA transcript whose expression is highly induced in the rat hippocampus by kainate-elicited seizures. Here, we report the cloning of a corresponding cDNA encoding a 55-amino-acid, serine-rich peptide which contains four predicted phosphorylation sites. The peptide was designated CaMK-related peptide (CARP) as it shares significant amino acid sequence identity with part of a novel putative calcium/calmodulin-dependent kinase (CaMK-VI) that was also cloned in this study. It appears that CARP and CaMK-VI are derived from the same gene through differential splicing. Intriguingly, CARP also exhibits 64% amino acid sequence identity with the C-terminal part of human doublecortin, encoded by a recently identified gene which is mutated in patients with X-linked lissencephaly and the double-cortex syndrome. In addition, the structure of CARP resembles the autoinhibitory, serine-rich N-terminal domain of CaMK-IV, suggesting a possible modulatory role of CARP with respect to CaMK activity. Northern blot analysis and in situ hybridization experiments showed that CARP mRNA is specifically induced by kainate-elicited seizures in the dentate gyrus and in the pyramidal layers CA1 and CA2, but not in CA3. In contrast, kainate-induced seizures did not change the level of expression of the CaMK-VI gene. We propose that CARP induction leads to the modulation of kinase activity in specific subregions of the rat hippocampus, providing a negative feedback mechanism for seizure-induced kinases.

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Available from: Edo Ronald de Kloet, Aug 20, 2014
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    • "The yeast 2-hybrid screen was performed with a Mouse Adult Brain_RP1 ULTImate Y2HTM Library and the bait CARP/ANIA-4 (GI:5468526) cloned into pB27 (N-LexA-CARP-C) (Hybrigenics-Services, Paris, France). CARP/ANIA-4 is a 55 amino acid peptide that covers the 50 C-terminal amino acids of DCL [28]. 79.3 million possible interactions were screened and a proteome-wide interaction map (PIM) biological score (Global PBS) were used to classify interactions into confidence categories [29,30]. "
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    ABSTRACT: Doublecortin-like (DCL) is a microtubule-binding protein crucial for neuroblastoma (NB) cell proliferation. We have investigated whether the anti-proliferative effect of DCL knockdown is linked to reduced mitochondrial activity. We found a delay in tumor development after DCL knockdown in vivo in doxycycline-inducible NB tumor xenografts. To understand the mechanisms underlying this tumor growth retardation we performed a series of in vitro experiments in NB cell lines. DCL colocalizes with mitochondria, interacts with the mitochondrial outer membrane protein OMP25/ SYNJ2BP and DCL knockdown results in decreased expression of genes involved in oxidative phosphorylation. Moreover, DCL knockdown decreases cytochrome c oxidase activity and ATP synthesis. We identified the C-terminal Serine/Proline-rich domain and the second microtubule-binding area as crucial DCL domains for the regulation of cytochrome c oxidase activity and ATP synthesis. Furthermore, DCL knockdown causes a significant reduction in the proliferation rate of NB cells under an energetic challenge induced by low glucose availability. Together with our previous studies, our results corroborate DCL as a key player in NB tumor growth in which DCL controls not only mitotic spindle formation and the stabilization of the microtubule cytoskeleton, but also regulates mitochondrial activity and energy availability, which makes DCL a promising molecular target for NB therapy.
    PLoS ONE 09/2013; 8(9):e75752. DOI:10.1371/journal.pone.0075752 · 3.23 Impact Factor
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    • "Mismatch controls yielded no signal (Fig. 3D). We have previously raised an antibody using synthetic CARP, a splice variant of the DCLK gene, as antigen (Vreugdenhil et al., 1999; Kruidering et al., 2001); Western blot analysis (Fig. 4) of embryonic brain homogenates probed with this antibody revealed a 40 kDa protein and no other DCLK-related immunoreactive bands, e.g. the 80 kDa DCLK-long (Burgess & Reiner, 2002) or the 80 kDa DCK2 protein (Edelman et al., 2005), indicating that this antibody recognizes specifically DCL during embryonic development. In line with our RT- PCR experiments, CARP protein in embryonic lysates was below detection level in our Western blot analysis. "
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    • "Generation and characterization of the anti-DCL antibody has been described before (Kruidering et al., 2001; Vreugdenhil et al., 2007). Briefly, anti-DCL was raised as a polyclonal antibody in rabbit against the complete 55 amino acid-long CARP peptide (MLELIEVNGTPGSQL- STPRSGKSPSPSPTSPGSLRKQRDLYRPLSSDDLDSVG- DSV; see Vreugdenhil et al., 1999) of which the C-terminal 49 amino acids are identical to the C-terminus of DCL (see Vreugdenhil et al., 2007). DCL has a predicted MW of 40 kD. "
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    ABSTRACT: During corticogenesis, radial glia-derived neural progenitors divide and migrate along radial fibers to their designated positions within the cortical plate. The microtubule-associated proteins doublecortin (DCX) and doublecortin-like (DCL) are critically involved in neuronal migration and division, and may function in a partially redundant pathway. Since little is known about the important early stages of corticogenesis, when neurogenesis is extensive, we addressed a possible differential role by examining spatiotemporal expression patterns of DCX, DCL, and the radial glia marker vimentin during murine development. We found expression patterns of DCL and DCX to differ remarkably prior to embryonic day (E)13. DCL was already expressed at E9 and largely overlapped with vimentin, whereas DCX expression started modestly from E10/E11 onward. DCL was mainly found in the ventricular zone, often in mitotic cells and in pial-oriented radial fibers. In contrast, DCX was expressed in tangential fibers in the outer cortical regions. After E13, DCX and DCL expression largely overlapped but DCL expression had disappeared from the ventricular zone. Also, DCL levels were attenuated, whereas DCX remained high beyond E17. In conclusion, DCX and DCL are differentially expressed, particularly during early corticogenesis, consistent with their different functional roles. Given its involvement in mitosis, DCL appears to have a unique role in the early neuroepithelium that is different from later developmental stages when DCX is coexpressed.
    The Journal of Comparative Neurology 04/2008; 507(4):1639-52. DOI:10.1002/cne.21646 · 3.23 Impact Factor
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