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PubMed2003

The DCX-domain tandems of doublecortin and doublecortin-like kinase.

The doublecortin-like domains (DCX), which typically occur in tandem, are novel microtubule-binding modules. DCX tandems are found in doublecortin, a 360-residue protein expressed in migrating neurons; the doublecortin-like kinase (DCLK); the product of the RP1 gene that is responsible for a form of inherited blindness; and several other proteins. Mutations in the gene encoding doublecortin cause lissencephaly in males and the 'double-cortex syndrome' in females. We here report a solution structure of the N-terminal DCX domain of human doublecortin and a 1.5 A resolution crystal structure of the equivalent domain from human DCLK. Both show a stable, ubiquitin-like tertiary fold with distinct structural similarities to GTPase-binding domains. We also show that the C-terminal DCX domains of both proteins are only partially folded. In functional assays, the N-terminal DCX domain of doublecortin binds only to assembled microtubules, whereas the C-terminal domain binds to both microtubules and unpolymerized tubulin.

Amino Acid Sequence↗

PubMed2003

Purification and crystallization of the N-terminal domain from the human doublecortin-like kinase.

The unique doublecortin-like tandem of two homologous domains is found in certain microtubule-associated proteins such as doublecortin (DCX) and doublecortin-like kinase (DCLK). It is responsible for interactions with tubulin/microtubules and regulates microtubule dynamics. Here, the expression and purification of the tandem from human DCLK (residues 49-280) and of the isolated domains (residues 49-154 and 176-280) and the successful crystallization of the N-terminal domain (N-DCLK) are reported. High-quality wild-type crystals were obtained and a complete native data set was collected to 1.5 A resolution. The crystals belong to space group C2, with unit-cell parameters a = 85.98, b = 29.62, c = 40.33 A, beta = 101.3 degrees. Crystals of SeMet-substituted N-DCLK (Leu120Met) were also obtained, but they exhibit the symmetry of space group P2(1), with unit-cell parameters a = 38.81, b = 29.43, c = 40.1 A, beta = 115.7 degrees.

Animals↗

PubMed1999

KIAA0369, doublecortin-like kinase, is expressed during brain development.

During embryonic development, the cerebral cortex attains its characteristic adult laminated structure. The finding that X-linked lissencephaly patients harbor mutations in the doublecortin gene implicated this gene product in the process of corticogenesis. An autosomal human gene, KIAA0369, with a high level of similarity to doublecortin, has been cloned from human adult brain. This gene product contains a kinase domain in addition to a doublecortin-like domain. In order to evaluate whether this doublecortin-like kinase also plays a role during brain development, we cloned and studied the expression pattern of the mouse homolog. Three cDNA products of this gene were cloned: one, doublecortin-like kinase, the second containing only the doublecortin-like region, and the third containing only the kinase domain, a homolog of the previously cloned rat CPG16 gene. We studied doublecortin-like kinase expression in mouse using Northern blot analysis, in situ hybridization, and Western blot analysis, and conclude that doublecortin-like kinase is expressed in multiple regions of embryonic brain including the developing cerebral cortex.

Amino Acid Sequence↗

PubMed2002

Alternative splice variants of doublecortin-like kinase are differentially expressed and have different kinase activities.

Alternative splicing of mRNA transcripts expands the range of protein products from a single gene locus. Several splice variants of DCLK (doublecortin-like kinase) have previously been reported. Here, we report the genomic organization underlying the splice variants of DCLK and examine the expression profile of two splice variants affecting the kinase domain of DCLK and CPG16 (candidate plasticity gene 16), one containing an Arg-rich domain and the other affecting the C terminus of the protein. These splice alternatives were differentially expressed in embryonic and adult brain. Both splice variants disrupted DCLK PEST domains; however, all splice variants remained sensitive to proteolysis by calpain. The adult-specific C-terminal splice variant of DCLK had reduced autophosphorylation activity, but similar kinase activity for myelin basic protein relative to the embryonic splice variant. The splice variant adding an Arg-rich domain gained an autophosphorylation site at Ser-382. Although this protein isoform was expressed mainly in the adult brain, the phosphorylated form was strongly enriched in embryonic brain and adult olfactory bulb, suggesting a possible role in migrating neurons.

Alternative Splicing↗

PubMed2001

Cleavage of doublecortin-like kinase by calpain releases an active kinase fragment from a microtubule anchorage domain.

Doublecortin-like kinase (DCLK) is widely expressed in postmitotic neurons throughout the embryonic nervous system. DCLK consists of an N-terminal doublecortin domain, responsible for its localization to microtubules, and a C-terminal serine-threonine kinase domain. Here we report that DCLK is a physiological substrate for the cysteine protease calpain. Cleavage of DCLK by calpain severs the kinase domain from its microtubule anchorage domain and releases it into the cytoplasm. The isolated kinase domain retains catalytic activity and is structurally similar to CPG16, a second product of the DCLK gene expressed in the adult brain that lacks the doublecortin domain. We propose that in neurons cleavage of DCLK by calpain represents a calcium responsive mechanism to regulate localization of the DCLK kinase domain.

Animals↗

PubMed2000

Doublecortin-like kinase is associated with microtubules in neuronal growth cones.

Doublecortin-like kinase (DCLK) shares sequence similarity to Doublecortin (DCX) in its N-terminal region. It contains the evolutionary conserved DC repeat motif as well a C-terminal kinase domain. Ectopic expression of DCLK in COS cells results in colocalization with microtubules, and phosphorylated DCLK copurifies with microtubules during assembly from embryonic brain extract. During brain development DCLK is expressed mainly in postmigratory neurons in a similar pattern to DCX. We demonstrate that DCLK is a microtubule-associated active protein kinase expressed in growth cones of postmitotic neurons.

Animals↗

PubMed2005

Expression of doublecortin (DCX) and doublecortin-like kinase (DCLK) within the developing chick brain.

Doublecortin (DCX) is a microtubule-associated protein widely expressed in the developing mammalian nervous system and important for neuronal migration. DCX is known to belong to a novel protein family defined by sequence homology and the presence of a conserved microtubule-binding domain, but the functions of other members of this family are still undefined. In this study, we describe the cloning of the chick ortholog of doublecortin-like kinase (DCLK), a member of this family, and assess the expression of DCX and DCLK in the layered regions of the developing chick brain. DCX and DCLK are widely expressed in pallial and subpallial structures, including the telencephalon, optic tectum, and cerebellum, in similar distribution patterns. In addition to their expression in migrating cells, both proteins were also detected in the ventricular zone and in postmigratory Purkinje cells. Finally, DCX and DCLK were found to be coexpressed in all areas examined. In postmigratory Purkinje cells, DCX and DCLK both colocalized to the cell membrane, although DCLK was also distributed more generally throughout the cell soma. These data are consistent with multiple roles for DCX and DCLK in the developing chicken brain and suggest that the chick cerebellum will be an intriguing system to explore the effects of DCX and DCLK on postmigratory neuronal function.

Amino Acid Sequence↗

PubMed2004

Functional differences between two DCLK splice variants.

Recently, we have cloned two splice variants of the doublecortin-like kinase (DCLK) gene, called DCLK-short-A and -B, both of which encode calcium/calmodulin-dependent protein kinase (CaMK)-like proteins with different C-terminal ends. Using in situ hybridization, we have found that both are highly expressed in limbic structures of the brain and that their expression differs in a number of brain areas. DCLK-short-A is relatively more strongly expressed than DCLK-short-B in the subependymal zone. The DCLK-short-B variant shows stronger expression in the cortex, the ventromedial and dorsomedial hypothalamic nuclei, the arcuate nucleus, the zona incerta and the subincertal nucleus. Also, within the hippocampus, the relative distribution of these two splice variants differs. DCLK-short-B expression compared to DCLK-short-A is highest in the CA1 area. The expression of the A variant is highest in the CA3/CA4 area. Additionally, DCLK-short-B is expressed at a higher level than DCLK-short-A in the substantia nigra and the mammillary nucleus. Both DCLK-short-A and -B were located in the cytoplasm, however DCLK-short-B was also found specifically in growth cone like structures and near the nucleus. Both DCLK-short proteins phosphorylate autocamtide and syntide, two highly specific CaMK substrates. Finally, removal of the C-terminal end of DCLK-short leads to a 10-fold increase of kinase activity, indicating that the different C-termini represent auto-inhibitory domains. Our results indicate that DCLK-short-A and -B control different neuronal processes that overlap with those controlled by CaMKs.

Alternative Splicing↗

PubMed1999

CPG16, a novel protein serine/threonine kinase downstream of cAMP-dependent protein kinase.

Gene expression is necessary for the formation and consolidation of long term memory in both invertebrates and vertebrates. Here, we describe the expression and characterization of candidate plasticity gene 16 (cpg16), a protein serine/threonine kinase that was previously isolated from rat hippocampus as a plasticity-related gene. CPG16, when expressed in and purified from bacteria and COS7 cells, was only capable of autophosphorylation and phosphorylation of myelin basic protein but failed to phosphorylate many other peptides and proteins in in vitro phosphorylation assays. Recombinant CPG16, when overexpressed and purified from COS7 cells, had a relatively low level of autophosphorylation activity. This activity was significantly stimulated when cAMP-elevating agents (forskolin, 8-bromo-cAMP) were added to the cells but not by any other extracellular stimuli tested, e.g. serum, phorbol esters, and a calcium ionophore. Although the stimulation of CPG16 activity was inhibited by the cAMP-dependent protein kinase inhibitor H-89, it did not serve as a direct substrate for this kinase. This suggests that CPG16 may be activated by a cAMP-stimulated protein kinase cascade. Immunolocalization studies in COS7 and NIH-3T3 cells showed mostly cytoplasmic localization of CPG16 that turned partially nuclear upon stimulation with 8-bromo-cAMP. Moreover, overexpression of CPG16 seems to partially inhibit cAMP-stimulated activity of the transcription factor CREB (cAMP response element-binding protein), suggesting its involvement in the down-regulation of cAMP-induced transcription. Thus, CPG16 is a protein serine/threonine kinase that may be involved in a novel signaling pathway downstream of cAMP-dependent protein kinase.

3T3 Cells↗

PubMed2001

zyg-8, a gene required for spindle positioning in C. elegans, encodes a doublecortin-related kinase that promotes microtubule assembly.

Proper spindle positioning is essential for spatial control of cell division. Here, we show that zyg-8 plays a key role in spindle positioning during asymmetric division of one-cell stage C. elegans embryos by promoting microtubule assembly during anaphase. ZYG-8 harbors a kinase domain and a domain related to Doublecortin, a microtubule-associated protein (MAP) affected in patients with neuronal migration disorders. Sequencing of zyg-8 mutant alleles demonstrates that both domains are essential for function. ZYG-8 binds to microtubules in vitro, colocalizes with microtubules in vivo, and promotes stabilization of microtubules to drug or cold depolymerization in COS-7 cells. Our findings demonstrate that ZYG-8 is a MAP crucial for proper spindle positioning in C. elegans, and indicate that the function of the Doublecortin domain in modulating microtubule dynamics is conserved across metazoan evolution.

Amino Acid Sequence↗

PubMed1999

Genomic structure, chromosomal mapping, and expression pattern of human DCAMKL1 (KIAA0369), a homologue of DCX (XLIS).

Human DCAMKL1, also known as KIAA0369, is a homologue of DCX (Xq22. 3), a gene associated with X-linked lissencephaly and subcortical band heterotopia. This suggests that DCAMKL1 may play a role in neuronal migration. The gene also shows similarity to Ca2+/calmodulin-dependent protein kinases. We have determined its genomic structure, regional mapping, and expression pattern in human tissues. DCAMKL1 consists of at least 18 exons ranging from 58 to 3359 bp in length. We have characterized the exon/intron borders, and primers were designed to amplify each individual exon for mutation analysis. DCAMKL1 was mapped to chromosome 13q13 by fluorescence in situ hybridization. Northern blot analysis showed DCAMKL1 to be predominantly expressed in human fetal brain as a major transcript of about 5.8 kb.

Brain↗

PubMed1999

DCAMKL1, a brain-specific transmembrane protein on 13q12.3 that is similar to doublecortin (DCX).

Mutations in the human doublecortin (DCX), a brain-specific putative signaling protein, cause X-linked lissencephaly and subcortical band heterotopia. A predicted 740-amino-acid protein from human brain has two distinct regions, an N-terminal 345-amino-acid region 78% similar to the DCX protein and a C-terminal 427-amino-acid region that contains two transmembrane domains and is 98% homologous to a rat Ca2+/calmodulin-dependent protein kinase. We have designated this protein DCAMKL1. It maps to chromosome 13q12.3-q13, within a 540-kb YAC clone containing markers D13S805 and D13S1164. Northern analysis detected three major transcript isoforms of the DCAMKL1 gene expressed differentially and predominantly in human fetal and adult brain and during mouse embryogenesis (11-17 dpc). These results and its homology with the DCX and Ca2+/calmodulin dependent kinase proteins suggest a likely role for DCAMKL1 transmembrane protein in developing and adult brain, possibly in a pathway of cortical development.

Amino Acid Sequence↗

PubMed2000

A novel migration-related gene product, doublecortin, in neuronal migration disorder of fetuses and infants with Zellweger syndrome.

Immunohistochemical expression of doublecortin (DCX), KIAA0369 (KIA) and LIS1 proteins as well as nestin and vimentin in the cerebral cortices of six patients with Zellweger syndrome (ZS), aged 19 gestational weeks (GW) to 8 months, was compared with that in nine controls, aged 12 GW to 8 months. DCX immunoreactivity was apparently reduced in ZS, particularly in the cortical plate of fetuses, and in the subependymal foci of heterotopic neurons of the infants. Reduced expression of DCX in ZS was confirmed by Western blot analysis. On the other hand, neuronal expression of nestin was high in the cortical plate, migrating cells of the white matter and germinal cells in the ventricular zone in fetuses with ZS. Immunoreactivities for KIA, LIS1 and vimentin in ZS were comparable to those of controls. Reduced expression of DCX may be responsible for the neuronal migration disorder, and increased expression of nestin may be another evidence for delayed neuronal maturation in ZS.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1998

Expression and chromosomal localization of KIAA0369, a putative kinase structurally related to Doublecortin.

Neuropathy in vertebrates can be a consequence of failure of genes involved in the nervous system to be expressed at the correct times and levels during embryonic life. Recently, a brain specific gene, Doublecortin, was cloned and was shown to have mutations in X-linked lissencephaly and double cortex syndrome. KIAA0369 is a putative kinase that is structurally related to Doublecortin. We compared the expression of KIAA0369 with that of Doublecortin, both of which were expressed specifically or predominantly in fetal brain among 20 different tissues examined. The deduced products of both genes contain a unique domain (the Doublecortin [DC] domain), but KIAA0369 also contains a calmodulin-dependent kinase (CaM kinase)-like domain following the DC domain. We found at least four splicing variants of KIAA0369: KIAA0369-AS (type A, short version), KIAA0369-AL (type A, long version), KIAA0369-BS (type B, short version), and KIAA0369-BL (type B, long version). KIAA0369-B, which lacked the DC domain and maintained the kinase domain, was expressed in adult as well as fetal brain, but the variants that included the DC domain, KIAA0369-A, were expressed predominantly in fetal brain. These results suggest that the DC domain plays an important role in the development of the nervous system. In the adult brain, KIAA0369 was expressed in all 15 different regions examined, more intensely in cerebral cortex, occipital pole, frontal lobe, amygdala, and hippocampus, and less intensely in corpus callosum and thalamus. The murine homologs of Doublecortin and KIAA0369 were not detectable in 7-day mouse embryos, but both genes were expressed extensively in 11-day embryos. Human KIAA0369 was mapped by fluorescence in situ hybridization (FISH) to chromosome 13q13-q14.1. The presence of genes related to neuropathy has been reported in this locus.

Adult↗

PubMed1999

High expression of doublecortin and KIAA0369 protein in fetal brain suggests their specific role in neuronal migration.

The X-linked subcortical laminar heterotopia and lissencephaly syndrome is a disorder of neuronal migration caused by a mutation in XLIS, a recently cloned gene on chromosome Xq22.3-q23. The predicted protein product for XLIS, doublecortin (DC), shows high homology to a putative calcium calmodulin-dependent kinase, KIAA0369 protein (KI). Here we identified DC and KI in the brains of human and rat fetuses by immunochemical and immunohistochemical means. In this study, Western blotting demonstrated that both DC and KI are specific to the nervous system and are abundant during the fetal period, around 20 gestational weeks in humans and embryonic days 17 to 20 in rats. Immunostaining of the developing neocortex disclosed localization of DC and KI immunoreactivities in neuronal cell bodies and processes in the zones of ongoing neuronal migration. Although KI showed a somewhat wider distribution than DC, the temporal and spatial patterns of their expression were similar. These results suggest that DC and KI participate in a common signaling pathway regulating neuronal migration.

Animals↗

PubMed2003

Catalytic and regulatory domains of doublecortin kinase-1.

Doublecortin kinase-1 (DCK1) is a newly described multidomain protein kinase with a sequence significantly similar to those of both CaM kinases (CaMKs) and doublecortin, the product of the gene mutated in X-linked lissencephaly/double cortex syndrome, a severe developmental disorder of the nervous system. Functional studies have revealed microtubule binding and polymerization activities of the doublecortin domain, yet little is known regarding the enzymatic properties and regulation of the kinase catalytic domain. We have identified and report here notable similarities as well as differences between the catalytic and regulatory properties of DCK1 and those of the CaMKs. Using synthetic peptide substrates modeled on synapsin I, a substrate recognition motif for DCK1 of Hyd-Arg-Arg-X-X-Ser/Thr-Hyd was derived. The similarity of this motif to that of CaMKI [Lee, J. C., Kwon, Y.-G., Lawrence, D. S., and Edelman, A. M. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6413-6417] is consistent with the 59% level of amino acid sequence similarity between their catalytic domains. DCK1 catalytic activity is enhanced by mutagenic introduction of negative charge at Thr-239, a residue in a position equivalent to that of Thr-177 of CaMKI, the activation loop site for regulation by CaM kinase kinase. Unlike CaMKs, DCK1 is not directly activated by Ca(2+)-bound CaM. However, truncation of a pseudosubstrate-like sequence in the C-terminus of DCK1 results in an approximately 6-fold enhancement of activity. Thus, DCK1 demonstrates the potential to be regulated by relief of autoinhibition in response to signal(s) distinct from Ca(2+)-bound CaM and potentially by activation loop phosphorylation and to phosphorylate intracellular targets at sites similar to those recognized by CaMK pathways.

Animals↗

PubMed2004

Doublecortin kinase-2, a novel doublecortin-related protein kinase associated with terminal segments of axons and dendrites.

The microtubule (MT)-associated DCX protein plays an essential role in the development of the mammalian cerebral cortex. We report on the identification of a protein kinase, doublecortin kinase-2 (DCK2), with a domain (DC) highly homologous to DCX. DCK2 has MT binding activity associated with its DC domain and protein kinase activity mediated by a kinase domain, organized in a structure in which the two domains are functionally independent. Overexpression of DCK2 stabilizes the MT cytoskeleton against cold-induced depolymerization. Autophosphorylation of DCK2 strongly reduces its affinity for MTs. DCK2 and DCX mRNAs are nervous system-specific and are expressed during the period of cerebrocortical lamination. DCX is down-regulated postnatally, whereas DCK2 persists in abundance into adulthood, suggesting that the DC sequence has previously unrecognized functions in the mature nervous system. In sympathetic neurons, DCK2 is localized to the cell body and to the terminal segments of axons and dendrites. DCK2 may represent a phosphorylation-dependent switch for the reversible control of MT dynamics in the vicinity of neuronal growth cones.

Amino Acid Sequence↗

PubMed2003

Doublecortin functions at the extremities of growing neuronal processes.

Type I lissencephaly is a cortical malformation disorder characterized by disorganized cortical layers and gyral abnormalities and associated with severe cognitive impairment and epilepsy. The exact pathophysiological mechanisms underlying the epilepsy and mental retardation in this and related disorders remain unknown. Two genes, LIS1 and doublecortin, have both been shown to be mutated in a large proportion of cases of type I lissencephaly and a milder allelic disorder, subcortical laminar heterotopia (SCLH). Studying the protein products of these genes and the biochemical pathways in which they belong is likely to yield important information concerning both normal and abnormal cortical development. The relationships between the LIS1 and Doublecortin proteins are not yet well defined, but both are believed to play a critical role in cortical neuronal migration. Lis1 is expressed from very early development in the mouse and in both proliferating cells and post-mitotic neurons of the cortex. This protein is likely to have multiple functions since it is a subunit of the enzyme platelet-activating factor acetylhydrolase, which degrades platelet activating factor, and has also been shown to be involved in microtubule dynamics, potentially influencing nuclear migration through its interaction with the dynein motor protein complex. Doublecortin on the other hand is exclusively expressed in post-mitotic neurons and is developmentally regulated. In young developing neurons Doublecortin has a specific subcellular localization at the ends of neuritic and leading processes. This localization, combined with our previous data showing that it is a microtubule-associated protein and that it interacts with adapter complexes involved in vesicle trafficking, suggests a role in the growth of neuronal processes, downstream of directional or guidance signals. The observations summarized here favor the suggestion that whereas LIS1 may play a role in nuclear migration, Doublecortin is instead restricted to functions at the leading edge of the cell.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗