Minibrain homologs in invertebrates

Dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs) are an emerging family of protein kinases that have been identified in all eukaryotic organisms examined to date. DYRK family members are involved in regulating key developmental and cellular processes such as neurogenesis, cell proliferation, cytokinesis and cellular differentiation. Two distinct subgroups exist, nuclear and cytosolic. In Drosophila, the founding family member minibrain, whose human orthologue maps to the Down syndrome critical region, belongs to the nuclear subclass and affects post-embryonic neurogenesis. dDYRK2, a cytosolic DYRK and the putative product of the smell-impaired smi35A gene, is described. This is the second such kinase described in Drosophila, but the first to be characterized at the molecular and biochemical level. dDYRK2 is an 81 kDa dual-specificity kinase that autophosphorylates on tyrosine and serine/threonine residues, but appears to phosphorylate exogenous substrates only on serine/threonine residues. It contains a YXY motif in the activation loop of the kinase domain in the same location as the TXY motif in mitogen-activated protein kinases. dDYRK2 is tyrosine-phosphorylated in vivo, and mutational analysis reveals that the activation loop tyrosines are phosphorylated and are essential for kinase activity. Finally, dDYRK2 is active at all stages of fly development, with elevated levels observed during embryogenesis and pupation (Lochhead, 2003).

To shed light on the cellular role of human DYRK1A and related genes three DYRK/minibrain-like genes were identified in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. These genes are widely expressed and they localize to distinct subcellular compartments. Deletion alleles were isolated in all three genes; loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led prompted an examination of the effects of overexpression of its C. elegans ortholog mbk-1. Animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, it was shown that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. These results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible (Raich, 2003).

In the newly fertilized Caenorhabditis elegans zygote, cytoplasmic determinants become localized asymmetrically along the anterior-posterior (A-P) axis of the embryo. The mitotic apparatus then orients so as to cleave the embryo into anterior and posterior blastomeres that differ in both size and developmental potential. A role is described for MBK-2, a member of the Dyrk family of protein kinases, in asymmetric cell division in C. elegans. In mbk-2 mutants, the initial mitotic spindle is misplaced and cytoplasmic factors, including the germline-specific protein PIE-1, are mislocalized. These findings support a model in which MBK-2 down-regulates the katanin-related protein MEI-1 to control spindle positioning and acts through distinct, as yet unknown factors, to control the localization of cytoplasmic determinants. These findings in conjunction with work from Schizosaccharomyces pombe indicate a possible conserved role for Dyrk family kinases in the regulation of spindle placement during cell division (Pang, 2004).

Toward an understanding of divergent compound eye development in drones and workers of the honeybee (Apis mellifera L.): A correlative analysis of morphology and gene expression

Eye development in insects is best understood in Drosophila melanogaster, but little is known for other holometabolous insects. Combining a morphological with a gene expression analysis, this study investigated eye development in the honeybee, putting emphasis on the sex-specific differences in eye size. Optic lobe development starts from an optic lobe anlage in the larval brain, which sequentially gives rise to the lobula, medulla, and lamina. The lamina differentiates in the last larval instar, when it receives optic nerve projections from the developing retina. The expression analysis focused on seven genes important for Drosophila eye development: eyes absent, sine oculis, embryonic lethal abnormal vision, minibrain, small optic lobes, epidermal growth factor receptor, and roughest. All except small optic lobes were more highly expressed in third-instar drone larvae, but then, in the fourth and fifth instar, their expression was sex-specifically modulated, showing shifts in temporal dynamics. The clearest differences were seen for small optic lobes, which is highly expressed in the developing eye of workers, and minibrain and roughest, which showed a strong expression peak coinciding with retina differentiation. A microarray analysis for optic lobe/retina complexes revealed the differential expression of several metabolism-related genes, as well as of two micro-RNAs. While major morphological differences were not seen in the developing eye structures before the pupal stage, the expression differences observed for the seven candidate genes and in the transcriptional microarray profiles indicate that molecular signatures underlying sex-specific optic lobe and retina development become established throughout the larval stages (Marco Antonio, 2016).

Minibrain homologs in chickens

The Minibrain (Mnb) gene encodes a new family of protein kinases that is evolutionarily conserved from insects to humans. In Drosophila, Mnb is involved in postembryonic neurogenesis. In humans, MNB has been mapped within the Down's Syndrome (DS) critical region of chromosome 21 and is overexpressed in DS embryonic brain. In order to study a possible role of Mnb on the neurogenesis of vertebrate brain, the chick Mnb ortholog has been cloned and the spatiotemporal expression of Mnb in proliferative regions of the nervous system has been studied. In early embryos, Mnb is expressed before the onset of neurogenesis in the three general locations where neuronal precursors are originated: neuroepithelia of the neural tube, neural crest, and cranial placodes. Mnb is transiently expressed during a single cell cycle of neuroepithelial progenitor (NEP) cells. Mnb expression precedes and widely overlaps with the expression of Tis21, an antiproliferative gene that has been reported to be expressed in the onset of neurogenic divisions of NEP cells. Mnb transcription begins in mitosis, continues during G1, and stops before S-phase. Very interestingly, Mnb mRNA wt was found to be asymmetrically localized during the mitosis of these cells and inherited by one of the sibling cells after division. It is proposed that Mnb defines a transition step between proliferating and neurogenic divisions of NEP cells (Hammerle, 2002).

Cloning and expression of mammalian Minibrain homologs

The presence of an extra copy of human chromosome 21 (trisomy 21), in particular, region 21q22.2, causes many phenotypes in Down syndrome, including mental retardation. To study genes potentially responsible for some of these phenotypes, a human candidate gene (DYRK) from 21q22.2 and its murine counterpart (Dyrk) were cloned. These are homologous to the Drosophila minibrain gene required for neurogenesis and to the rat Dyrk gene (dual specificity tyrosine phosphorylation regulated kinase). The three mammalian genes are highly conserved, greater than 99% identical at the protein level over their 763-amino-acid (aa) open reading frame; in addition, the mammalian genes are 83% identical over 414 aa to the smaller 542-aa mnb protein. The predicted human DYRK and murine Dyrk proteins both contain a nuclear targeting signal sequence, a protein kinase domain, a putative leucine zipper motif, and a highly conserved 13-consecutive-histidine repeat. Fluorescence in situ hybridization and regional mapping data localize DYRK between markers D21S336 and D21S337 in the 21q22.2 region. Northern blot analysis indicates that both human and murine genes encode approximately 6-kb transcripts. PCR screening of cDNA libraries derived from various human and murine tissues indicate that DYRK and Dyrk are expressed both during development and in the adult. In situ hybridization of Dyrk to mouse embryos (13, 15, and 17 days postcoitus) indicates a differential spatial and temporal pattern of expression, with the most abundant signal localized in brain gray matter, spinal cord, and retina. The observed expression pattern is coincident with many of the clinical findings in trisomy 21. Its chromosomal locus (21q22. 2), its homology to the mnb gene, and the in situ hybridization expression patterns of the murine Dyrk, combined with the fact that transgenic mice for a yeast artificial chromosome (YAC) to which DYRK maps, are mentally deficient suggest that DYRK may be involved in the abnormal neurogenesis found in Down's syndrome (Song, 1996).

A human homolog of Drosophila mnb was isolated from the Down syndrome (DS) critical region. Human MNB encodes a 6.1 kb transcript that is expressed in fetal brain, lung, kidney and liver. Using a human probe, two major transcripts (6.1 and 3.1 kb) have been identified in mouse and expression detected in situ in several regions of the mouse brain, including the olfactory bulb, the cerebellum, the cerebral cortex, the pyramidal cell layer of the hippocampus and several hypothalamic nuclei. This expression pattern corresponds to the regions of the brain that are abnormal in individuals with DS and suggests that overexpression of MNB could have detrimental consequences in DS patients (Guimera, 1996).

Exon trapping was used to identify portions of human chromosome 21-encoded genes. More than 600 potential exons on the chromosome have been cloned and characterised to date. A BLAST search of databases revealed that three of these trapped "exons" (hmc18a08, hmc18f10 and hmc27g09) show strong homology to different regions of the Drosophila mnb and rat Dyrk genes, indicating that these three exons may also be portions of a human homolog to the rat and Drosophila genes. With amplification by the polymerase chain reaction and hybridization analysis the human MNB gene was mapped on overlapping yeast artificial chromosomes 336G11 and 806A11 of chromosome 21q22.2 between markers D21S65 and ERG. The Dyrk gene, which encodes a dual specificity protein kinase, is a rat homolog of the Drosophila mnb gene. The kinase activity is dependent on tyrosine residues in the catalytic domain, and it has been speculated that the protein is involved in control of the cell cycle. Altered expression of the human MNB gene may be involved in the pathogenesis of certain phenotypes of Down's syndrome, including mental retardation (Chen, 1997).

To isolate genes responsible for some features of Down's syndrome, exon trapping experiments were carried out using a series of cosmid clones derived from "the Down's syndrome critical region" of chromosome 21. Six exons were trapped that are highly homologous to the sequence of Drosophila minibrain gene. Using one of these six exons as a probe, cDNA clones were isolated for a human homolog of Drosophila from a fetal brain cDNA library. Human MNB cDNA encodes a protein of 754 amino acids with a nuclear targeting sequence and a catalytic domain common to the serine/threonine-specific protein kinase. The human MNB protein strikingly resembles the recently discovered rat Dyrk protein kinase with a dual specificity. The MNB mRNA is expressed in various tissues including fetal and adult brains. The remarkable similarity of human MNB protein to Drosophila Mnb and rat Dyrk proteins implies that human MNB protein may play a significant role in a signaling pathway regulating nuclear functions of neuronal cell proliferation, contributing to certain features of Down's syndrome (Shindoh, 1997).

The Minibrain (Mnb) gene belongs to a new protein kinase family, which is evolutionarily conserved, and probably plays several roles during brain development and in adulthood. In Drosophila, mnb is involved in postembryonic neurogenesis and in learning/memory. In humans, MNB has been mapped within the Down syndrome critical region of chromosome 21 and is overexpressed in the Down syndrome embryonic brain. It has been widely proposed that MNB is involved in the neurobiological alterations associated with Down syndrome. Nevertheless, little is known about the functional role that MNB plays in vertebrate brain development. In early vertebrate embryos, Mnb is transiently expressed in neural progenitor cells during the transition from proliferating to neurogenic divisions. A second wave of Mnb expression, which takes place in the brain of intermediate and late vertebrate embryos, has been studied in detail. In these stages, MNB seems to be restricted to certain populations of neurons, since no consistent expression is detected in astroglial or oligodendroglial cells. Interestingly, MNB expression takes place at the time of dendritic tree differentiation and is initiated by a transient translocation from the cytoplasm to the nucleus. Afterwards, MNB protein is transported to the growing dendritic tree, where it colocalizes with Dynamin 1, a putative substrate of MNB kinases. It is proposed that MNB kinase is involved in the signalling mechanisms that regulate dendrite differentiation. This functional role helps to build a new hypothesis for the implication of MNB/DYRK1A in the developmental aetiology of Down syndrome neuropathologies (Hammerle, 2003 ).

Functional studies of mammalian Minibrain

Using Down's syndrome as a model for complex trait analysis, loci from chromosome 21q22.2 were sought which, when present in an extra dose, contribute to learning abnormalities. Low-copy-number transgenic mice were generated, containing four different yeast artificial chromosomes (YACs) that together cover approximately 2 megabases (Mb) of contiguous DNA from 21q22.2. Independent lines derived from each of these YAC transgenes were subjected to a series of behavioural and learning assays. Two of the four YACs caused defects in learning and memory in the transgenic animals, while the other two YACs had no effect. The most severe defects were caused by a 570-kb YAC; the interval responsible for these defects was narrowed to a 180-kb critical region as a consequence of YAC fragmentation. This region contains the human homolog of a Drosophila gene, minibrain, and strongly implicates it in learning defects associated with Down's syndrome (Smith, 1997).

DYRK1A is the human orthologue of the Drosophila minibrain, which is involved in postembryonic neurogenesis in flies. Because of its mapping position on chromosome 21 and the neurobehavioral alterations shown by mice overexpressing this gene, involvement of DYRK1A in some of the neurological defects of Down syndrome patients has been suggested. To gain insight into its physiological role, mice deficient in Dyrk1A function were generated by gene targeting. Dyrk1A(-/-) null mutants present a general growth delay and die during midgestation. Mice heterozygous for the mutation show decreased neonatal viability and a significant body size reduction from birth to adulthood. General neurobehavioral analysis revealed preweaning developmental delay of heterozygous mice and specific alterations in adults. Brains of heterozygous mice were decreased in size in a region-specific manner, although the cytoarchitecture and neuronal components in most areas were not altered. Cell counts showed increased neuronal densities in some brain regions and a specific decrease in the number of neurons in the superior colliculus, which exhibited a significant size reduction. These data provide evidence about the nonredundant, vital role of Dyrk1A and suggest a conserved mode of action that determines normal growth and brain size in both mice and flies (Fotaki, 2002).

The minibrain kinase (Mnbk)/dual specificity Yak 1-related kinase 1A (Dyrk1A) gene is implicated in the mental retardation associated with Down's syndrome. It encodes a proline-directed serine/threonine kinase whose function has yet to be defined. A solid-phase Mnbk/Dyrk1A kinase assay was used to aid in the search for the cellular Mnbk/Dyrk1A substrates. The assay revealed that rat brain contains two cytosolic proteins, one with a molecular mass of 100 kDa and one with a molecular mass of 140 kDa, that were prominently phosphorylated by Mnbk/Dyrk1A. The 100-kDa protein was purified and identified as dynamin 1. This conclusion was further supported by evidence that a recombinant glutathione S-transferase fusion protein containing dynamin isoform 1aa was phosphorylated by Mnbk/Dyrk1A. In addition to isoform 1aa, Mnbk/Dyrk1A also phosphorylates isoforms 1ab and 2aa but not human MxA protein when analyzed by the solid-phase kinase assay. Upon Mnbk/Dyrk1A phosphorylation, the interaction of dynamin 1 with the Src homology 3 domain of amphiphysin 1 is reduced. However, when Mnbk/Dyrk1A phosphorylation is allowed to proceed more extensively, the phosphorylation enhances rather than reduces the binding of dynamin 1 to amphiphysin 1. The result suggests that Mnbk/Dyrk1A can play a dual role in regulating the interaction of dynamin 1 with amphiphysin 1. Mnbk/Dyrk1A phosphorylation also reduces the interaction of dynamin with endophilin 1, whereas the same phosphorylation enhances the binding of dynamin 1 to Grb2. Nevertheless, the dual function of Mnbk/Dyrk1A phosphorylation is not observed for the interaction of dynamin 1 with endophilin 1 or Grb2. The interactions of dynamin with amphiphysin and endophilin are essential for the formation of endocytic complexes; these results suggest that Mnbk/Dyrk1A may function as a regulator controlling the assembly of endocytic apparatus (Chen-Hwang, 2002).

The Rho family of small GTPases regulates numerous signaling pathways that control the organization of the cytoskeleton, transcription factor activity, and many aspects of the differentiation of skeletal myoblasts. The kinase Mirk (minibrain-related kinase)/dyrk1B is induced by members of the Rho-family in myoblasts and that Mirk is active in skeletal muscle differentiation. Mirk is an arginine-directed serine/threonine kinase that is expressed at elevated levels in skeletal muscle compared with other normal tissues. A Mirk promoter construct is activated when C2C12 myoblasts are switched from growth to differentiation medium and is also activated by the Rho family members RhoA, Cdc42, and to a lesser degree Rac1, but not by MyoD or Myf5. Mirk protein levels increase following transient expression of constitutively active Cdc42-QL, RhoA-QL, or Rac1-QL in C2C12 cells. High concentrations of serum mitogens down-regulate Mirk through activation of the Ras-MEK-Erk pathway. As a result, Mirk transcription is induced by the MEK1 inhibitor PD98059 and by the switch from growth to differentiation medium. Mirk is induced with similar kinetics to another Rho-induced differentiation gene, myogenin. Mirk protein levels increased 10-fold within 24-48 h after primary cultured muscle cells; C2C12 mouse myoblasts or L6 rat myoblasts were induced to differentiate. Thus Mirk is induced following the commitment stage of myogenesis. Stable overexpression of Mirk enables myoblasts to fuse more rapidly when placed in differentiation medium. The function of Mirk in muscle differentiation was established by depletion of endogenous Mirk by small interfering RNA, which prevents myoblast fusion into myotubes and inhibits induction of markers of differentiation, including myogenin, fast twitch troponin T, and muscle myosin heavy chain. Other members of the dyrk/minibrain/HIPK family of kinases in lower organisms have been shown to regulate the transition from growth to differentiation, and Mirk is now shown to participate in skeletal muscle development (Deng, 2003 ).

Minibrain-related kinase (Mirk)/Dyrk1B is an arginine-directed serine/threonine kinase that is active in skeletal muscle development but is also expressed in various carcinomas. This study using yeast two-hybrid analysis identifies the Met adaptor protein Ran-binding protein M (RanBPM) as a Mirk-binding protein. The Mirk-RanBPM association was confirmed by glutathione S-transferase pull-down assays, co-immunoprecipitation studies, and in vivo cross-linking. Met plays an important role in tumor cell invasion and cell migration. RanBPM has been reported to bind to the tyrosine kinase domain of the hepatocyte growth factor (HGF) receptor Met, enhance Met downstream signaling, and enhance HGF-induced A704 kidney carcinoma cell invasion. A stable Mirk-inducible subline was made from nontransformed Mv1Lu lung epithelial cells; induction of Mirk inhibits the migration of these cells in wounding experiments and inhibits their invasion through polycarbonate Transwell filters. Furthermore the ability of Mirk to inhibit Mv1Lu cell migration is attenuated when cells are exposed to HGF or to elevated levels of transiently expressed RanBPM. RanBPM inhibits the kinase activity of Mirk/Dyrk1B and Dyrk1A. In addition, RanBPM and HGF inhibits the function of Mirk as a transcriptional coactivator. These findings suggest that Mirk plays a role in modulating cell migration through opposing the action of the Met signaling cascade adaptor protein RanBPM (Zou, 2003).

Autophosphorylation of a critical residue in the activation loop of several protein kinases is an essential maturation event required for full enzyme activity. However, the molecular mechanism by which this happens is unknown. This question was addressed for two dual-specificity tyrosine-phosphorylation-regulated protein kinases (DYRKs), since they autophosphorylate their activation loop on an essential tyrosine but phosphorylate their substrates on serine and threonine. Autophosphorylation of the critical activation-loop tyrosine is intramolecular and mediated by the nascent kinase passing through a transitory intermediate form. This DYRK intermediate differs in residue and substrate specificity, as well as sensitivity to small-molecule inhibitors, compared with its mature counterpart. The intermediate's characteristics are lost upon completion of translation, making the critical tyrosine autophosphorylation a "one-off" inceptive event. This mechanism is likely to be shared with other kinases (Lochhead, 2005).

Dual-specificity tyrosine-phosphorylated and regulated kinase 1A (Dyrk1A) is the human homologue of Drosophila Minibrain. In Drosophila, mnb is involved in postembryonic neurogenesis. In human, DYRK1A maps within the Down syndrome critical region of chromosome 21 and is overexpressed in Down syndrome embryonic brain. Despite its potential involvement in the neurobiological alterations observed in Down syndrome patients, the biological functions of the serine/threonine kinase DYRK1A have not yet been identified. DYRK1A overexpression potentiates nerve growth factor (NGF)-mediated PC12 neuronal differentiation by up-regulating the Ras/MAP kinase signaling pathway independently of its kinase activity. Furthermore, DYRK1A prolongs the kinetics of ERK activation by interacting with Ras, B-Raf, and MEK1 to facilitate the formation of a Ras/B-Raf/MEK1 multiprotein complex. These data indicate that DYRK1A may play a critical role in Ras-dependent transducing signals that are required for promoting or maintaining neuronal differentiation and suggest that overexpression of DYRK1A may contribute to the neurological abnormalities observed in Down syndrome patients (Kelly, 2005).

Protein interactions of Minibrain homologs

Minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase (Mnb/Dyrk1A) is a proline-directed serine/threonine kinase encoded in the Down syndrome critical region of human chromosome 21. This kinase has been shown to phosphorylate dynamin 1 and synaptojanin 1. Amphiphysin I (Amph I) is also a Mnb/Dyrk1A substrate. This kinase phosphorylated native Amph I in rodent brains and recombinant human Amph I expressed in Escherichia coli. Serine 293 (Ser-293) was identified as the major site, whereas serine 295 and threonine 310 were found as minor kinase sites. In cultured cells, recombinant Amph I was phosphorylated at Ser-293 by endogenous kinase(s). Because MAPK/ERK has been suggested to phosphorylate Amph I at Ser-293, whether Ser-293 is phosphorylated in vivo by MAPK/ERK or by Mnb/Dyrk1A was addressed. Overnight serum-withdrawal inactivated MAPK/ERK; nonetheless, Ser-293 was phosphorylated in Chinese hamster ovary and SY5Y cells. Epigallocatechin-3-gallate, a potent Mnb/Dyrk1A inhibitor in vitro, apparently reduced the phosphorylation at Ser-293, whereas PD98059, a potent MAPK/ERK inhibitor, did not. High frequency stimulation of mouse hippocampal slices reduced the phosphorylation at Ser-293, albeit in the midst of MAPK/ERK activation. The endophilin binding in vitro was inhibited by phosphorylating Amph I with Mnb/Dyrk1A. However, phosphorylation at Ser-293 did not appear to alter cellular distribution patterns of the protein. These results suggest that Mnb/Dyrk1A, not MAPK/ERK, is responsible for in vivo phosphorylation of Amph I at Ser-293 and that phosphorylation changes the recruitment of endophilin at the endocytic sites (Murakami, 2006).

The DYRKs (dual specificity tyrosine phosphorylation-regulated kinases) are a conserved family of protein kinases that autophosphorylate a tyrosine residue in their activation loop by an intra-molecular mechanism and phosphorylate exogenous substrates on serine/threonine residues. Little is known about the identity of true substrates for DYRK family members and their binding partners. To address this question, full-length dDYRK2 (Drosophila DYRK2) was used as bait in a yeast two-hybrid screen of a Drosophila embryo cDNA library. Of 14 independent dDYRK2 interacting clones identified, three were derived from the chromatin remodelling factor, SNR1 (Snf5-related 1), and three from the essential chromatin component, TRX (trithorax). The association of dDYRK2 with SNR1 and TRX was confirmed by co-immunoprecipitation studies. Deletion analysis showed that the C-terminus of dDYRK2 modulated the interaction with SNR1 and TRX. DYRK family member MNB (Minibrain) was also found to co-precipitate with SNR1 and TRX, associations that did not require the C-terminus of the molecule. dDYRK2 and MNB were also found to phosphorylate SNR1 at Thr102 in vitro and in vivo. This phosphorylation required the highly conserved DH-box (DYRK homology box) of dDYRK2, whereas the DH-box was not essential for phosphorylation by MNB. This is the first instance of phosphorylation of SNR1 or any of its homologues and implicates the DYRK family of kinases with a role in chromatin remodelling (Kinstrie, 2006. Full text of article).

In human, DYRK1A encodes a serine-threonine kinase but despite its potential involvement in the neurobiological alterations associated with Down syndrome. Its physiological function has not yet been defined. To gain some insight into its biological function, the yeast two-hybrid approach was used to identify binding partners of DYRK1A. The C-terminal region of DYRK1A interacts with a brain specific protein, phytanoyl-CoA alpha-hydroxylase-associated protein 1 (PAHX-AP1, also named PHYHIP) which interacts with phytanoyl-CoA alpha-hydroxylase (PAHX, also named PHYH), a Refsum disease gene product. This interaction was confirmed by co-immunoprecipitation of PC12 cells co-transfected with DYRK1A and PAHX-AP1. Furthermore, immunofluorescence analysis of PC12 cells co-transfected with both plasmids showed a re-distribution of DYRK1A from the nucleus to the cytoplasm where it co-localized with PAHX-AP1. Finally, in PC12 cells co-transfected with both plasmids, DYRK1A was no longer able to interact with the nuclear transcription factor CREB, thereby confirming that the intracellular localization of DYRK1A was changed from the nucleus to the cytoplasm in the presence of PAHX-AP1. Therefore, these data indicate that by inducing a re-localization of DYRK1A into the cytoplasm, PAHX-AP1 may contribute to new cellular functions of DYRK1A and suggest that PAHX-AP1 may be involved in the development of neurological abnormalities observed in Down syndrome patients (Bescond, 2005).

Lewy bodies (LBs) are pathological hallmarks of Parkinson disease (PD) but also occur in Alzheimer disease (AD) and dementia of LBs. Alpha-synuclein, the major component of LBs, is observed in the brain of Down syndrome (DS) patients with AD. Dyrk1A, a dual specificity tyrosine-regulated kinase (Dyrk) family member, is the mammalian ortholog of the Drosophila minibrain (Mnb) gene, essential for normal postembryonic neurogenesis. The Dyrk1A gene resides in the human chromosome 21q22.2 region, which is associated with DS anomalies, including mental retardation. This study examined whether Dyrk1A interacts with alpha-synuclein and subsequently affects intracellular alpha-synuclein inclusion formation in immortalized hippocampal neuronal (H19-7) cells. Dyrk1A selectively binds to alpha-synuclein in transformed and primary neuronal cells. Alpha-synuclein overexpression, followed by basic fibroblast growth factor-induced neuronal differentiation, resulted in cell death. It was observed that accompanying cell death was increased alpha-synuclein phosphorylation and intracytoplasmic aggregation. In addition, the transfection of kinase-inactive Dyrk1A or Dyrk1A small interfering RNA blocked alpha-synuclein phosphorylation and aggregate formation. In vitro kinase assay of anti-Dyrk1A immunocomplexes demonstrated that Dyrk1A could phosphorylate alpha-synuclein at Ser-87. Furthermore, aggregates formed by phosphorylated alpha-synuclein have a distinct morphology and are more neurotoxic compared with aggregates composed of unmodified wild type alpha-synuclein. These findings suggest alpha-synuclein inclusion formation regulated by Dyrk1A, potentially affecting neuronal cell viability (Kim, 2006).

MNB/DYRK1A is a proline-directed serine/threonine kinase implicated in Down syndrome (DS). In an earlier screening, two proteins from adult rat brain, one 100kDa and the other 140 kDa, were found to be prominently phosphorylated by the kinase. The 100-kDa protein was previously characterized as an isoform of dynamin 1. In this study, the 140-kDa protein was identified as synaptojanin 1 (SJ1). MNB/DYRK1A phosphorylates SJ1 at multiple sites and produces complex behaviors in binding to amphiphysin 1 and intersectin 1 (ITSN1). However, the phosphorylation has little effect on the phosphatidylinositol phosphatase activity of SJ1. These results suggest that MNB/DYRK1A is involved in regulating the recruitment activity but not the phosphatase activity of SJ1. These findings may be especially important in the etiology of DS because MNB/DYRK1A, SJ1, and ITSN1 are all located at or near the region of human chromosome 21, which is postulated to be involved in the disease (Adayev, 2006).

The precise regulation of programmed cell death is critical for the normal development of the nervous system. This study shows that DYRK1A (minibrain), a protein kinase essential for normal growth, is a negative regulator of the intrinsic apoptotic pathway in the developing retina. Evidence is provided that changes in Dyrk1A gene dosage in the mouse strongly alter the cellularity of inner retina layers and result in severe functional alterations. DYRK1A does not affect the proliferation or specification of retina progenitor cells, but rather regulates the number of cells that die by apoptosis. DYRK1A phosphorylates caspase-9 on threonine residue 125, and this phosphorylation event is crucial to protect retina cells from apoptotic cell death. The data suggest a model in which dysregulation of the apoptotic response in differentiating neurons participates in the neuropathology of diseases that display DYRK1A gene-dosage imbalance effects, such as Down's syndrome (Laguna, 2008).

The dual-specific kinase DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) is the mammalian orthologue of the Drosophila minibrain (MNB) protein kinase and executes diverse roles in neuronal development and adult brain physiology. DYRK1A is overexpressed in Down syndrome (DS) and has recently been implicated in several neurodegenerative diseases. In an attempt to elucidate the molecular basis of its involvement in cognitive and neurodegeneration processes, novel proteins were sought interacting with the kinase domain of DYRK1A in the adult mouse brain and septin 4 (SEPT4, also known as Pnutl2/CDCrel-2) was identified. SEPT4 is a member of the group III septin family of guanosine triphosphate hydrolases (GTPases), which has previously been found in neurofibrillary tangles of Alzheimer disease brains and in alpha-synuclein-positive cytoplasmic inclusions in Parkinson disease brains. In transfected mammalian cells, DYRK1A specifically interacts with and phosphorylates SEPT4. Phosphorylation of SEPT4 by DYRK1A was inhibited by harmine, which has recently been identified as the most specific inhibitor of DYRK1A. In support of a physiological relation in the brain, it was found that Dyrk1A and Sept4 are co-expressed and co-localized in neocortical neurons. These findings suggest that SEPT4 is a substrate of DYRK1A kinase and thus provide a possible link for the involvement of DYRK1A in neurodegenerative processes and in DS neuropathologies (Sitz, 2008).

Raf-MEK-extracellular signal-regulated kinase (Erk) signaling initiated by growth factor-engaged receptor tyrosine kinases (RTKs) is modulated by an intricate network of positive and negative feedback loops which determine the specificity and spatiotemporal characteristics of the intracellular signal. Well-known antagonists of RTK signaling are the Sprouty proteins. The activity of Sprouty proteins is modulated by phosphorylation. However, little is known about the kinases responsible for these posttranslational modifications. This study identifies DYRK1A as one of the protein kinases of Sprouty2. DYRK1A interacts with and regulates the phosphorylation status of Sprouty2. Moreover, Thr75 on Sprouty2 is identified as a DYRK1A phosphorylation site in vitro and in vivo. This site is functional, since its mutation enhances the repressive function of Sprouty2 on fibroblast growth factor (FGF)-induced Erk signaling. Further supporting the idea of a functional interaction, DYRK1A and Sprouty2 are present in protein complexes in mouse brain, where their expression overlaps in several structures. Moreover, both proteins copurify with the synaptic plasma membrane fraction of a crude synaptosomal preparation and colocalize in growth cones, pointing to a role in nerve terminals. These results suggest, therefore, that DYRK1A positively regulates FGF-mitogen-activated protein kinase signaling by phosphorylation-dependent impairment of the inhibitory activity of Sprouty2 (Aranda, 2008).

Attenuation of Notch signalling by the Down-syndrome-associated kinase DYRK1A

Notch signalling is used throughout the animal kingdom to spatially and temporally regulate cell fate, proliferation and differentiation. Its importance is reflected in the dramatic effects produced on both development and health by small variations in the strength of the Notch signal. The Down-syndrome-associated kinase DYRK1A is coexpressed with Notch in various tissues during embryonic development. DYRK1A moves to the nuclear transcription compartment where it interacts with the intracellular domain of Notch promoting its phosphorylation in the ankyrin domain and reducing its capacity to sustain transcription. DYRK1A attenuates Notch signalling in neural cells both in culture and in vivo, constituting a novel mechanism capable of modulating different developmental processes that can also contribute to the alterations observed during brain development in animal models of Down syndrome (Fernandez-Martinez, 2009).

The modulation of other signalling events by DYRK1A has been described previously, with the kinase activity of DYRK1A often being found to be dispensable for the regulation. By contrast, this study shows here that the attenuation of Notch signalling by DYRK1A is dependent on its kinase activity, compatible with previous findings indicating that the intracellular domain of Notch is a substrate of several kinases that can modulate its activity. Interestingly, the domains previously described as targets for phosphorylation TAD and OPA/PEST are dispensable for the regulation of Notch signalling by DYRK1A. Indeed, mapping analysis shows that DYRK1A phosphorylates the RAM-ANK domain (Fernandez-Martinez, 2009).

To identify the phosphorylation sites in Notch, the potential sites were examined based on the described consensus sequence (RPXS/TP), Notch crystal structure and their conservation in different species. Single substitutions in any of the putative 18 conserved serines and threonines that could be targets of DYRK1A did not reduce the attenuation caused by the kinase. These substitutions included the best conserved sites corresponding to described consensus sequence (RPXS/TP motifs) within the TPLH sequence at positions 4-7 of the ANK repeats 1, 3, 4 and 6 of Notch1. These results indicate that DYRK1A phosphorylates Notch at multiple sites. Double substitutions in different combinations of ANK repeats dramatically decreased Notch activity, but DYRK1A could still attenuate the diminished response. These data highlight the importance of the threonines in the maintenance of the ANK structure and preclude further analysis of multiple mutations in the context of DYRK1A activity. To overcome this problem, 2-D electrophoresis analysis was carried out, and it was confirmed that DYRK1A induces multiple phosphorylation events in the NICD as shown by the large shift observed when both proteins were coexpressed. Interestingly, whereas the phosphorylation of the PEST region by CDK8 targets the NICD to a degradation pathway, phosphorylation events mediated by DYRK1A do not affect the stability of the Notch protein. Thus, DYRK1A attenuates Notch activity by phosphorylation in multiple residues without affecting its stability (Fernandez-Martinez, 2009).

Although DYRK1A performs some of its activities in a kinase-independent manner, the data indicate that the relationship between DYRK1A and the NICD needs the kinase activity, suggesting an enzyme-substrate type interaction. Indeed, the data indicate that the interaction is very transient and thus, difficult to detect. The use of the kinase-inactive form of DYRK1A greatly favoured the detection of the complex, consistent with the formation of a stable, albeit unproductive, complex with Notch. Hence, it is concluded that DYRK1A and the NICD transiently associate in the nucleus, an association that is stabilized when the kinase is inactive (Fernandez-Martinez, 2009).

Besides the effect that DYRK1A exerts on the transcriptional activity of Notch signalling reporters, whether the expression of DYRK1A could affect some of the activities that Notch signalling performs in vivo was investigated. Notch signalling can prevent the maturation of neurons. Whether the reduction in neurite development in cells with an activated form of the NICD could be reversed by the presence of DYRK1A was examined. Indeed, it was found that the repression in neuritogenesis was released in the presence of DYRK1A, indicating that the response of the cell to Notch signalling was attenuated (Fernandez-Martinez, 2009).

As Notch signalling is used iteratively to control cell proliferation, determination or differentiation in the developing neural tube, it was used as an in vivo model to study the effect of DYRK1A on Notch signalling. The overexpression of DYRK1A in the developing neural tube repressed the expression of Hes5-1, a well known indicator of Notch signalling, confirming that DYRK1A is sufficient to attenuate endogenous Notch signalling in vivo (Fernandez-Martinez, 2009).

In Drosophila the lack of function mutant minibrain (mnb), the fly DYRK1A homologue, results in a reduced brain size because of a decrease of the generation of cells during postembryonic development and similarly, Dyrk1a+/– mice also have smaller brains. Although an abnormal increase in Notch signalling inhibits neuronal differentiation in mice, deficient Notch signalling also leads to a reduction in the number of neurons in the adult cause by the induction of precocious neuronal differentiation. Thus, these data are compatible with the finding that DIRK1A attenuates Notch signalling both in vitro and in vivo. Interestingly, the data may be also relevant for studies of Down syndrome (DS). Indeed, DYRK1A is one of the genes located in the Down syndrome critical region and its expression is upregulated in DS individuals. Notch signalling is also altered in the DS condition, although contrasting data have been obtained in studies in humans and mouse models. Although Notch signalling seems to be upregulated in the cortex of DS individuals, it is repressed in the cerebellum of Ts1Cje mice, a model for DS. Several factors could account for these apparently contradictory results. First, the human DS condition also produces an increase in the expression of the Notch receptor and changes in the expression of other Notch modifiers such as Dlx. The presence of DYRK1A in these cells, with a clear misbalance in gene expression, might result in an attenuation of an otherwise augmented Notch signalling and be coherent with the results in which Notch output is upregulated or downregulated. Nevertheless, it is believed that the clear effects of DYRK1A in the attenuation of Notch signalling that are describe in this study can have a higher impact on DS during embryonic development, when DYRK1A is prominently expressed in Notch expressing areas (Fernandez-Martinez, 2009).

In summary, DYRK1A is able to attenuate Notch signalling both in neuroblastoma cells and in vivo, providing further insight into the mechanisms by which neurogenesis and other cell decisions mediated by Notch signalling can be modulated both in physiological and pathological conditions. Indeed, its ability to downregulate Notch signalling could contribute to the severe alterations in the formation of certain brain regions observed in animal models and associated with the development of Down syndrome in humans. Finally, the widespread expression of DYRK1A opens up the possibility that it can also modulate Notch signalling in other tissues (Fernandez-Martinez, 2009).

Transient expression of Mnb/Dyrk1a couples cell cycle exit and differentiation of neuronal precursors by inducing p27KIP1 expression and suppressing NOTCH signaling

The decision of a neural precursor to stop dividing and begin its terminal differentiation at the correct place, and at the right time, is a crucial step in the generation of cell diversity in the nervous system. This study shows that the Down's syndrome candidate gene (Mnb/Dyrk1a) is transiently expressed in prospective neurons of vertebrate CNS neuroepithelia. The gain of function (GoF) of Mnb/Dyrk1a induces proliferation arrest. Conversely, its loss of function (LoF) caused over proliferation and cell death. It was found that MNB/DYRK1A is both necessary and sufficient to upregulate, at transcriptional level, the expression of the cyclin-dependent kinase inhibitor p27KIP1 in the embryonic chick spinal cord and mouse telencephalon, supporting a regulatory role for MNB/DYRK1A in cell cycle exit of vertebrate CNS neurons. All these actions required the kinase activity of MNB/DYRK1A. It was also observed that MNB/DYRK1A is co-expressed with the Notch ligand Delta1 in single neuronal precursors. Furthermore, MNB/DYRK1A was found to suppress Notch signaling, counteracting the pro-proliferative action of the Notch intracellular domain (NICD), stimulating Delta1 expression, and is required for the neuronal differentiation induced by the decrease in Notch signaling. Nevertheless, although Mnb/Dyrk1a GoF leads to extensive withdrawal of neuronal precursors from the cell cycle, it is insufficient to elicit their differentiation. Remarkably, a transient (ON/OFF) Mnb/Dyrk1a GoF efficiently induces neuronal differentiation. It is proposed that the transient expression of MNB/DYRK1A in neuronal precursors acts as a binary switch, coupling the end of proliferation and the initiation of neuronal differentiation by upregulating p27KIP1 expression and suppressing Notch signaling (Hämmerle, 2011).

Prefrontal deficits in a murine model overexpressing the down syndrome candidate gene dyrk1a

The gene Dyrk1a is the mammalian ortholog of Drosophila minibrain. Dyrk1a localizes in the Down syndrome (DS) critical region of chromosome 21q22.2 and is a major candidate for the behavioral and neuronal abnormalities associated with DS. Prefrontal cortex (PFC) malfunctions are a common denominator in several neuropsychiatric diseases, including DS, but the contribution of DYRK1A in PFC dysfunctions, in particular the synaptic basis for impairments of executive functions reported in DS patients, remains obscure. This study quantified synaptic plasticity, biochemical synaptic markers, and dendritic morphology of deep layer pyramidal PFC neurons in adult mBACtgDyrk1a transgenic mice that overexpress Dyrk1a under the control of its own regulatory sequences. It was found that overexpression of Dyrk1a largely increased the number of spines on oblique dendrites of pyramidal neurons, as evidenced by augmented spine density, higher PSD95 protein levels, and larger miniature EPSCs. The dendritic alterations were associated with anomalous NMDAR-mediated long-term potentiation and accompanied by a marked reduction in the pCaMKII/CaMKII ratio in mBACtgDyrk1a mice. Retrograde endocannabinoid-mediated long-term depression (eCB-LTD) was ablated in mBACtgDyrk1a mice. Administration of green tea extracts containing epigallocatechin 3-gallate, a potent DYRK1A inhibitor, to adult mBACtgDyrk1a mice normalized long-term potentiation and spine anomalies but not eCB-LTD. However, inhibition of the eCB deactivating enzyme monoacylglycerol lipase normalized eCB-LTD in mBACtgDyrk1a mice. These data shed light on previously undisclosed participation of DYRK1A in adult PFC dendritic structures and synaptic plasticity. Furthermore, they suggest its involvement in DS-related endophenotypes and identify new potential therapeutic strategies (Thomazeau, 2014).

Increased dosage of DYRK1A and DSCR1 delays neuronal differentiation in neocortical progenitor cells

Down's syndrome (DS), a major genetic cause of mental retardation, arises from triplication of genes on human chromosome 21. This study shows that DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A; Drosophila homolog - Minibrain) and DSCR1 (DS critical region 1; Drosophila homolog - Nebula/Sarah), two genes lying within human chromosome 21 and encoding for a serine/threonine kinase and calcineurin regulator, respectively, are expressed in neural progenitors in the mouse developing neocortex. Increasing the dosage of both proteins in neural progenitors leads to a delay in neuronal differentiation, resulting ultimately in alteration of their laminar fate. This defect is mediated by the cooperative actions of DYRK1A and DSCR1 in suppressing the activity of the transcription factor NFATc. In Ts1Cje mice, a DS mouse model, dysregulation of NFATc in conjunction with increased levels of DYRK1A and DSCR1 were observed. Furthermore, counteracting the dysregulated pathway ameliorates the delayed neuronal differentiation observed in Ts1Cje mice. In sum, these findings suggest that dosage of DYRK1A and DSCR1 is critical for proper neurogenesis through NFATc and provide a potential mechanism to explain the neurodevelopmental defects in DS (Kurabayashi, 2013).

minibrain: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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