Interactive Fly, Drosophila

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 ).

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

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).

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

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