Down's syndrome (DS) is a major cause of mental retardation, hypotonia and delayed development. Murine models of DS carrying large murine or human genomic fragments show motor alterations and memory deficits. The specific genes responsible for these phenotypic alterations have not yet been defined. DYRK1A, the human homolog of the Drosophila minibrain gene, maps to the DS critical region of human chromosome 21 and is overexpressed in DS fetal brain. DYRK1A encodes a serine-threonine kinase, probably involved in neuroblast proliferation. Mutant Drosophila minibrain flies have a reduction in both optic lobes and central brain, showing learning deficits and hypoactivity. We have generated transgenic mice (TgDyrk1A) overexpressing the full-length cDNA of Dyrk1A. TgDyrk1A mice exhibit delayed cranio-caudal maturation with functional consequences in neuromotor development. TgDyrk1A mice also show altered motor skill acquisition and hyperactivity, which is maintained to adulthood. In the Morris water maze, TgDyrk1A mice show a significant impairment in spatial learning and cognitive flexibility, indicative of hippocampal and prefrontal cortex dysfunction. In the more complex repeated reversal learning paradigm, this defect turned out to be specifically related to reference memory, whereas working memory was almost unimpaired. These alterations are comparable with those found in the partial trisomy chromosome 16 murine models of DS and suggest a causative role of DYRK1A in mental retardation and in motor anomalies of DS.

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Previous studies have shown that the skeletal dihydropyridine receptor (DHPR) pore subunit Ca V1.1 (␣1S) physically interacts with ryanodine receptor type 1 (RyR1), and a molecular signal is transmitted from ␣1S to RyR1 to trigger excitation-contraction (EC) coupling. We show that the ␤-subunit of the skeletal DHPR also binds RyR1 and participates in this signaling process. A novel binding site for the DHPR ␤1a-subunit was mapped to the M 3201 to W 3661 region of RyR1. In vitro binding experiments showed that the strength of the interaction is controlled by K 3495 KKRR R 3502 , a cluster of positively charged residues. Phenotypic expression of skeletal-type EC coupling by RyR1 with mutations in the K 3495 KKRR R 3502 cluster was evaluated in dyspedic myotubes. The results indicated that charge neutralization or deletion severely depressed the magnitude of RyR1-mediated Ca 2؉ transients coupled to voltage-dependent activation of the DHPR. Meantime the Ca 2؉ content of the sarcoplasmic reticulum was not affected, and the amplitude and activation kinetics of the DHPR Ca 2؉ currents were slightly affected. The data show that the DHPR ␤-subunit, like ␣1S, interacts directly with RyR1 and is critical for the generation of high-speed Ca 2؉ signals coupled to membrane depolarization. These findings indicate that EC coupling in skeletal muscle involves the interplay of at least two subunits of the DHPR, namely ␣1S and ␤1a, interacting with possibly different domains of RyR1.confocal imaging ͉ intracellular calcium ͉ skeletal muscle ͉ voltage-gated ion channels ͉ protein-protein interaction S keletal muscle cells respond to membrane action potentials with an elevation in cytosolic Ca 2ϩ that develops rapidly and is a graded function of voltage. The mechanism that couples muscle membrane excitation to the cytosolic Ca 2ϩ increase, also known as excitation-contraction (EC) coupling, is made possible by the strict functional and structural relationship of the dihydropyridine receptor (DHPR) and ryanodine receptor type 1 (RyR1) (1). Direct or indirect physical contact between these two Ca 2ϩ channels is thought to ensure that charge movements in the DHPR are transduced at high speed into a conformational change that opens the RyR1 channel, leading to a massive release of Ca 2ϩ from the sarcoplasmic reticulum (SR). Multiple approaches have suggested that the cytosolic loop linking repeats II and III of ␣1S binds RyR1 and͞or is responsible for the conformational change that opens the RyR1 channel (2-10). However, ␣1S domains outside the II-III loop also bind RyR1 (11) and, furthermore, the functional recovery produced by the II-III loop, in the absence of other skeletal domains, is incomplete (12). Thus, despite the structural and functional significance of the II-III loop, other molecular determinants may influence coupling between the skeletal DHPR and RyR1.The pore subunit of the DHPR is tightly bound to a ␤-subunit that modulates multiple processes ranging from the voltage dependence of the gating current to trafficking of ...

Dual-specificity tyrosine-phosphorylated and regulated kinase (DYRK) proteins are an evolutionarily conserved family of protein kinases, with members identified from yeast to humans, that participate in a variety of cellular processes. DYRKs are serine/threonine protein kinases that are activated by autophosphorylation on a tyrosine residue in the activation loop. The family member DYRK1A has been shown to phosphorylate several cytosolic proteins and a number of splicing and transcription factors, including members of the nuclear factor of activated T cells family. In the present study, we show that DYRK1A autophosphorylates, via an intramolecular mechanism, on Ser-520, in the PEST domain of the protein. We also show that phosphorylation of this residue, which we show is subjected to dynamic changes in vivo, mediates the interaction of DYRK1A with 14-3-3␤. A second 14-3-3 binding site is present within the N-terminal of the protein. In the context of the DYRK1A molecule, neither site can act independently of the other. Bacterially produced DYRK1A and the mutant DYRK1A/S520A have similar kinase activities, suggesting that Ser-520 phosphorylation does not affect the intrinsic kinase activity on its own. Instead, we demonstrate that this phosphorylation allows the binding of 14-3-3␤, which in turn stimulates the catalytic activity of DYRK1A. These findings provide evidence for a novel mechanism for the regulation of DYRK1A kinase activity. INTRODUCTIONDYRK1A belongs to a family of conserved protein kinases called dual-specificity tyrosine-phosphorylated and regulated kinase (DYRK), within the CMGC group (CDK, MAPK, GSK, and CLK families) of the eukaryote kinome. DYRK family members share a conserved kinase domain and an adjacent DYRK-homology domain, or DH-box (DDDNXDY), but they differ in their N-and C-terminal regions. From a phylogenetic viewpoint, the family can be classified into two subfamilies: a group of cytosolic DYRK proteins, which includes Schizosaccharomyces pombe Pom1p, Caenorhabditis elegans mbk-2, Drosophila melanogaster dDYRK2 and DYRK3, and vertebrate DYRK2, DYRK3, and DYRK4; and a subfamily of DYRKs that are considered mostly nuclear proteins, and which includes Saccharomyces cerevisiae Yak1p, Dictyostelium discoideum YakA, C. elegans mbk-1, D. melanogaster minibrain, and vertebrate DYRK1A and DYRK1B.The mammalian DYRK1A is ubiquitously expressed in adult and fetal tissues (Guimera et al., 1999;Okui et al., 1999) and shows a specific expression pattern in the central nervous system and during neurogenesis (Hammerle et al., 2002;Marti et al., 2003;Wegiel et al., 2004). Transgenic mice carrying extra copies of the gene exhibit learning defects and motor abnormalities (Smith et al., 1997;Altafaj et al., 2001), and it has been recently described that overexpression of Dyrk1A during mouse embryonic development leads to vascular defects (Arron et al., 2006). Heterozygous Dyrk1A mice also present a noticeable phenotype, with region-specific brain alterations (Fotaki et al., 2002). All these data strongl...