DYRK1A is a dual-specificity protein kinase that is thought to be involved in brain development. We identified a single phosphorylated amino acid residue in the DYRK substrate histone H3 (threonine 45) by mass spectrometry, phosphoamino acid analysis, and protein sequencing. Exchange of threonine 45 for alanine abolished phosphorylation of histone H3 by DYRK1A and by the related kinases DYRK1B, DYRK2, and DYRK3 but not by CLK3. In order to define the consensus sequence for the substrate specificity of DYRK1A, a library of 300 peptides was designed in variation of the H3 phosphorylation site. Evaluation of the phosphate incorporation into these peptides identified DYRK1A as a proline-directed kinase with a phosphorylation consensus sequence (RPX(S/T)P) similar to that of ERK2 (PX(S/T)P). A peptide designed after the optimal substrate sequence (DYRKtide) was efficiently phosphorylated by DYRK1A (K m ‫؍‬ 35 M) but not by ERK2. Both ERK2 and DYRK1A phosphorylated myelin basic protein, whereas only ERK2, but not DYRK1A, phosphorylated the mitogenactivated protein kinase substrate ELK-1. This marked difference in substrate specificity between DYRK1A and ERK2 can be explained by the requirement for an arginine at the P ؊3 site of DYRK substrates and its presumed interaction with aspartate 247 conserved in all DYRKs.DYRK1A is a nuclear protein kinase that is ubiquitously expressed in rat tissues (1, 2). We have recently characterized a closely related isoform, DYRK1B, that is predominantly expressed in testis and muscle (3). The homolog of DYRK1A and DYRK1B in Drosophila, the protein kinase MNB, is encoded by the minibrain gene whose mutation results in specific defects in the development of the central nervous system (4). The human gene for DYRK1A is located in the "Down syndrome critical region" of chromosome 21, and the similarity of DYRK1A and MNB suggests that the triplication of the DYRK1A gene may play a role in mental retardation of patients with Down syndrome (5-9).DYRK1A, DYRK1B, and MNB belong to a subfamily of protein kinases with structurally related catalytic domains and similar enzymatic properties (2, 10). At least 7 different DYRKrelated kinases exist in mammals, of which DYRK1A and DYRK1B are targeted to the nucleus, whereas DYRK2 and DYRK3 are located in the cytoplasm (2, 3).1 Members of the DYRK family have also been found in lower eukaryotes, such as Yak1p in Saccharomyces cerevisiae (11), Pom1p in Schizosaccharomyces pombe (12), and YAKA in Dictyostelium discoideum (13). Although mutations in YAK1, pom1, and yakA have diverse phenotypic consequences, it appears reasonable to generalize that DYRK-related kinases are involved in the regulation of growth and development.The enzymatic activity of DYRK1A has been shown to depend on the presence of tyrosine residues in the activation loop, a result that suggests an activation mechanism similar to that of the MAP 2 kinases (1). However, the participation of DYRK1A or other DYRK-related kinases in a particular signal transduction pathway has not been eluci...

Protein kinases of the DYRK ('dual-specificity tyrosine-regulated kinase') family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319-Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr). In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2. Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated. When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321. A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F mutant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.

Protein kinases of the DYRK (‘dual-specificity tyrosine-regulated kinase’) family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319–Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr). In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2. Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated. When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321. A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F mutant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.

No abstract

early and automatic neuropsychological processes may be influenced by altered dopaminergic functions but cannot be fully explained by these or by altered reinforcement and extinction processes. the reinforcement-extinction model is excellent for understanding certain causal pathways of attention-deficit/hyperactivity disorder (adhd), but it can hardly explain the heterogeneous developmental trajectories of adhd fully. it should be integrated into a multiple pathways model.