Protein Kinase DYRK1A Research Articles

P38α- and DYRK1A-dependent phosphorylation of caspase-9 at an inhibitory site in response to hyperosmotic stress

The cysteine aspartyl protease caspase-9 is a critical component of the intrinsic apoptotic pathway. Activation of caspase-9 is inhibited by phosphorylation at Thr125, which is catalysed by the mitogen-activated protein kinases (MAPKs) ERK1/2 in response to growth factors, by the cyclin-dependent protein kinase CDK1-cyclin B1 during mitosis, and at a basal level by the dual-specificity tyrosine-phosphorylation regulated protein kinase DYRK1A. Here we show that inhibitory phosphorylation of caspase-9 at Thr125 is induced in mammalian cells by hyperosmotic stress. This response does not require ERK1/2 or ERK5, but it is diminished by ablation of DYRK1A expression by siRNA or chemical inhibition of DYRK1A by harmine. Phosphorylation of Thr125 in response to hyperosmotic stress is also reduced by chemical inhibition of p38 MAPK and is abolished in p38α −/− mouse embryonic fibroblasts. These results show that both DYRK1A and p38α play roles in the inhibitory phosphorylation of caspase-9 following hyperosmotic stress and suggest a functional interaction between these protein kinases. Phosphorylation of caspase-9 at Thr125 may restrain apoptosis during the acute response to hyperosmotic stress.

Effect of hyperhomocysteinemia on the protein kinase DYRK1A in liver of mice

Hyperhomocysteinemia due to cystathionine beta synthase (CBS)-deficiency confers diverse clinical manifestations, notably liver diseases. Even if hyperhomocysteinemia in liver of CBS-deficient mice, a murine model of hyperhomocysteinemia, promotes mitochondrial oxidative stress and pro-apoptotic signals, protective signals may counteract these pro-apoptotic signals, leading to chronic inflammation. As DYRK1A, a serine/threonine kinase, has been described as a candidate antiapoptotic factor, we have analyzed the expression of DYRK1A in liver of CBS-deficient mice. We found that DYRK1A protein level was reduced in liver of CBS-deficient mice, which was not observed at the gene expression level. Moreover, the use of primary hepatocytes/Kupffer cells co-culture showed that degradation of DYRK1A induced by hyperhomocysteinemia requires calpain activation. Our results demonstrate a deleterious effect of hyperhomocysteinemia on DYRK1A protein expression, and emphasize the role of hyperhomocysteinemia on calpain activation.

The Protein Kinase DYRK1A Regulates Caspase-9-Mediated Apoptosis during Retina Development

The precise regulation of programmed cell death is critical for the normal development of the nervous system. We show here that DYRK1A (minibrain), a protein kinase essential for normal growth, is a negative regulator of the intrinsic apoptotic pathway in the developing retina. We provide evidence that changes in Dyrk1A gene dosage in the mouse strongly alter the cellularity of inner retina layers and result in severe functional alterations. We show that DYRK1A does not affect the proliferation or specification of retina progenitor cells, but rather regulates the number of cells that die by apoptosis. We demonstrate that DYRK1A phosphorylates caspase-9 on threonine residue 125, and that this phosphorylation event is crucial to protect retina cells from apoptotic cell death. Our 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.

The protein kinase Dyrk1A regulates cell number during the development of the central nervous system

International Journal of Developmental NeuroscienceVolume 26, Issue 8 p. 842-842 Article [P1.04]: The protein kinase Dyrk1A regulates cell number during the development of the central nervous system M.J. Barallobre, Corresponding Author M.J. Barallobre n/[email protected] Centre for Genomic Regulation, UPF and Centre for Biomedical Research on Rare Diseases (CIBERER), PRBB, SpainCorresponding author.Search for more papers by this authorA. Laguna, A. Laguna Centre for Genomic Regulation, UPF and Centre for Biomedical Research on Rare Diseases (CIBERER), PRBB, SpainSearch for more papers by this authorE. Ramirez-Bautista, E. Ramirez-Bautista Centre for Genomic Regulation, UPF and Centre for Biomedical Research on Rare Diseases (CIBERER), PRBB, SpainSearch for more papers by this authorM.L. Arbonés, M.L. Arbonés Centre for Genomic Regulation, UPF and Centre for Biomedical Research on Rare Diseases (CIBERER), PRBB, SpainSearch for more papers by this author M.J. Barallobre, Corresponding Author M.J. Barallobre n/[email protected] Centre for Genomic Regulation, UPF and Centre for Biomedical Research on Rare Diseases (CIBERER), PRBB, SpainCorresponding author.Search for more papers by this authorA. Laguna, A. Laguna Centre for Genomic Regulation, UPF and Centre for Biomedical Research on Rare Diseases (CIBERER), PRBB, SpainSearch for more papers by this authorE. Ramirez-Bautista, E. Ramirez-Bautista Centre for Genomic Regulation, UPF and Centre for Biomedical Research on Rare Diseases (CIBERER), PRBB, SpainSearch for more papers by this authorM.L. Arbonés, M.L. Arbonés Centre for Genomic Regulation, UPF and Centre for Biomedical Research on Rare Diseases (CIBERER), PRBB, SpainSearch for more papers by this author First published: 25 November 2008 https://doi.org/10.1016/j.ijdevneu.2008.09.054Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume26, Issue8Abstracts to the 17th Biennial Meeting of the International Society for Developmental Neuroscience, 1‐5 June 2008, Asilomar, USADecember 2008Pages 842-842 RelatedInformation

Sprouty2-Mediated Inhibition of Fibroblast Growth Factor Signaling Is Modulated by the Protein Kinase DYRK1A

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. We identify DYRK1A as one of the protein kinases of Sprouty2. We show that DYRK1A interacts with and regulates the phosphorylation status of Sprouty2. Moreover, we identify Thr75 on Sprouty2 as a DYRK1A phosphorylation site in vitro and in vivo. This site is functional, since its mutation enhanced 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. Our results suggest, therefore, that DYRK1A positively regulates FGF-mitogen-activated protein kinase signaling by phosphorylation-dependent impairment of the inhibitory activity of Sprouty2.

The protein kinase DYRK1A phosphorylates the splicing factor SF3b1/SAP155 at Thr434, a novel in vivo phosphorylation site

BackgroundThe U2 small nuclear ribonucleoprotein particle (snRNP) component SF3b1/SAP155 is the only spliceosomal protein known to be phosphorylated concomitant with splicing catalysis. DYRK1A is a nuclear protein kinase that has been localized to the splicing factor compartment. Here we describe the identification of DYRK1A as a protein kinase that phosphorylates SF3b1 in vitro and in cultivated cells.ResultsOverexpression of DYRK1A caused a markedly increased phosphorylation of SF3b1 in COS-7 cells as assessed by Western blotting with an antibody specific for phosphorylated Thr-Pro dipeptide motifs. Phosphopeptide mapping of metabolically labelled SF3b1 showed that the majority of the in vivo-phosphopeptides corresponded to sites also phosphorylated by DYRK1A in vitro. Phosphorylation with cyclin E/CDK2, a kinase previously reported to phosphorylate SF3b1, generated a completely different pattern of phosphopeptides. By mass spectrometry and mutational analysis of SF3b1, Thr434 was identified as the major phosphorylation site for DYRK1A. Overexpression of DYRK1A or the related kinase, DYRK1B, resulted in an enhanced phosphorylation of Thr434 in endogenous SF3b1 in COS-7 cells. Downregulation of DYRK1A in HEK293 cells or in HepG2 cells by RNA interference reduced the phosphorylation of Thr434 in SF3b1.ConclusionThe present data show that the splicing factor SF3b1 is a substrate of the protein kinase DYRK1A and suggest that DYRK1A may be involved in the regulation of pre mRNA-splicing.

Phosphorylation of splicing factor 3b1 (SF3b1) by the protein kinase DYRK1A

Phosphorylation of splicing factor 3b1 (SF3b1) by the protein kinase DYRK1A

Characterization of Cyclin L2, a Novel Cyclin with an Arginine/Serine-rich Domain: PHOSPHORYLATION BY DYRK1A AND COLOCALIZATION WITH SPLICING FACTORS

A novel method employing filter arrays of a cDNA expression library for the identification of substrates for protein kinases was developed. With this technique, we identified a new member of the cyclin family, cyclin L2, as a substrate of the nuclear protein kinase DYRK1A. Cyclin L2 contains an N-terminal cyclin domain and a C-terminal arginine/serine-rich domain (RS domain), which is a hallmark of many proteins involved in pre-mRNA processing. The gene for cyclin L2 encodes the full-length cyclin L2, which is predominantly expressed in testis, as well as a truncated splicing variant (cyclin L2S) that lacks the RS domain and is ubiquitously expressed in human tissues. Full-length cyclin L2, but not cyclin L2S, was associated with the cyclin-dependent kinase PITSLRE. Cyclin L2 interacted with splicing factor 2 in vitro and was co-localized with the splicing factor SC35 in the nuclear speckle compartment. Photobleaching experiments showed that a fusion protein of green fluorescent protein and cyclin L2 in nuclear speckles rapidly exchanged with unbleached molecules in the nucleus, similar to other RS domain-containing proteins. In striking contrast, the closely related green fluorescent protein-cyclin L1 was immobile in the speckle compartment. DYRK1A interacted with cyclin L2 in pull-down assays, and overexpression of DYRK1A stimulated phosphorylation of cyclin L2 in COS-7 cells. These data characterize cyclin L2 as a highly mobile component of nuclear speckles and suggest that DYRK1A may regulate splicing by phosphorylation of cyclin L2.

DYRK1A accumulates in splicing speckles through a novel targeting signal and induces speckle disassembly.

The protein kinase DYRK1A is distributed throughout the nucleoplasm, accumulating in speckle-like regions. We have found that this punctuated nuclear distribution is determined by the contribution of several elements. Although the nuclear import is mediated by two distinct nuclear localization signals, one at the N-terminus and the other located in the linker region, between subdomains X and XI of the catalytic domain, the accumulation in speckles that are SC35 positive depends on a sequence motif that is located C-terminal to the kinase domain and comprises a histidine tail. A similar sequence is also responsible for the targeting of cyclin T1. Therefore the histidine-rich region represents a novel splicing speckle targeting signal. Moreover, overexpression of DYRK1A induces speckle disassembly. Such disassembly is DYRK1A activity specific, since the overexpression of a DYRK1A kinase inactive mutant, the paralogous DYRK1B or a chimeric protein DYRK1B that has been directed to the speckles via the DYRK1A targeting signal, leaves the SC35 speckle pattern untouched. Thus DYRK1A protein kinase may play a role in regulating the biogenesis of the splicing speckle compartment.

Unusual function of the activation loop in the protein kinase DYRK1A

Protein kinases of the DYRK (dual-specificity tyrosine phosphorylation-regulated kinase) family require phosphorylation of a conserved tyrosine residue in the activation loop for full activity. Here we have characterized the role of conserved amino acids that are located in the vicinity of the phosphorylated tyrosine in DYRK1A (Tyr-321). Mutation of Gln-323, but not Asn-365 or Glu-366, to either alanine, glutamate, or asparagine reduced the in vitro-kinase activity of DYRK1A towards the peptide substrate, DYRKtide, to a similar degree (15–37% of wild type) as the mutation of the phosphorylation site itself (Y321F). Similarly, the in vivo-kinase activity of DYRK1A-Q323N and of DYRK1A-Y321F, as assessed by Ser-727 phosphorylation of signal transducer and activator of transcription 3 (STAT3) in COS-7 cells, was markedly reduced in comparison with wild type DYRK1A. These data show that the contribution of Gln-323 and Tyr-321 to the full catalytic activity of DYRK1A is a specific functional characteristic of the DYRK family.

Characterization of Caenorhabditis elegans homologs of the Down syndrome candidate gene DYRK1A.

The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYRK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that 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 us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our 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.

DYRK1 is a co-activator of FKHR (FOXO1a)-dependent glucose-6-phosphatase gene expression

Expression of glucose-6-phosphatase (G6Pase), one of the rate-limiting enzymes of hepatic gluconeogenesis, has recently been shown to be transactivated by the transcription factor FKHR. One of the proteins known to directly interact with FKHR is the nuclear protein kinase DYRK1A. In order to study the effects of DYRK1A on G6Pase gene expression, we generated a H4IIEC3 rat hepatoma cell line stably expressing DYRK1A by retroviral infection. Overexpression of DYRK1A increased the expression of G6Pase about threefold, as determined by Northern blotting. In transiently transfected HepG2 cells, co-expression of DYRK1A and a G6Pase promoter construct increased G6Pase promoter activity about twofold. This effect of DYRK1A was independent of its kinase activity, since a kinase-dead DYRK1A mutant as well as a point mutant of the phosphorylation site of DYRK1A in FKHR (Ser329Ala) failed to affect the effect of DYRK1A on the G6Pase expression. The effect of DYRK on the G6Pase promoter activity was produced by the isoforms DYRK1A and DYRK1B, which are localized in the nucleus, but not by DYRK2. Mutations of the FKHR-binding sites in the G6Pase promoter markedly reduced the effect of DYRK1 on the G6Pase promoter activity. In summary, the data suggest that DYRK1 is a specific co-activator of FKHR, independent of its kinase activity.

Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A

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.

Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A.

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.

Specificity Determinants of Substrate Recognition by the Protein Kinase DYRK1A

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 microM) but not by ERK2. Both ERK2 and DYRK1A phosphorylated myelin basic protein, whereas only ERK2, but not DYRK1A, phosphorylated the mitogen-activated 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.