Protein Kinase Dyrk1 Activates cAMP Response Element-binding Protein during Neuronal Differentiation in Hippocampal Progenitor Cells

Abstract

Dyrk is a dual specific protein kinase thought to be involved in normal embryo neurogenesis and brain development. Defects/imperfections in this kinase have been suggested to play an important role in the mental retardation of patients with Down's syndrome. The transcriptional factor cAMP response element-binding protein (CREB) has been implicated in the formation of many types of synaptic plasticity, such as learning and memory. In the present study we show that Dyrk1 activity is markedly induced during the differentiation of immortalized hippocampal progenitor (H19-7) cells. The addition of a neurogenic factor, basic fibroblast growth factor, to the H19-7 cells results in an increased specific binding of Dyrk1 to active CREB. In addition, Dyrk1 directly phosphorylates CREB, leading to the stimulation of subsequent CRE-mediated gene transcription during the neuronal differentiation in H19-7 cells. Blockade of Dyrk1 activation significantly inhibits the neurite outgrowth as well as CREB phosphorylation induced by basic fibroblast growth factor. These findings suggest that Dyrk1 activation and subsequent CREB phosphorylation is important in the neuronal differentiation of central nervous system hippocampal cells. Dyrk is a dual specific protein kinase thought to be involved in normal embryo neurogenesis and brain development. Defects/imperfections in this kinase have been suggested to play an important role in the mental retardation of patients with Down's syndrome. The transcriptional factor cAMP response element-binding protein (CREB) has been implicated in the formation of many types of synaptic plasticity, such as learning and memory. In the present study we show that Dyrk1 activity is markedly induced during the differentiation of immortalized hippocampal progenitor (H19-7) cells. The addition of a neurogenic factor, basic fibroblast growth factor, to the H19-7 cells results in an increased specific binding of Dyrk1 to active CREB. In addition, Dyrk1 directly phosphorylates CREB, leading to the stimulation of subsequent CRE-mediated gene transcription during the neuronal differentiation in H19-7 cells. Blockade of Dyrk1 activation significantly inhibits the neurite outgrowth as well as CREB phosphorylation induced by basic fibroblast growth factor. These findings suggest that Dyrk1 activation and subsequent CREB phosphorylation is important in the neuronal differentiation of central nervous system hippocampal cells. cAMP response element cAMP response element-binding protein central nervous system Down's syndrome enhanced chemiluminescence glutathione S-transferase hemagglutinin minibrain thymidine kinase basic fibroblast growth factor Tris-buffered saline with Tween epidermal growth factor polyacrylamide gel electrophoresis The regulation of gene expression by specific signal transduction pathways is tightly connected to the cell phenotype. The response elicited by a given transduction pathway varies according to the cell type. One major signal transduction system utilizes cAMP as a secondary messenger and has as its ultimate target a DNA control element, the cAMP response element (CRE)1(1Montminy M. Annu. Rev. Biochem. 1997; 66: 807-822Crossref PubMed Scopus (856) Google Scholar). The CRE-binding protein (CREB) is a transcription factor that activates CRE-mediated transcription. CREB activity is regulated by multiple kinases after various kinds of stimulation (2Shaywitz A.J. Greenberg M.E. Annu. Rev. Biochem. 1999; 68: 821-861Crossref PubMed Scopus (1764) Google Scholar). Specific roles for CREB in neuronal development and differentiation have been revealed through in vivo and in vitro manipulations of CREB function. For example, the expression of a dominant-interfering CREB within certain pituitary neurons causes them to develop abnormally and die perinatally (3Struthers R.S. Vale W.W. Arias C. Sawchenko P.E. Montminy M.R. Nature. 1991; 350: 622-624Crossref PubMed Scopus (257) Google Scholar). “Minibrain” (Mnb) is a mutant of Drosophila whose presence is exhibited by a specific and marked size reduction of the optic lobes and central hemispheres in the adult brain (4Tejedor F. Zhu X.R. Kaltenbach E. Ackermann A. Baumann A. Canal I. Heisenberg M. Fischbach K.F. Pongs O. Neuron. 1995; 14: 287-301Abstract Full Text PDF PubMed Scopus (308) Google Scholar). The Mnb gene encodes a Ser/Thr protein kinase that possesses a Tyr-X-Tyr sequence in the activation loop (5Shindoh N. Kudoh J. Maeda H. Yamaki A. Minoshima S. Shimizu Y. Shimizu N. Biochem. Biophys. Res. Commun. 1996; 225: 92-99Crossref PubMed Scopus (104) Google Scholar). At least 7 closely related homologous mammalian kinases have since been isolated, and a novel superfamily of protein kinases called Dyrk(s) has been established (6Becker W. Weber Y. Wetzel K. Eirmbter K. Tejedor F.J. Joost H.G. J. Biol. Chem. 1998; 273: 25893-25902Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Dyrks possess Ser/Thr phosphorylation activity as well as autophosphorylation activity on Tyr residues, suggesting that Dyrk seems to be a dual specificity kinase (5Shindoh N. Kudoh J. Maeda H. Yamaki A. Minoshima S. Shimizu Y. Shimizu N. Biochem. Biophys. Res. Commun. 1996; 225: 92-99Crossref PubMed Scopus (104) Google Scholar, 6Becker W. Weber Y. Wetzel K. Eirmbter K. Tejedor F.J. Joost H.G. J. Biol. Chem. 1998; 273: 25893-25902Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). The kinase activity of Dyrk is dependent on the Tyr-X-Tyr motif in the activation loop, suggesting the existence of a phosphorylation-dependent activation mechanism of Dyrk by certain upstream kinases (6Becker W. Weber Y. Wetzel K. Eirmbter K. Tejedor F.J. Joost H.G. J. Biol. Chem. 1998; 273: 25893-25902Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). The most homologous protein among the Dyrks in the Saccharomyces cerevisiae genome is YAK1, which has been characterized as a negative regulator of growth (7Garrett S. Menold M.M. Broach J.R. Mol. Cell. Biol. 1991; 11: 4045-4052Crossref PubMed Scopus (108) Google Scholar). Interestingly, human Dyrk1A is mapped to the Down's syndrome (DS) critical region on chromosome 21 (4-megabase region containing 60∼100 genes between the markers D21S17 and ETS2) (31Antonarakis S.E. Genomics. 1998; 51: 1-16Crossref PubMed Scopus (122) Google Scholar) and, thus, could be a candidate gene responsible for the mental retardation of DS patients (8Smith D.J. Stevens M.E. Sudanagunta S.P. Bronson R.T. Makhinson M. Watabe A.M. O'Dell T.J. Fung J. Weier H.U. Cheng J.F. Rubin E.M. Nat. Genet. 1997; 16: 28-36Crossref PubMed Scopus (264) Google Scholar). Thus, fromDrosophila to humans, it is suggested that Dyrk/Mnb is a key regulator of neuronal cell growth (36Miyata Y. Nishida E. Biochem. Biophys. Res. Commun. 1999; 266: 291-295Crossref PubMed Scopus (127) Google Scholar). Although the exact cellular function of the Dyrk kinases is still unknown, an understanding of the physiological role(s) of this protein kinase family will prove to be very relevant. Although little is known about the mechanism by which the overexpression of Dyrks interferes with normal development in DS patients, Dyrk genes are strongly implicated in normal neuronal development by a mechanism that may involve a signal transduction pathway (36Miyata Y. Nishida E. Biochem. Biophys. Res. Commun. 1999; 266: 291-295Crossref PubMed Scopus (127) Google Scholar). This study examined the functional role played by Dyrk1 during the neuronal differentiation in hippocampal progenitor cells. We found that Dyrk1 is activated and phosphorylates the transcription factor CREB during neuronal differentiation. In addition, Dyrk1 activation induces CRE-mediated gene transcription. These findings suggest that Dyrk1 may play an important role during neurogenic factor-induced differentiation of central nervous system (CNS) neuronal cells. The following materials were purchased. Peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulins (IgGs) were from Zymed Laboratories Inc. (San Francisco, CA); Dulbecco's modified Eagle's medium, fetal bovine serum, and cell culture reagents were from Life Technologies, Inc.; protein A-Sepharose was from Amersham Pharmacia Biotech; enhanced chemiluminescence (ECL) reagents and [γ-32P]ATP were from PerkinElmer Life Sciences; anti-Dyrk IgG was from Becton Dickinson; anti-CREB IgG was from NEN Cell Signaling (Beverly, MA); anti-phosphorylated CREB antibodies were from Upstate Biotechnology (Lake Placid, NY); synthetic dropout medium (SD/−T, SD/−L, SD/−HLT) and yeast extract peptone dextrose containing adenine were from Bio101 (Vista, CA); 3-amino-1,2,4-triazole was from Sigma; 5-bromo-4-chloro-3-indoryl-d-galactoside and luciferase assay kit were from Promega (Madison, WI); human fetal brain cDNA library was from CLONTECH (Palo Alto, Calif.); human basic FGF was from Bachem (Bubendorf, Switzerland). As gifts, pCRE-TK-Luc and pTK-Luc constructs were received from K. Saeki (Research Institute, International Medical Center of Japan). The plasmids encoding hemagglutinin (HA)-tagged pSVL-HA-Dyrk1A and mutant type K188R were provided by W. Becker (Institute of Pharmacology and Toxicology, RWTH, Aachen, Germany). The bait vector for yeast two-hybrid assay was constructed by subcloning the mutant CREB cDNA, in which RRPSY (amino acid 130–134) was replaced by RRSLY, into pHybTrp/Zeo vector. Human fetal cDNA library subcloned into prey vector (pACT2) was purchased from CLONTECH. The yeast strain L40, containing the reporter genes lacZ and HIS3downstream of the LexA promoter, was sequentially transformed with bait vector followed by cDNA library vectors and then plated on a synthetic medium containing 5 mm 3-amino-1,2,4-triazole and lacking histidine, leucine, and tryptophan residue. After incubating the plates for 10–14 days at 30 °C, the transformants were tested with a synthetic medium lacking histidine, leucine, and tryptophan residue and containing 50 µg/ml 5-bromo-4-chloro-3-indoryl-d-galactoside. After incubation for 2–3 days at 30 °C, the yeast colonies showing blue color were selected as positive clones. The positive clone plasmids were extracted from yeast in lysis buffer containing 2% Triton X-100, 1% SDS, 100 mm NaCl, 10 mm Tris, pH 8.0, and 1.0 mm EDTA and then transformed into Escherichia coli DH5α using electroporation. Sequences of the inserts in positive library plasmids were analyzed by automatic DNA sequencer (ALF express, Amersham Pharmacia Biotech). H19-7 cells were generated from rat embryonic hippocampal neurons (9Eves E.M. Tucker M.S. Roback J.D. Downen M. Rosner M.R. Wainer B.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4373-4377Crossref PubMed Scopus (140) Google Scholar). They were conditionally immortalized by stable transfection with temperature-sensitive SV40 large T antigen. They were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 100 units/ml penicillin-streptomycin under G418 selection throughout the experiments. To induce neuronal differentiation, the cells were placed in N2 medium and shifted to with as I. Rosner M.R. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). To the of Dyrk1 activation on the neuronal differentiation in H19-7 the cells were with a encoding type Dyrk1 at for the cells were to N2 medium, at for with and analyzed for with a cells were as cells with a and cell containing at least neurite whose was the cell To cell the cells were with saline and in lysis buffer mm Tris, pH containing Triton X-100, mm mm NaCl, µg/ml µg/ml mm mm 10 mm mm 5 10% and mm The cells were and the were after for 10 at at were using the protein assay The H19-7 cells were plated at a of cells well in the cell was the cells were with constructs using according to the luciferase reporter was with Dyrk1 mutant and the luciferase activity was using a luciferase assay kit and a Germany). In transfection the thymidine kinase was as a negative One of anti-CREB antibodies was at with of cell using lysis of a of protein A-Sepharose was to the cell and for at with The were and with cell lysis were by the in and were on The cell were through a 10% gel and to a Japan). The were in mm Tris, pH mm NaCl, and Tween containing for and then at in containing CREB, The was then in and with anti-mouse After the was with and with H19-7 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and to N2 medium differentiation for days at The cells were with 10 of and in lysis of protein was with Dyrk1 at The were to of protein A-Sepharose After the were in lysis and the kinase were at 30 for in of kinase buffer containing mm pH 5 5 of 10 5 of and 5 of glutathione S-transferase mutant as a The were by buffer and analyzed by followed by hippocampal H19-7 cells in response to at °C, the at which the large T is active M. J. E.M. M. D.J. M. Rosner M.R. Mol. Cell. Biol. 1996; 16: PubMed Scopus Google Scholar). In an we have that CREB phosphorylation and subsequent CRE-mediated gene transcription play an important role during neuronal differentiation in H19-7 cells and that the activation of novel protein pathways is for J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the differentiation of H19-7 residue in the CREB protein was and for after growth factor In addition, the activation of CREB kinases in response to was by using in vitro kinase J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the present in to the novel CREB we yeast two-hybrid by using mutant CREB mutant in which the of CREB been to as As a of the of human fetal brain cDNA we a of as well as for example, A. C. M. J. 1999; PubMed Scopus Google protein N. Mol. Cell. Biol. PubMed Scopus Google and protein revealed that of the encodes a clone of mammalian of yeast minibrain Dyrk1 was known to be important in the cell control in neuronal cells and implicated in the of DS mental we Dyrk1 as a target for we examined Dyrk1 to CREB in mammalian neuronal cells and the neurogenic growth factor the expression of Dyrk1 during neuronal differentiation. The H19-7 cell after the stimulation with neurogenic were antibodies and then with As in the expression of Dyrk1 protein with was induced by were in H19-7 cells. Although was formation the addition of to an of Dyrk1 binding to CREB. In the following the cells were with a encoding type Dyrk1 the cells were with under the cell were anti-CREB and the protein were analyzed using IgG with the stimulation the formation of binding between Dyrk1 and in H19-7 cells. encoding Dyrk1 was to the activation of the K188R Dyrk1 in which critical residue in the of Dyrk1 was to S. W. S. Joost H.G. Becker W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google was into the the specific binding of Dyrk1 to active CREB markedly in response to As a control for protein we the of CREB by In were present at the These results that Dyrk1 to active CREB in response to neurogenic growth factor. Dyrk1 is activated by phosphorylation in the activation loop of the H. Becker W. J. A. A. C. H. Joost H.G. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google we examined Dyrk1 is activated by After cells were with the cell were and the were analyzed using As in the phosphorylation of Dyrk1 increased in response to with the control the overexpression of Dyrk1 by transfection the Dyrk1 suggesting that the addition of induces Dyrk1 in CREB phosphorylation are known to be critical in the and regulation of and differentiation in neuronal cells A. H. Greenberg M.E. Mol. Cell. 1995; PubMed Scopus Google Scholar). H19-7 cells to and J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). At the induces at the the addition of FGF induces differentiation (9Eves E.M. Tucker M.S. Roback J.D. Downen M. Rosner M.R. Wainer B.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4373-4377Crossref PubMed Scopus (140) Google Scholar, E.M. J. Downen M. Tucker M.S. Wainer B.H. Rosner M.R. Res. PubMed Scopus Google Scholar). in to CREB phosphorylation by neurogenic the addition of induces CREB These findings from that stable CREB activation by is important in the of hippocampal progenitor cell to to neuronal cell J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To Dyrk1 activation is induced during neuronal differentiation in H19-7 the cells were with under under differentiation was with the cell using antibodies followed by an specific for residue to Dyrk1 with a stable activation of Dyrk1 within after stimulation a phosphorylation of Dyrk1 was the stimulation with the at In addition, the addition of at at to induce a phosphorylation of Dyrk These the activation of Dyrk1 in response to and neurogenic growth factor. In Dyrk1 activation may to neuronal differentiation by neurogenic in the H19-7 activation of Dyrk1 in response to neurogenic in H19-7 cells. H19-7 cells were with 50 10 for the The Dyrk1 was from cell and the protein were with The of Dyrk1 kinase was by using antibodies results are of at at at and at we active Dyrk1 could CREB. The H19-7 cell after were using and kinase were using protein with type CREB mutant CREB as a were by As in CREB phosphorylation was induced by transfection of Dyrk1 significantly the CREB in a of type Dyrk1 a of CREB phosphorylation with the control cells. encoding CREB in which residue was to was as a was phosphorylation of CREB In addition, the of Dyrk1 on the CREB phosphorylation was examined in H19-7 cells with the that to active the addition of induced a marked CREB the overexpression of Dyrk1 in a the CREB phosphorylation the stimulation with with the control cells These suggest that Dyrk1 directly phosphorylates the residue of transcription factor CREB during neuronal differentiation in H19-7 of Dyrk1 on CREB H19-7 cells were with with 5 of encoding the cells were with 10 of for under cell were by and analyzed by using the antibodies CREB, CREB. To Dyrk1 its on CRE-mediated gene transcription as well as on CREB the gene expression of was in response to of H19-7 cells with in the of CRE-mediated gene transcription in a and it a after J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To the role of Dyrk1 activation on CRE-mediated gene the cells were with pCRE-TK-Luc reporter an expression vector encoding Dyrk1 the addition of to an of CRE-mediated reporter luciferase the expression of Dyrk1 with the of critical residue significantly the activation of luciferase activity by findings that neurogenic causes the activation of CRE-mediated gene through the activation of Dyrk1 and subsequent CREB phosphorylation in embryonic hippocampal cells. The functional role of Dyrk1 activation during neuronal differentiation in H19-7 cells was After with most of the H19-7 cells neurite at at which the large is The cells were to be to stimulation by serum and to neuronal markers such as and brain type (9Eves E.M. Tucker M.S. Roback J.D. Downen M. Rosner M.R. Wainer B.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4373-4377Crossref PubMed Scopus (140) Google Scholar, E.M. J. Downen M. Tucker M.S. Wainer B.H. Rosner M.R. Res. PubMed Scopus Google Scholar). The of the H19-7 cells to in response to is to the response of hippocampal cells during since as a factor in certain such as the to Rosner M.R. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar). After a encoding type Dyrk1 was into the the formation of neurite outgrowth was As in the control cells a of cells in transfection experiments. cells in the mutant exhibited cells 7 Interestingly, transfection of type Dyrk1 on the neuronal differentiation in H19-7 cells 7 These results suggest that Dyrk activation is to play an important role in the differentiation of neuronal H19-7 cells. The of Dyrk1 kinases in signal transduction pathway has been The of the by protein kinase is an important mechanism that the specificity of cellular to various S. W. S. Joost H.G. Becker W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is important to the of Dyrk to its function in the regulation of cellular Dyrk was to be for the of neuronal cell types during neurogenesis in Drosophila (4Tejedor F. Zhu X.R. Kaltenbach E. Ackermann A. Baumann A. Canal I. Heisenberg M. Fischbach K.F. Pongs O. Neuron. 1995; 14: 287-301Abstract Full Text PDF PubMed Scopus (308) Google Scholar). with a Dyrk1 expression have a of neurons in of the adult brain and specific (4Tejedor F. Zhu X.R. Kaltenbach E. Ackermann A. Baumann A. Canal I. Heisenberg M. Fischbach K.F. Pongs O. Neuron. 1995; 14: 287-301Abstract Full Text PDF PubMed Scopus (308) Google Scholar). In the present study we examined the cellular Dyrk1 and its functional role during neuronal differentiation in hippocampal H19-7 cells. We have that Dyrk1 is activated by neurogenic by and that active Dyrk1 CREB phosphorylation and subsequent CRE-mediated gene transcription. strongly suggest that Dyrk1 may play an important role during neurogenic factor-induced differentiation in neuronal cells. a role was by the that the expression of Dyrk1 in a the formation of cells. We have that the Dyrk mutant could phosphorylation of CREB this mutant to CREB the in 5 be to the between and Dyrk1 for CREB. it that the mutant is then it by binding to the that activates Dyrk1 and with Dyrk1 could be that be activated at this such as then the Dyrk mutant The Dyrk1 protein such as a a present residues, and an activation loop between and H. Becker W. J. A. A. C. H. Joost H.G. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Dyrk1 is to the cell Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google the to control the expression of protein was found in the of cells (6Becker W. Weber Y. Wetzel K. Eirmbter K. Tejedor F.J. Joost H.G. J. Biol. Chem. 1998; 273: 25893-25902Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar). this kinase has been to be a dual specificity protein kinase regulated by phosphorylation in the activation loop H. Becker W. J. A. A. C. H. Joost H.G. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). This that Dyrk1 be a of a pathway Dyrk1 is involved in this mechanism has been The present showing as it that Dyrk1 is activated by and could CREB and subsequent CRE-mediated gene strongly the that Dyrk1 the expression of a certain is that gene for such a protein could the expression of genes and to the of in DS Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar). We found that the expression of Dyrk1 and that of Dyrk1 to be in H19-7 cells. This is in with that Dyrk1 is strongly in the brain from embryonic rat M. K. E. F. K. Y. Kudoh J. Shimizu N. Genomics. 1999; PubMed Scopus Google Scholar) and most in of such as in the and the C. M. Biochem. Biophys. Res. Commun. 1998; PubMed Scopus Google Scholar, C. Genomics. 1996; PubMed Scopus Google Scholar). activity a critical role in many of synaptic such as learning and A. W. J. PubMed Google Scholar). on the formation of that the of genes is with S.P. 1991; 14: Full Text PDF PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1992; 89: PubMed Scopus Google Scholar). The gene are thought to which the and functional of cells. Although the mechanism by which neuronal activity is to the in gene expression is CREB phosphorylation to be important in the expression of genes H. Greenberg M.E. PubMed Scopus Google Scholar, C. S. S.E. J. 14: PubMed Google Scholar). CREB has been implicated in the formation of memory. CREB in various that from in to in For example, of CREB activation by of has been found to a of in the M. Cell. Full Text PDF PubMed Scopus Google Scholar). with a in the CREB gene and Drosophila a of CREB show in on J. A.J. Cell. Full Text PDF PubMed Scopus Google Scholar, M. H. Cell. Full Text PDF PubMed Scopus Google Scholar, M. H. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). The the a critical role in the of of and The Dyrk1 in the the show in learning and as well as S. A. S. C. Scholar, M. C. E. J. J. M. J.R. J. C. Mol. Genet. PubMed Scopus Google Scholar). In addition, functional a of activity in the of S. A. S. C. Scholar). are in the expression of hippocampal synaptic and with show a reduction in hippocampal and a in suggesting that the overexpression of Dyrk may hippocampal development. Down's syndrome is a with DS have a of (31Antonarakis S.E. Genomics. 1998; 51: 1-16Crossref PubMed Scopus (122) Google Scholar). For example, with DS are at increased for and have increased to and with DS are at increased for with DS have of mental The secondary messenger cAMP a of physiological cellular and neuronal by the basic of gene expression a In addition, CREB phosphorylation to be important in the expression of various kinds of genes and in the formation of as on the findings that Dyrk1 activates CREB during neuronal differentiation in hippocampal of the upstream pathways activated by various neurogenic growth may into the mechanism of neuronal differentiation by Dyrk1 We are to H. and K. Y. for S. M. and for and J. for We W. Becker for the cDNA constructs encoding type and Dyrk1A and K. Saeki for vector.