G2Cdb::Gene report

Gene id
Gene symbol
Cdk5 (MGI)
Mus musculus
cyclin-dependent kinase 5
G00001416 (Homo sapiens)

Databases (10)

ENSMUSG00000028969 (Ensembl mouse gene)
12568 (Entrez Gene)
498 (G2Cdb plasticity & disease)
Gene Expression
NM_007668 (Allen Brain Atlas)
g00145 (BGEM)
12568 (Genepaint)
cdk5 (gensat)
123831 (OMIM)
Marker Symbol
MGI:101765 (MGI)
Protein Sequence
P49615 (UniProt)

Synonyms (1)

  • Crk6

Alleles (1)

Literature (161)

Pubmed - other

  • Neuronal nuclear organization is controlled by cyclin-dependent kinase 5 phosphorylation of Ras Guanine nucleotide releasing factor-1.

    Kesavapany S, Pareek TK, Zheng YL, Amin N, Gutkind JS, Ma W, Kulkarni AB, Grant P and Pant HC

    Cytoskeletal Protein Regulation Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.

    RasGRF1 is a member of the Ras guanine nucleotide exchange factor (RasGEF) family of proteins which are directly responsible for the activation of Ras and Rac GTPases. Originally identified as a phosphoprotein, RasGRF1 has been shown to be phosphorylated by protein kinase A and more recently, by the non-receptor tyrosine kinases Ack1 and Src. In this report we show that RasGRF1 interacts with and is phosphorylated by Cdk5 on serine 731 to regulate its steady state levels in mammalian cells as well as in neurons. Phosphorylation on this site by Cdk5 leads to RasGRF1 degradation through a calpain-dependent mechanism. Additionally, cortical neurons from Cdk5 knockout mice have higher levels of RasGRF1 which are reduced when wild-type Cdk5 is transfected into these neurons. In mitotic cells, nuclei become disorganized when RasGRF1 is overexpressed and this is rescued when RasGRF1 is co-expressed with active Cdk5. When RasGRF1 levels are elevated in neurons through overexpression of either the wild-type RasGRF1, or the phosphorylation mutant of RasGRF1 and by the transfection of a dominant negative Cdk5 construct, nuclei appeared condensed and fragmented. On the other hand, a reduction of RasGRF1 levels through p35/Cdk5 overexpression also leads to nuclear condensation in neurons. These data show that phosphorylation of RasGRF1 by Cdk5 tightly regulates its levels, which is essential for proper cellular organization.


  • Cdk5 interacts with Hif-1α in neurons: a new hypoxic signalling mechanism?

    Antoniou X, Gassmann M and Ogunshola OO

    Institute of Veterinary Physiology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland.

    The cyclin dependent kinase 5 (Cdk5)/p35 complex is essential for regulation of cell survival during development and in models of neuronal excitotoxicity. Dysregulation of Cdk5, by cleavage of its neuronal specific activators p35 and p39, has been implicated in various neurodegenerative disorders such as Alzheimer's disease, however targets of the complex that regulate neuronal survival physiologically and/or during pathogenesis are largely unknown. Since hypoxia is a key feature in the pathogenesis of several neuronal disorders we investigated a role for Cdk5/p35 in the neuronal hypoxic response. Our data show that hypoxia modulates the p35/Cdk5 complex in primary cortical neurons at the transcriptional and protein level. Furthermore hypoxic induction of Cdk5 activity correlates with Hif-1α stabilisation, and direct interaction between these proteins can occur. Importantly, we demonstrate that Cdk5-mediated signaling is involved in Hif-1α stabilisation since inhibition of Cdk5 by roscovitine abrogates Hif-1α accumulation and induces cell death. Taken together our results show that the Cdk5/p35 complex may significantly contribute to modulation of Hif-1α stabilisation and impact neuronal survival during oxygen deprivation. Thus this study highlights a new hypoxia-mediated signaling pathway and implicates the cytoskeleton as a potential regulator of Hif-1α.

    Brain research 2011;1381;1-10

  • A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

    Diez-Roux G, Banfi S, Sultan M, Geffers L, Anand S, Rozado D, Magen A, Canidio E, Pagani M, Peluso I, Lin-Marq N, Koch M, Bilio M, Cantiello I, Verde R, De Masi C, Bianchi SA, Cicchini J, Perroud E, Mehmeti S, Dagand E, Schrinner S, Nürnberger A, Schmidt K, Metz K, Zwingmann C, Brieske N, Springer C, Hernandez AM, Herzog S, Grabbe F, Sieverding C, Fischer B, Schrader K, Brockmeyer M, Dettmer S, Helbig C, Alunni V, Battaini MA, Mura C, Henrichsen CN, Garcia-Lopez R, Echevarria D, Puelles E, Garcia-Calero E, Kruse S, Uhr M, Kauck C, Feng G, Milyaev N, Ong CK, Kumar L, Lam M, Semple CA, Gyenesei A, Mundlos S, Radelof U, Lehrach H, Sarmientos P, Reymond A, Davidson DR, Dollé P, Antonarakis SE, Yaspo ML, Martinez S, Baldock RA, Eichele G and Ballabio A

    Telethon Institute of Genetics and Medicine, Naples, Italy.

    Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

    Funded by: Medical Research Council: MC_U127527203; Telethon: TGM11S03

    PLoS biology 2011;9;1;e1000582

  • Actin interaction and regulation of cyclin-dependent kinase 5/p35 complex activity.

    Xu J, Tsutsumi K, Tokuraku K, Estes KA, Hisanaga S and Ikezu T

    Center for Neurodegenerative Disorders, Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska, USA.

    Cyclin-dependent kinase 5 (Cdk5) plays a critical role during neurodevelopment, synaptic plasticity, and neurodegeneration. Cdk5 activity depends on association with neuronal proteins p35 and p25, a proteolytic product of p35. Cdk5 regulates the actin cytoskeletal dynamics that are essential for neuronal migration, neuritic growth, and synaptogenesis. However, little is known about the interaction of actin and Cdk5 and its effect on neuronal Cdk5 activity. In a previous study, we observed that Cdk5/p35 activity is negatively correlated with co-immunoprecipitated F-actin (filamentous actin) amounts in the mouse brain, and suggested that F-actin inhibits the formation of the Cdk5/p35 complex [Journal of Neuroscience (2008) vol. 28, p. 14511]. The experiments reported here were undertaken to elucidate the relationship between actin and the formation of the Cdk5/p35 complex and its activity. Instead of an F-actin-mediated inhibition, we propose that G-actin (globular actin) in the F-actin preparations is responsible for inhibiting Cdk5/p35 and Cdk5/p25 kinase activity. We found that F-actin binds to p35 but not p25 or Cdk5. We have shown that G-actin binds directly to Cdk5 without disrupting the formation of the Cdk5/p35 or Cdk5/p25 complexes. G-actin potently suppressed Cdk5/p35 and Cdk5/p25 activity when either histone H1 or purified human tau protein were used as substrates, indicating a substrate-independent inhibitory effect of G-actin on Cdk5 activity. Finally, G-actin suppressed the activity of Cdk5 immunoprecipitated from wild type and p35-deficient mouse brain, suggesting that G-actin suppresses endogenous Cdk5 activity in a p35-independent manner. Together, these results suggest a novel mechanism of actin cytoskeletal regulation of Cdk5/p35 activity.

    Funded by: NIMH NIH HHS: R01 MH083523, R01 MH083523-01, R01 MH083523-02, R01 MH083523-03

    Journal of neurochemistry 2011;116;2;192-204

  • Expression of cyclin E in postmitotic neurons during development and in the adult mouse brain.

    Ikeda Y, Matsunaga Y, Takiguchi M and Ikeda MA

    Department of Histology and Cell Biology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan. yayoi@yokohama-cu.ac.jp

    Cyclin E, a member of the G1 cyclins, is essential for the G1/S transition of the cell cycle in cultured cells, but its roles in vivo are not fully defined. The present study characterized the spatiotemporal expression profile of cyclin E in two representative brain regions in the mouse, the cerebral and cerebellar cortices. Western blotting showed that the levels of cyclin E increased towards adulthood. In situ hybridization and immunohistochemistry showed the distributions of cyclin E mRNA and protein were comparable in the cerebral cortex and the cerebellum. Immunohistochemistry for the proliferating cell marker, proliferating cell nuclear antigen (PCNA) revealed that cyclin E was expressed by both proliferating and non-proliferating cells in the cerebral cortex at embryonic day 12.5 (E12.5) and in the cerebellum at postnatal day 1 (P1). Subcellular localization in neurons was examined using immunofluorescence and western blotting. Cyclin E expression was nuclear in proliferating neuronal precursor cells but cytoplasmic in postmitotic neurons during embryonic development. Nuclear cyclin E expression in neurons remained faint in newborns, increased during postnatal development and was markedly decreased in adults. In various adult brain regions, cyclin E staining was more intense in the cytoplasm than in the nucleus in most neurons. These data suggest a role for cyclin E in the development and function of the mammalian central nervous system and that its subcellular localization in neurons is important. Our report presents the first detailed analysis of cyclin E expression in postmitotic neurons during development and in the adult mouse brain.

    Gene expression patterns : GEP 2011;11;1-2;64-71

  • Cyclin-dependent kinase 5 activity is required for T cell activation and induction of experimental autoimmune encephalomyelitis.

    Pareek TK, Lam E, Zheng X, Askew D, Kulkarni AB, Chance MR, Huang AY, Cooke KR and Letterio JJ

    Division of Pediatric Hematology/Oncology, Department of Pediatrics, University Hospitals Case Medical Center and The Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA.

    Cyclin-dependent kinase 5 (Cdk5) is a ubiquitously expressed serine/threonine kinase. However, a requirement for Cdk5 has been demonstrated only in postmitotic neurons where there is abundant expression of its activating partners p35 and/or p39. Although hyperactivation of the Cdk5-p35 complex has been found in a variety of inflammatory neurodegenerative disorders, the potential contribution of nonneuronal Cdk5-p35 activity has not been explored in this context. We describe a previously unknown function of the Cdk5-p35 complex in T cells that is required for induction of experimental autoimmune encephalomyelitis (EAE). T cell receptor (TCR) stimulation leads to a rapid induction of Cdk5-p35 expression that is required for T lymphocyte activation. Chimeric mice lacking Cdk5 gene expression in hematopoietic tissues (Cdk5(-/-C)) are resistant to induction of EAE, and adoptive transfer of either Cdk5(-/-C) or p35(-/-) encephalitogenic lymphocytes fails to transfer disease. Moreover, our data reveal a novel mechanism involving Cdk5-mediated phosphorylation of the actin modulator coronin 1a on threonine 418. Cdk5-deficient lymphocytes lack this posttranslational modification of coronin 1a and exhibit defective TCR-induced actin polarization and reduced migration toward CCL-19. These data define a distinct role for Cdk5 in lymphocyte biology and suggest that inhibition of this kinase may be beneficial in the treatment of T cell-mediated inflammatory disorders.

    The Journal of experimental medicine 2010;207;11;2507-19

  • Control of activating transcription factor 4 (ATF4) persistence by multisite phosphorylation impacts cell cycle progression and neurogenesis.

    Frank CL, Ge X, Xie Z, Zhou Y and Tsai LH

    Massachusetts Institute of Technology, the Howard Hughes Medical Institute, Cambridge, Massachusetts 02139, the Stanley Center for Psychiatric Research, Cambridge, Massachusetts 02139, USA.

    Organogenesis is a highly integrated process with a fundamental requirement for precise cell cycle control. Mechanistically, the cell cycle is composed of transitions and thresholds that are controlled by coordinated post-translational modifications. In this study, we describe a novel mechanism controlling the persistence of the transcription factor ATF4 by multisite phosphorylation. Proline-directed phosphorylation acted additively to regulate multiple aspects of ATF4 degradation. Stabilized ATF4 mutants exhibit decreased β-TrCP degron phosphorylation, β-TrCP interaction, and ubiquitination, as well as elicit early G(1) arrest. Expression of stabilized ATF4 also had significant consequences in the developing neocortex. Mutant ATF4 expressing cells exhibited positioning and differentiation defects that were attributed to early G(1) arrest, suggesting that neurogenesis is sensitive to ATF4 dosage. We propose that precise regulation of the ATF4 dosage impacts cell cycle control and impinges on neurogenesis.

    Funded by: Howard Hughes Medical Institute

    The Journal of biological chemistry 2010;285;43;33324-37

  • Silencing of CDK5 reduces neurofibrillary tangles in transgenic alzheimer's mice.

    Piedrahita D, Hernández I, López-Tobón A, Fedorov D, Obara B, Manjunath BS, Boudreau RL, Davidson B, Laferla F, Gallego-Gómez JC, Kosik KS and Cardona-Gómez GP

    Cellular and Molecular Neurobiology Area, Viral Vector Core and Gene Therapy, Group of Neuroscience of Antioquia, Faculty of Medicine, Sede de Investigación Universitaria, University of Antioquia, AA 1226 Medellin, Colombia.

    Alzheimer's disease is a major cause of dementia for which treatments remain unsatisfactory. Cyclin-dependent kinase 5 (CDK5) is a relevant kinase that has been hypothesized to contribute to the tau pathology. Several classes of chemical inhibitors for CDK5 have been developed, but they generally lack the specificity to distinguish among various ATP-dependent kinases. Therefore, the efficacy of these compounds when tested in animal models cannot definitively be attributed to an effect on CDK5. However, RNA interference (RNAi) targeting of CDK5 is specific and can be used to validate CDK5 as a possible treatment target. We delivered a CDK5 RNAi by lentiviral or adenoassociated viral vectors and analyzed the results in vitro and in vivo. Silencing of CDK5 reduces the phosphorylation of tau in primary neuronal cultures and in the brain of wild-type C57BL/6 mice. Furthermore, the knockdown of CDK5 strongly decreased the number of neurofibrillary tangles in the hippocampi of triple-transgenic mice (3×Tg-AD mice). Our data suggest that this downregulation may be attributable to the reduction of the CDK5 availability in the tissue, without affecting the CDK5 kinase activity. In summary, our findings validate CDK5 as a reasonable therapeutic target for ameliorating tau pathology.

    Funded by: NHLBI NIH HHS: T32 HL007121; NIA NIH HHS: R01 AG029802, R01 AG029802-01, R21 AG024024063, R21 AG024063, R21 AG024063-01; NINDS NIH HHS: NS 50210, P01 NS050210

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;42;13966-76

  • Phosphorylation of p27Kip1 at Thr187 by cyclin-dependent kinase 5 modulates neural stem cell differentiation.

    Zheng YL, Li BS, Rudrabhatla P, Shukla V, Amin ND, Maric D, Kesavapany S, Kanungo J, Pareek TK, Takahashi S, Grant P, Kulkarni AB and Pant HC

    Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-4130, USA.

    Cyclin-dependent kinase 5 (Cdk5) plays a key role in the development of the mammalian nervous system; it phosphorylates a number of targeted proteins involved in neuronal migration during development to synaptic activity in the mature nervous system. Its role in the initial stages of neuronal commitment and differentiation of neural stem cells (NSCs), however, is poorly understood. In this study, we show that Cdk5 phosphorylation of p27(Kip1) at Thr187 is crucial to neural differentiation because 1) neurogenesis is specifically suppressed by transfection of p27(Kip1) siRNA into Cdk5(+/+) NSCs; 2) reduced neuronal differentiation in Cdk5(-/-) compared with Cdk5(+/+) NSCs; 3) Cdk5(+/+) NSCs, whose differentiation is inhibited by a nonphosphorylatable mutant, p27/Thr187A, are rescued by cotransfection of a phosphorylation-mimicking mutant, p27/Thr187D; and 4) transfection of mutant p27(Kip1) (p27/187A) into Cdk5(+/+) NSCs inhibits differentiation. These data suggest that Cdk5 regulates the neural differentiation of NSCs by phosphorylation of p27(Kip1) at theThr187 site. Additional experiments exploring the role of Ser10 phosphorylation by Cdk5 suggest that together with Thr187 phosphorylation, Ser10 phosphorylation by Cdk5 promotes neurite outgrowth as neurons differentiate. Cdk5 phosphorylation of p27(Kip1), a modular molecule, may regulate the progress of neuronal differentiation from cell cycle arrest through differentiation, neurite outgrowth, and migration.

    Molecular biology of the cell 2010;21;20;3601-14

  • Anti-diabetic drugs inhibit obesity-linked phosphorylation of PPARgamma by Cdk5.

    Choi JH, Banks AS, Estall JL, Kajimura S, Boström P, Laznik D, Ruas JL, Chalmers MJ, Kamenecka TM, Blüher M, Griffin PR and Spiegelman BM

    Department of Cancer Biology and Division of Metabolism and Chronic Disease, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.

    Obesity induced in mice by high-fat feeding activates the protein kinase Cdk5 (cyclin-dependent kinase 5) in adipose tissues. This results in phosphorylation of the nuclear receptor PPARgamma (peroxisome proliferator-activated receptor gamma), a dominant regulator of adipogenesis and fat cell gene expression, at serine 273. This modification of PPARgamma does not alter its adipogenic capacity, but leads to dysregulation of a large number of genes whose expression is altered in obesity, including a reduction in the expression of the insulin-sensitizing adipokine, adiponectin. The phosphorylation of PPARgamma by Cdk5 is blocked by anti-diabetic PPARgamma ligands, such as rosiglitazone and MRL24. This inhibition works both in vivo and in vitro, and is completely independent of classical receptor transcriptional agonism. Similarly, inhibition of PPARgamma phosphorylation in obese patients by rosiglitazone is very tightly associated with the anti-diabetic effects of this drug. All these findings strongly suggest that Cdk5-mediated phosphorylation of PPARgamma may be involved in the pathogenesis of insulin-resistance, and present an opportunity for development of an improved generation of anti-diabetic drugs through PPARgamma.

    Funded by: Intramural NIH HHS; NCRR NIH HHS: S10 RR027270; NIDDK NIH HHS: DK087853, DK31405, K99 DK087853, R00 DK087853, R01 DK031405, R37 DK031405, R37 DK031405-30; NIGMS NIH HHS: R01 GM084041, R01 GM084041-03, R01-GM084041; NIMH NIH HHS: U54 MH084512, U54 MH084512-020010, U54-MH084512

    Nature 2010;466;7305;451-6

  • Mice lacking p35 display hyperactivity and paradoxical response to psychostimulants.

    Krapacher FA, Mlewski EC, Ferreras S, Pisano V, Paolorossi M, Hansen C and Paglini G

    Laboratory of Neurobiology and Cell Biology, Instituto de Investigación Médica Mercedes y Martín Ferreyra (INIMEC-CONICET), 5016 Córdoba, Argentina.

    Cyclin-dependent kinase 5/p35 kinase complex plays a critical role in dopaminergic neurotransmission. Dysregulation of dopamine (DA) signaling is associated with neurological and neuropsychiatric disorders. As cyclin-dependent kinase 5 (Cdk5) requires association with p35 for its proper activation, we hypothesized that dysregulation of Cdk5 activity might have an effect on striatal-mediated behavior. We used a mutant mouse, deficient in p35 protein (p35 KO), which displayed reduced Cdk5 activity. Throughout behavioral and biochemical characterization of naïve and psychostimulant-treated mice, we demonstrated that only juvenile p35 KO mice displayed spontaneous hyperactivity, responded with a paradoxical hypolocomotor effect to psychostimulant drugs and exhibited deficit on proper behavioral inhibition. Strong immunolabeling for tyrosine-hydroxylase and high striatal DA synthesis and contents with a low DA turnover, which were reverted by psychostimulants, were also found in mutant mice. Our results demonstrate that p35 deficiency is critically involved in the expression of a hyperactive behavioral phenotype with hyper-functioning of the dopaminergic system, emphasizing the importance of proper Cdk5 kinase activity for normal motor and emotional features. Thus, p35 KO mice may be another useful animal model for understanding cellular and molecular events underlying attention deficit hyperactivity disorder-like disorders.

    Journal of neurochemistry 2010;114;1;203-14

  • Myocardin-related transcription factors regulate the Cdk5/Pctaire1 kinase cascade to control neurite outgrowth, neuronal migration and brain development.

    Mokalled MH, Johnson A, Kim Y, Oh J and Olson EN

    Department of Molecular Biology, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9148, USA.

    Numerous motile cell functions depend on signaling from the cytoskeleton to the nucleus. Myocardin-related transcription factors (MRTFs) translocate to the nucleus in response to actin polymerization and cooperate with serum response factor (Srf) to regulate the expression of genes encoding actin and other components of the cytoskeleton. Here, we show that MRTF-A (Mkl1) and MRTF-B (Mkl2) redundantly control neuronal migration and neurite outgrowth during mouse brain development. Conditional deletion of the genes encoding these Srf coactivators disrupts the formation of multiple brain structures, reflecting a failure in neuronal actin polymerization and cytoskeletal assembly. These abnormalities were accompanied by dysregulation of the actin-severing protein gelsolin and Pctaire1 (Cdk16) kinase, which cooperates with Cdk5 to initiate a kinase cascade that governs cytoskeletal rearrangements essential for neuron migration and neurite outgrowth. Thus, the MRTF/Srf partnership interlinks two key signaling pathways that control actin treadmilling and neuronal maturation, thereby fulfilling a regulatory loop that couples cytoskeletal dynamics to nuclear gene transcription during brain development.

    Funded by: NHLBI NIH HHS: R01 HL077439, R01 HL093039, R37 HL053351

    Development (Cambridge, England) 2010;137;14;2365-74

  • Cdk5-mediated phosphorylation of delta-catenin regulates its localization and GluR2-mediated synaptic activity.

    Poore CP, Sundaram JR, Pareek TK, Fu A, Amin N, Mohamed NE, Zheng YL, Goh AX, Lai MK, Ip NY, Pant HC and Kesavapany S

    Department of Biochemistry, Neurobiology Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

    Cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation plays an important role in proper synaptic function and transmission. Loss of Cdk5 activity results in abnormal development of the nervous system accompanied by massive disruptions in cortical migration and lamination, therefore impacting synaptic activity. The Cdk5 activator p35 associates with delta-catenin, the synaptic adherens junction protein that serves as part of the anchorage complex of AMPA receptor at the postsynaptic membrane. However, the implications of Cdk5-mediated phosphorylation of delta-catenin have not been fully elucidated. Here we show that Cdk5-mediated phosphorylation of delta-catenin regulates its subcellular localization accompanied by changes in dendritic morphogenesis and synaptic activity. We identified two Cdk5 phosphorylation sites in mouse delta-catenin, serines 300 and 357, and report that loss of Cdk5 phosphorylation of delta-catenin increased its localization to the membrane. Furthermore, mutations of the serines 300 and 357 to alanines to mimic nonphosphorylated delta-catenin resulted in increased dendritic protrusions accompanied by increased AMPA receptor subunit GluR2 localization at the membrane. Consistent with these observations, loss of Cdk5 phosphorylation of delta-catenin increased the AMPA/NMDA ratio. This study reveals how Cdk5 phosphorylation of the synaptic mediator protein delta-catenin can alter its localization at the synapse to impact neuronal synaptic activity.

    Funded by: Intramural NIH HHS: Z99 NS999999

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;25;8457-67

  • Cdk5 nuclear localization is p27-dependent in nerve cells: implications for cell cycle suppression and caspase-3 activation.

    Zhang J, Li H and Herrup K

    Department of Cell Biology and Neuroscience, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854-8082, USA.

    Initiation of a cell cycle in an adult neuron leads to cell death, placing great importance on the mechanisms that normally suppress the neuronal cell cycle. We have previously shown that the cyclin-dependent kinase Cdk5 is an important part of this process, but only when it is present in the nucleus. We report here that Cdk5 nuclear localization relies on its binding to the cyclin-dependent kinase inhibitor p27. Cdk5 has no intrinsic nuclear localization signal; in the absence of p27, two weak nuclear export signals that bind CRM1 cause it to shuttle to the cytoplasm. When a neuron is subjected to stress, such as exposure to beta-amyloid, the Cdk5-p27 interaction is lost, reducing Cdk5 levels in the nucleus and depriving the neuron of a major cell cycle suppression mechanism. Caspase-3 is activated within hours, but death is not immediate; elevated levels of cytoplasmic Cdk5 appear to retard neuronal death by a mechanism that may involve Bcl2. These data suggest a model in which Cdk5 exerts a double protective function in neurons: chronically suppressing the cell cycle when located in the nucleus and transiently delaying cell death in the cytoplasm.

    Funded by: NINDS NIH HHS: R01 NS020591, R01 NS020591-26A2, R01NS020591

    The Journal of biological chemistry 2010;285;18;14052-61

  • Protein kinase Czeta regulates Cdk5/p25 signaling during myogenesis.

    de Thonel A, Ferraris SE, Pallari HM, Imanishi SY, Kochin V, Hosokawa T, Hisanaga S, Sahlgren C and Eriksson JE

    Turku Centre for Biotechnology, University of Turku and Abo Akademi University, 20521 Turku, Finland.

    Atypical protein kinase Czeta (PKCzeta) is emerging as a mediator of differentiation. Here, we describe a novel role for PKCzeta in myogenic differentiation, demonstrating that PKCzeta activity is indispensable for differentiation of both C2C12 and mouse primary myoblasts. PKCzeta was found to be associated with and to regulate the Cdk5/p35 signaling complex, an essential factor for both neuronal and myogenic differentiation. Inhibition of PKCzeta activity prevented both myotube formation and simultaneous reorganization of the nestin intermediate filament cytoskeleton, which is known to be regulated by Cdk5 during myogenesis. p35, the Cdk5 activator, was shown to be a specific phosphorylation target of PKCzeta. PKCzeta-mediated phosphorylation of Ser-33 on p35 promoted calpain-mediated cleavage of p35 to its more active and stable fragment, p25. Strikingly, both calpain activation and the calpain-mediated cleavage of p35 were shown to be PKCzeta-dependent in differentiating myoblasts. Overall, our results identify PKCzeta as a controller of myogenic differentiation by its regulation of the phosphorylation-dependent and calpain-mediated p35 cleavage, which is crucial for the amplification of the Cdk5 activity that is required during differentiation.

    Molecular biology of the cell 2010;21;8;1423-34

  • Cdk5 suppresses the neuronal cell cycle by disrupting the E2F1-DP1 complex.

    Zhang J, Li H, Yabut O, Fitzpatrick H, D'Arcangelo G and Herrup K

    Department of Cell Biology and Neuroscience, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA.

    Neurons that reenter a cell cycle after maturation are at increased risk for death, yet the mechanisms by which a normal neuron suppresses the cycle remain mostly unknown. Our laboratory has shown that cyclin-dependent kinase 5 (Cdk5) is a potent cell cycle suppressor, and we report here on the molecular basis of this activity. Cell cycle suppression by Cdk5 requires its binding to the p35 activator protein. The related p39 and p25 proteins cannot serve as substitutes. Unexpectedly, Cdk5 enzymatic activity is not required to perform this function. Rather, the link to cell cycle regulation is made through the formation of a previously unknown complex consisting of the p35-Cdk5 dimer and E2F1. Formation of this complex excludes the E2F1 cofactor, DP1, thus inhibiting E2F1 binding to the promoters of various cell cycle genes. This anti-cell cycle activity is most likely a neuroprotective function of Cdk5.

    Funded by: NINDS NIH HHS: NS20591, R01 NS020591, R01 NS020591-26A2

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;15;5219-28

  • Neurodegeneration in an Abeta-induced model of Alzheimer's disease: the role of Cdk5.

    Lopes JP, Oliveira CR and Agostinho P

    Center for Neuroscience and Cell Biology, Faculty of Medicine, Biochemistry Institute, University of Coimbra, 3004 Coimbra, Portugal.

    Cdk5 dysregulation is a major event in the neurodegenerative process of Alzheimer's disease (AD). In vitro studies using differentiated neurons exposed to Abeta exhibit Cdk5-mediated tau hyperphosphorylation, cell cycle re-entry and neuronal loss. In this study we aimed to determine the role of Cdk5 in neuronal injury occurring in an AD mouse model obtained through the intracerebroventricular (icv) injection of the Abeta(1-40) synthetic peptide. In mice icv-injected with Abeta, Cdk5 activator p35 is cleaved by calpains, leading to p25 formation and Cdk5 overactivation. Subsequently, there was an increase in tau hyperphosphorylation, as well as decreased levels of synaptic markers. Cell cycle reactivation and a significant neuronal loss were also observed. These neurotoxic events in Abeta-injected mice were prevented by blocking calpain activation with MDL28170, which was administered intraperitoneally (ip). As MDL prevents p35 cleavage and subsequent Cdk5 overactivation, it is likely that this kinase is involved in tau hyperphosphorylation, cell cycle re-entry, synaptic loss and neuronal death triggered by Abeta. Altogether, these data demonstrate that Cdk5 plays a pivotal role in tau phosphorylation, cell cycle induction, synaptotoxicity, and apoptotic death in postmitotic neurons exposed to Abeta peptides in vivo, acting as a link between diverse neurotoxic pathways of AD.

    Aging cell 2010;9;1;64-77

  • Conditional deletion of neuronal cyclin-dependent kinase 5 in developing forebrain results in microglial activation and neurodegeneration.

    Takahashi S, Ohshima T, Hirasawa M, Pareek TK, Bugge TH, Morozov A, Fujieda K, Brady RO and Kulkarni AB

    Functional Genomics Section, Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA.

    Neuronal migration disorders are often identified in patients with epilepsy refractory to medical treatment. The prolonged or repeated seizures are known to cause neuronal death; however, the mechanism underlying seizure-induced neuronal death remains to be elucidated. An essential role of cyclin-dependent kinase 5 (Cdk5) in brain development has been demonstrated in Cdk5(-/-) mice, which show neuronal migration defects and perinatal lethality. Here, we show the consequences of Cdk5 deficiency in the postnatal brain by generating Cdk5 conditional knockout mice, in which Cdk5is selectively eliminated from neurons in the developing forebrain. The conditional mutant mice were viable, but exhibited complex neurological deficits including seizures, tremors, and growth retardation. The forebrain not only showed disruption of layering, but also neurodegenerative changes accompanied by neuronal loss and microglial activation. The neurodegenerative changes progressed with age and were accompanied by up-regulation of the neuronal tissue-type plasminogen activator, a serine protease known to mediate microglial activation. Thus age-dependent neurodegeneration in the Cdk5 conditional knockout mouse brain invoked a massive inflammatory reaction. These findings indicate an important role of Cdk5 in inflammation, and also provide a mouse model to examine the possible involvement of inflammation in the pathogenesis of progressive cognitive decline in patients with neuronal migration disorders.

    The American journal of pathology 2010;176;1;320-9

  • Direct and indirect roles of cyclin-dependent kinase 5 as an upstream regulator in the c-Jun NH2-terminal kinase cascade: relevance to neurotoxic insults in Alzheimer's disease.

    Sun KH, Lee HG, Smith MA and Shah K

    Department of Chemistry and Purdue Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA.

    Significant increase in JNK, c-Jun, and Cdk5 activities are reported in Alzheimer's disease (AD). Inhibition of c-Jun prevents neuronal cell death in in vivo AD models, highlighting it as a major JNK effector. Both JNK and Cdk5 promote neurodegeneration upon deregulation; however, Cdk5 has not been mechanistically linked to JNK or c-Jun. This study presents the first mechanism showing Cdk5 as a major regulator of the JNK cascade. Deregulated Cdk5 induces biphasic activation of JNK pathway. The first phase revealed c-Jun as a direct substrate of Cdk5, whose activation is independent of reactive oxygen species (ROS) and JNK. In the second phase, Cdk5 activates c-Jun via ROS-mediated activation of JNK. Rapid c-Jun activation is supported by in vivo data showing c-Jun phosphorylation in cerebral cortex upon p25 induction in transgenic mice. Cdk5-mediated biphasic activation of c-Jun highlights c-Jun, rather than JNK, as an important therapeutic target, which was confirmed in neuronal cells. Finally, Cdk5 inhibition endows superior protection against neurotoxicity, suggesting that Cdk5 is a preferable therapeutic target for AD relative to JNK and c-Jun.

    Funded by: NIA NIH HHS: R01 AG028679, R01AG028679

    Molecular biology of the cell 2009;20;21;4611-9

  • The Cdk5/p35 kinases modulate leptin-induced STAT3 signaling.

    He Y, Kastin AJ, Hsuchou H and Pan W

    Blood-Brain Barrier Group, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808, USA.

    Cyclin-dependent kinase (Cdk) 5 is ubiquitously expressed in the brain and plays an essential role in central nervous system development and synaptic plasticity. The p35 kinase is a neuronal specific activator of Cdk5. Here, we show for the first time that Cdk5 activation modulates leptin signaling. P35 and its metabolite p25 were colocalized with the leptin receptor ObR in selective neurons in the hypothalamus. Overexpression of p35 alone was sufficient to induce the transcriptional activation of signal transducer and activator of transcription 3 (STAT3) in a cellular model. In retinoic acid-differentiated SH-SY5Y neuronal cells where ObRb was induced, leptin increased the expression of Cdk5, p35, and p25 kinases. The time course of induction coincided with that of phosphorylated (p)-STAT3. When Cdk5 activity was inhibited, either by roscovitine or overexpression of dominant negative Cdk5, there was a reduction of pSTAT3 activation. The results show that the activation of Cdk5 by p35 sustained leptin-induced pSTAT3 at 3-6 h. Thus, p35 is a novel modulator of leptin-induced STAT3 signaling.

    Funded by: NIDDK NIH HHS: DK54880, R01 DK054880-10; NINDS NIH HHS: NS45751, NS46528, NS62291, R01 NS045751-06, R01 NS046528-05, R01 NS062291-01A1

    Journal of molecular neuroscience : MN 2009;39;1-2;49-58

  • Analysis of early nephron patterning reveals a role for distal RV proliferation in fusion to the ureteric tip via a cap mesenchyme-derived connecting segment.

    Georgas K, Rumballe B, Valerius MT, Chiu HS, Thiagarajan RD, Lesieur E, Aronow BJ, Brunskill EW, Combes AN, Tang D, Taylor D, Grimmond SM, Potter SS, McMahon AP and Little MH

    NHMRC Principal Research Fellow, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Australia.

    While nephron formation is known to be initiated by a mesenchyme-to-epithelial transition of the cap mesenchyme to form a renal vesicle (RV), the subsequent patterning of the nephron and fusion with the ureteric component of the kidney to form a patent contiguous uriniferous tubule has not been fully characterized. Using dual section in situ hybridization (SISH)/immunohistochemistry (IHC) we have revealed distinct distal/proximal patterning of Notch, BMP and Wnt pathway components within the RV stage nephron. Quantitation of mitoses and Cyclin D1 expression indicated that cell proliferation was higher in the distal RV, reflecting the differential developmental programs of the proximal and distal populations. A small number of RV genes were also expressed in the early connecting segment of the nephron. Dual ISH/IHC combined with serial section immunofluorescence and 3D reconstruction revealed that fusion occurs between the late RV and adjacent ureteric tip via a process that involves loss of the intervening ureteric epithelial basement membrane and insertion of cells expressing RV markers into the ureteric tip. Using Six2-eGFPCre x R26R-lacZ mice, we demonstrate that these cells are derived from the cap mesenchyme and not the ureteric epithelium. Hence, both nephron patterning and patency are evident at the late renal vesicle stage.

    Funded by: NIDDK NIH HHS: DK070136, DK070251, F32DK060319, R37 DK054364, U01 DK070136

    Developmental biology 2009;332;2;273-86

  • Cortical interneurons require p35/Cdk5 for their migration and laminar organization.

    Rakić S, Yanagawa Y, Obata K, Faux C, Parnavelas JG and Nikolić M

    Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, UK.

    Projection neurons and interneurons populate the cerebral cortex in a layer-specific manner. Here, we studied the role of Cyclin-dependent kinase 5 (Cdk5) and its activator p35 in cortical interneuron migration and disposition in the cortex. We found that mice lacking p35 (p35(-/-)) show accumulation of interneurons in the upper part of the cortex. We also observed an inverted distribution of both early- and late-born interneurons, with the former showing a preference for the upper and the latter for the lower aspects of the cortex. We investigated the causes of the altered laminar organization of interneurons in p35(-/-) mice and found a cell-autonomous delay in their tangential migration that may prevent them from reaching correct positions. Incomplete splitting of the preplate in p35(-/-) mice, which causes accumulation of cells in the superficial layer and defects in the "inward" and "outward" components of their radial movement, may also account for the altered final arrangement of interneurons. We, therefore, propose that p35/Cdk5 plays a key role in guiding cortical interneurons to their final positions in the cortex.

    Funded by: Wellcome Trust: 069441, 074549

    Cerebral cortex (New York, N.Y. : 1991) 2009;19;8;1857-69

  • The Cdk5 inhibitor roscovitine strongly inhibits glucose uptake in 3T3-L1 adipocytes without altering GLUT4 translocation from internal pools to the cell surface.

    Muruáis G, Lalioti V and Sandoval IV

    Centro de Biología Molecular Severo Ochoa, Centro de Investigaciones Biomédicas en Red, Enfermedades Hepáticas y Digestivas, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain.

    Glucose entry into mammalian cells is facilitated by a family of glucose transport proteins known as GLUTs. Treatment of 3T3-L1 adipocytes with the Cdk5 inhibitor roscovitine strongly inhibits insulin-stimulated/GLUT4-mediated glucose transport. Inhibition of glucose uptake occurs within 2-6 min of the addition of roscovitine and is slowly reversed. The roscovitine treatment interferes with neither the translocation nor the insertion of GLUT4 into the plasma membrane. These studies support recent evidence showing that insulin-stimulated Cdk5 is implicated in the regulation of GLUT4-mediated glucose uptake in 3T3-L1 adipocytes.

    Journal of cellular physiology 2009;220;1;238-44

  • Phosphorylation of the homer-binding domain of group I metabotropic glutamate receptors by cyclin-dependent kinase 5.

    Orlando LR, Ayala R, Kett LR, Curley AA, Duffner J, Bragg DC, Tsai LH, Dunah AW and Young AB

    Department of Neurology, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Boston, 02129, USA.

    Phosphorylation of neurotransmitter receptors can modify their activity and regulate neuronal excitability. Cyclin-dependent kinase 5 (cdk5) is a proline-directed serine/threonine kinase involved not only in neuronal development, but also in synaptic function and plasticity. Here we demonstrate that group I metabotropic glutamate receptors (mGluRs), which modulate post-synaptic signaling by coupling to intracellular signal transduction pathways, are phosphorylated by cdk5. In vitro kinase assays reveal that cdk5 phosphorylates mGluR5 within the domain of the receptor that interacts with the scaffolding protein homer. Using a novel phosphospecific mGluR antibody, we show that the homer-binding domain of both mGluR1 and mGluR5 are phosphorylated in vivo, and that inhibition of cdk5 with siRNA decreases the amount of phosphorylated receptor. Furthermore, kinetic binding analysis, by surface plasmon resonance, indicates that phosphorylation of mGluR5 enhances its association with homer. Homer protein complexes in the post-synaptic density, and their disruption by an activity-dependent short homer 1a isoform, have been shown to regulate the trafficking and signaling of the mGluRs and impact many neuroadaptive processes. Phosphorylation of the mGluR homer-binding domain, in contrast to homer 1a induction, provides a novel mechanism for potentially regulating a subset of homer interactions.

    Funded by: NIA NIH HHS: AG13617, R37 AG013617; NINDS NIH HHS: NS049006

    Journal of neurochemistry 2009;110;2;557-69

  • Regulation of hippocampal and behavioral excitability by cyclin-dependent kinase 5.

    Hawasli AH, Koovakkattu D, Hayashi K, Anderson AE, Powell CM, Sinton CM, Bibb JA and Cooper DC

    Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

    Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that has been implicated in learning, synaptic plasticity, neurotransmission, and numerous neurological disorders. We previously showed that conditional loss of Cdk5 in adult mice enhanced hippocampal learning and plasticity via modulation of calpain-mediated N-methyl-D-aspartic acid receptor (NMDAR) degradation. In the present study, we characterize the enhanced synaptic plasticity and examine the effects of long-term Cdk5 loss on hippocampal excitability in adult mice. Field excitatory post-synaptic potentials (fEPSPs) from the Schaffer collateral CA1 subregion of the hippocampus (SC/CA1) reveal that loss of Cdk5 altered theta burst topography and enhanced post-tetanic potentiation. Since Cdk5 governs NMDAR NR2B subunit levels, we investigated the effects of long-term Cdk5 knockout on hippocampal neuronal excitability by measuring NMDAR-mediated fEPSP magnitudes and population-spike thresholds. Long-term loss of Cdk5 led to increased Mg(2+)-sensitive potentials and a lower threshold for epileptiform activity and seizures. Biochemical analyses were performed to better understand the role of Cdk5 in seizures. Induced-seizures in wild-type animals led to elevated amounts of p25, the Cdk5-activating cofactor. Long-term, but not acute, loss of Cdk5 led to decreased p25 levels, suggesting that Cdk5/p25 may be activated as a homeostatic mechanism to attenuate epileptiform activity. These findings indicate that Cdk5 regulates synaptic plasticity, controls neuronal and behavioral stimulus-induced excitability and may be a novel pharmacological target for cognitive and anticonvulsant therapies.

    Funded by: NICHD NIH HHS: R21 HD065290, R21 HD065290-01, R21 HD065290-02; NIDA NIH HHS: K01 DA017750, R01 DA024040; NIMH NIH HHS: K08 MH065975, R01 MH081164, R01 MH081164-01A2, R01 MH081164-02, R01 MH081164-03, R01 MH081164-04, R01 MH081164-05

    PloS one 2009;4;6;e5808

  • The neuroprotective effects of cyclin-dependent kinase-5 inhibition in mice with Niemann-Pick disease type C.

    Hao Y, Pan D, Zhang M, Xu J, Li L, Wei J and Wang X

    Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Wuhan, 430030, China. youguohao6@yahoo.com.cn

    In order to investigate the neuroprotective effects of cyclin-dependent kinase-5 (cdk-5) inhibition in mice with Niemann-Pick disease type C (NPC) (npc(-/-)), recombinant adeno-associated virus (rAAV) carrying the small interfering RNA (siRNA) specific for cdk-5 gene was injected into 3-day-old npc(-/-) mice intracerebroventricularly. The rAAV-GFP-injected age-matched npc(-/-) mice and non-surgery age-matched npc(-/-) mice were employed as controls (n=6-10/group). From the 4th to 8th week after the treatment, mice were weighed, and evaluated for limb motor activity by using the coat hanger test once a week. Eight-week-old npc(-/-) mice were sacrificed by decapitation, and brains were quickly dissected and halved sagittally. Immunohistochemistry, Western blotting, and HE staining were used to evaluate the neuropathology in npc(-/-) mice. The results showed that rAAV-cdk-5-siRNA-GFP significantly reduced the number of axonal spheroids, delayed the death of Purkinje neurons, ameliorated motor defects in npc(-/-) mice, and significantly attenuated the hyperphosphorylation of tau proteins. These data suggested that inhibition of cdk-5 activity has neuroprotective effect on neurons in NPC mice.

    Journal of Huazhong University of Science and Technology. Medical sciences = Hua zhong ke ji da xue xue bao. Yi xue Ying De wen ban = Huazhong keji daxue xuebao. Yixue Yingdewen ban 2009;29;3;324-9

  • [Neuronal migration and Cdk5].

    Ohshima T


    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 2009;54;7;796-801

  • [Role of Cdk5 in axon guidance and synapse development].

    Yamashita N, Nakamura F and Goshima Y


    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 2009;54;7;802-7

  • The atypical kinase Cdk5 is activated by insulin, regulates the association between GLUT4 and E-Syt1, and modulates glucose transport in 3T3-L1 adipocytes.

    Lalioti V, Muruais G, Dinarina A, van Damme J, Vandekerckhove J and Sandoval IV

    Department of Cell Biology and Immunology, Centro de Biologia Molecular Severo Ochoa, Universidad Autónoma de Madrid, 28049 Madrid, Spain.

    Here, we report that Cdk5 activation is stimulated by insulin and plays a key role in the regulation of GLUT4-mediated glucose uptake in 3T3-L1 adipocytes. Insulin activation of Cdk5 requires PI3K signaling. Insulin-activated Cdk5 phosphorylates E-Syt1, a 5 C2-domain protein-related to the synaptotagmins that is induced during adipocyte differentiation. Phosphorylated E-Syt1 associates with GLUT4, an event inhibited by the Cdks inhibitor roscovitine. Cdk5 silencing inhibits glucose uptake by 3T3-L1 adipocytes. These studies elucidate a previously unknown activity of Cdk5 and demonstrate the involvement of this kinase in the regulation of insulin-dependent glucose uptake in adipocytes.

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;11;4249-53

  • Activation of cyclin-dependent kinase 5 is a consequence of cell death.

    Ye Y, Tinari A, Malorni W, Lockshin RA and Zakeri Z

    Department of Biology, Queens College and Graduate Center of the City University of New York, Flushing, NY 11367, USA.

    Cyclin-dependent kinase 5 (Cdk5) is similar to other Cdks but is activated during cell differentiation and cell death rather than cell division. Since activation of Cdk5 has been reported in many situations leading to cell death, we attempted to determine if it was required for any form of cell death. We found that Cdk5 is activated during apoptotic deaths and that the activation can be detected even when the cells continue to secondary necrosis. This activation can occur in the absence of Bim, calpain, or neutral cathepsins. The kinase is typically activated by p25, derived from p35 by calpain-mediated cleavage, but inhibition of calpain does not affect cell death or the activation of Cdk5. Likewise, RNAi-forced suppression of the synthesis of Cdk5 does not affect the incidence or kinetics of cell death. We conclude that Cdk5 is activated as a consequence of metabolic changes that are common to many forms of cell death. Thus its activation suggests processes during cell death that will be interesting or important to understand, but activation of Cdk5 is not necessary for cells to die.

    Journal of biomedicine & biotechnology 2009;2009;805709

  • CDK5-dependent phosphorylation of the Rho family GTPase TC10(alpha) regulates insulin-stimulated GLUT4 translocation.

    Okada S, Yamada E, Saito T, Ohshima K, Hashimoto K, Yamada M, Uehara Y, Tsuchiya T, Shimizu H, Tatei K, Izumi T, Yamauchi K, Hisanaga S, Pessin JE and Mori M

    Department of Medicine, Gunma University Graduate School of Medicine, Showa-machi, Maebashi, Gunma 371-0034, Japan. okadash@showa.gunma-u.ac.jp

    Insulin stimulation results in the activation of cyclin-dependent kinase-5 (CDK5) in lipid raft domains via a Fyn-dependent phosphorylation on tyrosine residue 15. In turn, activated CDK5 phosphorylates the Rho family GTP-binding protein TC10alpha on threonine 197 that is sensitive to the CDK5 inhibitor olomoucine and blocked by small interfering RNA-mediated knockdown of CDK5. The phosphorylation deficient mutant T197A-TC10alpha was not phosphorylated and excluded from the lipid raft domain, whereas the phosphorylation mimetic mutant (T197D-TC10alpha) was lipid raft localized. Insulin resulted in the GTP loading of T197D-TC10alpha but not T197A-TC10alpha and in parallel, T197D-TC10alpha but not T197A-TC10alpha depolymerized cortical actin and inhibited insulin-stimulated GLUT4 translocation. These data demonstrate that CDK5-dependent phosphorylation maintains TC10alpha in lipid raft compartments thereby disrupting cortical actin, whereas subsequent dephosphorylation of TC10alpha through inactivation of CDK5 allows for the re-assembly of F-actin. Because cortical actin reorganization is required for insulin-stimulated GLUT4 translocation, these data are consistent with a CDK5-dependent TC10alpha cycling between lipid raft and non-lipid raft compartments.

    The Journal of biological chemistry 2008;283;51;35455-63

  • Regulation of neural migration by the CREB/CREM transcription factors and altered Dab1 levels in CREB/CREM mutants.

    Díaz-Ruiz C, Parlato R, Aguado F, Ureña JM, Burgaya F, Martínez A, Carmona MA, Kreiner G, Bleckmann S, Del Río JA, Schütz G and Soriano E

    Institute for Research in Biomedicine-Barcelona (IRB), Department of Cell Biology and CIBERNED (ISCIII), University of Barcelona, Barcelona Science Park, Lab A1-S1, Josep Samitier 1-5, Barcelona 08028, Spain.

    The family of CREB transcription factors is involved in a variety of biological processes including the development and plasticity of the nervous system. To gain further insight into the roles of CREB family members in the development of the embryonic brain, we examined the migratory phenotype of CREB1(Nescre)CREM(-/-) mutants. We found that the lack of CREB/CREM genes is accompanied by anatomical defects in specific layers of the olfactory bulb, hippocampus and cerebral cortex. These changes are associated with decreased Dab1 expression in CREB1(Nescre)CREM(-/-) mutants. Our results indicate that the lack of CREB/CREM genes, specifically in neural and glial progenitors, leads to migration abnormalities during brain development, suggesting that unidentified age-dependent factors modulate the role of CREB/CREM genes in neural development.

    Molecular and cellular neurosciences 2008;39;4;519-28

  • Cdk5 is essential for adult hippocampal neurogenesis.

    Lagace DC, Benavides DR, Kansy JW, Mapelli M, Greengard P, Bibb JA and Eisch AJ

    Department of Psychiatry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9070, USA.

    The molecular factors regulating adult neurogenesis must be understood to harness the therapeutic potential of neuronal stem cells. Although cyclin-dependent kinase 5 (Cdk5) plays a critical role in embryonic corticogenesis, its function in adult neurogenesis is unknown. Here, we assessed the role of Cdk5 in the generation of dentate gyrus (DG) granule cell neurons in adult mice. Cre recombinase-mediated conditional knockout (KO) of Cdk5 from stem cells and their progeny in the DG subgranular zone (SGZ) prevented maturation of new neurons. In addition, selective KO of Cdk5 from mature neurons throughout the hippocampus reduced the number of immature neurons. Furthermore, Cdk5 gene deletion specifically from DG granule neurons via viral-mediated gene transfer also resulted in fewer immature neurons. In each case, the total number of proliferating cells was unaffected, indicating that Cdk5 is necessary for progression of adult-generated neurons to maturity. This role for Cdk5 in neurogenesis was activating-cofactor specific, as p35 KO but not p39 KO mice also had fewer immature neurons. Thus, Cdk5 has an essential role in the survival, but not proliferation, of adult-generated hippocampal neurons through both cell-intrinsic and cell-extrinsic mechanisms.

    Funded by: NIDA NIH HHS: DA10044, DA16672, K02 DA023555, P01 DA010044, R01 DA016672, R01 DA016765, R21 DA023701; NIMH NIH HHS: MH074866, MH079710, P50 MH074866, R01 MH079710

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;47;18567-71

  • Striatal dysregulation of Cdk5 alters locomotor responses to cocaine, motor learning, and dendritic morphology.

    Meyer DA, Richer E, Benkovic SA, Hayashi K, Kansy JW, Hale CF, Moy LY, Kim Y, O'Callaghan JP, Tsai LH, Greengard P, Nairn AC, Cowan CW, Miller DB, Antich P and Bibb JA

    Departments of Psychiatry and Radiology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9070, USA.

    Motor learning and neuro-adaptations to drugs of abuse rely upon neuronal signaling in the striatum. Cyclin-dependent kinase 5 (Cdk5) regulates striatal dopamine neurotransmission and behavioral responses to cocaine. Although the role for Cdk5 in neurodegeneration in the cortex and hippocampus and in hippocampal-dependent learning has been demonstrated, its dysregulation in the striatum has not been examined. Here we show that strong activation of striatal NMDA receptors produced p25, the truncated form of the Cdk5 co-activator p35. Furthermore, inducible overexpression of p25 in the striatum prevented locomotor sensitization to cocaine and attenuated motor coordination and learning. This corresponded with reduced dendritic spine density, increased neuro-inflammation, altered dopamine signaling, and shifted Cdk5 specificity with regard to physiological and aberrant substrates, but no apparent loss of striatal neurons. Thus, dysregulation of Cdk5 dramatically affects striatal-dependent brain function and may be relevant to non-neurodegenerative disorders involving dopamine neurotransmission.

    Funded by: Howard Hughes Medical Institute; NHLBI NIH HHS: HL077101; NIDA NIH HHS: DA10044, DA16672, P01 DA008227-17, P01 DA010044, T32-DA7290; NIMH NIH HHS: MH074866, MH079710-0; NINDS NIH HHS: NS051874

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;47;18561-6

  • Cdk5 regulates accurate maturation of newborn granule cells in the adult hippocampus.

    Jessberger S, Aigner S, Clemenson GD, Toni N, Lie DC, Karalay O, Overall R, Kempermann G and Gage FH

    Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California, USA. jessberger@cell.biol.ethz.ch

    Newborn granule cells become functionally integrated into the synaptic circuitry of the adult dentate gyrus after a morphological and electrophysiological maturation process. The molecular mechanisms by which immature neurons and the neurites extending from them find their appropriate position and target area remain largely unknown. Here we show that single-cell-specific knockdown of cyclin-dependent kinase 5 (cdk5) activity in newborn cells using a retrovirus-based strategy leads to aberrant growth of dendritic processes, which is associated with an altered migration pattern of newborn cells. Even though spine formation and maturation are reduced in cdk5-deficient cells, aberrant dendrites form ectopic synapses onto hilar neurons. These observations identify cdk5 to be critically involved in the maturation and dendrite extension of newborn neurons in the course of adult neurogenesis. The data presented here also suggest a mechanistic dissociation between accurate dendritic targeting and subsequent synapse formation.

    PLoS biology 2008;6;11;e272

  • Tyrosine hydroxylase expression and Cdk5 kinase activity in ataxic cerebellum.

    Cheung KJ, Rosales JL, Lee BC, Jeong YG and Lee KY

    Department of Cell Biology and Anatomy, Southern Alberta Cancer Research Institute and Hotchkiss Brain Institute, The University of Calgary, Heritage Medical Research Building, Calgary, AB, Canada, T2N4N1.

    Ataxia has been associated with abnormalities in neuronal differentiation and migration, which are regulated by Cyclin-dependent kinase 5 (Cdk5). The cerebellum of mice lacking Cdk5 or its activator, p35, resembles those of ataxic reeler and scrambler mice, suggesting that Cdk5 may contribute to ataxic pathology. As with other ataxic mice, the pogo/pogo mouse shows aberrant cerebellar tyrosine hydroxylase (TH) expression. Since Cdk5 phosphorylates and upregulates TH expression, we sought to analyze (i) Cdk5 activity in the pogo cerebellum, which exhibits abnormal TH expression, and (ii) TH expression in the cerebellum of p35-/- and p39-/- mice, which display reduced Cdk5 activity. Interestingly, we found that increased TH expression in the pogo cerebellum coincided with reduced Cdk5 activity. However, reduced Cdk5 activity in both p35-/- and p39-/- cerebellum did not correspond to defects in TH expression. Together, these suggest that abnormal TH expression in the cerebellum might be regulated by mechanisms other than Cdk5 activity.

    Funded by: Canadian Institutes of Health Research: 82788-1

    Molecular and cellular biochemistry 2008;318;1-2;7-12

  • Dopaminergic and glutamatergic signaling crosstalk in Huntington's disease neurodegeneration: the role of p25/cyclin-dependent kinase 5.

    Paoletti P, Vila I, Rifé M, Lizcano JM, Alberch J and Ginés S

    Departament de Biologia Cellular, Immunologia i Neurociències, Facultat de Medicina, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Universitat de Barcelona, E-08036 Barcelona, Spain.

    Altered glutamatergic and dopaminergic signaling has been proposed as contributing to the specific striatal cell death observed in Huntington's disease (HD). However, the precise mechanisms by which mutant huntingtin sensitize striatal cells to dopamine and glutamate inputs remain unclear. Here, we demonstrate in knock-in HD striatal cells that mutant huntingtin enhances dopamine-mediated striatal cell death via dopamine D(1) receptors. Moreover, we show that NMDA receptors specifically potentiate the vulnerability of mutant huntingtin striatal cells to dopamine toxicity as pretreatment with NMDA increased D(1)R-induced cell death in mutant but not wild-type cells. As potential underlying mechanism of increased striatal vulnerability, we identified aberrant cyclin-dependent kinase 5 (Cdk5) activation. We demonstrate that enhanced Cdk5 phosphorylation and increased calpain-mediated conversion of the Cdk5 activator p35 into p25 may account for the deregulation of Cdk5 associated to dopamine and glutamate receptor activation in knock-in HD striatal cells. Moreover, supporting a detrimental role of Cdk5 in striatal cell death, neuronal loss can be widely prevented by roscovitine, a potent Cdk5 inhibitor. Significantly, reduced Cdk5 expression together with enhanced Cdk5 phosphorylation and p25 accumulation also occurs in the striatum of mutant Hdh(Q111) mice and HD human brain suggesting the relevance of deregulated Cdk5 pathway in HD pathology. These findings provide new insights into the molecular mechanisms underlying the selective vulnerability of striatal cells in HD and identify p25/Cdk5 as an important mediator of dopamine and glutamate neurotoxicity associated to HD.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;40;10090-101

  • Cerebellar development transcriptome database (CDT-DB): profiling of spatio-temporal gene expression during the postnatal development of mouse cerebellum.

    Sato A, Sekine Y, Saruta C, Nishibe H, Morita N, Sato Y, Sadakata T, Shinoda Y, Kojima T and Furuichi T

    Laboratory of Molecular Neurogenesis, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan.

    A large amount of genetic information is devoted to brain development and functioning. The neural circuit of the mouse cerebellum develops through a series of cellular and morphological events (including neuronal proliferation and migration, axogenesis, dendritogenesis, synaptogenesis and myelination) all within three weeks of birth. All of these events are controlled by specific gene groups, whose temporal and spatial expression profiles must be encoded in the genome. To understand the genetic basis underlying cerebellar circuit development, we analyzed gene expression (transcriptome) during the developmental stages on a genome-wide basis. Spatio-temporal gene expression data were collected using in situ hybridization for spatial (cellular and regional) resolution and fluorescence differential display, GeneChip, microarray and RT-PCR for temporal (developmental time series) resolution, and were annotated using Gene Ontology (controlled terminology for genes and gene products) and anatomical context (cerebellar cell types and circuit structures). The annotated experimental data were integrated into a knowledge resource database, the Cerebellar Development Transcriptome Database (CDT-DB http://www.cdtdb.brain.riken.jp), with seamless links to the relevant information at various bioinformatics database websites. The CDT-DB not only provides a unique informatics tool for mining both spatial and temporal pattern information on gene expression in developing mouse brains, but also opens up opportunities to elucidate the transcriptome for cerebellar development.

    Neural networks : the official journal of the International Neural Network Society 2008;21;8;1056-69

  • Deregulated Cdk5 promotes oxidative stress and mitochondrial dysfunction.

    Sun KH, de Pablo Y, Vincent F and Shah K

    Department of Chemistry and Purdue Cancer Center, Purdue University, West Lafayette, IN 47907, USA.

    Oxidative stress is one of the earliest events in Alzheimer's disease (AD). A chemical genetic screen revealed that deregulated cyclin-dependent kinase 5 (Cdk5) may cause oxidative stress by compromising the cellular anti-oxidant defense system. Using novel Cdk5 modulators, we show the mechanism by which Cdk5 can induce oxidative stress in the disease's early stage and cell death in the late stage. Cdk5 dysregulation upon neurotoxic insults results in reactive oxygen species (ROS) accumulation in neuronal cells because of the inactivation of peroxiredoxin I and II. Sole temporal activation of Cdk5 also increases ROS, suggesting its major role in this process. Cdk5 inhibition rescues mitochondrial damage upon neurotoxic insults, thereby revealing Cdk5 as an upstream regulator of mitochondrial dysfunction. As mitochondrial damage results in elevated ROS and Ca(2+) levels, both of which activate Cdk5, we propose that a feedback loop occurs in late stage of AD and leads to cell death (active Cdk5 --> ROS --> excess ROS --> mitochondrial damage --> ROS --> hyperactive Cdk5 --> severe oxidative stress and cell injury --> cell death). Cdk5 inhibition upon neurotoxic insult prevents cell death significantly, supporting this hypothesis. As oxidative stress and mitochondrial dysfunction play pivotal roles in promoting neurodegeneration, Cdk5 could be a viable therapeutic target for AD.

    Journal of neurochemistry 2008;107;1;265-78

  • Suppression of mutant Huntingtin aggregate formation by Cdk5/p35 through the effect on microtubule stability.

    Kaminosono S, Saito T, Oyama F, Ohshima T, Asada A, Nagai Y, Nukina N and Hisanaga S

    Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan.

    Huntington's disease (HD) is a polyglutamine [poly(Q)] disease with an expanded poly(Q) stretch in the N terminus of the huntingtin protein (htt). A major pathological feature of HD neurons is inclusion bodies, detergent-insoluble aggregates composed of poly(Q)-expanded mutant htt (mhtt). Misfolding of mhtt is thought to confer a toxic property via formation of aggregates. Although toxic molecular species are still debated, it is important to clarify the aggregation mechanism to understand the pathogenesis of mhtt. We show Cdk5/p35 suppresses the formation of mhtt inclusion bodies in cell lines and primary neurons. Although we expressed the N-terminal exon 1 fragment of htt lacking phosphorylation sites for Cdk5 in COS-7 cells, the kinase activity of Cdk5 was required for the suppression. Furthermore, Cdk5/p35 suppressed inclusion formation of atrophin-1, another poly(Q) protein, raising the possibility that Cdk5/p35 generally suppresses inclusion formation of poly(Q) proteins. Microtubules (MTs) were a downstream component of Cdk5/p35 in the suppression of inclusion formation; Cdk5/p35 disrupted MTs, which were required for the formation of inclusions. Moreover, stabilization of MTs by Taxol induced inclusions even with overexpression of Cdk5/p35. The formation of inclusions was also regulated by manipulating the Cdk5/p35 activity in primary rat or mouse cortical neuron cultures. These results indicate that Cdk5-dependent regulation of MT organization is involved in the development of aggregate formation and subsequent pathogenesis of poly(Q) diseases. This Cdk5 inhibition of htt aggregates is a novel mechanism different from htt phosphorylation and interaction with Cdk5 reported previously (Luo et al., 2005; Anne et al., 2007).

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;35;8747-55

  • Nuclear localization of Cdk5 is a key determinant in the postmitotic state of neurons.

    Zhang J, Cicero SA, Wang L, Romito-Digiacomo RR, Yang Y and Herrup K

    Department of Cell Biology and Neuroscience, Rutgers University, 604 Allison Road, Piscataway, NJ 08854, USA.

    Cyclin-dependent kinase 5 (Cdk5) is a nontraditional Cdk that is primarily active in postmitotic neurons. Its best known substrates are cytoskeletal proteins. Less appreciated is its role in the maintenance of a postmitotic state. We show here that in cycling cells (NIH 3T3), the localization of Cdk5 changes from predominantly nuclear to cytoplasmic as cells reenter a cell cycle after serum starvation. Similarly, when beta-amyloid peptide is used to stimulate cultured primary neurons to reenter a cell cycle, they too show a loss of nuclear Cdk5. Blocking nuclear export pharmacologically abolishes cell cycle reentry in wild-type but not Cdk5(-/-) neurons, suggesting a Cdk5-specific effect. Cdk5 overexpression targeted to the nucleus of Cdk5(-/-) neurons effectively blocks the cell cycle, but cytoplasmic targeting is ineffective. Further, in both human Alzheimer's disease as well as in the R1.40 mouse Alzheimer's model and the E2f1(-/-) mouse, neurons expressing cell cycle markers consistently show reduced nuclear Cdk5. Thus, both in vivo and in vitro, neurons that reenter a cell cycle lose nuclear Cdk5. We propose that the nuclear Cdk5 plays an active role in allowing neurons to remain postmitotic as they mature and that loss of nuclear Cdk5 leads to cell cycle entry.

    Funded by: NINDS NIH HHS: NS20591, R01 NS020591, R01 NS020591-25

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;25;8772-7

  • Cyclin-dependent kinase 5 supports neuronal survival through phosphorylation of Bcl-2.

    Cheung ZH, Gong K and Ip NY

    Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.

    Accumulating evidence indicates that deregulation of cyclin-dependent kinase 5 (Cdk5) activity is associated with apoptosis in various neurodegenerative disease models. Interestingly, recent studies suggest that Cdk5 may also favor neuronal survival. Nonetheless, whether Cdk5 is directly required for neuronal survival during development remains enigmatic. In the current study, we established the pivotal role of Cdk5 in neuronal survival during development by demonstrating that reduction or absence of Cdk5 activity markedly exacerbated neuronal death in cultures and in vivo. Interestingly, the antiapoptotic protein Bcl-2 (B-cell lymphoma protein 2) was identified as a novel substrate of Cdk5. We found that Cdk5-mediated phosphorylation of Bcl-2 at Ser70 was required for the neuroprotective effect of Bcl-2. Intriguingly, inhibition of this phosphorylation conferred proapoptotic property to Bcl-2. Furthermore, overexpression of a Bcl-2 mutant lacking the Cdk5 phosphorylation site abolished the protective effect of Cdk5 re-expression in Cdk5(-/-) neurons, suggesting that Ser70 phosphorylation of Bcl-2 contributed to Cdk5-mediated neuronal survival. Our observations revealed that Cdk5-mediated Bcl-2 phosphorylation is pivotal for the antiapoptotic effect of Bcl-2 and contributes to the maintenance of neuronal survival by Cdk5. Our study has also identified Cdk5 as a regulator of Bcl-2 function in neuronal apoptosis.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;19;4872-7

  • Conditional deletion of cyclin-dependent kinase 5 in primary sensory neurons leads to atypical skin lesions.

    Saikkonen B, Pareek TK, Agarwal N, Molinolo A, Kriete M and Kulkarni AB

    National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892-4395, USA.

    The key role of Cyclin-dependent kinase 5 (Cdk5) in neuronal function has been well established but understanding of its importance in sensory pathways is in its infancy. Recently we described the important role of Cdk5 in pain signaling. Our studies indicated that conditional deletion of Cdk5 in small sensory neurons causes hypoalgesia. In current study, we identified development of atypical non-healing skin lesions in these mutant mice during the general colony maintenance. Detailed examination of these lesions clearly distinguishes them from ulcerative dermatitis. Here we hypothesize that these skin lesions are due to general sensation loss in these mice as evident from deep skin scratches that turn into unhealed wounds.

    Cell cycle (Georgetown, Tex.) 2008;7;6;750-3

  • Transcriptional regulation of beta-secretase by p25/cdk5 leads to enhanced amyloidogenic processing.

    Wen Y, Yu WH, Maloney B, Bailey J, Ma J, Marié I, Maurin T, Wang L, Figueroa H, Herman M, Krishnamurthy P, Liu L, Planel E, Lau LF, Lahiri DK and Duff K

    Taub Institute at Columbia University Medical Center, New York, NY 10032, USA.

    Cyclin-dependent kinase 5 (cdk5) has been implicated in Alzheimer's disease (AD) pathogenesis. Here, we demonstrate that overexpression of p25, an activator of cdk5, led to increased levels of BACE1 mRNA and protein in vitro and in vivo. A p25/cdk5 responsive region containing multiple sites for signal transducer and activator of transcription (STAT1/3) was identified in the BACE1 promoter. STAT3 interacts with the BACE1 promoter, and p25-overexpressing mice had elevated levels of pSTAT3 and BACE1, whereas cdk5-deficient mice had reduced levels. Furthermore, mice with a targeted mutation in the STAT3 cdk5 responsive site had lower levels of BACE1. Increased BACE levels in p25 overexpressing mice correlated with enhanced amyloidogenic processing that could be reversed by a cdk5 inhibitor. These data demonstrate a pathway by which p25/cdk5 increases the amyloidogenic processing of APP through STAT3-mediated transcriptional control of BACE1 that could have implications for AD pathogenesis.

    Funded by: NIA NIH HHS: AG172116, AG18379, AG18884, P01 AG017216, P01 AG017216-020004, R01 AG018379, R01 AG018884; NINDS NIH HHS: NS48447, P01 NS048447, P01 NS048447-030005

    Neuron 2008;57;5;680-90

  • Negative regulation of cyclin-dependent kinase 5 targets by protein kinase C.

    Sahin B, Hawasli AH, Greene RW, Molkentin JD and Bibb JA

    Department of Psychiatry, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9070, United States.

    Cyclin-dependent kinase 5 (Cdk5) is a proline-directed protein serine/threonine kinase essential for brain development and implicated in synaptic plasticity, dopaminergic neurotransmission, drug addiction, and neurodegenerative disorders. Relatively little is known about the molecular mechanisms that regulate the activity of Cdk5 in vivo. In order to determine whether protein kinase C (PKC) regulates Cdk5 activity in the central nervous system, the phosphorylation levels of two Cdk5 substrates were evaluated under conditions of altered PKC activity in vivo. Treatment of acute striatal slices with a PKC-activating phorbol ester caused a time- and dose-dependent decrease in the levels of phospho-Ser6 inhibitor-1, phospho-Ser67 inhibitor-1, and phospho-Thr75 dopamine- and cAMP-regulated phosphoprotein, Mr 32,000 (DARPP-32). This effect was reversed by the PKC inhibitor, Ro-32-0432. Moreover, phospho-Ser6 inhibitor-1, phospho-Ser67 inhibitor-1, and phospho-Thr75 DARPP-32 levels were elevated in brain tissue from mice lacking the gene for PKC-alpha. PKC did not phosphorylate Cdk5 or its cofactor, p25, in vitro. Striatal levels of the Cdk5 cofactor, p35, did not change in response to phorbol ester treatment. Furthermore, Cdk5 immunoprecipitated from striatal slices treated with phorbol ester had unaltered activity toward a control substrate in vitro. These results suggest that PKC exerts its effects on the phosphorylation state of Cdk5 substrates through an indirect mechanism that may involve the regulatory binding partners of Cdk5 other than its neuronal cofactors.

    Funded by: NHLBI NIH HHS: P50 HL077101, P50 HL077101-01; NIDA NIH HHS: DA16672, R01 DA016672, R01 DA016672-05; NIMH NIH HHS: MH67777, R01 MH067777, R01 MH067777-05, R01 MH080297; NINDS NIH HHS: R01 NS073855

    European journal of pharmacology 2008;581;3;270-5

  • Interplay between cyclin-dependent kinase 5 and glycogen synthase kinase 3 beta mediated by neuregulin signaling leads to differential effects on tau phosphorylation and amyloid precursor protein processing.

    Wen Y, Planel E, Herman M, Figueroa HY, Wang L, Liu L, Lau LF, Yu WH and Duff KE

    Taub Institute at Columbia University Medical Center, New York State Psychiatric Institute, New York, New York 10032, USA.

    Cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase 3beta (GSK3beta) have been implicated in pathogenic processes associated with Alzheimer's disease because both kinases regulate tau hyperphosphorylation and enhance amyloid precursor protein (APP) processing leading to an increase in amyloid beta (Abeta) production. Here we show that young p25 overexpressing mice have enhanced cdk5 activity but reduced GSK3beta activity attributable to phosphorylation at the inhibitory GSK3beta-serine 9 (GSK3beta-S9) site. Phosphorylation at this site was mediated by enhanced activity of the neuregulin receptor complex, ErbB, and activation of the downstream phosphatidylinositol 3 kinase/Akt pathway. Young p25 mice had elevated Abeta peptide levels, but phospho-tau levels were decreased overall. Thus, cdk5 appears to play a dominant role in the regulation of amyloidogenic APP processing, whereas GSK3beta plays a dominant role in overall tau phosphorylation. In older mice, GSK3beta inhibitory phosphorylation at S9 was reduced relative to young mice. Abeta peptide levels were still elevated but phospho-tau levels were either unchanged or showed a trend to increase, suggesting that GSK3beta activity increases with aging. Inhibition of cdk5 by a specific inhibitor reduced cdk5 activity in p25 mice, leading to reduced Abeta production in both young and old mice. However, in young mice, cdk5 inhibition reversed GSK3beta inhibition, leading to an increase in overall tau phosphorylation. When cdk5 inhibitor was administered to very old, nontransgenic mice, inhibition of cdk5 reduced Abeta levels, and phospho-tau levels showed a trend to increase. Thus, cdk5 inhibitors may not be effective in targeting tau phosphorylation in the elderly.

    Funded by: NIA NIH HHS: AG172116; NINDS NIH HHS: NS48447

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;10;2624-32

  • Cdk5 regulates differentiation of oligodendrocyte precursor cells through the direct phosphorylation of paxillin.

    Miyamoto Y, Yamauchi J, Chan JR, Okada A, Tomooka Y, Hisanaga S and Tanoue A

    Department of Pharmacology, National Research Institute for Child Health and Development, Okura, Setagaya, Tokyo 157-8535, Japan.

    Oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes (OLs) in order to form myelin, which is required for the rapid propagation of action potentials in the vertebrate nervous system. In spite of the considerable clinical importance of myelination, little is known about the basic molecular mechanisms underlying OL differentiation and myelination. Here, we show that cyclin-dependent kinase (Cdk) 5 is activated following the induction of differentiation, and that the Cdk5 inhibitor roscovitine inhibits OL differentiation. The complexity of the OL processes is also diminished after knocking down endogenous Cdk5 using RNAi. We also show that the focal adhesion protein paxillin is directly phosphorylated at Ser244 by Cdk5. Transfection of a paxillin construct harboring a Ser244 to Ala mutation dramatically inhibits its morphological effects. Importantly, phosphorylation of paxillin at Ser244 reduces its interaction with focal adhesion kinase (FAK). Taken together, these results suggest that phosphorylation of paxillin by Cdk5 is a key mechanism in OL differentiation and may ultimately regulate myelination.

    Journal of cell science 2007;120;Pt 24;4355-66

  • Cdk5 promotes synaptogenesis by regulating the subcellular distribution of the MAGUK family member CASK.

    Samuels BA, Hsueh YP, Shu T, Liang H, Tseng HC, Hong CJ, Su SC, Volker J, Neve RL, Yue DT and Tsai LH

    Howard Hughes Medical Institute, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

    Synaptogenesis is a highly regulated process that underlies formation of neural circuitry. Considerable work has demonstrated the capability of some adhesion molecules, such as SynCAM and Neurexins/Neuroligins, to induce synapse formation in vitro. Furthermore, Cdk5 gain of function results in an increased number of synapses in vivo. To gain a better understanding of how Cdk5 might promote synaptogenesis, we investigated potential crosstalk between Cdk5 and the cascade of events mediated by synapse-inducing proteins. One protein recruited to developing terminals by SynCAM and Neurexins/Neuroligins is the MAGUK family member CASK. We found that Cdk5 phosphorylates and regulates CASK distribution to membranes. In the absence of Cdk5-dependent phosphorylation, CASK is not recruited to developing synapses and thus fails to interact with essential presynaptic components. Functional consequences include alterations in calcium influx. Mechanistically, Cdk5 regulates the interaction between CASK and liprin-alpha. These results provide a molecular explanation of how Cdk5 can promote synaptogenesis.

    Funded by: Howard Hughes Medical Institute; NINDS NIH HHS: NS051874, R01 NS051874, R01 NS051874-10A1, R01 NS051874-11, R01 NS051874-12; PHS HHS: R01

    Neuron 2007;56;5;823-37

  • Kinase activities increase during the development of tauopathy in htau mice.

    Kelleher I, Garwood C, Hanger DP, Anderton BH and Noble W

    King's College London, Department of Neuroscience, MRC Centre for Neurodegeneration Research, Institute of Psychiatry, De Crespigny Park, London, UK.

    Hyperphosphorylated tau aggregates are the core constituent of neurofibrillary tangles. Recent research has shown a division between the presence of tangles, neurodegeneration and subsequent memory impairment, raising the possibility that an earlier pre-aggregated form of tau may be toxic. To gain further insight into the relationship between abnormal forms of tau, we have analyzed pathological changes in tau during tauopathy development in tangle-forming transgenic mice. In addition, we have quantified changes in the endogenous levels of a panel of protein kinases. We show progressive increases in aggregated tau and disease-specific conformational change, with hyperphosphorylation occurring in an age-dependent manner at specific sites. There were significant correlations between specific phosphorylation changes and amounts of aggregated tau and and abnormal tau conformations. Of the protein kinases tested, we found increases in phosphorylated (activated) p38 and the cyclin-dependent kinase-5 neuronal activators, p35 and p25, with aging, in the htau line, but not in non-tangle-forming control mice. Changes in tau kinases correlated with the amount of tau present in abnormal conformations and with insoluble tau in htau mice. These data suggest that cdk5 and p38 may be associated with pathological changes in wild-type human tau during the progressive development of tauopathy.

    Journal of neurochemistry 2007;103;6;2256-67

  • Regulation of the interaction of Disabled-1 with CIN85 by phosphorylation with Cyclin-dependent kinase 5.

    Sato Y, Taoka M, Sugiyama N, Kubo K, Fuchigami T, Asada A, Saito T, Nakajima K, Isobe T and Hisanaga S

    Department of Biological Science, Tokyo Metropolitan University, Minami-osawa, Hachioji, Tokyo 192-0397, Japan.

    Disabled-1 (Dab1) is an adaptor protein mediating Reelin signaling in neuronal migration during brain development. Cyclin-dependent kinase 5 (Cdk5)-p35 is a proline-directed Ser/Thr kinase also involved in neuronal migration. The interaction between Dab1 and Cdk5 is in need of investigation. Dab1 was phosphorylated at Ser400 and Ser491 by Cdk5 in vivo. We search for proteins that interact with Dab1 in a phosphorylation-dependent manner at these sites, and identified CIN85, an SH3-containing adaptor protein involved in endocytosis, and CPalpha/CPbeta, which are subunits of barbed end F-actin-capping proteins (CP), as proteins bound to unphosphorylated Dab1 by mass spectrometric analysis. It was shown that the PTPAPR sequence of Dab1, conforming to the PxxxPR atypical SH3-binding motif, was the binding site for SH3 domains of CIN85. The results that phosphorylation at Ser491 close to the PTPAPR sequence inhibited association with CIN85 may provide a mechanism regulating the interaction between the PxxxPR motif proteins and SH3 domains of CIN85 family proteins. Together with previous results that CIN85 regulates actin assembly, present results raise the possibility that Cdk5 modulates actin dynamics through regulation of CIN85-Dab1 interaction by the Dab1 phosphorylation.

    Genes to cells : devoted to molecular & cellular mechanisms 2007;12;12;1315-27

  • Cdk5 modulates cocaine reward, motivation, and striatal neuron excitability.

    Benavides DR, Quinn JJ, Zhong P, Hawasli AH, DiLeone RJ, Kansy JW, Olausson P, Yan Z, Taylor JR and Bibb JA

    Department of Psychiatry, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    Cyclin-dependent kinase 5 (Cdk5) regulates dopamine neurotransmission and has been suggested to serve as a homeostatic target of chronic psychostimulant exposure. To study the role of Cdk5 in the modulation of the cellular and behavioral effects of psychoactive drugs of abuse, we developed Cre/loxP conditional knock-out systems that allow temporal and spatial control of Cdk5 expression in the adult brain. Here, we report the generation of Cdk5 conditional knock-out (cKO) mice using the alphaCaMKII promoter-driven Cre transgenic line (CaMKII-Cre). In this model system, loss of Cdk5 in the adult forebrain increased the psychomotor-activating effects of cocaine. Additionally, these CaMKII-Cre Cdk5 cKO mice show enhanced incentive motivation for food as assessed by instrumental responding on a progressive ratio schedule of reinforcement. Behavioral changes were accompanied by increased excitability of medium spiny neurons in the nucleus accumbens (NAc) in Cdk5 cKO mice. To study NAc-specific effects of Cdk5, another model system was used in which recombinant adeno-associated viruses expressing Cre recombinase caused restricted loss of Cdk5 in NAc neurons. Targeted knock-out of Cdk5 in the NAc facilitated cocaine-induced locomotor sensitization and conditioned place preference for cocaine. These results suggest that Cdk5 acts as a negative regulator of neuronal excitability in the NAc and that Cdk5 may govern the behavioral effects of cocaine and motivation for reinforcement.

    Funded by: NIDA NIH HHS: DA011717, DA016672, DA020199; NIMH NIH HHS: MH063128; NINDS NIH HHS: NS048911

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;47;12967-76

  • Cyclin-dependent kinase 5 is required for control of neuroblast migration in the postnatal subventricular zone.

    Hirota Y, Ohshima T, Kaneko N, Ikeda M, Iwasato T, Kulkarni AB, Mikoshiba K, Okano H and Sawamoto K

    Department of Developmental and Regenerative Biology, Institute of Molecular Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan.

    At the lateral wall of the lateral ventricles in the adult rodent brain, neuroblasts form an extensive network of elongated cell aggregates called chains in the subventricular zone and migrate toward the olfactory bulb. The molecular mechanisms regulating this migration of neuroblasts are essentially unknown. Here, we report a novel role for cyclin-dependent kinase 5 (Cdk5), a neuronal protein kinase, in this process. Using in vitro and in vivo conditional knock-out experiments, we found that Cdk5 deletion impaired the chain formation, speed, directionality, and leading process extension of the neuroblasts in a cell-autonomous manner. These findings suggest that Cdk5 plays an important role in neuroblast migration in the postnatal subventricular zone.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;47;12829-38

  • Regulation of spine development by semaphorin3A through cyclin-dependent kinase 5 phosphorylation of collapsin response mediator protein 1.

    Yamashita N, Morita A, Uchida Y, Nakamura F, Usui H, Ohshima T, Taniguchi M, Honnorat J, Thomasset N, Takei K, Takahashi T, Kolattukudy P and Goshima Y

    Department of Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan.

    Collapsin response mediator protein 1 (CRMP1) is one of the CRMP family members that mediates signal transduction of axonal guidance and neuronal migration. We show here evidence that CRMP1 is involved in semaphorin3A (Sema3A)-induced spine development in the cerebral cortex. In the cultured cortical neurons from crmp1+/- mice, Sema3A increased the density of clusters of synapsin I and postsynaptic density-95, but this increase was markedly attenuated in crmp1-/- mice. This attenuation was also seen in cyclin-dependent kinase 5 (cdk5)-/- neurons. Furthermore, the introduction of wild-type CRMP1 but not CRMP1-T509A/S522A, (Thr 509 and Ser 522 were replaced by Ala), a mutant that cannot be phosphorylated by Cdk5, into crmp1-/- neurons rescued the defect in Sema3A responsiveness. The Golgi-impregnation method showed that the crmp1-/- layer V cortical neurons showed a lower density of synaptic bouton-like structures and that this phenotype had genetic interaction with sema3A. These findings suggest that Sema3A-induced spine development is regulated by phosphorylation of CRMP1 by Cdk5.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;46;12546-54

  • Targeted deletion of the mitogen-activated protein kinase kinase 4 gene in the nervous system causes severe brain developmental defects and premature death.

    Wang X, Nadarajah B, Robinson AC, McColl BW, Jin JW, Dajas-Bailador F, Boot-Handford RP and Tournier C

    Faculty of Life Sciences, Wellcome Trust Center for Cell-Matrix Research, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom.

    The c-Jun NH2-terminal protein kinase (JNK) is a mitogen-activated protein kinase (MAPK) involved in the regulation of various physiological processes. Its activity is increased upon phosphorylation by the MAPK kinases MKK4 and MKK7. The early embryonic death of mice lacking an mkk4 or mkk7 gene has provided genetic evidence that MKK4 and MKK7 have nonredundant functions in vivo. To elucidate the physiological role of MKK4, we generated a novel mouse model in which the mkk4 gene could be specifically deleted in the brain. At birth, the mutant mice were indistinguishable from their control littermates, but they stopped growing a few days later and died prematurely, displaying severe neurological defects. Decreased JNK activity in the absence of MKK4 correlated with impaired phosphorylation of a subset of physiologically relevant JNK substrates and with altered gene expression. These defects resulted in the misalignment of the Purkinje cells in the cerebellum and delayed radial migration in the cerebral cortex. Together, our data demonstrate for the first time that MKK4 is an essential activator of JNK required for the normal development of the brain.

    Funded by: Biotechnology and Biological Sciences Research Council: BBS/B/0224X; Medical Research Council: G0001285

    Molecular and cellular biology 2007;27;22;7935-46

  • A stream of cells migrating from the caudal telencephalon reveals a link between the amygdala and neocortex.

    Remedios R, Huilgol D, Saha B, Hari P, Bhatnagar L, Kowalczyk T, Hevner RF, Suda Y, Aizawa S, Ohshima T, Stoykova A and Tole S

    Department of Biological Sciences, B-304, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.

    The amygdaloid complex consists of diverse nuclei that belong to distinct functional systems, yet many issues about its development are poorly understood. Here, we identify a stream of migrating cells that form specific amygdaloid nuclei in mice. In utero electroporation showed that this caudal amygdaloid stream (CAS) originated in a unique domain at the caudal telencephalic pole that is contiguous with the dorsal pallium, which was previously thought to generate only neocortical cells. The CAS and the neocortex share mechanisms for specification (transcription factors Tbr1, Lhx2 and Emx1/2) and migration (reelin and Cdk5). Reelin, a critical cue for migration in the neocortex, and Cdk5, which is specifically required for migration along radial glia in the neocortex, were both selectively required for the normal migration of the CAS, but not for that of other amygdaloid nuclei. This is first evidence of a dorsal pallial contribution to the amygdala, demonstrating a developmental and mechanistic link between the amygdala and the neocortex.

    Funded by: Wellcome Trust: 056684/Z/99/Z

    Nature neuroscience 2007;10;9;1141-50

  • Cdk5 selectively affects the migration of different populations of neurons in the developing spinal cord.

    Yip YP, Capriotti C, Drill E, Tsai LH and Yip JW

    Department of Neurobiology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15261, USA.

    It has been shown that cyclin-dependent kinase 5 (Cdk5) is crucial for neuronal migration and survival in the brain. However, the role of Cdk5 in neuronal migration in the spinal cord has never been investigated. The present study is the first to show that Cdk5 affects the migration of different populations of neurons in the developing spinal cord. In the absence of Cdk5, at least four neuronal populations failed to migrate to their final destinations: sympathetic and parasympathetic preganglionic neurons, as well as dorsally originating and ventrally originating (U-shaped group) diaphorase-positive dorsal horn interneurons. In contrast, the migration of somatic motor neurons and various types of ventral and dorsal interneurons was unaffected by the absence of Cdk5. Moreover, our results suggest that Cdk5-dependent migration in the developing spinal cord is axon- or glial fiber-mediated. Finally, our results show that sympathetic preganglionic neurons and somatic motor neurons in Cdk5-deficient mice continue to extend processes and project toward their normal target areas, suggesting that Cdk5 has no obvious effects on axonal outgrowth and guidance mechanisms of these two neuronal populations in spinal cord development.

    Funded by: NIMH NIH HHS: MH62180

    The Journal of comparative neurology 2007;503;2;297-307

  • Stabilization and activation of p53 induced by Cdk5 contributes to neuronal cell death.

    Lee JH, Kim HS, Lee SJ and Kim KT

    Department of Life Science, Division of Molecular and Life Science, Systems-Biodynamics NCRC, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea.

    The p53 tumor suppressor protein is a key regulator of cellular functions including responses to numerous stress signals, and triggers apoptosis in many cell types, including neurons. The major mechanisms known to regulate p53 stabilization and activation include phosphorylation and ubiquitin ligase-mediated proteasomal degradation. Cyclin-dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase, is most active in the central nervous system and plays a variety of roles in neuronal degeneration. Here, we demonstrate for the first time that Cdk5 interacts with p53 and increases its stability through posttranslational regulation, leading to accumulation of p53, particularly in the nucleus. We show that Cdk5 phosphorylates p53 on Ser15, Ser33 and Ser46 in vitro, and that increased Cdk5 activity in the nucleus mediates these phosphorylation events in response to genotoxic and oxidative stresses. Cdk5 mediates disruption of the interaction between p53 and Hdm2 (also known as Mdm2), and prevents Hdm2-induced p53 ubiquitylation and downregulation. Cdk5 additionally enhances phosphorylation-dependent binding of the p300 coactivator, inducing acetylation of p53. Cdk5-stabilized p53 protein is transcriptionally active, resulting in the induction of pro-apoptotic genes and subsequent mitochondria-mediated apoptosis in response to genotoxic or oxidative stress. Collectively, these novel findings help define the mechanisms underlying neuronal apoptosis occurring as a result of Cdk5-mediated p53 stabilization and transcriptional activation.

    Journal of cell science 2007;120;Pt 13;2259-71

  • Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation.

    Hawasli AH, Benavides DR, Nguyen C, Kansy JW, Hayashi K, Chambon P, Greengard P, Powell CM, Cooper DC and Bibb JA

    Department of Psychiatry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390, USA.

    Learning is accompanied by modulation of postsynaptic signal transduction pathways in neurons. Although the neuronal protein kinase cyclin-dependent kinase 5 (Cdk5) has been implicated in cognitive disorders, its role in learning has been obscured by the perinatal lethality of constitutive knockout mice. Here we report that conditional knockout of Cdk5 in the adult mouse brain improved performance in spatial learning tasks and enhanced hippocampal long-term potentiation and NMDA receptor (NMDAR)-mediated excitatory postsynaptic currents. Enhanced synaptic plasticity in Cdk5 knockout mice was attributed to reduced NR2B degradation, which caused elevations in total, surface and synaptic NR2B subunit levels and current through NR2B-containing NMDARs. Cdk5 facilitated the degradation of NR2B by directly interacting with both it and its protease, calpain. These findings reveal a previously unknown mechanism by which Cdk5 facilitates calpain-mediated proteolysis of NR2B and may control synaptic plasticity and learning.

    Funded by: NIDA NIH HHS: K01 DA017750; NIMH NIH HHS: K08 MH065975, K08 MH065975-04, K08 MH065975-05

    Nature neuroscience 2007;10;7;880-886

  • Regulation of protein phosphatase inhibitor-1 by cyclin-dependent kinase 5.

    Nguyen C, Nishi A, Kansy JW, Fernandez J, Hayashi K, Gillardon F, Hemmings HC, Nairn AC and Bibb JA

    Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    Inhibitor-1, the first identified endogenous inhibitor of protein phosphatase 1 (PP-1), was previously reported to be a substrate for cyclin-dependent kinase 5 (Cdk5) at Ser67. Further investigation has revealed the presence of an additional Cdk5 site identified by mass spectrometry and confirmed by site-directed mutagenesis as Ser6. Basal levels of phospho-Ser6 inhibitor-1, as detected by a phosphorylation state-specific antibody against the site, existed in specific regions of the brain and varied with age. In the striatum, basal in vivo phosphorylation and dephosphorylation of Ser6 were mediated by Cdk5, PP-2A, and PP-1, respectively. Additionally, calcineurin contributed to dephosphorylation under conditions of high Ca2+. In biochemical assays the function of Cdk5-dependent phosphorylation of inhibitor-1 at Ser6 and Ser67 was demonstrated to be an intramolecular impairment of the ability of inhibitor-1 to be dephosphorylated at Thr35; this effect was recapitulated in two systems in vivo. Dephosphorylation of inhibitor-1 at Thr35 is equivalent to inactivation of the protein, as inhibitor-1 only serves as an inhibitor of PP-1 when phosphorylated by cAMP-dependent kinase (PKA) at Thr35. Thus, inhibitor-1 serves as a critical junction between kinase- and phosphatase-signaling pathways, linking PP-1 to not only PKA and calcineurin but also Cdk5.

    Funded by: NHLBI NIH HHS: HL077101; NIDA NIH HHS: DA10044, DA16672, P01 DA010044; NIMH NIH HHS: MH072062, MH67777

    The Journal of biological chemistry 2007;282;22;16511-20

  • Cdk5 is required for multipolar-to-bipolar transition during radial neuronal migration and proper dendrite development of pyramidal neurons in the cerebral cortex.

    Ohshima T, Hirasawa M, Tabata H, Mutoh T, Adachi T, Suzuki H, Saruta K, Iwasato T, Itohara S, Hashimoto M, Nakajima K, Ogawa M, Kulkarni AB and Mikoshiba K

    Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan. ohshima@brain.riken.go.jp

    The mammalian cerebral cortex consists of six layers that are generated via coordinated neuronal migration during the embryonic period. Recent studies identified specific phases of radial migration of cortical neurons. After the final division, neurons transform from a multipolar to a bipolar shape within the subventricular zone-intermediate zone (SVZ-IZ) and then migrate along radial glial fibres. Mice lacking Cdk5 exhibit abnormal corticogenesis owing to neuronal migration defects. When we introduced GFP into migrating neurons at E14.5 by in utero electroporation, we observed migrating neurons in wild-type but not in Cdk5(-/-) embryos after 3-4 days. Introduction of the dominant-negative form of Cdk5 into the wild-type migrating neurons confirmed specific impairment of the multipolar-to-bipolar transition within the SVZ-IZ in a cell-autonomous manner. Cortex-specific Cdk5 conditional knockout mice showed inverted layering of the cerebral cortex and the layer V and callosal neurons, but not layer VI neurons, had severely impaired dendritic morphology. The amount of the dendritic protein Map2 was decreased in the cerebral cortex of Cdk5-deficient mice, and the axonal trajectory of cortical neurons within the cortex was also abnormal. These results indicate that Cdk5 is required for proper multipolar-to-bipolar transition, and a deficiency of Cdk5 results in abnormal morphology of pyramidal neurons. In addition, proper radial neuronal migration generates an inside-out pattern of cerebral cortex formation and normal axonal trajectories of cortical pyramidal neurons.

    Development (Cambridge, England) 2007;134;12;2273-82

  • The presence of active Cdk5 associated with p35 in astrocytes and its important role in process elongation of scratched astrocyte.

    He Y, Li HL, Xie WY, Yang CZ, Yu AC and Wang Y

    Neuroscience Research Institute, Department of Neurobiology, the Key Laboratory for Neuroscience, Peking University, Beijing 100083, People's Republic of China.

    Cyclin-dependent kinase 5 (Cdk5) is a unique member of the Cdk family; its kinase activity requires association with its activator, p35 or p39. p35 is the strongest and best characterized activator. Previous studies showed that p35 is a neuron-specific protein that restricts Cdk5 activity in neurons. However, a high expression level of Cdk5 is found in astrocytes, which raises the possibility that astrocytic Cdk5 is functional. Here we show the presence of functional Cdk5 associated with p35 in astrocytes and demonstrate its important role in process elongation of scratched astrocytes. We found that p35 and glial fibrillary acidic protein (GFAP) were co-localized in primary cultured and acute isolated brain cells. Cdk5 could form an immunocomplex with p35 and its activity was shown in pure primary cultured astrocytes. p35 was upregulated in astrocytes injured by scratching, concomitantly with upregulation of Cdk5 kinase activity. Pretreatment of the scratched astrocytes with a Cdk5 inhibitor, roscovitine, could delay wound healing by inhibiting the reorganization of tubulin, GFAP, and the extension of hypertrophic processes. Moreover, overexpression of dominant negative Cdk5 could shorten the length of extending protrusion of reactive astrocytes. Thus, our findings demonstrated that functional Cdk5, associated with p35, was expressed in astrocytes and its activity could be upregulated in reactive astrocytes, a new role of Cdk5 that has never been reported in the nervous system. The present study may provide new insight for understanding the multifunctional protein complex Cdk5/p35 in the nervous system.

    Glia 2007;55;6;573-83

  • Modulation of Reelin signaling by Cyclin-dependent kinase 5.

    Ohshima T, Suzuki H, Morimura T, Ogawa M and Mikoshiba K

    Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, Wako, Saitama, Japan. ohshima@brain.riken.go.jp

    The Reelin signaling and Cyclin-dependent kinase 5 (Cdk5) both regulate neuronal positioning in the developing brain. Using double-transgenic mice, we have previously shown that these two signaling pathways lie in parallel fashion and have a genetic interaction. Disabled-1 (Dab1), an adapter protein, mediates Reelin signaling and becomes tyrosine-phosphorylated on the binding of Reelin to its receptors. Several isoforms of Dab1 are expressed in embryonic mouse brain, and p80 [Dab1(555)] is the major protein translated. In the present study, we investigated whether Cdk5-mediated phosphorylation of Dab1 modulates Reelin signaling. Cdk5 phosphorylates p80 Dab1 at multiple sites in its carboxyl-terminal region, and tyrosine phosphorylation of p80 Dab1 by Fyn tyrosine kinase is attenuated by this Cdk5-mediated phosphorylation in vitro. Tyrosine phosphorylation of p80 Dab1 induced by exogenous Reelin is enhanced in Cdk5-deficient neurons, corroborating the inhibitory effect of Cdk5-mediated Ser/Thr phosphorylation on tyrosine phosphorylation of p80 Dab1. Another isoform, p45 Dab1 [Dab1(271)], however, is phosphorylated by Cdk5 at one serine residue within a unique carboxyl-terminal region, and its serine phosphorylation enhances tyrosine phosphorylation by Fyn and results in progressive degradation of p45 Dab1. These results indicate that Cdk5 modulates Reelin signaling through the Ser/Thr phosphorylation of Dab1 differently in an isoform-specific manner.

    Brain research 2007;1140;84-95

  • Phosphorylation of adult type Sept5 (CDCrel-1) by cyclin-dependent kinase 5 inhibits interaction with syntaxin-1.

    Taniguchi M, Taoka M, Itakura M, Asada A, Saito T, Kinoshita M, Takahashi M, Isobe T and Hisanaga S

    Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan. taniguti-makoto@c.metro-u.ac.jp

    Increasing evidence implicates cyclin-dependent kinase 5 (Cdk5) in neuronal synaptic function. We searched for Cdk5 substrates in synaptosomal fractions prepared from mouse brains. Mass spectrometric analysis after two-dimensional SDS-PAGE identified several synaptic proteins phosphorylated by Cdk5-p35; one protein identified was Sept5 (CDCrel-1). Although septins were isolated originally as cell division-related proteins in yeast, Sept5 is expressed predominantly in neurons and is implicated in exocytosis. We confirmed that Sept5 is phosphorylated by Cdk5-p35 in vitro and identified Ser17 of adult type Sept5 (Sept5_v1) as a major phosphorylation site. We found that Ser17 of Sept5_v1 is phosphorylated in mouse brains. Coimmunoprecipitation from synaptosomal fractions and glutathione S-transferase-syntaxin-1A pulldown assays of Sept5_v1 expressed in COS-7 cells showed that phosphorylation of Sept5_v1 by Cdk5-p35 decreases the binding to syntaxin-1. These results indicate that the interaction of Sept5 with syntaxin-1 is regulated by the phosphorylation of Sept5_v1 at Ser17 by Cdk5-p35.

    The Journal of biological chemistry 2007;282;11;7869-76

  • AKT and CDK5/p35 mediate brain-derived neurotrophic factor induction of DARPP-32 in medium size spiny neurons in vitro.

    Bogush A, Pedrini S, Pelta-Heller J, Chan T, Yang Q, Mao Z, Sluzas E, Gieringer T and Ehrlich ME

    Farber Institute for Neurosciences and Department of Neurology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

    Mature striatal medium size spiny neurons express the dopamine and cyclic AMP-regulated phosphoprotein, 32 kDa (DARPP-32), but little is known about the mechanisms regulating its levels or the specification of fully differentiated neuronal subtypes. Cell extrinsic molecules that increase DARPP-32 mRNA and/or protein levels include brain-derived neurotrophic factor (BDNF), retinoic acid, and estrogen. DARPP-32 induction by BDNF in vitro requires phosphatidylinositide 3-kinase (PI3K), but inhibition of phosphorylation of protein kinase B/Akt does not entirely abolish expression of DARPP-32. Moreover, the requirement for Akt has not been established. Using pharmacologic inhibitors of PI3K, Akt, and cyclin-dependent kinase 5 (cdk5) and constitutively active and dominant negative PI3K, Akt, cdk5, and p35 viruses in cultured striatal neurons, we measured BDNF-induced levels of DARPP-32 protein and/or mRNA. We demonstrated that both the PI3K/Akt/mammalian target of rapamycin and the cdk5/p35 signal transduction pathways contribute to the induction of DARPP-32 protein levels by BDNF and that the effects are on both the transcriptional and translational levels. It also appears that PI3K is upstream of cdk5/p35, and its activation can lead to an increase in p35 protein levels. These data support the presence of multiple signal transduction pathways mediating expression of DARPP-32 in vitro, including a novel, important pathway via by which PI3K regulates the contribution of cdk5/p35.

    Funded by: NIA NIH HHS: AG023695; NINDS NIH HHS: R01NS045942, R01NS048254

    The Journal of biological chemistry 2007;282;10;7352-9

  • Phosphorylation of protein phosphatase 1 by cyclin-dependent protein kinase 5 during nerve growth factor-induced PC12 cell differentiation.

    Li T, Chalifour LE and Paudel HK

    Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Canada.

    The transcription factor Egr-1 activates cyclin-dependent protein kinase 5 (Cdk5) during nerve growth factor (NGF)-induced differentiation of PC12 cells into neurons (Harada, T. Morooka, T., Ogawa, S., and Nishida, E. (2001) Nat. Cell Biol. 3, 453-459). The downstream target of Cdk5 in the Egr-1/Cdk5 pathway is not clear. In this study, we observed that phosphorylation of protein phosphatase 1 (PP1) on Thr(320) is reduced in brain extracts from Egr-1(-/-) mice, indicating that a kinase downstream of Egr-1 phosphorylates PP1. In HEK 293 cells co-transfected with PP1 and Cdk5, Cdk5 phosphorylates PP1. In vitro, Cdk5 purified from bovine brain phosphorylates bacterially expressed recombinant PP1. In NGF-treated PC12 cells, inhibition of Cdk5 by olomoucine or silencing Cdk5 expression by small interfering RNA strategy, suppresses PP1 phosphorylation. Silencing Cdk5 expression by small interfering RNA also blocks NGF-induced neurite outgrowth. Overexpression of PP1 (wild type) promotes NGF-induced differentiation of PC12 cells, whereas that of PP1 (T320A) has no effect. Our data indicate that PP1 is a downstream target of the NGF/Egr-1/Cdk5 pathway during NGF-induced differentiation of PC12 cells and suggest that PP1 phosphorylation promotes neuronal differentiation.

    The Journal of biological chemistry 2007;282;9;6619-28

  • Cyclin-dependent kinase 5 modulates nociceptive signaling through direct phosphorylation of transient receptor potential vanilloid 1.

    Pareek TK, Keller J, Kesavapany S, Agarwal N, Kuner R, Pant HC, Iadarola MJ, Brady RO and Kulkarni AB

    Functional Genomics Section, Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.

    Transient receptor potential vanilloid 1 (TRPV1), a ligand-gated cation channel highly expressed in small-diameter sensory neurons, is activated by heat, protons, and capsaicin. The phosphorylation of TRPV1 provides a versatile regulation of intracellular calcium levels and is critical for TRPV1 function in responding to a pain stimulus. We have previously reported that cyclin-dependent kinase 5 (Cdk5) activity regulates nociceptive signaling. In this article we report that the Cdk5-mediated phosphorylation of TRPV1 at threonine-407 can modulate agonist-induced calcium influx. Inhibition of Cdk5 activity in cultured dorsal root ganglia neurons resulted in a significant reduction of TRPV1-mediated calcium influx, and this effect could be reversed by restoring Cdk5 activity. Primary nociceptor-specific Cdk5 conditional-knockout mice showed reduced TRPV1 phosphorylation, resulting in significant hypoalgesia. Thus, the present study indicates that Cdk5-mediated TRPV1 phosphorylation is important in the regulation of pain signaling.

    Funded by: Intramural NIH HHS

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;2;660-5

  • Cdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanism.

    Fu WY, Chen Y, Sahin M, Zhao XS, Shi L, Bikoff JB, Lai KO, Yung WH, Fu AK, Greenberg ME and Ip NY

    Department of Biochemistry, Biotechnology Research Institute and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.

    The development of dendritic spines is thought to be crucial for synaptic plasticity. Dendritic spines are retracted upon Eph receptor A4 (EphA4) activation, but the mechanisms that control this process are not well understood. Here we report an important function of cyclin-dependent kinase 5 (Cdk5) in EphA4-dependent spine retraction in mice. We found that blocking Cdk5 activity inhibits ephrin-A1-triggered spine retraction and reduction of mEPSC frequency at hippocampal synapses. The activation of EphA4 resulted in the recruitment of Cdk5 to EphA4, leading to the tyrosine phosphorylation and activation of Cdk5. EphA4 and Cdk5 then enhanced the activation of ephexin1, a guanine-nucleotide exchange factor that regulates activation of the small Rho GTPase RhoA. The association between EphA4 and ephexin1 was significantly reduced in Cdk5(-/-) brains and Cdk5-dependent phosphorylation of ephexin1 was required for the ephrin-A1-mediated regulation of spine density. These findings suggest that ephrin-A1 promotes EphA4-dependent spine retraction through the activation of Cdk5 and ephexin1, which in turn modulates actin cytoskeletal dynamics.

    Funded by: NICHD NIH HHS: HD18655, K08 HD01384; NINDS NIH HHS: NS045500

    Nature neuroscience 2007;10;1;67-76

  • p35/Cyclin-dependent kinase 5 is required for protection against beta-amyloid-induced cell death but not tau phosphorylation by ceramide.

    Seyb KI, Ansar S, Li G, Bean J, Michaelis ML and Dobrowsky RT

    Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS 66045, USA.

    Ceramide is a bioactive sphingolipid that can prevent calpain activation and beta-amyloid (A beta) neurotoxicity in cortical neurons. Recent evidence supports A beta induction of a calpain-dependent cleavage of the cyclin-dependent kinase 5 (cdk5) regulatory protein p35 that contributes to tau hyperphosphorylation and neuronal death. Using cortical neurons isolated from wild-type and p35 knockout mice, we investigated whether ceramide required p35/cdk5 to protect against A beta-induced cell death and tau phosphorylation. Ceramide inhibited A beta-induced calpain activation and cdk5 activity in wild-type neurons and protected against neuronal death and tau hyperphosphorylation. Interestingly, A beta also increased cdk5 activity in p35-/- neurons, suggesting that the alternate cdk5 regulatory protein, p39, might mediate this effect. In p35 null neurons, ceramide blocked A beta-induced calpain activation but did not inhibit cdk5 activity or cell death. However, ceramide blocked tau hyperphosphorylation potentially via inhibition of glycogen synthase kinase-3beta. These data suggest that ceramide can regulate A beta cell toxicity in a p35/cdk5-dependent manner.

    Journal of molecular neuroscience : MN 2007;31;1;23-35

  • Identifying novel substrates for mouse Cdk5 kinase using the yeast Saccharomyces cerevisiae.

    Horiuchi Y, Asada A, Hisanaga S, Toh-e A and Nishizawa M

    Department of Microbiology and Immunology, Keio University School of Medicine, Shinjuku, Tokyo 160-8582, Japan.

    Among the mammalian Cdk family members, Cdk5, activated by the binding of p35, plays an important role in the control of neurogenesis, and its deregulation is thought to be one of the causes of neurodegenerative diseases. Overproduction of Cdk5 and p35 in yeast cells causes growth arrest, probably because of hyperphosphorylation of yeast proteins. We screened mouse brain cDNA that could recover the growth of yeast cells overproducing Cdk5 and p35, hoping that such cDNA encodes a substrate or inhibitor of Cdk5. Mouse brain cDNA library was introduced into a yeast strain in which Cdk5, p35 and mouse cDNA were over-expressed under the control of the GAL promoter, and cDNA plasmids were isolated from the transformants that recovered growth on galactose medium. The analysis of those plasmids revealed that they harbored cDNA that encodes neuronal proteins including SCLIP and CRMP-1, and those with unknown function. We found that Cdk5 could phosphorylate SCLIP and CRMP-1 in vitro and the two proteins in cultured cells showed a mobility shift depending on Cdk5 activity and the presence of specific Ser or Thr residues, indicating that SCLIP and CRMP-1 are likely substrates for Cdk5 in vitro and in cultured cells. Further screening with these systems will enable us to identify more novel substrates and regulators of Cdk5/p35, which will lead to the exploration of Cdk5 function in diverse cellular systems.

    Genes to cells : devoted to molecular & cellular mechanisms 2006;11;12;1393-404

  • The c-Jun N-terminal kinase activator dual leucine zipper kinase regulates axon growth and neuronal migration in the developing cerebral cortex.

    Hirai S, Cui DF, Miyata T, Ogawa M, Kiyonari H, Suda Y, Aizawa S, Banba Y and Ohno S

    Department of Molecular Biology, Graduate School of Medical Science, Yokohama City University, Yokohama 236-0004, Japan. sh3312@med.yokohama-cu.ac.jp

    Mammalian corticogenesis substantially depends on migration and axon projection of newborn neurons that are coordinated by a yet unidentified molecular mechanism. Dual leucine zipper kinase (DLK) induces activation of c-Jun N-terminal kinase (JNK), a molecule that regulates morphogenesis in various organisms. We show here, using gene targeting in mice, that DLK is indispensable for establishing axon tracts, especially those originating from neocortical pyramidal neurons of the cerebrum. Direct and quantitative analysis of radial migration of pyramidal neurons using slice culture and a time-lapse imaging system revealed that acceleration around the subplate was affected by DLK gene disruption and by administration of a JNK inhibitor. Phosphorylation of JNK substrates, including c-Jun and doublecortin, and of JNK itself at the activation loop were partially affected in brains of DLK-deficient mouse embryos. These data suggest that DLK plays a significant role in the coordinated regulation of radial migration and axon projection by modulating JNK activity.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;46;11992-2002

  • COUP-TFI is required for the formation of commissural projections in the forebrain by regulating axonal growth.

    Armentano M, Filosa A, Andolfi G and Studer M

    TIGEM (Telethon Institute of Genetics and Medicine Disorders Program, Via P. Castellino 111, 80131 Napoli, Italy.

    The transcription factor COUP-TFI (NR2F1), an orphan member of the nuclear receptor superfamily, is an important regulator of neurogenesis, cellular differentiation and cell migration. In the forebrain, COUP-TFI controls the connectivity between thalamus and cortex and neuronal tangential migration in the basal telencephalon. Here, we show that COUP-TFI is required for proper axonal growth and guidance of all major forebrain commissures. Fibres of the corpus callosum, the hippocampal commissure and the anterior commissure project aberrantly and fail to cross the midline in COUP-TFI null mutants. Moreover, hippocampal neurons lacking COUP-TFI have a defect in neurite outgrowth and show an abnormal axonal morphology. To search for downstream effectors, we used microarray analysis and showed that, in the absence of COUP-TFI, expression of various cytoskeleton molecules involved in neuronal morphogenesis is affected. Diminished protein levels of the microtubule-associated protein MAP1B and increased levels of the GTP-binding protein RND2 were confirmed in the developing cortex in vivo and in primary hippocampal neurons in vitro. Therefore, based on morphological studies, gene expression profiling and primary cultured neurons, the present data uncover a previously unappreciated intrinsic role for COUP-TFI in axonal growth in vivo and supply one of the premises for COUP-TFI coordination of neuronal morphogenesis in the developing forebrain.

    Funded by: Telethon: TGM06A04, TGM06S01

    Development (Cambridge, England) 2006;133;21;4151-62

  • p25/cyclin-dependent kinase 5 induces production and intraneuronal accumulation of amyloid beta in vivo.

    Cruz JC, Kim D, Moy LY, Dobbin MM, Sun X, Bronson RT and Tsai LH

    Department of Pathology, Harvard Medical School and Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA.

    Aberrant processing of the amyloid precursor protein (APP) and the subsequent accumulation of amyloid beta (Abeta) peptide has been widely established as a central event in Alzheimer's disease (AD) pathogenesis. The sequential cleavage steps required for the generation of Abeta are well outlined; however, there is a relative dearth of knowledge pertaining to signaling pathways and molecular mechanisms that can modulate this process. Here, we demonstrate a novel role for p25/cyclin-dependent kinase 5 (Cdk5) in regulating APP processing, Abeta peptide generation, and intraneuronal Abeta accumulation in inducible p25 transgenic and compound PD-APP transgenic mouse models that demonstrate deregulated Cdk5 activity and a neurodegenerative phenotype. Induction of p25 resulted in enhanced forebrain Abeta levels before any evidence of neuropathology in these mice. Intracellular Abeta accumulated in perinuclear regions and distended axons within the forebrains of these mice. Evidence for modulations in axonal transport or beta-site APP cleaving enzyme 1 protein levels and activity are presented as mechanisms that may account for the Abeta accumulation caused by p25/Cdk5 deregulation. Collectively, these findings delineate a novel pathological mechanism involving aberrant APP processing by p25/Cdk5 and have important implications in AD pathogenesis.

    Funded by: NINDS NIH HHS: NS051874

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;41;10536-41

  • The roles of cyclin-dependent kinase 5 and glycogen synthase kinase 3 in tau hyperphosphorylation.

    Plattner F, Angelo M and Giese KP

    Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, United Kingdom. f.plattner@ucl.ac.uk

    Hyperphosphorylation of the microtubule-associated protein tau is a characteristic feature of neurodegenerative tauopathies including Alzheimer disease. Over-activation of proline-directed kinases, such as cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3), has been implicated in the aberrant phosphorylation of tau at proline-directed sites. In this study we tested the roles of Cdk5 and GSK3 in tau hyperphosphorylation in vivo using transgenic mice with p25-induced Cdk5 over-activation. We found that over-activation of Cdk5 in young transgenic animals does not induce tau hyperphosphorylation at sites recognized by the antibodies AT8, AT100, PHF-1, and TG3. In fact, we observed that Cdk5 over-activation leads to inhibition of GSK3. However, in old transgenic animals the inhibition of GSK3 is lost and results in increased GSK3 activity, which coincides with tau hyperphosphorylation at the AT8 and PHF-1 sites. Pharmacological inhibition of GSK3 in old transgenic mice by chronic treatment with lithium leads to a reduction of the age-dependent increase in tau hyperphosphorylation. Furthermore, we found that Cdk5, GSK3, and PP2A co-immunoprecipitate, suggesting a functional association of these molecules. Together, these results reveal the role of GSK3 as a key mediator of tau hyperphosphorylation, whereas Cdk5 acts as a modulator of tau hyperphosphorylation via the inhibitory regulation of GSK3. Furthermore, these findings suggest that disruption of regulation of GSK3 activity underlies tau hyperphosphorylation in neurodegenerative tauopathies. Hence, GSK3 may be a prime target for therapeutic intervention in tauopathies including Alzheimer disease.

    Funded by: Medical Research Council: G0400983

    The Journal of biological chemistry 2006;281;35;25457-65

  • Role of heat-shock factor 2 in cerebral cortex formation and as a regulator of p35 expression.

    Chang Y, Ostling P, Akerfelt M, Trouillet D, Rallu M, Gitton Y, El Fatimy R, Fardeau V, Le Crom S, Morange M, Sistonen L and Mezger V

    Biologie Moléculaire du Stress, Centre National de la Recherche Scientifique (CNRS) UMR8541, Ecole Normale Supérieure, Paris, France.

    Heat-shock factors (HSFs) are associated with multiple developmental processes, but their mechanisms of action in these processes remain largely enigmatic. Hsf2-null mice display gametogenesis defects and brain abnormalities characterized by enlarged ventricles. Here, we show that Hsf2-/- cerebral cortex displays mispositioning of neurons of superficial layers. HSF2 deficiency resulted in a reduced number of radial glia fibers, the architectural guides for migrating neurons, and of Cajal-Retzius cells, which secrete the positioning signal Reelin. Therefore, we focused on the radial migration signaling pathways. The levels of Reelin and Dab1 tyrosine phosphorylation were reduced, suggesting that the Reelin cascade is affected in Hsf2-/- cortices. The expression of p35, an activator of cyclin-dependent kinase 5 (Cdk5), essential for radial migration, was dependent on the amount of HSF2 in gain- and loss-of-function systems. p39, another Cdk5 activator, displayed reduced mRNA levels in Hsf2-/- cortices, which, together with the lowered p35 levels, decreased Cdk5 activity. We demonstrate in vivo binding of HSF2 to the p35 promoter and thereby identify p35 as the first target gene for HSF2 in cortical development. In conclusion, HSF2 affects cellular populations that assist in radial migration and directly regulates the expression of p35, a crucial actor of radial neuronal migration.

    Genes & development 2006;20;7;836-47

  • BGEM: an in situ hybridization database of gene expression in the embryonic and adult mouse nervous system.

    Magdaleno S, Jensen P, Brumwell CL, Seal A, Lehman K, Asbury A, Cheung T, Cornelius T, Batten DM, Eden C, Norland SM, Rice DS, Dosooye N, Shakya S, Mehta P and Curran T

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States.

    Funded by: NINDS NIH HHS: 5R37NS036558, N01-NS-0-2331, R37 NS036558

    PLoS biology 2006;4;4;e86

  • Calpain-regulated p35/cdk5 plays a central role in dopaminergic neuron death through modulation of the transcription factor myocyte enhancer factor 2.

    Smith PD, Mount MP, Shree R, Callaghan S, Slack RS, Anisman H, Vincent I, Wang X, Mao Z and Park DS

    Ottawa Health Research Institute, Ottawa, Ontario, K1H 8M5, Canada.

    The mechanisms underlying dopamine neuron loss in Parkinson's disease (PD) are not clearly defined. Here, we delineate a pathway by which dopaminergic loss induced by 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP) is controlled in vivo. We reported previously that calpains play a central required role in dopamine loss after MPTP treatment. Here, we provide evidence that the downstream effector pathway of calpains is through cyclin-dependent kinase 5 (cdk5)-mediated modulation of the transcription factor myocyte enhancer factor 2 (MEF2). We show that MPTP-induced conversion of the cdk5 activator p35 to a pathogenic p25 form is dependent on calpain activity in vivo. In addition, p35 deficiency attenuates MPTP-induced dopamine neuron loss and behavioral outcome. Moreover, MEF2 is phosphorylated on Ser444, an inactivating site, after MPTP treatment. This phosphorylation is dependent on both calpain and p35 activity, consistent with the model that calpain-mediated activation of cdk5 results in phosphorylation of MEF2 in vivo. Finally, we provide evidence that MEF2 is critical for dopaminergic loss because "cdk5 phosphorylation site mutant" of MEF2D provides neuroprotection in an MPTP mouse model of PD. Together, these data indicate that calpain-p35-p25/cdk5-mediated inactivation of MEF2 plays a critical role in dopaminergic loss in vivo.

    Funded by: NICHD NIH HHS: HD39446; NINDS NIH HHS: NS048254

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;2;440-7

  • Cdk5 phosphorylates and stabilizes p27kip1 contributing to actin organization and cortical neuronal migration.

    Kawauchi T, Chihama K, Nabeshima Y and Hoshino M

    Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

    p27(kip1), a cyclin-dependent kinase (CDK) inhibitor (CKI), generally suppresses CDK activity in proliferating cells. Although another role of p27 in cell migration has been recently suggested in vitro, the physiological importance of p27 in cell migration remains elusive, as p27-deficient mice have not shown any obvious migration-defect-related phenotypes. Here, we show that Cdk5, an unconventional neuronal CDK, phosphorylates and stabilizes p27 as an upstream regulator, maintaining the amount of p27 in post-mitotic neurons. In vivo RNA interference (RNAi) experiments showed that reduced amounts of p27 caused inhibition of cortical neuronal migration and decreased the amount of F-actin in the processes of migrating neurons. The Cdk5-p27 pathway activates an actin-binding protein, cofilin, which is also shown to be involved in cortical neuronal migration in vivo. Our findings shed light on a previously unknown new relationship between CDK and CKI in G0-arrested cells that regulates cytoskeletal reorganization and neuronal migration during corticogenesis.

    Nature cell biology 2006;8;1;17-26

  • p53, Apaf-1, caspase-3, and -9 are dispensable for Cdk5 activation during cell death.

    Lin L, Ye Y and Zakeri Z

    Department of Biology, Queens College and Graduate Center of the City University of New York, 65-30 Kissena Blvd, Flushing, NY 11367, USA.

    Cyclin-dependent kinase 5 (Cdk5) is a member of the cyclin-dependent kinase family that is mostly seen in neurons, does not vary with cell cycle, and is activated in many neurodegenerative disorders and other non-neuronal pathologies, but its relationship to non-neuronal apoptosis is not understood, nor is the control of the activation of Cdk5 by its activators. The most widely studied activator of Cdk5, p35, is cleaved to p25 by calpain, an event that has been linked with activation of Cdk5 and neuronal death. Here we report that calpain-mediated Cdk5/p25 activation accompanies non-neuronal as well as neuronal cell death, suggesting that the p35/calpain/p25/Cdk5 activation sequence is a general feature of cell death. We further demonstrate that Cdk5 can be activated in the absence of p53, Apaf-1, caspase-9, and -3 during cell death, indicating that its activation relates more to cell death than to a specific pathway of apoptosis.

    Cell death and differentiation 2006;13;1;141-50

  • Cyclin-dependent kinase 5 is essential for neuronal cell cycle arrest and differentiation.

    Cicero S and Herrup K

    Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.

    Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase with significant homology to cell cycle-related Cdks but is not believed to be active in a typical cell cycle. In Cdk5-deficient embryos and Cdk5 chimeras, migration and survival of postmitotic neurons is compromised in a cell-autonomous manner. In the present study, we show that loss of Cdk5 leads to both failure of neuronal differentiation and loss of cell cycle control. Using specific cytoskeletal proteins as indices of neuronal differentiation, we find that Cdk5-deficient neurons are significantly arrested or delayed in their developmental program both in vivo and in vitro. For example, immunocytochemistry of embryonic day 16 (E16) cortex reveals that the expression of microtubule-associated protein 2c (Map-2c), a marker of mature neurons, is nearly absent in Cdk5(-/-) cells that have migrated to the cortical plate while these same cells continue to express nestin. Similarly, in vitro, Map-2-positive cells are rare in cultures from E16 Cdk5(-/-) embryos. Cell cycle control is also deficient in Cdk5(-/-) cells. In vivo, neurons engaged in cell cycle activities are found in the cortical plate, and, in vitro, class III beta-tubulin-positive cells continue to label with bromodeoxyuridine even after 5 d of incubation. Transfection of a wild-type Cdk5 construct reveals that cell cycle control can be regained in Cdk5(-/-) cells by overexpression of Cdk5. These data indicate that Cdk5 is necessary for both neuronal differentiation and cell cycle inhibition.

    Funded by: NIA NIH HHS: P50-AG08012; NINDS NIH HHS: R01-NS20591

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;42;9658-68

  • Aberrant motor axon projection, acetylcholine receptor clustering, and neurotransmission in cyclin-dependent kinase 5 null mice.

    Fu AK, Ip FC, Fu WY, Cheung J, Wang JH, Yung WH and Ip NY

    Department of Biochemistry, Biotechnology Research Institute, and Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.

    Cyclin-dependent kinase (Cdk)5 is a key regulator of neural development. We have previously demonstrated that Cdk5/p35 are localized to the postsynaptic muscle and are implicated in the regulation of neuregulin/ErbB signaling in myotube culture. To further elucidate whether Cdk5 activity contributes to neuromuscular junction (NMJ) development in vivo, the NMJ of Cdk5-/- mice was examined. Consistent with our previous demonstration that Cdk5 phosphorylates ErbB2/3 to regulate its tyrosine phosphorylation, we report here that the phosphorylation of ErbB2 and ErbB3 and the ErbB2 kinase activity are reduced in Cdk5-deficient muscle. In addition, Cdk5-/- mice also display morphological abnormalities at the NMJ pre- and postsynaptically. Whereas the outgrowth of the main nerve trunk is grossly normal, the intramuscular nerve projections exhibit profuse and anomalous branching patterns in the Cdk5-/- embryos. The central band of acetylcholine receptor (AChR) clusters is also wider in Cdk5-/- diaphragms, together with the absence of S100 immunoreactivity along the phrenic nerve during late embryonic stages. Moreover, we unexpectedly discovered that the agrin-induced formation of large AChR clusters is significantly increased in primary muscle cultures prepared from Cdk5-null mice and in C2C12 myotubes when Cdk5 activity was suppressed. These abnormalities are accompanied by elevated frequency of miniature endplate potentials in Cdk5-null diaphragm. Taken together, our findings reveal the essential role of Cdk5 in regulating the development of motor axons and neuromuscular synapses in vivo.

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;42;15224-9

  • Cdk5-dependent regulation of glucose-stimulated insulin secretion.

    Wei FY, Nagashima K, Ohshima T, Saheki Y, Lu YF, Matsushita M, Yamada Y, Mikoshiba K, Seino Y, Matsui H and Tomizawa K

    Department of Physiology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558, Japan.

    Tight glycemic control in individuals with diabetes mellitus is essential to prevent or delay its complications. Present treatments to reduce hyperglycemia mainly target the ATP-sensitive K(+) (K(ATP)) channel of pancreatic beta cells to increase insulin secretion. These current approaches are often associated with the side effect of hypoglycemia. Here we show that inhibition of the activity of cyclin-dependent kinase 5 (Cdk5) enhanced insulin secretion under conditions of stimulation by high glucose but not low glucose in MIN6 cells and pancreatic islets. The role of Cdk5 in regulation of insulin secretion was confirmed in pancreatic beta cells deficient in p35, an activator of Cdk5. p35-knockout mice also showed enhanced insulin secretion in response to a glucose challenge. Cdk5 kinase inhibition enhanced the inward whole-cell Ca(2+) channel current and increased Ca(2+) influx across the L-type voltage-dependent Ca(2+) channel (L-VDCC) upon stimulation with high glucose in beta cells, but had no effect on Ca(2+) influx without glucose stimulation. The inhibitory regulation by Cdk5 on the L-VDCC was attributed to the phosphorylation of loop II-III of the alpha(1C) subunit of L-VDCC at Ser783, which prevented the binding to SNARE proteins and subsequently resulted in a decrease of the activity of L-VDCC. These results suggest that Cdk5/p35 may be a drug target for the regulation of glucose-stimulated insulin secretion.

    Nature medicine 2005;11;10;1104-8

  • Differential roles of nuclear and cytoplasmic cyclin-dependent kinase 5 in apoptotic and excitotoxic neuronal death.

    O'Hare MJ, Kushwaha N, Zhang Y, Aleyasin H, Callaghan SM, Slack RS, Albert PR, Vincent I and Park DS

    Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa Health Research Institute, Ottawa, Ontario, K1H 8M5, Canada.

    Cyclin-dependent kinase 5 (cdk5) is a member of the cyclin-dependent kinase family whose activity is localized mainly to postmitotic neurons attributable to the selective expression of its activating partners p35 and p39. Deregulation of cdk5, as a result of calpain cleavage of p35 to a smaller p25 form, has been suggested to be a central component of neuronal death underlying numerous neurodegenerative diseases. However, the relevance of cdk5 in apoptotic death that relies on the mitochondrial pathway is unknown. Furthermore, evidence that cdk5 can also promote neuronal survival has necessitated a more complex understanding of cdk5 in the control of neuronal fate. Here we explore each of these issues using apoptotic and excitotoxic death models. We find that apoptotic death induced by the DNA-damaging agent camptothecin is associated with early transcription-mediated loss of p35 and with late production of p25 that is dependent on Bax, Apaf1, and caspases. In contrast, during excitotoxic death induced by glutamate, neurons rapidly produce p25 independent of the mitochondrial pathway. Analysis of the localization of p35 and p25 revealed that p35 is mainly cytoplasmic, whereas p25 accumulates selectively in the nucleus. By targeting a dominant-negative cdk5 to either the cytoplasm or nucleus, we show that cdk5 has a death-promoting activity within the nucleus and that this activity is required in excitotoxic death but not apoptotic death. Moreover, we also find that cdk5 contributes to pro-survival signaling selectively within the cytoplasm, and manipulation of this signal can modify death induced by both excitotoxicity and DNA damage.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;39;8954-66

  • Multiple cyclin-dependent kinases signals are critical mediators of ischemia/hypoxic neuronal death in vitro and in vivo.

    Rashidian J, Iyirhiaro G, Aleyasin H, Rios M, Vincent I, Callaghan S, Bland RJ, Slack RS, During MJ and Park DS

    Ottawa Health Research Institute, Neuroscience Group, Ottawa, ON, Canada K1H 8M5.

    The mechanisms involving neuronal death after ischemic/hypoxic insult are complex, involving both rapid (excitotoxic) and delayed (apoptotic-like) processes. Recent evidence suggests that cell cycle regulators such as cyclin-dependent kinases are abnormally activated in neuropathological conditions, including stroke. However, the function of this activation is unclear. Here, we provide evidence that inhibition of the cell cycle regulator, Cdk4, and its activator, cyclinD1, plays critical roles in the delayed death component of ischemic/hypoxic stress by regulating the tumor suppressor retinoblastoma protein. In contrast, the excitotoxic component of ischemia/hypoxia is predominately regulated by Cdk5 and its activator p35, components of a cyclin-dependent kinase complex associated with neuronal development. Hence, our data both characterize the functional significance of the cell cycle Cdk4 and neuronal Cdk5 signals as well as define the pathways and circumstances by which they act to control ischemic/hypoxic damage.

    Funded by: NIA NIH HHS: AG12721, R01 AG012721

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;39;14080-5

  • Implication of cyclin-dependent kinase 5 in the development of psychological dependence on and behavioral sensitization to morphine.

    Narita M, Shibasaki M, Nagumo Y, Narita M, Yajima Y and Suzuki T

    Department of Toxicology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Shinagawa-ku, Tokyo, Japan. narita@hoshi.ac.jp

    In the present study, we investigated the role of cyclin-dependent kinase 5 (cdk5) in the brain dynamics changed by repeated in vivo treatment with morphine. The level of phosphorylated-cdk5 was significantly increased in the cingulate cortex of mice showing the morphine-induced rewarding effect. Under these conditions, roscovitine, a cdk5 inhibitor, given intracerebroventricularly (i.c.v.) caused a dose-dependent and significant inhibition of the morphine-induced rewarding effect. In addition, the dose-response effect of the morphine-induced rewarding effect was dramatically attenuated in cdk5 heterozygous (+/-) knockout mice. Furthermore, the development of behavioral sensitization by intermittent administration of morphine was virtually abolished in cdk5 (+/-) mice. These findings suggest that the induction and/or activation of cdk5 are implicated in the development of psychological dependence on morphine.

    Journal of neurochemistry 2005;93;6;1463-8

  • Proteomic analysis of in vivo phosphorylated synaptic proteins.

    Collins MO, Yu L, Coba MP, Husi H, Campuzano I, Blackstock WP, Choudhary JS and Grant SG

    Division of Neuroscience, University of Edinburgh, Edinburgh EH8 9JZ, UK.

    In the nervous system, protein phosphorylation is an essential feature of synaptic function. Although protein phosphorylation is known to be important for many synaptic processes and in disease, little is known about global phosphorylation of synaptic proteins. Heterogeneity and low abundance make protein phosphorylation analysis difficult, particularly for mammalian tissue samples. Using a new approach, combining both protein and peptide immobilized metal affinity chromatography and mass spectrometry data acquisition strategies, we have produced the first large scale map of the mouse synapse phosphoproteome. We report over 650 phosphorylation events corresponding to 331 sites (289 have been unambiguously assigned), 92% of which are novel. These represent 79 proteins, half of which are novel phosphoproteins, and include several highly phosphorylated proteins such as MAP1B (33 sites) and Bassoon (30 sites). An additional 149 candidate phosphoproteins were identified by profiling the composition of the protein immobilized metal affinity chromatography enrichment. All major synaptic protein classes were observed, including components of important pre- and postsynaptic complexes as well as low abundance signaling proteins. Bioinformatic and in vitro phosphorylation assays of peptide arrays suggest that a small number of kinases phosphorylate many proteins and that each substrate is phosphorylated by many kinases. These data substantially increase existing knowledge of synapse protein phosphorylation and support a model where the synapse phosphoproteome is functionally organized into a highly interconnected signaling network.

    The Journal of biological chemistry 2005;280;7;5972-82

  • Increased activity of cyclin-dependent kinase 5 leads to attenuation of cocaine-mediated dopamine signaling.

    Takahashi S, Ohshima T, Cho A, Sreenath T, Iadarola MJ, Pant HC, Kim Y, Nairn AC, Brady RO, Greengard P and Kulkarni AB

    Functional Genomics Section, Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Bethesda, MD 20892, USA.

    Cocaine, a drug of abuse, increases synaptic dopamine levels in the striatum by blocking dopamine reuptake at axon terminals. Cyclin-dependent kinase 5 (Cdk5) and its activator p35, proteins involved in phosphorylation of substrates in postmitotic neurons, have been found to be up-regulated after chronic exposure to cocaine. To further examine the effects of Cdk5 and p35 induction on striatal dopamine signaling, we generated two independent transgenic mouse lines in which Cdk5 or p35 was overexpressed specifically in neurons. We report here that increased Cdk5 activity, as a result of p35 but not of Cdk5 overexpression, leads to attenuation of cocaine-mediated dopamine signaling. Increased Cdk5-mediated phosphorylation of dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa (DARPP-32) at Thr-75, was accompanied by decreased phosphorylation of DARPP-32 at Thr-34. Increased Cdk5-mediated phosphorylation of extracellular signal-regulated kinase kinase 1 at Thr-286 was accompanied by decreased activation of extracellular signal-regulated kinase 1/2. These effects contributed to attenuation of cocaine-induced phosphorylation of cAMP response element-binding protein as well as a lesser induction of c-fos in the striatum. These results support the idea that Cdk5 activity is involved in altered gene expression after chronic exposure to cocaine and hence impacts the long-lasting changes in neuronal function underlying cocaine addiction.

    Funded by: NIDA NIH HHS: DA10044, P01 DA010044; NIDCR NIH HHS: Z01DE00664-05

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;5;1737-42

  • Semaphorin3A signalling is mediated via sequential Cdk5 and GSK3beta phosphorylation of CRMP2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer's disease.

    Uchida Y, Ohshima T, Sasaki Y, Suzuki H, Yanai S, Yamashita N, Nakamura F, Takei K, Ihara Y, Mikoshiba K, Kolattukudy P, Honnorat J and Goshima Y

    Department of Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan.

    Collapsin response mediating protein-2 (CRMP2) has been identified as an intracellular protein mediating Semaphorin3A (Sema3A), a repulsive guidance molecule. In this study, we demonstrate that cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3beta (GSK3beta) plays a critical role in Sema3A signalling. In In vitro kinase assay, Cdk5 phosphorylated CRMP2 at Ser522, while GSK3beta did not induce any phosphorylation of CRMP2. Phosphorylation by GSK3beta was exclusively observed in Cdk5-phosphorylated CRMP2, but barely in CRMP2T509A. These results indicate that Cdk5 primarily phosphorylates CRMP2 at Ser522 and GSK3beta secondarily phosphorylates at Thr509. The dual-phosphorylated CRMP2, but not non-phosphorylated or single-phosphorylated CRMP2, is recognized with the antibody 3F4, which is highly reactive with the neurofibrillary tangles of Alzheimer's disease. 3F4 recognized the CRMP2 in the wild-type but not cdk5-/- mouse embryonic brain lysates. The phosphorylation of CRMP2 at Ser522 caused reduction of its affinity to tubulin. In dorsal root ganglion neurones, Sema3A stimulation enhanced the levels of the phosphorylated form of CRMP2 detected by 3F4. Over-expression of CRMP2 mutant substituting either Ser522 or Thr509 to Ala attenuates Sema3A-induced growth cone collapse response. These results suggest that the sequential phosphorylation of CRMP is an important process of Sema3A signalling and the same mechanism may have some relevance to the pathological aggregation of the microtubule-associated proteins.

    Genes to cells : devoted to molecular & cellular mechanisms 2005;10;2;165-79

  • Cdk5/p35 phosphorylates mSds3 and regulates mSds3-mediated repression of transcription.

    Li Z, David G, Hung KW, DePinho RA, Fu AK and Ip NY

    Department of Biochemistry, Biotechnology Research Institute and Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.

    Cyclin-dependent kinase 5 (Cdk5), a serine/threonine kinase that displays kinase activity predominantly in neurons, is activated by two non-cyclin activators, p35 or p39. Here, we report a physical and functional interaction between the Cdk5/p35 complex and mouse Sds3 (mSds3), an essential component of mSin3-histone deacetylase (HDAC) co-repressor complex. mSds3 binds to p35 both in vitro and in vivo, enabling active Cdk5 to phosphorylate mSds3 at serine 228. A mSds3 S228A mutant retained mSin3 binding activity, but its dimerization was not greatly enhanced by p35 when compared with wild type. Notably, p35 overexpression augmented mSds3-mediated transcriptional repression in vitro. Interestingly, mutational studies revealed that the ability of exogenous mSds3 to rescue cell growth and viability in mSds3 null cells correlates with its ability to be phosphorylated by Cdk5. The identification of mSds3 as a substrate of the Cdk5/p35 complex reveals a new regulatory mechanism in controlling the mSin3-HDAC transcriptional repressor activity and provides a new potential therapeutic means to inhibit specific HDAC activities in disease.

    Funded by: NCI NIH HHS: R01CA86379

    The Journal of biological chemistry 2004;279;52;54438-44

  • GTP-dependent secretion from neutrophils is regulated by Cdk5.

    Rosales JL, Ernst JD, Hallows J and Lee KY

    Department of Cell Biology and Anatomy, Cancer Biology and Neuroscience Research Groups, The University of Calgary, Calgary, Alberta T2N 4N1, Canada. rosales@ucalgary.ca

    We have previously shown evidence for the existence of a calcium-independent, GTP-regulated mechanism of secretion from neutrophils, but this secretory mechanism remains to be fully elucidated. Cyclin-dependent kinase 5 (Cdk5), the various substrates of which include Munc18 and synapsin 1, has been implicated in neuronal secretion. Although the Cdk5 activator, p35, and Cdk5-p35 activity are primarily associated with neurons, we report here that p35 also exists in neutrophils and that an active Cdk5-p35 complex is present in these cells. Cdk5-p35 activity in human neutrophils is mostly localized in secretory granules, which show an increase in Cdk5-p35 level and activity upon GTP stimulation. The potent Cdk5 inhibitor, roscovitine, completely blocks GTP-stimulated granule Cdk5 activity, which accompanies lactoferrin secretion from neutrophil-specific granules. Roscovitine also inhibits GTP-induced lactoferrin secretion and surface localization of the secretion markers, CD63 and CD66b, to a certain extent. Furthermore, neutrophils from wild-type mice treated with roscovitine and neutrophils from p35(-/-) mice exhibit comparable surface expression levels of both CD63 and CD66b upon GTP stimulation. Although our data suggest that other molecules control GTP-induced secretion from neutrophils, it is clear that Cdk5-p35 is required to elicit the maximum GTP-induced secretory response. Our observation that multiple proteins in neutrophil granules serve as specific substrates of Cdk5 further supports the premise that the kinase is a key component of the GTP-regulated secretory apparatus in neutrophils.

    The Journal of biological chemistry 2004;279;52;53932-6

  • Cyclin-dependent kinase 5 phosphorylates serine 31 of tyrosine hydroxylase and regulates its stability.

    Moy LY and Tsai LH

    Department of Pathology and Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA. lily_moy@hms.harvard.edu

    Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its activity is regulated by phosphorylation in the N-terminal regulatory domain. The proline-directed serine/threonine kinase cyclin-dependent kinase 5 (cdk5) plays an important role in diverse neuronal processes. In the present study, we identify TH as a novel substrate of cdk5. We show that cdk5 phosphorylates TH at serine 31 and that this phosphorylation is associated with an increase in total TH activity. In transgenic mice with increased cdk5 activity, the immunoreactivity for phosphorylated TH at Ser-31 is enhanced in neurons of the substantia nigra, a brain region enriched with TH-positive neurons. In addition, we demonstrate that co-expression of cdk5 and its regulatory activator p35 with TH increases the stability of TH. Consistent with these findings, TH protein levels are reduced in cdk5 knock-out mice. Importantly, the TH activity and protein turnover of the phosphorylation-defective mutant TH S31A was not altered by cdk5 activity. Taken together, these data suggest that cdk5 phosphorylation of TH is an important regulator of TH activity through stabilization of TH protein levels.

    Funded by: NIA NIH HHS: AG000111-10

    The Journal of biological chemistry 2004;279;52;54487-93

  • Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas.

    Watahiki A, Waki K, Hayatsu N, Shiraki T, Kondo S, Nakamura M, Sasaki D, Arakawa T, Kawai J, Harbers M, Hayashizaki Y and Carninci P

    Genome Science Laboratory, RIKEN, Wako main campus, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan.

    It is becoming increasingly clear that alternative splicing enables the complex development and homeostasis of higher organisms. To gain a better understanding of how splicing contributes to regulatory pathways, we have developed an alternative splicing library approach for the identification of alternatively spliced exons and their flanking regions by alternative splicing sequence enriched tags sequencing. Here, we have applied our approach to mouse melan-c melanocyte and B16-F10Y melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those encoding important regulatory factors such as cyclin D2, Ilk, MAPK12, MAPK14, RAB4, melastatin 1 and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line-specific exons for Tmc6, Abi1, Sorbs1, Ndel1 and Snx16. Thus, the ASL approach proved effective in identifying splicing events, which suggest that alternative splicing is important in melanoma development.

    Nature methods 2004;1;3;233-9

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Cdk5 regulates activation and localization of Src during corneal epithelial wound closure.

    Gao CY, Stepp MA, Fariss R and Zelenka P

    National Eye Institute, NIH, Building 7, 7 Memorial Drive MSC 0704, Bethesda, MD 20892, USA.

    Recent studies have shown that Cdk5, a member of the cyclin-dependent-kinase family, regulates adhesion and migration in a mouse corneal epithelial cell line. Here, we extend these findings to corneal wound healing in vivo and examine the mechanism linking Cdk5 to cytoskeletal reorganization and migration. Cdk5 was overexpressed in the corneal epithelium of transgenic mice under control of the ALDH3 promoter. Elevated Cdk5 expression retarded corneal debridement wound closure in these animals and suppressed remodeling of the actin cytoskeleton. Conversely, the Cdk5 inhibitor, olomoucine, accelerated debridement wound healing in organ cultured eyes of normal mice, caused migrating cells to separate from the epithelial cell sheet, and increased the level of activated Src(pY416) along the wound edge. To explore the relationship between Cdk5 and Src in greater detail, we examined scratch-wounded cultures of corneal epithelial cells. Src was activated in cells along the wound edge and blocking this activation with the Src kinase inhibitor, PP1, inhibited wound closure by 85%. Inhibiting Cdk5 activity with olomoucine or a dominant negative construct, Cdk5T33, increased the concentration of Src(pY416), shifted its subcellular localization to the cell periphery and enhanced wound closure. Cdk5(pY15), an activated form of Cdk5, also appeared along the wound edge. Inhibiting Src activity with PP1 blocked the appearance of Cdk5(pY15), suggesting that Cdk5 phosphorylation is Src dependent. Cdk5 and Src co-immunoprecipitated from scratch-wounded cultures, demonstrating that both kinases are part of an intracellular protein complex. These findings indicate that Cdk5 exerts its effects on cell migration during corneal epithelial wound healing by regulating the activation and localization of Src.

    Journal of cell science 2004;117;Pt 18;4089-98

  • Cyclin-dependent kinase 5 associated with p39 promotes Munc18-1 phosphorylation and Ca(2+)-dependent exocytosis.

    Lilja L, Johansson JU, Gromada J, Mandic SA, Fried G, Berggren PO and Bark C

    Department of Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, SE-171 76 Stockholm, Sweden.

    Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine protein kinase that requires association with a regulatory protein, p35 or p39, to form an active enzyme. Munc18-1 plays an essential role in membrane fusion, and its function is regulated by phosphorylation. We report here that both p35 and p39 were expressed in insulin-secreting beta-cells, where they exhibited individual subcellular distributions and associated with membranous organelles of different densities. Overexpression of Cdk5, p35, or p39 showed that Cdk5 and p39 augmented Ca(2+)-induced insulin exocytosis. Suppression of p39 and Cdk5, but not of p35, by antisense oligonucleotides selectively inhibited insulin exocytosis. Transient transfection of primary beta-cells with Munc18-1 templates mutated in potential Cdk5 or PKC phosphorylation sites, in combination with Cdk5 and the different Cdk5 activators, suggested that Cdk5/p39-promoted Ca(2+)-dependent insulin secretion from primary beta-cells by phosphorylating Munc18-1 at a biochemical step immediately prior to vesicle fusion.

    Funded by: NIDDK NIH HHS: DK58505

    The Journal of biological chemistry 2004;279;28;29534-41

  • Cyclin-dependent kinase 5 phosphorylates signal transducer and activator of transcription 3 and regulates its transcriptional activity.

    Fu AK, Fu WY, Ng AK, Chien WW, Ng YP, Wang JH and Ip NY

    Department of Biochemistry, Biotechnology Research Institute and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.

    The activity of cyclin-dependent kinase 5 (Cdk5) depends on the association with one of its activators, p35 and p39, which are prominently expressed in the nervous system. Studies on the repertoire of protein substrates for Cdk5 have implicated the involvement of Cdk5 in neuronal migration and synaptic plasticity. Our recent analysis of the sequence of signal transducer and activator of transcription (STAT)3, a key transcription factor, reveals the presence of potential Cdk5 phosphorylation site. We report here that the Cdk5/p35 complex associates with STAT3 and phosphorylates STAT3 on the Ser-727 residue in vitro and in vivo. Intriguingly, whereas the Ser phosphorylation of STAT3 can be detected in embryonic and postnatal brain and muscle of wild-type mice, it is essentially absent from those of Cdk5-deficient embryos. In addition, treatment of cultured myotubes with neuregulin enhances the Ser phosphorylation of STAT3 and transcription of STAT3 target genes, such as c-fos and junB, in a Cdk5-dependent manner. Both the DNA-binding activity of STAT3 and the transcription of specific target genes, such as fibronectin, are reduced in Cdk5-deficient muscle. Taken together, these results reveal a physiological role of Cdk5 in regulating STAT3 phosphorylation and modulating its transcriptional activity.

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;17;6728-33

  • Perinatal abrogation of Cdk5 expression in brain results in neuronal migration defects.

    Hirasawa M, Ohshima T, Takahashi S, Longenecker G, Honjo Y, Veeranna, Pant HC, Mikoshiba K, Brady RO and Kulkarni AB

    Functional Genomics Unit, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.

    Cyclin-dependent kinase 5 (Cdk5) is essential for the proper development of the CNS, as is evident from the perinatal lethality of conventional Cdk5 knockout (Cdk5-/-) mice. Cdk5 is also implicated in numerous complex functions of the adult CNS such as synaptic transmission, synaptic plasticity, and neuronal signaling. To elucidate the molecular roles of Cdk5 in the adult CNS, we have abrogated neuronal expression of Cdk5 in perinatal mice by using a cre-loxP system. The Cdk5-loxP flanked mice were crossed with the cre-transgenic mice in which the cre expression is driven by the murine neurofilament-heavy chain promoter, resulting in generation of viable Cdk5 conditional knockout mice with the restricted deletion of the Cdk5 gene in specific neurons beginning around embryonic day 16.5. Twenty-five percent of the Cdk5 conditional knockout mice carrying the heterozygous cre allele had neuronal migration defects confined to brain areas where neuronal migration continues through the perinatal period. These results indicate that abrogation of Cdk5 expression in mature neurons results in a viable mouse model that offers further opportunities to investigate the molecular roles of Cdk5 in the adult CNS.

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;16;6249-54

  • Effects of cyclin-dependent kinase-5 activity on apoptosis and tau phosphorylation in immortalized mouse brain cortical cells.

    Shelton SB, Krishnamurthy P and Johnson GV

    Department of Psychiatry, University of Alabama at Birmingham, Birmingham, Alabama 35294-0001, USA.

    Cyclin-dependent kinase-5 (CDK5), a unique CDK family member, is active primarily in the central nervous system (CNS). Previous studies suggest that CDK5 is proapoptotic and contributes to tau hyperphosphorylation and neurodegeneration in Alzheimer's disease. The objective of this study was to examine CDK5 effects on apoptotic progression and tau phosphorylation. Immortalized embryonic mouse brain cortical cells were used to establish a stable cell line that overexpressed wild-type human tau. In these studies, thapsigargin, which induces endoplasmic reticulum stress and can cause accumulation of misfolded proteins, was used to induce apoptosis. Caspase-3 activity and poly-(ADP-ribose)-polymerase (PARP) cleavage, as measures of apoptosis, were significantly increased 24 and 48 hr after thapsigargin treatment, and these events were unaffected by tau expression. Although transient coexpression of CDK5 and its activator, p25, increased CDK5 activity greater than tenfold, increases in caspase-3 activity in response to thapsigargin treatment were unaffected by the presence of CDK5/p25. Tau phosphorylation at the PHF-1 epitope, but not the Tau-1 epitope, was increased significantly in CDK5/p25-transfected cells compared to cells transfected with dominant negative CDK5 (DNCDK5). The PHF-1 epitope remained phosphorylated until 48 hr after thapsigargin treatment in the CDK5/p25-transfected cells. Over the course of apoptosis in this model, phosphorylation of the Tau-1 epitope was unaffected in cells transfected with DNCDK5, vector, or CDK5/p25. In summary, these results demonstrate that CDK5 does not have a significant impact on tau phosphorylation and thapsigargin-induced apoptosis in this neuronal cell model.

    Funded by: NINDS NIH HHS: NS35060

    Journal of neuroscience research 2004;76;1;110-20

  • Cdk5/p35 expression in the mouse ovary.

    Lee KY, Rosales JL, Lee BC, Chung SH, Fukui Y, Lee NS, Lee KY and Jeong YG

    Department of Anatomy, College of Medicine, Konyang University, Nonsan 320-711, Korea.

    Cyclin-dependent kinase 5 (Cdk5) is primarily associated with brain development but it is also implicated in lens and muscle differentiation. We found that Cdk5 is also expressed in mouse ovary, and explored the possibility that it plays a role in that tissue. We show by Western blotting and immunohistochemistry that the known Cdk5 activator, p35, is also present in the mouse ovary. Cdk5 and p35 were detected in oocytes at all stages of the follicle. While Cdk5 was present in the cytoplasm and nucleus of the oocyte, p35 was observed only in the cytoplasm. Both proteins were detected in the cytoplasm of luteinized cells in the corpus luteum. Immunoprecipitation and histone H1 kinase assays revealed that they form an ovarian complex with considerable kinase activity. Phosphorylation assays showed that several ovarian proteins are substrates for Cdk5/p35 in vitro. Together our findings suggest that p35-associated Cdk5 activity plays an important role in the ovary, where it may regulate cell differentiation and apoptosis as it does in the brain.

    Molecules and cells 2004;17;1;17-22

  • Cyclin-dependent kinase 5 regulates dopaminergic and glutamatergic transmission in the striatum.

    Chergui K, Svenningsson P and Greengard P

    Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.

    Dopaminergic and glutamatergic neurotransmissions in the striatum play an essential role in motor- and reward-related behaviors. Dysfunction of these neurotransmitter systems has been found in Parkinson's disease, schizophrenia, and drug addiction. Cyclin-dependent kinase 5 (CDK5) negatively regulates postsynaptic signaling of dopamine in the striatum. This kinase also reduces the behavioral effects of cocaine. Here we demonstrate that, in addition to a postsynaptic role, CDK5 negatively regulates dopamine release in the striatum. Inhibitors of CDK5 increase evoked dopamine release in a way that is additive to that of cocaine. This presynaptic action of CDK5 also regulates glutamatergic transmission. Indeed, inhibition of CDK5 increases the activity and phosphorylation of N-methyl-d-aspartate receptors, and these effects are reduced by a dopamine D1 receptor antagonist. Using mice with a point mutation of the CDK5 site of the postsynaptic protein DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, molecular mass of 32 kDa), in the absence or in the presence of a dopamine D1 receptor antagonist, we provide evidence that CDK5 inhibitors potentiate dopaminergic transmission at both presynaptic and postsynaptic locations. These findings, together with the known ability of CDK5 inhibitors to prevent degeneration of dopaminergic neurons, suggest that this class of compounds could potentially be used as a novel treatment for disorders associated with dopamine deficiency, such as Parkinson's disease.

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;7;2191-6

  • Cdk5 phosphorylation of FAK regulates centrosome-associated miocrotubules and neuronal migration.

    Xie Z and Tsai LH

    Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA. zhigang_xie@hms.harvard.edu

    Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. Unlike other Cdks that promote cell cycle, Cdk5 is activated in postmitotic neurons and critically regulates neuronal migration by phosphorylating its substrates during brain development. Recently, we found that Cdk5 phosphorylates focal adhesion kinase (FAK) at Serine 732 in vitro and is responsible for this phosphorylation in the developing brain. Our experiments using a phospho-specific antibody and an S732-unphosphorylatable mutant FAK suggest that S732 phosphorylation may regulate a centrosome-associated microtubule structure to promote nuclear translocation, a critical step in neuronal migration. S732 phosphorylation does not directly impact on the kinase activity of FAK, but appears to prevent the accumulation of FAK at the centrosome. Our study reveals a similarity between Cdk5 and Cdk1 in the regulation of neuronal migration and cell division, respectively. In addition, our study implicates FAK in a signaling pathway that directly regulates microtubules.

    Cell cycle (Georgetown, Tex.) 2004;3;2;108-10

  • Cyclin-dependent kinase 5 phosphorylates the N-terminal domain of the postsynaptic density protein PSD-95 in neurons.

    Morabito MA, Sheng M and Tsai LH

    Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA. Maria.Morabito@umassmed.edu

    PSD-95 (postsynaptic density 95) is a postsynaptic scaffolding protein that links NMDA receptors to the cytoskeleton and signaling molecules. The N-terminal domain of PSD-95 is involved in the synaptic targeting and clustering of PSD-95 and in the clustering of NMDA receptors at synapses. The N-terminal domain of PSD-95 contains three consensus phosphorylation sites for cyclin-dependent kinase 5 (cdk5), a proline-directed serine-threonine kinase essential for brain development and implicated in synaptic plasticity, dopamine signaling, cocaine addiction, and neurodegenerative disorders. We report that PSD-95 is phosphorylated in the N-terminal domain by cdk5 in vitro and in vivo, and that this phosphorylation is not detectable in brain lysates of cdk5-/- mice. N-terminal phosphorylated PSD-95 is found in PSD fractions together with cdk5 and its activator, p35, suggesting a role for phosphorylated PSD-95 at synapses. In heterologous cells, coexpression of active cdk5 reduces the ability of PSD-95 to multimerize and to cluster neuronal ion channels, two functions attributed to the N-terminal domain of PSD-95. Consistent with these observations, the lack of cdk5 activity in cultured neurons results in larger clusters of PSD-95. In cdk5-/- cortical neurons, more prominent PSD-95 immunostained clusters are observed than in wild-type neurons. In hippocampal neurons, the expression of DNcdk5 (inactive form of cdk5) or of the triple alanine mutant (T19A, S25A, S35A) full-length PSD-95 results in increased PSD-95 cluster size. These results identify cdk5-dependent phosphorylation of the N-terminal domain of PSD-95 as a novel mechanism for regulating the clustering of PSD-95. Moreover, these observations support the possibility that cdk5-dependent phosphorylation of PSD-95 dynamically regulates the clustering of PSD-95/NMDA receptors at synapses, thus providing a possible mechanism for rapid changes in density and/or number of receptor at synapses.

    Funded by: NIMH NIH HHS: MH62161; NINDS NIH HHS: NS37007

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;4;865-76

  • Cdk5 phosphorylation of doublecortin ser297 regulates its effect on neuronal migration.

    Tanaka T, Serneo FF, Tseng HC, Kulkarni AB, Tsai LH and Gleeson JG

    Department of Neurosciences, University of California, San Diego, La Jolla, 92093, USA.

    Mutations in the doublecortin (DCX) gene in human or targeted disruption of the cdk5 gene in mouse lead to similar cortical lamination defects in the developing brain. Here we show that Dcx is phosphorylated by Cdk5. Dcx phosphorylation is developmentally regulated and corresponds to the timing of expression of p35, the major activating subunit for Cdk5. Mass spectrometry and Western blot analysis indicate phosphorylation at Dcx residue Ser297. Phosphorylation of Dcx lowers its affinity to microtubules in vitro, reduces its effect on polymerization, and displaces it from microtubules in cultured neurons. Mutation of Ser297 blocks the effect of Dcx on migration in a fashion similar to pharmacological inhibition of Cdk5 activity. These results suggest that Dcx phosphorylation by Cdk5 regulates its actions on migration through an effect on microtubules.

    Funded by: NINDS NIH HHS: R01 NS041537; PHS HHS: N5047101

    Neuron 2004;41;2;215-27

  • Divergent roles of GSK3 and CDK5 in APP processing.

    Ryder J, Su Y, Liu F, Li B, Zhou Y and Ni B

    Neuroscience Discovery Research, Lilly Research Laboratories, Indianapolis, IN 46285, USA. ryder_john_w@lilly.com

    Glycogen synthase kinase-3 (GSK3) and cyclin-dependent kinase 5 (CDK5) are related serine/threonine kinases that have been well studied for their role in tau hyperphosphorylation, however, little is known about their significance in APP processing. Here we report that GSK3 and CDK5 are involved in APP processing in a divergent manner. Specific inhibition of cellular GSK3 by lithium or GSK3beta antisense elicits a reduction in Abeta. Conversely, negative modulation of cellular CDK5 activity by CDK5 inhibitor, roscovitine, or CDK5 antisense stimulates Abeta production. Neither GSK3 nor CDK5 inhibition by these means significantly affected cellular APP levels or APP maturation. Moreover, oral administration of lithium significantly reduces Abeta production whereas direct ICV administration of roscovitine augmented Abeta production in the brains of PDAPP (APP(V717F)) mice. Our data support a function for both GSK3 and CDK5 in APP processing, further implicating these two kinases in the pathogenesis of Alzheimer's disease.

    Biochemical and biophysical research communications 2003;312;4;922-9

  • Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles.

    Tomizawa K, Sunada S, Lu YF, Oda Y, Kinuta M, Ohshima T, Saito T, Wei FY, Matsushita M, Li ST, Tsutsui K, Hisanaga S, Mikoshiba K, Takei K and Matsui H

    Department of Physiology, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan. tomikt@md.okayama-u.ac.jp

    It has been thought that clathrin-mediated endocytosis is regulated by phosphorylation and dephosphorylation of many endocytic proteins, including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependent cophosphorylation of amphiphysin I and dynamin I plays a critical role in such processes. Cdk5 inhibitors enhanced the electric stimulation-induced endocytosis in hippocampal neurons, and the endocytosis was also enhanced in the neurons of p35-deficient mice. Cdk5 phosphorylated the proline-rich domain of both amphiphysin I and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylation of amphiphysin I inhibited the association with beta-adaptin. Furthermore, the phosphorylation of dynamin I blocked its binding to amphiphysin I. The phosphorylation of each protein reduced the copolymerization into a ring formation in a cell-free system. Moreover, the phosphorylation of both proteins completely disrupted the copolymerization into a ring formation. Finally, phosphorylation of both proteins was undetectable in p35-deficient mice.

    The Journal of cell biology 2003;163;4;813-24

  • Decreased cyclin-dependent kinase 5 (cdk5) activity is accompanied by redistribution of cdk5 and cytoskeletal proteins and increased cytoskeletal protein phosphorylation in p35 null mice.

    Hallows JL, Chen K, DePinho RA and Vincent I

    The Nathan Shock Center of Excellence in the Basic Biology of Aging and Department of Pathology, University of Washington, Seattle, Washington 98195, USA.

    Cdk5/p35 has been implicated in cytoskeletal protein phosphorylation in normal brain and in many human neurodegenerative disorders. Yet, mouse models of cdk5/p35 hyperactivity have not yielded corresponding changes in cytoskeletal protein phosphorylation. To elucidate the relationship between p35, cdk5, and the neuronal cytoskeleton, we deleted the p35 gene in mice having a pure C57BL/6 background. We found that p35 deficiency leads to a 38% reduction of cdk5 activity in adult brain. In addition, loss of p35 causes an anterograde redistribution of cdk5 toward peripheral neuronal processes. The unusual presence of nonphosphorylated neurofilament (NF) in aberrant axon fascicles and the relocation of tau and MAP2B from cell bodies and proximal neuronal processes to more distal sites of the neuropil in p35-/- mouse brain implicate p35 in neuronal trafficking, particularly in dynein-driven retrograde transport. In many axons of normal brain, cdk5 fails to colocalize with phosphorylated cytoskeletal protein epitopes. This observation, together with an unexpected increase of NF, tau, and MAP2B phosphoepitopes accompanying the decreased cdk5 activity in p35-/- mice, supports the idea that cdk5 does not phosphorylate cytoskeletal proteins directly. Rather, in structures where cdk5 does colocalize with phosphorylated cytoskeletal protein epitopes, it may function as a negative regulator of other proline-directed kinases that directly phosphorylate the proteins. Evidence for increased glycogen synthase kinase 3beta (GSK3beta) activity in p35-/- mice suggests that GSK3beta may be one such kinase regulated by cdk5. Our studies illustrate that p35 regulates the subcellular distribution of cdk5 and cytoskeletal proteins in neurons and that cdk5 has a hierarchical role in regulating the phosphorylation and function of cytoskeletal proteins.

    Funded by: NIA NIH HHS: AG00057, AG12721

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2003;23;33;10633-44

  • Cyclin-dependent kinase 5 is a mediator of dopaminergic neuron loss in a mouse model of Parkinson's disease.

    Smith PD, Crocker SJ, Jackson-Lewis V, Jordan-Sciutto KL, Hayley S, Mount MP, O'Hare MJ, Callaghan S, Slack RS, Przedborski S, Anisman H and Park DS

    Neuroscience Group, Ottawa Health Research Institute, Ottawa, ON, Canada K1H 8M5.

    Recent evidence indicates that cyclin-dependent kinases (CDKs, cdks) may be inappropriately activated in several neurodegenerative conditions. Here, we report that cdk5 expression and activity are elevated after administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin that damages the nigrostriatal dopaminergic pathway. Supporting the pathogenic significance of the cdk5 alterations are the findings that the general cdk inhibitor, flavopiridol, or expression of dominant-negative cdk5, and to a lesser extent dominant-negative cdk2, attenuates the loss of dopaminergic neurons caused by MPTP. In addition, CDK inhibition strategies attenuate MPTP-induced hypolocomotion and markers of striatal function independent of striatal dopamine. We propose that cdk5 is a key regulator in the degeneration of dopaminergic neurons in Parkinson's disease.

    Funded by: NINDS NIH HHS: R01 NS041202, R01 NS041202-02

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;23;13650-5

  • Aberrant Cdk5 activation by p25 triggers pathological events leading to neurodegeneration and neurofibrillary tangles.

    Cruz JC, Tseng HC, Goldman JA, Shih H and Tsai LH

    Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. jonathan_cruz@hms.harvard.edu

    Cyclin-dependent kinase 5 (Cdk5) and its regulatory subunit p35 are integral players in the proper development of the mammalian central nervous system. Proteolytic cleavage of p35 generates p25, leading to aberrant Cdk5 activation. The accumulation of p25 is implicated in several neurodegenerative diseases. In primary neurons, p25 causes apoptosis and tau hyperphosphorylation. Current mouse models expressing p25, however, fail to rigorously recapitulate these phenotypes in vivo. Here, we generated inducible transgenic mouse lines overexpressing p25 in the postnatal forebrain. Induction of p25 preferentially directed Cdk5 to pathological substrates. These animals exhibited neuronal loss in the cortex and hippocampus, accompanied by forebrain atrophy, astrogliosis, and caspase-3 activation. Endogenous tau was hyperphosphorylated at many epitopes, aggregated tau accumulated, and neurofibrillary pathology developed progressively in these animals. Our cumulative findings provide compelling evidence that in vivo deregulation of Cdk5 by p25 plays a causative role in neurodegeneration and the development of neurofibrillary pathology.

    Neuron 2003;40;3;471-83

  • Puralpha is essential for postnatal brain development and developmentally coupled cellular proliferation as revealed by genetic inactivation in the mouse.

    Khalili K, Del Valle L, Muralidharan V, Gault WJ, Darbinian N, Otte J, Meier E, Johnson EM, Daniel DC, Kinoshita Y, Amini S and Gordon J

    Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania 19122, USA. kamel.khalili@temple.edu

    The single-stranded DNA- and RNA-binding protein, Puralpha, has been implicated in many biological processes, including control of transcription of multiple genes, initiation of DNA replication, and RNA transport and translation. Deletions of the PURA gene are frequent in acute myeloid leukemia. Mice with targeted disruption of the PURA gene in both alleles appear normal at birth, but at 2 weeks of age, they develop neurological problems manifest by severe tremor and spontaneous seizures and they die by 4 weeks. There are severely lower numbers of neurons in regions of the hippocampus and cerebellum of PURA(-/-) mice versus those of age-matched +/+ littermates, and lamination of these regions is aberrant at time of death. Immunohistochemical analysis of MCM7, a protein marker for DNA replication, reveals a lack of proliferation of precursor cells in these regions in the PURA(-/-) mice. Levels of proliferation were also absent or low in several other tissues of the PURA(-/-) mice, including those of myeloid lineage, whereas those of PURA(+/-) mice were intermediate. Evaluation of brain sections indicates a reduction in myelin and glial fibrillary acidic protein labeling in oligodendrocytes and astrocytes, respectively, indicating pathological development of these cells. At postnatal day 5, a critical time for cerebellar development, Puralpha and Cdk5 were both at peak levels in bodies and dendrites of Purkinje cells of PURA(+/+) mice, but both were absent in dendrites of PURA(-/-) mice. Puralpha and Cdk5 can be coimmunoprecipitated from brain lysates of PURA(+/+) mice. Immunohistochemical studies reveal a dramatic reduction in the level of both phosphorylated and nonphosphorylated neurofilaments in dendrites of the Purkinje cell layer and of synapse formation in the hippocampus. Overall results are consistent with a role for Puralpha in developmentally timed DNA replication in specific cell types and also point to a newly emerging role in compartmentalized RNA transport and translation in neuronal dendrites.

    Molecular and cellular biology 2003;23;19;6857-75

  • Cyclin-dependent kinase-5 is involved in neuregulin-dependent activation of phosphatidylinositol 3-kinase and Akt activity mediating neuronal survival.

    Li BS, Ma W, Jaffe H, Zheng Y, Takahashi S, Zhang L, Kulkarni AB and Pant HC

    Laboratory of Neurochemistry, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20892-4130, USA.

    The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays an important role in mediating survival signals in wide variety of neurons and cells. Recent studies show that Akt also regulates metabolic pathways to regulate cell survival. In this study, we reported that cyclin-dependent kinase-5 (Cdk5) regulates Akt activity and cell survival through the neuregulin-mediated PI 3-kinase signaling pathway. We found that brain extracts of Cdk5-/-mice display a lower PI 3-kinase activity and phosphorylation of Akt compared with that in wild type mice. Moreover, we demonstrated that Cdk5 phosphorylated Ser-1176 in the neuregulin receptor ErbB2 and phosphorylated Thr-871 and Ser-1120 in the ErbB3 receptor. We identified the Ser-1120 sequence RSRSPR in ErbB3 as a novel phosphorylation consensus sequence of Cdk5. Finally, we found that Cdk5 activity is involved in neuregulin-induced Akt activity and neuregulin-mediated neuronal survival. These findings suggest that Cdk5 may exert a key role in promoting neuronal survival by regulating Akt activity through the neuregulin/PI 3-kinase signaling pathway.

    The Journal of biological chemistry 2003;278;37;35702-9

  • Pak1 and its T212 phosphorylated form accumulate in neurones and epithelial cells of the developing rodent.

    Zhong JL, Banerjee MD and Nikolic M

    Developmental Neurobiology MRC Centre, King's College London, London, United Kingdom.

    The serine/threonine kinase Pak1 is a target of the RhoGTPases Rac and Cdc42 and an important regulator of cell morphology and migration. Recent work from several laboratories has indicated that Pak1 controls microtubule dynamics as well as the organisation of F-actin microfilaments. Pak1 is phosphorylated on T212 by the p35/Cdk5 or cyclin B1/Cdc2 kinase in postmitotic neurones and mitotic cells, respectively. To understand its function during development, we have carried out a detailed temporal and spatial analysis of Pak1 expression and phosphorylation on T212. In the embryonic forebrain, Pak1 and Pak1T212(PO4) were seen to accumulate in the corpus callosum, intermediate zone, lateral olfactory tracts, and anterior commissures. Epithelial cells of the mouse embryo lung, kidney, intestine, and skin also exhibited high levels of Pak1 and Pak1T212(PO4), suggesting a previously unsuspected role in epithelial differentiation. Pak1T212(PO4) was undetectable in all adult tissues. Together, these data indicate a specific, developmentally regulated role of the Pak1 kinase.

    Developmental dynamics : an official publication of the American Association of Anatomists 2003;228;1;121-7

  • Serine 732 phosphorylation of FAK by Cdk5 is important for microtubule organization, nuclear movement, and neuronal migration.

    Xie Z, Sanada K, Samuels BA, Shih H and Tsai LH

    Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.

    The serine/threonine kinase Cdk5 plays an essential role in neuronal positioning during corticogenesis, but the underlying mechanisms are unknown. In nonneuronal cells, the tyrosine kinase FAK is a major regulator of cell motility through focal adhesions. It is unclear whether FAK plays a role in brain development. Here, we show that FAK phosphorylation by Cdk5 at S732 is important for microtubule organization, nuclear movement, and neuronal migration. In cultured neurons, S732-phosphorylated FAK is enriched along a centrosome-associated microtubule fork that abuts the nucleus. Overexpression of the nonphosphorylatable mutant FAK S732A results in disorganization of the microtubule fork and impairment of nuclear movement in vitro, and neuronal positioning defects in vivo. These observations are reminiscent of what is seen in the Cdk5-deficient mice. Taken together, these results suggest that Cdk5 phosphorylation of FAK is critical for neuronal migration through regulation of a microtubule fork important for nuclear translocation.

    Funded by: NINDS NIH HHS: NS37007

    Cell 2003;114;4;469-82

  • Mutations in dynein link motor neuron degeneration to defects in retrograde transport.

    Hafezparast M, Klocke R, Ruhrberg C, Marquardt A, Ahmad-Annuar A, Bowen S, Lalli G, Witherden AS, Hummerich H, Nicholson S, Morgan PJ, Oozageer R, Priestley JV, Averill S, King VR, Ball S, Peters J, Toda T, Yamamoto A, Hiraoka Y, Augustin M, Korthaus D, Wattler S, Wabnitz P, Dickneite C, Lampel S, Boehme F, Peraus G, Popp A, Rudelius M, Schlegel J, Fuchs H, Hrabe de Angelis M, Schiavo G, Shima DT, Russ AP, Stumm G, Martin JE and Fisher EM

    Department of Neurodegenerative Disease, Institute of Neurology, National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, UK.

    Degenerative disorders of motor neurons include a range of progressive fatal diseases such as amyotrophic lateral sclerosis (ALS), spinal-bulbar muscular atrophy (SBMA), and spinal muscular atrophy (SMA). Although the causative genetic alterations are known for some cases, the molecular basis of many SMA and SBMA-like syndromes and most ALS cases is unknown. Here we show that missense point mutations in the cytoplasmic dynein heavy chain result in progressive motor neuron degeneration in heterozygous mice, and in homozygotes this is accompanied by the formation of Lewy-like inclusion bodies, thus resembling key features of human pathology. These mutations exclusively perturb neuron-specific functions of dynein.

    Science (New York, N.Y.) 2003;300;5620;808-12

  • Tau phosphorylation by cyclin-dependent kinase 5/p39 during brain development reduces its affinity for microtubules.

    Takahashi S, Saito T, Hisanaga S, Pant HC and Kulkarni AB

    Functional Genomics Unit, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA.

    The microtubule-associated protein tau is a developmentally regulated neuronal phosphoprotein. The phosphorylation of tau reduces its ability to bind and stabilize axonal microtubules during axonal growth. Although tau is phosphorylated by cyclin-dependent kinase 5 (Cdk5) in vitro, its in vivo roles remain unclear. Here, we show that tau is phosphorylated by Cdk5/p39 during brain development, resulting in a reduction of its affinity for microtubules. The activity of Cdk5 is tightly regulated by association with its neuronal activators, p35 or p39. The p35 and p39 expression levels were investigated in the developing mouse brain; the p39 expression level was higher in embryonic hind brain and spinal cord and in postnatal cerebral cortex, whereas that of p35 was most prominent in cerebral cortex at earlier stages of development. The ability of Cdk5 to phosphorylate tau was higher when in association with p39 than in association with p35. Tau phosphorylation at Ser-202 and Thr-205 was decreased in Cdk5-/- mouse brain but not in p35-/- mouse brain, suggesting that Cdk5/p39 is responsible for the in vivo phosphorylation of tau at these sites. Our data suggest that tau phosphorylation by Cdk5 may provide the neuronal microtubules with dynamic properties in a region-specific and developmentally regulated manner.

    The Journal of biological chemistry 2003;278;12;10506-15

  • Reelin activates SRC family tyrosine kinases in neurons.

    Bock HH and Herz J

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, 75390, Dallas, TX, USA. hans.bock@utsouthwestern.edu

    Background: Reelin is a large signaling molecule that regulates the positioning of neurons in the mammalian brain. Transmission of the Reelin signal to migrating embryonic neurons requires binding to the very-low-density lipoprotein receptor (VLDLR) and the apolipoprotein E receptor-2 (apoER2). This induces tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1), which interacts with a shared sequence motif in the cytoplasmic tails of both receptors. However, the kinases that mediate Dab1 tyrosine phosphorylation and the intracellular pathways that are triggered by this event remain unknown.

    Results: We show that Reelin activates members of the Src family of non-receptor tyrosine kinases (SFKs) and that this activation is dependent on the Reelin receptors apoER2 and VLDLR and the adaptor protein Dab1. Dab1 is tyrosine phosphorylated by SFKs, and the kinases themselves can be further activated by phosphorylated Dab1. Increased Dab1 protein expression in fyn-deficient mice implies a response to impaired Reelin signaling that is also observed in mice lacking Reelin or its receptors. However, fyn deficiency alone does not compound the neuronal positioning defect of vldlr- or apoer2-deficient mice, and this finding suggests functional compensation by other SFKs.

    Conclusions: Our results show that Dab1 is a physiological substrate as well as an activator of SFKs in neurons. Based on genetic evidence gained from multiple strains of mutant mice with defects in Reelin signaling, we conclude that activation of SFKs is a normal part of the cellular Reelin response.

    Funded by: NHLBI NIH HHS: HL20948, HL63762, R37 HL063762; NINDS NIH HHS: NS43408

    Current biology : CB 2003;13;1;18-26

  • CDK5 regulates cell adhesion and migration in corneal epithelial cells.

    Gao C, Negash S, Guo HT, Ledee D, Wang HS and Zelenka P

    National Eye Institute, NIH, Bethesda, MD. Yang Ming University, Taipei, Taiwan.

    CDK5 and its activator, p35, are expressed in mouse corneal epithelium and can be coimmunoprecipited from corneal epithelial cell lysates. Immunostaining shows CDK5 and p35 in all layers of the corneal epithelium, especially along the basal side of the basal cells. Stable transfection of corneal epithelial cells with CDK5, which increases CDK5 kinase activity by approximately 33%, also increases the number of cells adhering to fibronectin and the strength of adhesion. CDK5 kinase activity seems to be required for this effect, because the kinase inactive mutation, CDK5-T33, either reduces adhesion or has no significant effect, depending on the level of expression. Using an in vitro scrape wound in confluent cultures of stably transfected cells to examine the effect of CDK5 on cell migration, we show that reoccupation of the wound area is significantly decreased by CDK5 and increased by CDK5-T33. These findings indicate that CDK5 may be an important regulator of adhesion and migration of corneal epithelial cells.

    Molecular cancer research : MCR 2002;1;1;12-24

  • Identification of candidate lung cancer susceptibility genes in mouse using oligonucleotide arrays.

    Lemon WJ, Bernert H, Sun H, Wang Y and You M

    Division of Human Cancer Genetics, The Ohio State University Comprehensive Cancer Center, 420 West 12th Avenue, Columbus, Ohio 43210, USA.

    We applied microarray gene expression profiling to lungs from mouse strains having variable susceptibility to lung tumour development as a means to identify, within known quantitative trait loci (QTLs), candidate genes responsible for susceptibility or resistance to lung cancer. At least eight chromosomal regions of mice have been mapped and verified to be linked with lung tumour susceptibility or resistance. In this study, high density oligonucleotide arrays were used to measure the relative expression levels of >36 000 genes and ESTs in lung tissues of A/J, BALB/cJ, SM/J, C3H/HeJ, and C57BL/6J mice. A number of differentially expressed genes were found in each of the lung cancer susceptibility QTLs. Bioinformatic analysis of the differentially expressed genes located within QTLs produced 28 susceptibility candidates and 22 resistance candidates. These candidates may be extremely helpful in the ultimate identification of the precise genes responsible for lung tumour susceptibility or resistance in mice and, through follow up, humans. Complete data sets are available at http://thinker.med.ohio-state.edu.

    Funded by: NCI NIH HHS: P30CA16058, R01CA58554, R01CA78797

    Journal of medical genetics 2002;39;9;644-55

  • Fyn and Cdk5 mediate semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex.

    Sasaki Y, Cheng C, Uchida Y, Nakajima O, Ohshima T, Yagi T, Taniguchi M, Nakayama T, Kishida R, Kudo Y, Ohno S, Nakamura F and Goshima Y

    Department of Molecular Pharmacology and Neurobiology, Yokohama City University School of Medicine, Yokohama, Japan. yukio.sasaki@pharmac.med.yokohama-cu.ac.jp

    Semaphorin-3A (Sema3A), a member of class 3 semaphorins, regulates axon and dendrite guidance in the nervous system. How Sema3A and its receptors plexin-As and neuropilins regulate neuronal guidance is unknown. We observed that in fyn- and cdk5-deficient mice, Sema3A-induced growth cone collapse responses were attenuated compared to their heterologous controls. Cdk5 is associated with plexin-A2 through the active state of Fyn. Sema3A promotes Cdk5 activity through phosphorylation of Tyr15, a phosphorylation site with Fyn. A Cdk5 mutant (Tyr15 to Ala) shows a dominant-negative effect on the Sema3A-induced collapse response. The sema3A gene shows strong interaction with fyn for apical dendrite guidance in the cerebral cortex. We propose a signal transduction pathway in which Fyn and Cdk5 mediate neuronal guidance regulated by Sema3A.

    Neuron 2002;35;5;907-20

  • Brn-1 and Brn-2 share crucial roles in the production and positioning of mouse neocortical neurons.

    Sugitani Y, Nakai S, Minowa O, Nishi M, Jishage K, Kawano H, Mori K, Ogawa M and Noda T

    Department of Cell Biology, JFCR-Cancer Institute, Tokyo 170-8455, Japan.

    Formation of highly organized neocortical structure depends on the production and correct placement of the appropriate number and types of neurons. POU homeodomain proteins Brn-1 and Brn-2 are coexpressed in the developing neocortex, both in the late precursor cells and in the migrating neurons. Here we show that double disruption of both Brn-1 and Brn-2 genes in mice leads to abnormal formation of the neocortex with dramatically reduced production of layer IV-II neurons and defective migration of neurons unable to express mDab1. These data indicate that Brn-1 and Brn-2 share roles in the production and positioning of neocortical neuron development.

    Genes & development 2002;16;14;1760-5

  • Cyclin-dependent kinase 5 phosphorylates disabled 1 independently of Reelin signaling.

    Keshvara L, Magdaleno S, Benhayon D and Curran T

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

    Two major signaling pathways that control neuronal positioning during brain development have been uncovered as a result of genetic and biochemical studies on neurological mouse mutants. Mice deficient in Reelin, Disabled 1 (Dab1), or both the very low-density lipoprotein receptor (VLDLR) and the apolipoprotein E receptor 2 (ApoER2) exhibit identical neuroanatomic defects in laminar structures throughout the brain. These proteins function as components of the Reelin signaling pathway. Reelin is a secreted glycoprotein that binds to VLDLR and ApoER2, inducing tyrosine phosphorylation of Dab1, an intracellular adapter protein. Neuronal migration is also regulated by cyclin-dependent kinase 5 (Cdk5) and its activating subunits p35 and p39. Mice deficient in Cdk5, p35, or both p35 and p39 exhibit lamination defects that are similar but not identical to those observed in mice with a defect in the Reelin signaling pathway. Cdk5 phosphorylates proteins that maintain cytoskeletal structures and promote cell motility. To explore the possibility that Cdk5 influences the Reelin pathway, we sought to determine whether Dab1 is a substrate for Cdk5. Here we show that Cdk5 phosphorylates Dab1 on serine 491 in vitro and in vivo, independently of Reelin signaling. We also show that ectopic neurons in Cdk5-deficient mice exhibit reduced levels of Reelin signaling during later stages of cortical development, although Cdk5 is not required for Reelin-induced tyrosine phosphorylation of Dab1. Although the functional significance of Dab1 serine phosphorylation is unclear, our results suggest that there is biochemical cross-talk between two signaling pathways that control cell positioning.

    Funded by: NCI NIH HHS: P30 CA21765; NINDS NIH HHS: R01-NS36558

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;12;4869-77

  • Cdk5 regulates cell-matrix and cell-cell adhesion in lens epithelial cells.

    Negash S, Wang HS, Gao C, Ledee D and Zelenka P

    National Eye Institute, NIH, Bethesda, MD 20892, USA.

    Cdk5 is a member of the cyclin-dependent kinase family, which is expressed predominantly in terminally differentiated neurons. Lower levels of Cdk5 are also found in a wide variety of cell types, including the lens. Although Cdk5 has been shown to play an important role in neuronal migration and neurite outgrowth, its function in non-neuronal cells is not known. Therefore, this study was undertaken to explore the role of Cdk5 in the lens. Results showed that, within the adult mouse lens, Cdk5 was localized to the cytoplasm, especially along the lateral membranes of differentiating primary fiber cells, which suggests a role in cell-cell adhesion. Staining at the tips of elongating fiber cells was also particularly strong, suggesting a role in cell-matrix adhesion. To examine the possible role of Cdk5 in lens epithelial cell adhesion, we stably transfected N/N1003A rabbit lens epithelial cells with cDNAs for Cdk5 or a dominant-negative mutation, Cdk5-T33. Attachment to a fibronectin matrix, as measured with substrate-coated cell adhesion strips, was increased by Cdk5 overexpression, while an equivalent overexpression of Cdk5-T33 had no effect. Cdk5 also increased the rate of cell attachment and spreading as measured by electric cell-substrate impedance sensing (ECIS). In addition, Cdk5 overexpression decreased cell-cell adhesion as measured by a cell aggregation assay. These findings suggest that Cdk5 plays a role in regulating both cell-matrix and cell-cell interactions in the lens.

    Journal of cell science 2002;115;Pt 10;2109-17

  • Cyclin-dependent kinase 5/p35 contributes synergistically with Reelin/Dab1 to the positioning of facial branchiomotor and inferior olive neurons in the developing mouse hindbrain.

    Ohshima T, Ogawa M, Takeuchi K, Takahashi S, Kulkarni AB and Mikoshiba K

    Laboratory for Developmental Neurobiology, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan. ohshima@brain.riken.go.jp

    Cyclin-dependent kinase 5 (Cdk5)/p35 is a serine/threonine kinase, and its activity is detected primarily in postmitotic neurons. Mice lacking Cdk5/p35 display migration defects of the cortical neurons in the cerebrum and cerebellum. In this study, we demonstrate that although most brainstem nuclei are found in their proper positions, the motor nucleus of the facial nerve is ectopically located and neurons of the inferior olive fail to position correctly, resulting in the lack of their characteristic structures in the hindbrain of Cdk5-/- mice. Despite the defective migration of these neurons, axonal exits of the facial nerve from brainstem and projections of the inferior cerebellar axons appear unchanged in Cdk5-/- mice. Defective neuronal migration in Cdk5-/- hindbrain was rescued by the neuron-specific expression of Cdk5 transgene. Because developmental defects of these structures have been reported in reeler and Dab1 mutant mice, we analyzed the double-null mutants of p35 and Dab1 and found more extensive ectopia of VII motor nuclei in these mice. These results indicate that Cdk5/p35 and Reelin signaling regulates the selective mode of neuronal migration in the developing mouse hindbrain.

    Funded by: NIDCR NIH HHS: Z01DE00694-010DIR

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;10;4036-44

  • Axonopathy, tau abnormalities, and dyskinesia, but no neurofibrillary tangles in p25-transgenic mice.

    Bian F, Nath R, Sobocinski G, Booher RN, Lipinski WJ, Callahan MJ, Pack A, Wang KK and Walker LC

    CNS Pharmacology, Pfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, USA. feng.bian@pfizer.com

    Neurofibrillary tangles, one of the pathologic hallmarks of Alzheimer's disease (AD), are composed of abnormally polymerized tau protein. The hyperphosphorylation of tau alters its normal cellular function and is thought to promote the formation of neurofibrillary tangles. Growing evidence suggests that cyclin-dependent kinase 5 (cdk5) plays a role in tau phosphorylation, but the function of the enzyme in tangle formation remains uncertain. In AD, cdk5 is constitutively activated by p25, a highly stable, 25kD protein thought to be increased in the AD brain. To test the hypothesis that p25/cdk5 interactions promote neurofibrillary pathology, we created transgenic mouse lines that overexpress the human p25 protein specifically in neurons. Mice with high transgenic p25 expression have augmented cdk5 activity and develop severe hindlimb semiparalysis and mild forelimb dyskinesia beginning at approximately 3 months of age. Immunohistochemical and ultrastructural analyses showed widespread axonal degeneration with focal accumulation of tau in various regions of the brain and, to a lesser extent, the spinal cord. However, there was no evidence of neurofibrillary tangles in neuronal somata or axons, nor were paired helical filaments evident ultrastructurally. These studies confirm that p25 overexpression can lead to tau abnormalities and axonal degeneration in vivo but do not support the hypothesis that p25-related induction of cdk5 is a primary event in the genesis of neurofibrillary tangles.

    The Journal of comparative neurology 2002;446;3;257-66

  • Cyclin-dependent kinase 5 is required for associative learning.

    Fischer A, Sananbenesi F, Schrick C, Spiess J and Radulovic J

    Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, 37075 Goettingen, Germany. fischer@mail.em.mpg.de

    Transient stressful experiences may persistently facilitate associative and nonassociative learning, possibly through alterations of gene expression. Here we identify, by subtractive hybridization, differential expression of the Cdk5 gene in response to stress. The Cdk5 protein is selectively induced in the fibers of septohippocampal cholinergic neurons but not in other regions of prominent Cdk5 production. This upregulation is accompanied by increased Cdk5 kinase activity, which is blocked completely by the Cdk5 inhibitor butyrolactone I. Microinjection of butyrolactone I into the lateral septum and hippocampus prevents the acquisition of conditioned context-dependent fear as well as its stress-induced facilitation. By demonstrating that a transient increase of Cdk5 activity within the septohippocampal system is required for associative learning, an important novel role of Cdk5 has been identified.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;9;3700-7

  • Cyclin dependent kinase 5 and its interacting proteins in cell death induced in vivo by cyclophosphamide in developing mouse embryos.

    Zhu Y, Lin L, Kim S, Quaglino D, Lockshin RA and Zakeri Z

    Department of Biology, Queens College and Graduate Center of City University of New York, Flushing, New York, NY 11367, USA.

    Activation or inactivation of members of the cyclin-dependent kinase family is important during cell cycle progression. However, Cdk5, a member of this family that was originally identified because of its high structural homology to Cdc2, is activated during cell differentiation and cell death but not during cell cycle progression. We previously demonstrated a correlation between the up-regulation of Cdk5 protein and kinase activity and cell death during development and pathogenesis. We report here that cyclophosphamide (CP) induces massive apoptotic cell death in mouse embryos and that Cdk5 is expressed in apoptotic cells displaying fragmented DNA. During CP-induced cell death, Cdk5 protein expression is substantially increased as detected by immunohistochemistry but not by Western blot, while its mRNA level remains the same as control, and its kinase activity is markedly elevated. The up-regulation of Cdk5 during CP-induced cell death is not due to de novo protein synthesis. We also examined p35, a regulatory protein of Cdk5 in neuronal differentiation. Using a yeast two-hybrid system, we isolated p35, a neuronal differentiation specific protein, as a protein that interacts with Cdk5 in CP-treated embryos. p35 mRNA level does not change, but the protein expression of p25, a truncated form of p35, is elevated during cell death in vivo, as established here, as well as during cell death in vitro. Our results suggest a role for Cdk5 and its regulatory proteins during CP induced cell death. These results further support the view that Cdk5 and its regulation may be key players in the execution of cell death regardless of how the cell dies, whether through biological mechanisms, disease states such as Alzheimer's disease, or induction by CP.

    Cell death and differentiation 2002;9;4;421-30

  • ik3-2, a relative to ik3-1/cables, is associated with cdk3, cdk5, and c-abl.

    Sato H, Nishimoto I and Matsuoka M

    Department of Pharmacology, Keio University School of Medicine, Tokyo, Japan.

    A cDNA coding for ik3-2 (designated as ik3-2 from an interactor-2 with cdk3) was cloned by cross-hybridization with ik3-1 and RT-PCR. Analysis of amino acid sequence indicated that ik3-2 has the C-terminal cyclin-box-like region highly homologous to that of ik3-1 (identity in amino acids: 78%). On the other hand, the remainder of ik3-2 gene is not so similar to that of ik3-1. There are several regions other than the C-terminal cyclin-box-like region that are conserved between ik3-1 and ik3-2. In vivo binding assay indicated that like ik3-1, ik3-2 binds to cdk3, cdk5, and c-abl, although ik3-2 binds to cdk3 weakly as compared with ik3-1. The C-terminal cyclin-box-like region of ik3-2 (123 amino acids) is able to be associated with cdk5. Accordingly, ik3-2 is very similar to ik3-1 concerning its molecular interaction with other molecules, suggesting that ik3-2 function in the same biological field as ik3-1. Northern blot analysis indicated that ik3-2 is expressed ubiquitously all over tissues.

    Biochimica et biophysica acta 2002;1574;2;157-63

  • Cyclin-dependent kinase 5 prevents neuronal apoptosis by negative regulation of c-Jun N-terminal kinase 3.

    Li BS, Zhang L, Takahashi S, Ma W, Jaffe H, Kulkarni AB and Pant HC

    Laboratory of Neurochemistry, NINDS, NIH, Bethesda, MD 20892-4130, USA.

    Cyclin-dependent kinase 5 (cdk5) is a serine/threonine kinase activated by associating with its neuron-specific activators p35 and p39. Analysis of cdk5(-/-) and p35(-/-) mice has demonstrated that both cdk5 and p35 are essential for neuronal migration, axon pathfinding and the laminar configuration of the cerebral cortex, suggesting that the cdk5-p35 complex may play a role in neuron survival. However, the targets of cdk5 that regulate neuron survival are unknown. Here, we show that cdk5 directly phosphorylates c-Jun N-terminal kinase 3 (JNK3) on Thr131 and inhibits its kinase activity, leading to reduced c-Jun phosphorylation. Expression of cdk5 and p35 in HEK293T cells inhibits c-Jun phosphorylation induced by UV irradiation. These effects can be restored by expression of a catalytically inactive mutant form of cdk5. Moreover, cdk5-deficient cultured cortical neurons exhibit increased sensitivity to apoptotic stimuli, as well as elevated JNK3 activity and c-Jun phosphorylation. Taken together, these findings show that cdk5 may exert its role as a key element by negatively regulating the c-Jun N-terminal kinase/stress-activated protein kinase signaling pathway during neuronal apoptosis.

    The EMBO journal 2002;21;3;324-33

  • Phosphorylation of Pak1 by the p35/Cdk5 kinase affects neuronal morphology.

    Rashid T, Banerjee M and Nikolic M

    Molecular and Developmental Neurobiology Medical Research Council Centre, New Hunt's House, King's College London, London, SE1 1UL, United Kingdom.

    The small GTPase Rac and its effectors, the Pak1 and p35/Cdk5 kinases, have been assigned important roles in regulating cytoskeletal dynamics in neurons. Our previous work revealed that the neuronal p35/Cdk5 kinase associates with Pak1 in a RacGTP-dependent manner, causing hyperphosphorylation and down-regulation of Pak1 kinase activity. We have now demonstrated direct phosphorylation of Pak1 on threonine 212 by the p35/Cdk5 kinase. In neuronal growth cones, Pak1 phosphorylated on Thr-212 localized to actin and tubulin-rich areas, suggesting a role in regulating growth cone dynamics. The expression of a non-phosphorylatable Pak1 mutant (Pak1A212) induced dramatic neurite disorganization. We also observed a strong association between p35/Cdk5 and the Pak1 C-terminal kinase domain. Overall, our data show that in neurons, membrane-associated, active Pak1 is regulated by the p35/Cdk5 kinase both by association and phosphorylation, which is essential for the proper regulation of the cytoskeleton during neurite outgrowth and remodeling.

    The Journal of biological chemistry 2001;276;52;49043-52

  • Neocortical cell migration: GABAergic neurons and cells in layers I and VI move in a cyclin-dependent kinase 5-independent manner.

    Gilmore EC and Herrup K

    Department of Neurosciences, School of Medicine, Case Western Reserve University, and Alzheimer Research Laboratory, University Hospitals of Cleveland, Cleveland, Ohio 44106.

    The adult mammalian cerebral cortex arises from a complex series of neuronal migrations. The primitive layer known as the preplate is split into an outer marginal zone and an inner subplate by invading cortical plate neurons in an "inside-out" pattern of layering with respect to time of neuronal origin. In cyclin-dependent kinase 5 (Cdk5)-deficient mice (cdk5(-/-)), the earliest born cortical neurons split the preplate, but later born neurons arrest below the subplate, resulting in an ectopic "outside-in" layer of neurons normally destined for layers II-V. We have pursued this analysis in cdk5(-/-) <--> wild-type chimeric mice coupled with experiments in cell culture. In vitro migration assays show no difference in migrational ability between embryonic cdk5(-/-) and wild-type neurons. In cdk5(-/-) chimeras, layers I and VI are made up of both mutant and wild-type genotype neurons, whereas layers II-V contain predominantly wild-type cells. In addition, a thin layer of neurons is found below layer VI, made up of cdk5(-/-) cells; bromodeoxyuridine labeling suggests that these neurons were destined for layers II-V. Scattered cdk5(-/-) cells are found throughout layers II-V, but these neurons are always found to be GABAergic. The findings suggest that Cdk5 is not required for migration of either the deepest cortical plate neurons or the GABAergic neurons from the ganglionic eminences. The migration of layer II-V pyramidal neurons, however, is intrinsically blocked by Cdk5 deficiency, thus suggesting that different neuronal cell types use distinct mechanisms of migration.

    Funded by: NINDS NIH HHS: NS20591

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;24;9690-700

  • A dual-color FISH gene map of the proximal region of rat Chromosome 4 and comparative analysis in human and mouse.

    Walentinsson A, Helou K and Levan G

    Department of Cell and Molecular Biology-Genetics, Göteborg University, Box 462, SE 405 30 Gothenburg, Sweden. Anna.Walentinsson@gen.gu.se

    The development and refinement of the rat genome map is a prerequisite for a continued qualified and fruitful use of this model system for the study of complex traits. In two distinct rat cancer models, recurrent amplification affecting the proximal region of rat Chr 4 was detected. To further characterize this region, we turned to the evolutionarily conserved chromosome segments in human Chr 7 and mouse Chrs 5 and 6 to identify functional and positional candidate genes. By means of single- and dual-color FISH on metaphase, prometaphase, and interphase chromatin, 15 genes in rat Chr 4q11-q23 (Cdk5, Hgf, Dmtf1, Abcb1, Cyp51, Cdk6, Tac1, Asns, Cav1, Met, Wnt2, Cftr, Smoh, Braf, Arhgef5) were mapped and aligned. In the course of this work, six cancer-related rat genes were isolated de novo and partly sequenced. Ten loci were also mapped by FISH in the mouse. The map provides the framework for a more detailed genetic characterization of individual tumor amplicons, but may also be valuable for the analysis of this region in other rat models of human complex disease. In addition, our data facilitate the analysis of events in mammalian chromosomal evolution affecting the region. In a comparison with human sequence data, we found that there is considerable conservation in this region both in gene order and in distances between genes. There is a single evolutionary breakpoint between rat and mouse and two between rat and human. Since our analysis shows that the three breaks all occurred in different positions, they must be independent of one another. The data tend to support the notion that the genomic configuration in rat Chr 4 is ancestral compared with that in humans and mice.

    Mammalian genome : official journal of the International Mammalian Genome Society 2001;12;12;900-8

  • p35 and p39 are essential for cyclin-dependent kinase 5 function during neurodevelopment.

    Ko J, Humbert S, Bronson RT, Takahashi S, Kulkarni AB, Li E and Tsai LH

    Department of Pathology and Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

    Cyclin-dependent kinase 5 (Cdk5) plays a pivotal role in brain development and neuronal migration. Cdk5 is abundant in postmitotic, terminally differentiated neurons. The ability of Cdk5 to phosphorylate substrates is dependent on activation by its neuronal-specific activators p35 and p39. There exist striking differences in the phenotypic severity of Cdk5-deficient mice and p35-deficient mice. Cdk5-null mutants show a more severe disruption of lamination in the cerebral cortex, hippocampus, and cerebellum. In addition, Cdk5-null mice display perinatal lethality, whereas p35-null mice are viable. These discrepancies have been attributed to the function of other Cdk5 activators, such as p39. To understand the roles of p39 and p35, we created p39-null mice and p35/p39 compound-mutant mice. Interestingly, p39-null mice show no obvious detectable abnormalities, whereas p35(-/-)p39(-/-) double-null mutants are perinatal lethal. We show here that the p35(-/-)p39(-/-) mutants exhibit phenotypes identical to those of the Cdk5-null mutant mice. Other compound-mutant mice with intermediate phenotypes allow us to determine the distinct and redundant functions between p35 and p39. Our data strongly suggest that p35 and p39 are essential for Cdk5 activity during the development of the nervous system. Thus, p35 and p39 are likely to be the principal, if not the only, activators of Cdk5.

    Funded by: NIGMS NIH HHS: GM53049

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;17;6758-71

  • Cdk5 is involved in neuregulin-induced AChR expression at the neuromuscular junction.

    Fu AK, Fu WY, Cheung J, Tsim KW, Ip FC, Wang JH and Ip NY

    Department of Biochemistry, Biotechnology Research Institute, Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.

    Here we describe an important involvement of Cdk5/p35 in regulating the gene expression of acetylcholine receptor (AChR) at the neuromuscular synapse. Cdk5 and p35 were prominently expressed in embryonic muscle, and concentrated at the neuromuscular junction in adulthood. Neuregulin increased the p35-associated Cdk5 kinase activity in the membrane fraction of cultured C2C12 myotubes. Co-immunoprecipitation studies revealed the association between Cdk5, p35 and ErbB receptors in muscle and cultured myotubes. Inhibition of Cdk5 activity not only blocked the NRG-induced AChR transcription, but also attenuated ErbB activation in cultured myotubes. In light of our finding that overexpression of p35 alone led to an increase in AChR promoter activity in muscle, Cdk5 activation is sufficient to mediate the up-regulation of AChR gene expression. Taken together, these results reveal the unexpected involvement of Cdk5/p35 in neuregulin signaling at the neuromuscular synapse.

    Nature neuroscience 2001;4;4;374-81

  • Deregulation of Cdk5 in a mouse model of ALS: toxicity alleviated by perikaryal neurofilament inclusions.

    Nguyen MD, Larivière RC and Julien JP

    Centre for Research in Neurosciences, McGill University, Research Institute of the McGill University, Health Centre, H3G 1A4, Montreal, Quebec, Canada.

    Recent studies suggest that increased activity of cyclin-dependent kinase 5 (Cdk5) may contribute to neuronal death and cytoskeletal abnormalities in Alzheimer's disease. We report here such deregulation of Cdk5 activity associated with the hyperphosphorylation of tau and neurofilament (NF) proteins in mice expressing a mutant superoxide dismutase (SOD1(G37R)) linked to amyotrophic lateral sclerosis (ALS). A Cdk5 involvement in motor neuron degeneration is supported by our analysis of three SOD1(G37R) mouse lines exhibiting perikaryal inclusions of NF proteins. Our results suggest that perikaryal accumulations of NF proteins in motor neurons may alleviate ALS pathogenesis by acting as a phosphorylation sink for Cdk5 activity, thereby reducing the detrimental hyperphosphorylation of tau and other neuronal substrates.

    Neuron 2001;30;1;135-47

  • Synergistic contributions of cyclin-dependant kinase 5/p35 and Reelin/Dab1 to the positioning of cortical neurons in the developing mouse brain.

    Ohshima T, Ogawa M, Veeranna, Hirasawa M, Longenecker G, Ishiguro K, Pant HC, Brady RO, Kulkarni AB and Mikoshiba K

    Laboratory for Developmental Neurobiology, Cell Culture Development, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan. ohshima@brain.riken.go.jp

    Cyclin-dependent kinase (Cdk) 5 is a unique member of the Cdk family, because Cdk5 kinase activity is detected only in the nervous tissue. Two neuron-specific activating subunits of Cdk5, p35 and p39, have been identified. Overlapping expression pattern of these isoforms in the embryonic mouse brain and the significant residual Cdk5 kinase activity in brain homogenate of the p35-/- mice indicate the redundant functions of the Cdk5 activators in vivo. Severe neuronal migration defects in p35-/-Cdk5 +/- mice further support the idea that the redundant expression of the Cdk5 activators may cause a milder phenotype in p35-/- mice compared with Cdk5-/- mice. Mutant mice lacking either Cdk5 or p35 exhibit certain similarities with Reelin/Dab1-mutant mice in the disorganization of cortical laminar structure in the brain. To elucidate the relationship between Cdk5/p35 and Reelin/Dab1 signaling, we generated mouse lines that have combined defects of these genes. The addition of heterozygosity of either Dab1 or Reelin mutation to p35-/- causes the extensive migration defects of cortical neurons in the cerebellum. In the double-null mice of p35 and either Dab1 or Reelin, additional migration defects occur in the Purkinje cells in the cerebellum and in the pyramidal neurons in the hippocampus. These additional defects in neuronal migration in mice lacking both Cdk5/p35 and Reelin/Dab1 indicate that Cdk5/p35 may contribute synergistically to the positioning of the cortical neurons in the developing mouse brain.

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;5;2764-9

  • Coexpression of human cdk5 and its activator p35 with human protein tau in neurons in brain of triple transgenic mice.

    Van den Haute C, Spittaels K, Van Dorpe J, Lasrado R, Vandezande K, Laenen I, Geerts H and Van Leuven F

    Experimental Genetics Group, Center for Human Genetics, Flemish Institute for Biotechnology (VIB), Gasthuisberg O&N 06, Leuven, B-3000, Belgium.

    The potential contribution of cyclin-dependent protein kinase 5 (cdk5) to hyperphosphorylate protein tau, as claimed in Alzheimer's disease, was investigated in vivo. We generated single, double, and triple transgenic mice that coexpress human cdk5 and its activator p35 as well as human protein tau in cerebral neurons. Whereas expression and increased cdk5-kinase activity was obtained, as measured in vitro and demonstrated in vivo, neither murine nor human protein tau was appreciably phosphorylated in the brain of double and triple transgenic mice. These mice behaved and reproduced normally. Silver impregnation and immunohistochemistry of brain sections demonstrated that neurofilament proteins became redistributed in apical dendrites of cortical neurons. This suggested a cytoskeletal effect, but no other relevant brain pathology became apparent. These observations indicate that cdk5/p35 is not a major protein tau kinase and that cdk5/p35 did not cause neurodegeneration in mouse brain, as opposed to cdk5/p25.

    Neurobiology of disease 2001;8;1;32-44

  • Tbr1 regulates differentiation of the preplate and layer 6.

    Hevner RF, Shi L, Justice N, Hsueh Y, Sheng M, Smiga S, Bulfone A, Goffinet AM, Campagnoni AT and Rubenstein JL

    Nina Ireland Laboratory of, Developmental Neurobiology, Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94143, USA.

    During corticogenesis, early-born neurons of the preplate and layer 6 are important for guiding subsequent neuronal migrations and axonal projections. Tbr1 is a putative transcription factor that is highly expressed in glutamatergic early-born cortical neurons. In Tbr1-deficient mice, these early-born neurons had molecular and functional defects. Cajal-Retzius cells expressed decreased levels of Reelin, resulting in a reeler-like cortical migration disorder. Impaired subplate differentiation was associated with ectopic projection of thalamocortical fibers into the basal telencephalon. Layer 6 defects contributed to errors in the thalamocortical, corticothalamic, and callosal projections. These results show that Tbr1 is a common genetic determinant for the differentiation of early-born glutamatergic neocortical neurons and provide insights into the functions of these neurons as regulators of cortical development.

    Funded by: NIMH NIH HHS: K02 MH01046; NINDS NIH HHS: NS01973, R01 NS34661; Telethon: F.2

    Neuron 2001;29;2;353-66

  • Neuronal cyclin-dependent kinase 5 activity is critical for survival.

    Tanaka T, Veeranna, Ohshima T, Rajan P, Amin ND, Cho A, Sreenath T, Pant HC, Brady RO and Kulkarni AB

    Functional Genomics Unit, Gene Targeting Facility, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Cyclin-dependent kinase 5 (Cdk5) null mice exhibit a unique phenotype characterized by perinatal mortality, disrupted cerebral cortical layering attributable to abnormal neuronal migration, lack of cerebellar foliation, and chromatolytic changes of neurons in the brainstem and the spinal cord. Because Cdk5 is expressed in both neurons and astrocytes, it has been unclear whether this phenotype is primarily attributable to defects in neurons or in astrocytes. Herein we report reconstitution of Cdk5 expression in neurons in Cdk5 null mice and its effect on the null phenotype. Unlike the Cdk5 null mice, the reconstituted Cdk5 null mice that express the Cdk5 transgene under the p35 promoter (TgKO mice) were viable and fertile. Because Cdk5 expression is mainly limited to neurons in these mice and rescues the defects in the nervous system of the Cdk5 null phenotype, it clearly demonstrates that Cdk5 activity is necessary for normal development and survival of p35-expressing neurons.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;2;550-8

  • NUDEL is a novel Cdk5 substrate that associates with LIS1 and cytoplasmic dynein.

    Niethammer M, Smith DS, Ayala R, Peng J, Ko J, Lee MS, Morabito M and Tsai LH

    Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachussetts 02115, USA.

    Disruption of one allele of the LIS1 gene causes a severe developmental brain abnormality, type I lissencephaly. In Aspergillus nidulans, the LIS1 homolog, NUDF, and cytoplasmic dynein are genetically linked and regulate nuclear movements during hyphal growth. Recently, we demonstrated that mammalian LIS1 regulates dynein functions. Here we characterize NUDEL, a novel LIS1-interacting protein with sequence homology to gene products also implicated in nuclear distribution in fungi. Like LIS1, NUDEL is robustly expressed in brain, enriched at centrosomes and neuronal growth cones, and interacts with cytoplasmic dynein. Furthermore, NUDEL is a substrate of Cdk5, a kinase known to be critical during neuronal migration. Inhibition of Cdk5 modifies NUDEL distribution in neurons and affects neuritic morphology. Our findings point to cross-talk between two prominent pathways that regulate neuronal migration.

    Neuron 2000;28;3;697-711

  • Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth.

    Zukerberg LR, Patrick GN, Nikolic M, Humbert S, Wu CL, Lanier LM, Gertler FB, Vidal M, Van Etten RA and Tsai LH

    Howard Hughes Medical Institute, Department of Pathology, Massachusetts General Hospital, Boston 02114, USA.

    Cyclin-dependent kinase 5 (Cdk5) is a small serine/threonine kinase that plays a pivotal role during development of the CNS. Cables, a novel protein, interacts with Cdk5 in brain lysates. Cables also binds to and is a substrate of the c-Abl tyrosine kinase. Active c-Abl kinase leads to Cdk5 tyrosine phosphorylation, and this phosphorylation is enhanced by Cables. Phosphorylation of Cdk5 by c-Abl occurs on tyrosine 15 (Y15), which is stimulatory for p35/Cdk5 kinase activity. Expression of antisense Cables in primary cortical neurons inhibited neurite outgrowth. Furthermore, expression of active Abl resulted in lengthening of neurites. The data provide evidence for a Cables-mediated interplay between the Cdk5 and c-Abl signaling pathways in the developing nervous system.

    Funded by: NINDS NIH HHS: R01-NS37007

    Neuron 2000;26;3;633-46

  • Regulation of N-cadherin-mediated adhesion by the p35-Cdk5 kinase.

    Kwon YT, Gupta A, Zhou Y, Nikolic M and Tsai LH

    Health Advances, Inc., Wellesley, Massachusetts 02181, USA.

    Background: The p35-Cdk5 kinase has been implicated in a variety of functions in the central nervous system (CNS), including axon outgrowth, axon guidance, fasciculation, and neuronal migration during cortical development. In p35(-/-) mice, embryonic cortical neurons are unable to migrate past their predecessors, leading to an inversion of cortical layers in the adult cortex.

    Results: In order to identify molecules important for p35-Cdk5-dependent function in the cortex, we screened for p35-interacting proteins using the two-hybrid system. In this study, we report the identification of a novel interaction between p35 and the versatile cell adhesion signaling molecule beta-catenin. The p35 and beta-catenin proteins interacted in vitro and colocalized in transfected COS cells. In addition, the p35-Cdk5 kinase was associated with a beta-catenin-N-cadherin complex in the cortex. In N-cadherin-mediated aggregation assays, inhibition of Cdk5 kinase activity using the Cdk5 inhibitor roscovitine led to the formation of larger aggregates of embryonic cortical neurons. This finding was recapitulated in p35(-/-) cortical neurons, which aggregated to a greater degree than wild-type neurons. In addition, introduction of active p35-Cdk5 kinase into COS cells led to a decreased beta-catenin-N-cadherin interaction and loss of cell adhesion.

    Conclusions: The association between p35-Cdk5 and an N-cadherin adhesion complex in cortical neurons and the modulation of N-cadherin-mediated aggregation by p35-Cdk5 suggests that the p35-Cdk5 kinase is involved in the regulation of N-cadherin-mediated adhesion in cortical neurons.

    Funded by: NINDS NIH HHS: NS37007

    Current biology : CB 2000;10;7;363-72

  • Mouse cyclin-dependent kinase (Cdk) 5 is a functional homologue of a yeast Cdk, pho85 kinase.

    Nishizawa M, Kanaya Y and Toh-E A

    Department of Microbiology, Keio University School of Medicine, Shinjuku, Tokyo 160-8582, Japan. mas@mc.med.keio.ac.jp

    Mouse cyclin-dependent kinase (Cdk) 5 and yeast Pho85 kinase share similarities in structure as well as in the regulation of their activity. We found that mouse Cdk5 kinase produced in pho85Delta mutant cells could suppress some of pho85Delta mutant phenotypes including failure to grow on nonfermentable carbon sources, morphological defects, and growth defect caused by Pho4 or Clb2 overproduction. We also demonstrated that Cdk5 coimmunoprecipitated with Pho85-cyclins including Pcl1, Pcl2, Pcl6, Pcl9, and Pho80, and that the immunocomplex could phosphorylate Pho4, a native substrate of Pho85 kinase. Thus mouse Cdk5 is a functional homologue of yeast Pho85 kinase.

    The Journal of biological chemistry 1999;274;48;33859-62

  • Mutant mice with scrambled brains: understanding the signaling pathways that control cell positioning in the CNS.

    Rice DS and Curran T

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA. dennis.rice@stjude.org

    Funded by: NCI NIH HHS: P30 CA21765; NEI NIH HHS: F32 EY06972; NINDS NIH HHS: R01 NS36558

    Genes & development 1999;13;21;2758-73

  • Migration defects of cdk5(-/-) neurons in the developing cerebellum is cell autonomous.

    Ohshima T, Gilmore EC, Longenecker G, Jacobowitz DM, Brady RO, Herrup K and Kulkarni AB

    Functional Genomics Unit, Gene Targeting Facility, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Cyclin-dependent kinase 5 (Cdk5) is a member of the family of cell cycle-related kinases. Previous neuropathological analysis of cdk5(-/-) mice showed significant changes in CNS development in regions from cerebral cortex to brainstem. Among the defects in these animals, a disruption of the normal pattern of cell migrations in cerebellum was particularly apparent, including a pronounced abnormality in the location of cerebellar Purkinje cells. Complete analysis of this brain region is hampered in the mutant because most of cerebellar morphogenesis occurs after birth and the cdk5(-/-) mice die in the perinatal period. To overcome this disadvantage, we have generated chimeric mice by injection of cdk5(-/-) embryonic stem cells into host blastocysts. Analysis of the cerebellum from the resulting cdk5(-/-) left arrow over right arrow cdk5(+/+) chimeric mice shows that the abnormal location of the mutant Purkinje cells is a cell-autonomous defect. In addition, significant numbers of granule cells remain located in the molecular layer, suggesting a failure to complete migration from the external to the internal granule cell layer. In contrast to the Purkinje and granule cell populations, all three of the deep cerebellar nuclear cell groupings form correctly and are composed of cells of both mutant and wild-type genotypes. Despite similarities of the cdk5(-/-) phenotype to that reported in reeler and mdab-1(-/-) (scrambler/yotari) mutant brains, reelin and disabled-1 mRNA were found to be normal in cdk5(-/-) brain. Together, the data further support the hypothesis that Cdk5 activity is required for specific components of neuronal migration that are differentially required by different neuronal cell types and by even a single neuronal cell type at different developmental stages.

    Funded by: NINDS NIH HHS: NS20591

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1999;19;14;6017-26

  • Comparison of munc-18 and cdk5 expression in the nervous system during mouse embryogenesis.

    Han SH and Morgan JI

    Department of Developmental Neurobiology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.

    Cyclin-dependent kinase-5 (Cdk5) and its neuron-specific activator, p35, are essential for the proper migration of neurons. While the defects in p35 null mice are largely confined to the cerebral cortex, the anomalies in cdk5 nullizygous mice are also evident in the hippocampus and cerebellum. This suggested that additional cyclin-like activators, such as Munc-18, must be coexpressed with Cdk5 in some migrating neurons. Therefore, the expression patterns of munc-18 and cdk5 were determined in the developing mouse nervous system by in situ hybridization. In the embryonic day 11.5-13.5 developing neocortex, cdk5 was expressed in the proliferative zone and also in migratory and postmitotic neurons. In contrast, munc-18 messenger RNA was only detected in postmigratory, differentiated neurons. In the cerebellum and the hippocampus, cdk5 was expressed in proliferative, migrating and postmigratory neurons, while munc-18 was expressed in migrating and postmigratory neurons. This supports the hypothesis that Munc-18 could compensate for the loss of p35 in migrating neurons in the hippocampus and cerebellum, but not the cerebral cortex. Munc-18 levels increased substantially during late embryogenesis and into adulthood. Therefore, the function of Munc-18 is most likely relevant to mature neurons and any redundancy with p35 in migration is probably fortuitous.

    Funded by: NCI NIH HHS: P30 CA21765

    Neuroscience 1999;94;3;955-64

  • Cyclin-dependent kinase 5-deficient mice demonstrate novel developmental arrest in cerebral cortex.

    Gilmore EC, Ohshima T, Goffinet AM, Kulkarni AB and Herrup K

    Department of Neuroscience, Case Western Reserve Medical School, Cleveland, Ohio 44106, USA.

    The cerebral cortex of mice with a targeted disruption in the gene for cyclin-dependent kinase 5 (cdk5) is abnormal in its structure. Bromodeoxyuridine labeling reveals that the normal inside-out neurogenic gradient is inverted in the mutants; earlier born neurons are most often found superficial to those born later. Despite this, the early preplate layer separates correctly and neurons with a normal, pyramidal morphology can be found between true marginal zone and subplate. Consistent with their identity as layer VI corticothalamic neurons, they can be labeled by DiI injections into thalamus. The DiI injections also reveal that the trajectories of the cdk5(-/-) thalamocortical axons are oblique and cut across the entire cortical plate, instead of being oriented tangentially in the subcortical white matter. We propose a model in which the cdk5(-/-) defect blocks cortical development at a heretofore undescribed intermediate stage, after the splitting of the preplate, but before the migration of the full complement of cortical neurons.

    Funded by: NINDS NIH HHS: NS20591

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1998;18;16;6370-7

  • A novel disruption of cortical development in p35(-/-) mice distinct from reeler.

    Kwon YT and Tsai LH

    Howard Hughes Medical Institute and the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    The p35/cdk5 neuronal-specific kinase complex has been shown to play an important role in the laminar configuration of cortical neurons. Mice lacking either p35 or cdk5 exhibit a disrupted cortical lamination pattern. We showed previously that instead of the normal "inside-out" layering pattern of cortical neurons, cortical neurons are layered from "outside-in" in p35 mutant mice. To gain insight into the mechanisms that underlie these defects, we examined the organization of landmark structures formed during cortical development and the migratory behavior of p35(-/-) cortical neurons by using bromodeoxyuridine labeling. In the present study, we show that reelin localization in the marginal zone is normal in p35 mutant mice. Furthermore, the preplate splits into the marginal zone and subplate properly, a developmental event that fails to occur in reeler mice. Finally, the migration of the earliest born cortical plate neurons is normal in p35 mutant mice; cortical neurons subsequently generated remain underneath these neurons. These data suggest that the p35/cdk5 kinase is required for cortical plate neurons to migrate past preexisting neurons and take up superficial positions to constitute the inside-outside layering order of cortical lamination.

    Funded by: NIGMS NIH HHS: GM53049

    The Journal of comparative neurology 1998;395;4;510-22

  • The identification and characterization of expression of Pftaire-1, a novel Cdk family member, suggest its function in the mouse testis and nervous system.

    Besset V, Rhee K and Wolgemuth DJ

    Department of Genetics and Development, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

    We have isolated a murine cDNA encoding for a novel putative Cdk-related protein kinase, which has been named Pftaire-1, by screening a testis cDNA library for new serine/threonine kinases. Pftaire-1 showed 50% and 49% amino acid identity with Cdk5 and Pctaire-3, respectively, and contains the eleven subdomains characteristic of the protein kinases. By northern blot analysis we detected two transcripts of approximately 5.5 and 4.9 kb in size. These transcripts were expressed at low level in all murine tissues tested, except in the brain, testis and embryo, where high expression was detected. Cellular localization of the mRNAs by in situ hybridization analysis shows that Pftaire-1 is expressed in late pachytene spermatocytes in the testis and in post mitotic neuronal cells both in the brain and the embryo, suggesting a role of Pftaire-1 both in the process of meiosis as well as neuron differentiation and/or function.

    Funded by: NICHD NIH HHS: HD 07968

    Molecular reproduction and development 1998;50;1;18-29

  • Phosphorylation of neurofilament heavy-chain side-arm fragments by cyclin-dependent kinase-5 and glycogen synthase kinase-3alpha in transfected cells.

    Bajaj NP and Miller CC

    Department of Neurology, Institute of Psychiatry, London, England, U.K.

    The side-arm domain of neurofilament heavy-chain (NF-H) is heavily phosphorylated in axons. Much of this phosphate is located within a multiphosphorylation repeat (MPR) domain situated toward the carboxy terminus of the molecule. The MPR domain contains the repeat motif KSP of which there are two broad categories, KSPXX and KSPXK. In mouse NF-H, the KSPXK repeats are situated toward the latter part of the MPR domain. We have expressed in mammalian cells fragments of mouse NF-H side-arm containing all of the MPR domain, the latter part of the MPR domain containing the KSPXK repeats, and the complementary amino-terminal part of the MPR domain, which contains the KSPXX repeats. By cotransfecting these fragments with the neurofilament kinases cyclin-dependent kinase-5 (cdk-5)/p35 and glycogen synthase kinase-3alpha (GSK-3alpha), we show that cdk-5 induces cellular phosphorylation of the KSPXK-containing fragment of NF-H. Using the transfected fragments, we also map the epitopes for several commonly utilised NF-H monoclonal antibodies and describe the effects that phosphorylation by cdk-5 and GSK-3alpha have on their reactivities.

    Funded by: Wellcome Trust

    Journal of neurochemistry 1997;69;2;737-43

  • A novel cdc2-related protein kinase expressed in the nervous system.

    Lazzaro MA, Albert PR and Julien JP

    Centre for Research in Neuroscience, McGill University and Montreal General Hospital Research Institute, Quebec, Canada.

    We report the cloning and characterization of a cDNA encoding a cdc2-related protein kinase, named PFTAIRE, that is expressed primarily in the postnatal and adult nervous system. We have demonstrated by in situ hybridization and indirect immunofluorescence that several populations of terminally differentiated neurons and some neuroglia expressed PFTAIRE mRNA and protein. In neurons, PFTAIRE protein was localized in the nucleus and cytoplasm of cell bodies. The anatomical, cellular, and ontogenic patterns of PFTAIRE expression in the nervous system differed from those of p34cdc2 and cdk5, which are expressed in brain and several other mitotic tissues. Proteins of approximately 58-60 kDa coprecipitated specifically with PFTAIRE from cytosolic protein preparations of adult mouse brain and transfected cells. These proteins appeared to be the major endogenous substrates associated with this kinase activity. The temporal and spatial expression patterns of PFTAIRE in the postnatal and adult nervous system suggest that PFTAIRE kinase activity may be associated with the postmitotic and differentiated state of cells in the nervous system and that its function may be distinct from those of p34cdc2 and cdk5.

    Journal of neurochemistry 1997;69;1;348-64

  • Cyclin dependent kinase 5, cdk5, is a positive regulator of myogenesis in mouse C2 cells.

    Lazaro JB, Kitzmann M, Poul MA, Vandromme M, Lamb NJ and Fernandez A

    Cell Biology Unit, CRBM, CNRS-INSERM, Montpellier, France.

    We have examined the expression, activity and localization of cyclin dependent kinase 5 (cdk5), during myogenesis. Cdk5 protein was found expressed in adult mouse muscle. In murine C2 cells, both the protein level and kinase activity of cdk5 showed a marked increase during early myogenesis with a peak between 36 and 48 hours of differentiation, decreasing as myotubes fuse after 60 to 72 hours. This increase in cdk5 protein level was specific for differentiation and not simply related to cell cycle arrest since it was not observed in fibroblasts grown for 48 hours in low serum medium. Indirect immunofluorescence using monospecific purified anti-cdk5 antibodies showed a low level cytoplasmic staining in proliferative myoblasts, a rapid increase in nuclear staining during the initial 12 hours of differentiation and a predominant nuclear staining in myotubes. Microinjection of plasmids encoding wild-type cdk5 into C2 myoblasts enhanced differentiation as assessed by both myogenin and troponin T expression after 48 hours of differentiation. In contrast, microinjection of plasmids encoding a dominant negative mutant of cdk5 inhibited the onset of differentiation. These data imply a previously unsuspected role for cdk5 protein kinase as a positive modulator of early myogenesis.

    Journal of cell science 1997;110 ( Pt 10);1251-60

  • Cyclin-dependent kinase 5 is associated with apoptotic cell death during development and tissue remodeling.

    Zhang Q, Ahuja HS, Zakeri ZF and Wolgemuth DJ

    Department of Genetics, Columbia University College of Physicians and Surgeons, New York, New York 10032.

    In a series of studies to more precisely localize the cellular sites of expression of the cyclin-dependent kinase (Cdk) family members in reproductive organs, we observed a striking expression of Cdk5 in atretic follicles in the ovary, particularly in granulosa cells that appeared to be dying. We determined that these granulosa cells were undergoing apoptotic cell death using the in situ DNA fragmentation assay. To extend the generality of the association of Cdk5 with apoptotic cells, we examined its expression as it correlated with the detection of apoptosis in a number of developmental paradigms, including regions of the embryonic nervous system, the developing eye, and the developing limb. Finally, the association of apoptosis and Cdk5 expression and associated kinase activity was examined in the limb and in an induced cell death system, that of androgen withdrawal-induced regression of the prostate gland in male mice. These observations provide new insight into the possible function of this novel Cdk during both differentiation and apoptotic cell death.

    Funded by: NICHD NIH HHS: P50 HD05077, T32 HD07093; PHS HHS: NIA KO4; ...

    Developmental biology 1997;183;2;222-33

  • Association of cyclin-dependent kinase 5 and its activator p35 with apoptotic cell death.

    Ahuja HS, Zhu Y and Zakeri Z

    Department of Biology, Queens College, Flushing, New York 11367, USA.

    We have investigated the role of cyclin-dependent kinases in cell death and found that the expression of cyclin-dependent kinase 5 (Cdk5) is associated with apoptotic cell death in both adult and embryonic tissues. By double labeling immunohistochemistry and confocal microscopy, we specifically associated the expression of Cdk5 to dying cells. The association of Cdks with cell death is unique to Cdk5 as this association is not found with the other Cdks (Cdk 1-8) and cell death. The differential increase in Cdk5 expression is at the level of protein only, and no differences can be detected at the level of mRNA Using both limbs of mutant mice detective in the pattern of interdigital cell death and limbs with increased interdigital cell death by retinoic acid treatment, we confirmed the specificity of Cdk5 protein expression in dying cells. To investigate the regulation of Cdk5 during cell death, we examined the expression of a regulatory protein of Cdk5, p35, and found p35 to be expressed in the dying cells as well. Similar to Cdk5, there is also no specific differential expression of the p35 mRNA in dying cells. Our results suggest a role for Cdk5 and p35 proteins in cell death. This protein complex may function in the rearrangement of the cytoskeleton during apoptosis.

    Funded by: NIA NIH HHS: K04, AG00631

    Developmental genetics 1997;21;4;258-67

  • Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death.

    Ohshima T, Ward JM, Huh CG, Longenecker G, Veeranna, Pant HC, Brady RO, Martin LJ and Kulkarni AB

    National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892, USA.

    Although cyclin-dependent kinase 5 (Cdk5) is closely related to other cyclin-dependent kinases, its kinase activity is detected only in the postmitotic neurons. Cdk5 expression and kinase activity are correlated with the extent of differentiation of neuronal cells in developing brain. Cdk5 purified from nervous tissue phosphorylates neuronal cytoskeletal proteins including neurofilament proteins and microtubule-associated protein tau in vitro. These findings indicate that Cdk5 may have unique functions in neuronal cells, especially in the regulation of phosphorylation of cytoskeletal molecules. We report here generation of Cdk5(-/-) mice through gene targeting and their phenotypic analysis. Cdk5(-/-) mice exhibit unique lesions in the central nervous system associated with perinatal mortality. The brains of Cdk5(-/-) mice lack cortical laminar structure and cerebellar foliation. In addition, the large neurons in the brain stem and in the spinal cord show chromatolytic changes with accumulation of neurofilament immunoreactivity. These findings indicate that Cdk5 is an important molecule for brain development and neuronal differentiation and also suggest that Cdk5 may play critical roles in neuronal cytoskeleton structure and organization.

    Proceedings of the National Academy of Sciences of the United States of America 1996;93;20;11173-8

  • Intracellular localization of cyclin-dependent kinase 5 (CDK5) in mouse neuron: CDK5 is located in both nucleus and cytoplasm.

    Ino H and Chiba T

    Third Department of Anatomy, Chiba University School of Medicine, Japan.

    Cyclin-dependent kinase 5 (CDK5) is one of the cyclin-dependent kinases, and is expressed in mature neurons. CDK5 has been postulated to be a neurofilament or tau protein kinase, because it phosphorylates neurofilaments and tau protein (microtubule-associated protein tau) in vitro. It has been reported that CDK5 was immunohistochemically detected only in axons of neurons. We here report the immunohistochemical study of CDK5 using two distinct antibodies, one recognizing the N-terminal of CDK5 and the other the C-terminal. Immunoreactivity of CDK5 was found not only in axons, but also intensively in nuclei, though not in nucleoli, of neurons in the mouse central and peripheral nervous systems. The nuclear CDK5 possibly has a physiological function distinct from the neurofilament or tau protein kinase.

    Brain research 1996;732;1-2;179-85

  • Promoter region of the mouse cyclin-dependent kinase 5-encoding gene.

    Ishizuka T, Ino H, Sawa K, Suzuki N and Tatibana M

    Department of Biochemistry, Chiba University, School of Medicine, Japan.

    While cyclin-dependent kinases, such as CDC2 and CDK2, are key regulators of cell-cycle progression, cyclin-dependent kinase 5 (CDK5) is highly expressed in mature neurons with no evident cell-cycle regulation. The 5'-region of the mouse CDK5 gene was isolated and sequenced. The isolated clone included exons 1 through 7. The 5'-flanking region has a high G+C content. There is no TATA box around the transcriptional start points (tsp), as determined by primer extension analysis. One CCAAT box, one AP-1-binding site, two AP-2-binding sites, and one cAMP-responsive element are located upstream from the tsp. Promoter/cat fusion assays showed that the 5.8-kb fragment of this 5'-flanking sequence possessed the promoter activities expressing cat in rat PC12 pheochromocytoma cells. The effect of deletions of the promoter suggested the presence of two negative control elements located from -2.9 kb to -546 bp, and from -212 to -155 upstream from the 5' end of the tsp. Two positive elements from bp -300 to -212 and from -155 to -41 were also detected. In the element from bp -300 to -212, there was a putative NF-IL6-binding sequence. Thus, the CDK5 promoter region contains multiple positive and negative cis-acting regulatory elements, an arrangement which suggests that the regulation of transcription of CDK5 is under complex control.

    Gene 1995;166;2;267-71

  • Molecular cloning and chromosomal mapping of the mouse cyclin-dependent kinase 5 gene.

    Ohshima T, Nagle JW, Pant HC, Joshi JB, Kozak CA, Brady RO and Kulkarni AB

    Unit on Mouse Genetics and Human Disease Models, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in neurons. In vitro, Cdk5 purified from the nervous tissue phosphorylates both high-molecular-weight neurofilament and microtubule-associated tau. The mouse gene encoding Cdk5 (Cdk5) was found to be 5 kb in length and divided into 12 exons. All of the exon-intron junctions matched the expected consensus sequence with the exception of the splice junction for intron 9, which has AT and AC dinucleotides instead of the usual GT and AG bordering sequence. In the 5'-flanking region of mouse Cdk5, several putative promoter elements were present, including AP1, Sp1, PuF, and TATA motifs. A metal regulatory element was also identified at position -207 to -201. Nucleotide sequence analysis of mouse Cdk5 showed high identity to the homologues of other vertebrate species, indicating that this kinase is highly conserved during evolution. Mouse Cdk5 was mapped to the centromeric region of mouse chromosome 5.

    Genomics 1995;28;3;585-8

  • Expression of CDK5 (PSSALRE kinase), a neural cdc2-related protein kinase, in the mature and developing mouse central and peripheral nervous systems.

    Ino H, Ishizuka T, Chiba T and Tatibana M

    Department of Anatomy, Chiba University School of Medicine, Japan.

    CDK5 is a cdc2-related protein kinase that is known to be highly expressed in mature brain. In this study, we obtained a mouse CDK5 cDNA by screening an adult mouse cDNA library. Northern blot analysis demonstrated that the mouse CDK5 mRNA was expressed especially highly in brain, and moderately in kidney, testis and ovary. In brain the expression of CDK5 is already seen at embryonal 12.5 days (E12.5), and it gradually increases through the embryonal stage. After birth, the expression is maintained at a high level to adulthood. In situ hybridization demonstrated that the expression of CDK5 mRNA was distributed in neurons throughout the brain, spinal cord and peripheral ganglia, especially in the hippocampal pyramidal cells, cerebellar Purkinje cells, cortical neurons, olfactory mitral cells, mesencephalic and motor trigeminal nuclei and trigeminal ganglion. In any portion, no apparent expression was observed in glia. During development, the expression of CDK5 was already seen at E12.5 intensely in trigeminal and dorsal root ganglia, and moderately and diffusely in the central nervous system. The expression pattern of CDK5 is quite in contrast with that of CDC2. The fact that CDK5 is expressed in terminally differentiated non-dividing neurons predicts an alternative function(s) in addition to controlling the cell cycle.

    Brain research 1994;661;1-2;196-206

  • p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5.

    Tsai LH, Delalle I, Caviness VS, Chae T and Harlow E

    Massachusetts General Hospital Cancer Center, Charlestown 02129.

    Cyclin-dependent kinase 5 (Cdk5) was originally isolated through its structural homology to human Cdc2, a key regulator of cell-cycle progression. In tissue samples from adult mice, Cdk5 protein is found at the highest level in brain, at an intermediate level in testis, and at low or undetectable levels in all other tissues, but brain is the only tissue that shows Cdk5 histone H1 kinase activity. No equivalent kinase activity has been found in tissue culture cell lines despite high levels of Cdk5. This raised the possibility that a Cdk5 regulatory subunit was responsible for the activation of Cdk5 in brain. Here we describe the cloning and characterization of a regulatory subunit for Cdk5 known as p35. p35 displays a neuronal cell-specific pattern of expression, it associates physically with Cdk5 in vivo and activates the Cdk5 kinase. p35 differs from the mammalian cyclins and thus represents a new type of regulatory subunit for cyclin-dependent kinase activity.

    Nature 1994;371;6496;419-23

  • Chromosomal mapping of human CDK2, CDK4, and CDK5 cell cycle kinase genes.

    Demetrick DJ, Zhang H and Beach DH

    Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, NY 11724.

    Cyclin dependent kinases (CDK's) are kinases that interact with cyclins and regulate cell division. Genomic clones encoding human CDK2, CDK4, and CDK5 were obtained and mapped to their respective chromosomal loci using fluorescence in situ hybridization on human lymphocyte metaphase spreads. Interestingly, CDK2 and CDK4 were located at the same position, 12q13, and CDK5 was mapped to 7q36. 12q13 has been shown to be associated with chromosome alterations such as amplifications and translocations in solid tumors. 7q36 does not appear to be a major site of chromosome alterations in tumors. As CDK2 and CDK4 appear to be important in regulating the human cell cycle, it is possible that the alterations of the 12q13 locus in tumors may involve changes in the regulation of CDK2 and CDK4 genes.

    Funded by: NIGMS NIH HHS: GM 39620

    Cytogenetics and cell genetics 1994;66;1;72-4

  • Activity and expression pattern of cyclin-dependent kinase 5 in the embryonic mouse nervous system.

    Tsai LH, Takahashi T, Caviness VS and Harlow E

    Massachusetts General Hospital Cancer Center, Charlestown 02129.

    Cyclin-dependent kinase 5 (cdk5) was originally isolated on the basis of its close primary sequence homology to the human cdc2 serine/threonine kinase, the prototype of the cyclin-dependent kinases. While kinase activities of both cdc2 and cdk2 are detected in proliferating cells and are essential for cells to progress through the key transition points of the cell cycle, cdk5 kinase activity has been observed only in lysates of adult brain. In this study, we compared the activity and expression of cdk5 with that of cdc2 and cdk2 in the embryonic mouse forebrain. The expression and activity of cdk5 increased progressively as increasing numbers of cells exited the proliferative cycle. In contrast, the expression and activity of cdc2 and cdk2 were maximum at gestational day 11 (E11) when the majority of cells were proliferating and fell to barely detectable levels at E17 at the end of the cytogenetic period. Immunohistochemical studies showed that cdk5 is expressed in postmitotic neurons but not in glial cells or mitotically active cells. Expression of cdk5 was concentrated in fasciculated axons of postmitotic neurons. In contrast to other cell division cycle kinases to which it is closely related, cdk5 appears not to be expressed in dividing cells in the developing brain. These observations suggest that cdk5 may have a role in neuronal differentiation but not in the cell division cycle in the embryonic nervous system.

    Development (Cambridge, England) 1993;119;4;1029-40

  • Novel CDC2-related protein kinases produced in murine hematopoietic stem cells.

    Ershler M, Nagorskaya TV, Visser JW and Belyavsky AV

    W. A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.

    The polymerase chain reaction with degenerate primers was used for the amplification of cDNA encoding CDC2-related protein kinase (PK) sequences from murine hematopoietic stem cells. In total, nine different PK-encoding sequences were obtained. At least four of them encode previously unknown PKs.

    Gene 1993;124;2;305-6

  • A nomenclature proposal for the chromogranin/secretogranin proteins.

    Eiden LE, Huttner WB, Mallet J, O'Connor DT, Winkler H and Zanini A

    Neuroscience 1987;21;3;1019-21

Gene lists (6)

Gene List Source Species Name Description Gene count
L00000001 G2C Mus musculus Mouse PSD Mouse PSD adapted from Collins et al (2006) 1080
L00000008 G2C Mus musculus Mouse PSP Mouse PSP adapted from Collins et al (2006) 1121
L00000060 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus (ortho) 748
L00000062 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus 984
L00000070 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list (ortho) 1461
L00000072 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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