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Yuwen Hwang - One of the best experts on this subject based on the ideXlab platform.

  • phosphorylation by DYRK1A of clathrin coated vesicle associated proteins identification of the substrate proteins and the effects of phosphorylation
    PLOS ONE, 2012
    Co-Authors: Noriko Murakami, David C Bolton, Elizabeth Kida, Wen Xie, Yuwen Hwang
    Abstract:

    DYRK1A phosphorylated multiple proteins in the clathrin-coated vesicle (CCV) preparations obtained from rat brains. Mass spectrometric analysis identified MAP1A, MAP2, AP180, and α- and β-adaptins as the phosphorylated proteins in the CCVs. Each protein was subsequently confirmed by [(32)P]-labeling and immunological methods. The DYRK1A-mediated phosphorylation released the majority of MAP1A and MAP2 and enhanced the release of AP180 and adaptin subunits from the CCVs. Furthermore, DYRK1A displaced adaptor proteins physically from CCVs in a kinase-concentration dependent manner. The clathrin heavy chain release rate, in contrast, was not affected by DYRK1A. Surprisingly, the DYRK1A-mediated phosphorylation of α- and β-adaptins led to dissociation of the AP2 complex, and released only β-adaptin from the CCVs. AP180 was phosphorylated by DYRK1A also in the membrane-free fractions, but α- and β-adaptins were not. DYRK1A was detected in the isolated CCVs and was co-localized with clathrin in neurons from mouse brain sections and from primary cultured rat hippocampus. Previously, we proposed that DYRK1A inhibits the onset of clathrin-mediated endocytosis in neurons by phosphorylating dynamin 1, amphiphysin 1, and synaptojanin 1. Current results suggest that besides the inhibition, DYRK1A promotes the uncoating process of endocytosed CCVs.

  • harmine is an atp competitive inhibitor for dual specificity tyrosine phosphorylation regulated kinase 1a DYRK1A
    Archives of Biochemistry and Biophysics, 2011
    Co-Authors: Tatyana Adayev, Jerzy Wegiel, Yuwen Hwang
    Abstract:

    Harmine is a β-carboline alkaloid. The compound is a potent inhibitor of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), a kinase implicated in Down syndrome. In this study, we show that harmine functions as an ATP-competitive inhibitor against DYRK1A. Our conclusion is supported by kinetic analysis of harmine inhibition as well as by the characterization of a DYRK1A mutation conferring significant resistance to harmine. The mutation, V306A, is located next to the highly conserved D307 residue in kinases known to coordinate the phosphate groups of ATP through a Mg²+ ion. The V306A mutation offers harmine resistance by differentially altering DYRK1A affinity for harmine and ATP. The V306A mutation causes no apparent alteration to DYRK1A activity except for the reduction in ATP affinity. This deficiency could be fully compensated by supplying ATP with a concentration in the physiological range. Our results reveal that harmine inhibits DYRK1A activity by interacting with residues in the ATP-binding pocket and displacing ATP. Our results also suggest that harmine will be a good lead compound for further designing of selective ATP-competitive DYRK1A inhibitors through exploration of the ATP-binding pocket of DYRK1A.

  • mnb DYRK1A phosphorylation regulates the interactions of synaptojanin 1 with endocytic accessory proteins
    Biochemical and Biophysical Research Communications, 2006
    Co-Authors: Tatyana Adayev, Noriko Murakami, Mochou Chenhwang, Rong Wang, Yuwen Hwang
    Abstract:

    Abstract MNB/DYRK1A is a proline-directed serine/threonine kinase implicated in Down syndrome (DS). In an earlier screening, two proteins from adult rat brain, one 100 kDa and the other 140 kDa, were found to be prominently phosphorylated by the kinase. The 100-kDa protein was previously characterized as an isoform of dynamin 1. In this study, we identified the 140-kDa protein as synaptojanin 1 (SJ1). MNB/DYRK1A phosphorylates SJ1 at multiple sites and produces complex behaviors in binding to amphiphysin 1 and intersectin 1 (ITSN1). However, the phosphorylation has little effect on the phosphatidylinositol phosphatase activity of SJ1. These results suggest that MNB/DYRK1A is involved in regulating the recruitment activity but not the phosphatase activity of SJ1. Our findings may be especially important in the etiology of DS because MNB/DYRK1A, SJ1, and ITSN1 are all located at or near the region of human chromosome 21, which is postulated to be involved in the disease.

  • phosphorylation of amphiphysin i by minibrain kinase dual specificity tyrosine phosphorylation regulated kinase a kinase implicated in down syndrome
    Journal of Biological Chemistry, 2006
    Co-Authors: Noriko Murakami, Wen Xie, Mochou Chenhwang, Andrzej Wieraszko, Yuwen Hwang
    Abstract:

    Minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase (Mnb/DYRK1A) is a proline-directed serine/threonine kinase encoded in the Down syndrome critical region of human chromosome 21. This kinase has been shown to phosphorylate dynamin 1 and synaptojanin 1. Here we report that amphiphysin I (Amph I) is also a Mnb/DYRK1A substrate. This kinase phosphorylated native Amph I in rodent brains and recombinant human Amph I expressed in Escherichia coli. Serine 293 (Ser-293) was identified as the major site, whereas serine 295 and threonine 310 were found as minor kinase sites. In cultured cells, recombinant Amph I was phosphorylated at Ser-293 by endogenous kinase(s). Because mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) has been suggested to phosphorylate Amph I at Ser-293, our efforts addressed whether Ser-293 is phosphorylated in vivo by MAPK/ERK or by Mnb/DYRK1A. Overnight serum-withdrawal inactivated MAPK/ERK; nonetheless, Ser-293 was phosphorylated in Chinese hamster ovary and SY5Y cells. Epigallocatechin-3-gallate, a potent Mnb/DYRK1A inhibitor in vitro, apparently reduced the phosphorylation at Ser-293, whereas PD98059, a potent MAPK/ERK inhibitor, did not. High frequency stimulation of mouse hippocampal slices reduced the phosphorylation at Ser-293, albeit in the midst of MAPK/ERK activation. The endophilin binding in vitro was inhibited by phosphorylating Amph I with Mnb/DYRK1A. However, phosphorylation at Ser-293 did not appear to alter cellular distribution patterns of the protein. Our results suggest that Mnb/DYRK1A, not MAPK/ERK, is responsible for in vivo phosphorylation of Amph I at Ser-293 and that phosphorylation changes the recruitment of endophilin at the endocytic sites.

Walter Becker - One of the best experts on this subject based on the ideXlab platform.

  • the adaptor protein dcaf7 mediates the interaction of the adenovirus e1a oncoprotein with the protein kinases DYRK1A and hipk2
    Scientific Reports, 2016
    Co-Authors: Florian Glenewinkel, Joe S. Mymryk, Michael J Cohen, Cason R King, Sophie Kaspar, Simone Bamberglemper, Walter Becker
    Abstract:

    DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation.

  • Selectivity Profiling and Biological Activity of Novel β-Carbolines as Potent and Selective DYRK1 Kinase Inhibitors
    2015
    Co-Authors: Katharina Rüben, Anne Wurzlbauer, Agnes Walte, Wolfgang Sippl, Franz Bracher, Walter Becker
    Abstract:

    DYRK1A is a pleiotropic protein kinase with diverse functions in cellular regulation, including cell cycle control, neuronal differentiation, and synaptic transmission. Enhanced activity and overexpression of DYRK1A have been linked to altered brain development and function in Down syndrome and neurodegenerative diseases such as Alzheimer’s disease. The β-carboline alkaloid harmine is a high affinity inhibitor of DYRK1A but suffers from the drawback of inhibiting monoamine oxidase A (MAO-A) with even higher potency. Here we characterized a series of novel harmine analogs with minimal or absent MAO-A inhibitory activity. We identified several inhibitors with submicromolar potencies for DYRK1A and selectivity for DYRK1A and DYRK1B over the related kinases DYRK2 and HIPK2. An optimized inhibitor, AnnH75, inhibited CLK1, CLK4, and haspin/GSG2 as the only off-targets in a panel of 300 protein kinases. In cellular assays, AnnH75 dose-dependently reduced the phosphorylation of three known DYRK1A substrates (SF3B1, SEPT4, and tau) without negative effects on cell viability. AnnH75 inhibited the cotranslational tyrosine autophosphorylation of DYRK1A and threonine phosphorylation of an exogenous substrate protein with similar potency. In conclusion, we have characterized an optimized β-carboline inhibitor as a highly selective chemical probe that complies with desirable properties of drug-like molecules and is suitable to interrogate the function of DYRK1A in biological studies.

  • Analysis of kinase inhibitor interactions.
    2015
    Co-Authors: Katharina Rüben, Anne Wurzlbauer, Agnes Walte, Wolfgang Sippl, Franz Bracher, Walter Becker
    Abstract:

    A-D, Predicted binding modes of AnnH75 at DYRK1A and related kinases. The inhibitor is colored cyan and kinases are depicted as ribbon structures. Only relevant amino acid residues in the ATP binding pocket are shown for clarity. In B-D, residues different from DYRK1A are colored green and labeled. The distances of the two hydrogen bonds (red line) and the distance between the cyano group and the glycine of the P-loop (G166 in DYRK1A) are given in Angstrom. (DYRK1A, PDB ID 3ANR; DYRK2 PDB ID 4AZF; HIPK2, homology model, CLK1, PDB ID 2VAG). E, Comparison of amino acid residues relevant for AnnH75 binding. In the left, the relationship of the kinases is illustrated by the sequence identity of their catalytic domains. Residues generally conserved in protein kinases (G166, K188, D307) and the gatekeeper residue (F238) are highlighted by bold print. Residues different from DYRK1A are shown with their position in the sequence. Differences correlating with kinase resistance to AnnH75 are highlighted in red.

  • the down syndrome related protein kinase DYRK1A phosphorylates p27kip1 and cyclin d1 and induces cell cycle exit and neuronal differentiation
    Cell Cycle, 2014
    Co-Authors: Ulf Soppa, Francisco J Tejedor, Julian Schumacher, Victoria Florencio Ortiz, Tobias Pasqualon, Walter Becker
    Abstract:

    A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G1 phase. Sustained overe...

  • harmine specifically inhibits protein kinase DYRK1A and interferes with neurite formation
    FEBS Journal, 2009
    Co-Authors: Nora Gockler, Guillermo Jofre, Chrisovalantis Papadopoulos, Ulf Soppa, Francisco J Tejedor, Walter Becker
    Abstract:

    DYRK1A is a dual-specificity protein kinase that autophosphorylates a conserved tyrosine residue in the activation loop but phosphorylates exogenous substrates only at serine or threonine residues. Tyrosine autophosphorylation of DYRKs is a one-off event that takes place during translation and induces the activation of the kinase. Here we characterize the beta-carboline alkaloid harmine as a potent and specific inhibitor of DYRK1A both in vitro and in cultured cells. Comparative in vitro assays of four kinases of the DYRK family showed that harmine inhibited substrate phosphorylation by DYRK1A more potently than it inhibited substrate phosphorylation by the closely related kinase DYRK1B [half maximal inhibitory concentrations (IC(50)) of 33 nm versus 166 nm, respectively] and by the more distant members of the family, DYRK2 and DYRK4 (1.9 microm and 80 microm, respectively). Much higher concentrations of harmine were required to suppress tyrosine autophosphorylation of the translational intermediate of DYRK1A in a bacterial in vitro translation system (IC(50) = 1.9 microm). Importantly, harmine inhibited the phosphorylation of a specific substrate by DYRK1A in cultured cells with a potency similar to that observed in vitro (IC(50) = 48 nm), without negative effects on the viability of the cells. Overexpression of the DYRK1A gene on chromosome 21 has been implicated in the altered neuronal development observed in Down syndrome. Here, we show that harmine interferes with neuritogenesis in cultured hippocampal neurons. In summary, our data show that harmine inhibits DYRK1A substrate phosphorylation more potently than it inhibits tyrosine autophosphorylation, and provide evidence for a role of DYRK1A in the regulation of neurite formation.

Nathalie Janel - One of the best experts on this subject based on the ideXlab platform.

  • Molecular Rescue of DYRK1A Overexpression in Cystathionine Beta Synthase-Deficient Mouse Brain by Enriched Environment Combined with Voluntary Exercise
    Journal of Molecular Neuroscience, 2015
    Co-Authors: Benoit Souchet, Jeanmaurice Delabar, Alizée Latour, Fabrice Daubigney, Jean-louis Paul, Nathalie Janel
    Abstract:

    Hyperhomocysteinemia resulting from cystathionine beta synthase (CBS) deficiency can produce cognitive dysfunction. We recently found that CBS-deficient mice exhibit increased expression of the serine/threonine kinase dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A) in the brain. When dysregulated, DYRK1A contributes to the neurodegeneration, neuronal death, and loss of function observed in neurodegenerative diseases. However, brain plasticity can be improved by interventions like enriched environment combined with voluntary exercise (EE/VE). The present study sought to assess the effects of EE/VE on molecular mechanisms linked to DYRK1A overexpression in the brain of CBS-deficient mice. EE/VE was applied to 3-month-old female CBS-deficient mice for 1 month. Without intervention, CBS-deficient mice exhibited increased DYRK1A and decreased brain-derived neurotrophic factor (BDNF) levels in the cortex and hippocampus. However, EE/VE rescued these altered DYRK1A and BDNF levels in the hippocampus of CBS-deficient mice. We conclude that exercise combined with enriched environment can restore the altered molecular mechanisms in the brain of CBS-deficient mice.

  • prefrontal deficits in a murine model overexpressing the down syndrome candidate gene DYRK1A
    The Journal of Neuroscience, 2014
    Co-Authors: Aurore Thomazeau, Benoit Souchet, Fayçal Guedj, Olivier Lassalle, Jillian Iafrati, Nathalie Janel
    Abstract:

    The gene DYRK1A is the mammalian ortholog of Drosophila minibrain. DYRK1A localizes in the Down syndrome (DS) critical region of chromosome 21q22.2 and is a major candidate for the behavioral and neuronal abnormalities associated with DS. PFC malfunctions are a common denominator in several neuropsychiatric diseases, including DS, but the contribution of DYRK1A in PFC dysfunctions, in particular the synaptic basis for impairments of executive functions reported in DS patients, remains obscure. We quantified synaptic plasticity, biochemical synaptic markers, and dendritic morphology of deep layer pyramidal PFC neurons in adult mBACtgDYRK1A transgenic mice that overexpress DYRK1A under the control of its own regulatory sequences. We found that overexpression of DYRK1A largely increased the number of spines on oblique dendrites of pyramidal neurons, as evidenced by augmented spine density, higher PSD95 protein levels, and larger miniature EPSCs. The dendritic alterations were associated with anomalous NMDAR-mediated long-term potentiation and accompanied by a marked reduction in the pCaMKII/CaMKII ratio in mBACtgDYRK1A mice. Retrograde endocannabinoid-mediated long-term depression (eCB-LTD) was ablated in mBACtgDYRK1A mice. Administration of green tea extracts containing epigallocatechin 3-gallate, a potent DYRK1A inhibitor, to adult mBACtgDYRK1A mice normalized long-term potentiation and spine anomalies but not eCB-LTD. However, inhibition of the eCB deactivating enzyme monoacylglycerol lipase normalized eCB-LTD in mBACtgDYRK1A mice. These data shed light on previously undisclosed participation of DYRK1A in adult PFC dendritic structures and synaptic plasticity. Furthermore, they suggest its involvement in DS-related endophenotypes and identify new potential therapeutic strategies.

  • Mice Deficient in Cystathionine Beta Synthase Display Increased DYRK1A and SAHH Activities in Brain
    Journal of Molecular Neuroscience, 2013
    Co-Authors: Chris Planque, Clémentine Ripoll, Christophe Noll, Jean M Delabar, Julien Dairou, Linh-chi Bui, Fayçal Guedj, Nathalie Janel
    Abstract:

    Hyperhomocysteinemia is associated with brain disease. However, biological actions linking hyperhomocysteinemia to neuronal abnormalities are not well understood. We recently found a relationship between DYRK1A protein expression, a serine/threonine kinase that might be responsible for cognitive functions in Down’s syndrome, and hepatic S -adenosylhomocysteine hydrolase (SAHH) activity, which plays a key role in S -adenosylmethionine-dependent methylation reactions. Considering the role of methylation and DYRK1A in cognitive functions, the aim of this study was to investigate the relationship between DYRK1A and SAHH activity in brain of hyperhomocysteinemic mice. We found an increase in DYRK1A protein expression and activity in brain of hyperhomocysteinemic mice, concomitant with an increased SAHH activity. The effect of overexpression of protein DYRK1A on SAHH activity was confirmed in brain of DYRK1A transgenic mice, and additionally we found a positive correlation between DYRK1A and SAHH activity. These observations suggest a potential effect of DYRK1A on brain phenotypes linked to hyperhomocysteinemia.

Maria L Arbones - One of the best experts on this subject based on the ideXlab platform.

  • abnormal mineralization of the ts65dn down syndrome mouse appendicular skeleton begins during embryonic development in a DYRK1A independent manner
    Mechanisms of Development, 2015
    Co-Authors: Joshua D Blazek, Clara S. Moore, Ahmed M Malik, Maeve Tischbein, Maria L Arbones, Randall J. Roper
    Abstract:

    Abstract The relationship between gene dosage imbalance and phenotypes associated with Trisomy 21, including the etiology of abnormal bone phenotypes linked to Down syndrome (DS), is not well understood. The Ts65Dn mouse model for DS exhibits appendicular skeletal defects during adolescence and adulthood but the developmental and genetic origin of these phenotypes remains unclear. It is hypothesized that the postnatal Ts65Dn skeletal phenotype originates during embryonic development and results from an increased DYRK1A gene copy number, a gene hypothesized to play a critical role in many DS phenotypes. Ts65Dn embryos exhibit a lower percent bone volume in the E17.5 femur when compared to euploid embryos. Concomitant with gene copy number, qPCR analysis revealed a  ~1.5 fold increase in DYRK1A transcript levels in the Ts65Dn E17.5 embryonic femur as compared to euploid. Returning DYRK1A copy number to euploid levels in Ts65Dn, DYRK1A+/− embryos did not correct the trisomic skeletal phenotype but did return DYRK1A gene transcript levels to normal. The size and protein expression patterns of the cartilage template during embryonic bone development appear to be unaffected at E14.5 and E17.5 in trisomic embryos. Taken together, these data suggest that the dosage imbalance of genes other than DYRK1A is involved in the development of the prenatal bone phenotype in Ts65Dn embryos.

  • the protein kinase DYRK1A regulates caspase 9 mediated apoptosis during retina development
    Developmental Cell, 2008
    Co-Authors: Ariadna Laguna, Jeanmaurice Delabar, Sergi Aranda, Mariajose Barallobre, R Barhoum, Eduardo Fernandez, Vassiliki Fotaki, Susana De La Luna, Pedro De La Villa, Maria L Arbones
    Abstract:

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

  • DYRK1A haploinsufficiency affects viability and causes developmental delay and abnormal brain morphology in mice
    Molecular and Cellular Biology, 2002
    Co-Authors: Vassiliki Fotaki, Mara Dierssen, Soledad Alcantara, Salvador Martinez, Eulalia Marti, Caty Casas, Joana Visa, Eduardo Soriano, Xavier Estivill, Maria L Arbones
    Abstract:

    DYRK1A is the human orthologue of the Drosophila minibrain (mnb) gene, which is involved in postembryonic neurogenesis in flies. Because of its mapping position on chromosome 21 and the neurobehavioral alterations shown by mice overexpressing this gene, involvement of DYRK1A in some of the neurological defects of Down syndrome patients has been suggested. To gain insight into its physiological role, we have generated mice deficient in DYRK1A function by gene targeting. DYRK1A / null mutants presented a general growth delay and died during midgestation. Mice heterozygous for the mutation (DYRK1A / ) showed decreased neonatal viability and a significant body size reduction from birth to adulthood. General neurobehavioral analysis revealed preweaning developmental delay of DYRK1A / mice and specific alterations in adults. Brains of DYRK1A / mice were decreased in size in a region-specific manner, although the cytoarchitecture and neuronal components in most areas were not altered. Cell counts showed increased neuronal densities in some brain regions and a specific decrease in the number of neurons in the superior colliculus, which exhibited a significant size reduction. These data provide evidence about the nonredundant, vital role of DYRK1A and suggest a conserved mode of action that determines normal growth and brain size in both mice and flies. Dual-specificity tyrosine-regulated kinases (DYRKs) are a novel subfamily of protein kinases that catalyze their autophosphorylation on tyrosine residues and the phosphorylation of serine/threonine residues on exogenous substrates (5, 7, 19). Their kinase activity depends on the presence of a YXY motif in the activation loop of the catalytic domain (20), which is located at the same position as the characteristic TXY motif of the mitogen-activated protein kinases (MAPKs), indicating a possible involvement of these proteins in signal transduction pathways similar to those of the MAPKs (26).

Noriko Murakami - One of the best experts on this subject based on the ideXlab platform.

  • phosphorylation by DYRK1A of clathrin coated vesicle associated proteins identification of the substrate proteins and the effects of phosphorylation
    PLOS ONE, 2012
    Co-Authors: Noriko Murakami, David C Bolton, Elizabeth Kida, Wen Xie, Yuwen Hwang
    Abstract:

    DYRK1A phosphorylated multiple proteins in the clathrin-coated vesicle (CCV) preparations obtained from rat brains. Mass spectrometric analysis identified MAP1A, MAP2, AP180, and α- and β-adaptins as the phosphorylated proteins in the CCVs. Each protein was subsequently confirmed by [(32)P]-labeling and immunological methods. The DYRK1A-mediated phosphorylation released the majority of MAP1A and MAP2 and enhanced the release of AP180 and adaptin subunits from the CCVs. Furthermore, DYRK1A displaced adaptor proteins physically from CCVs in a kinase-concentration dependent manner. The clathrin heavy chain release rate, in contrast, was not affected by DYRK1A. Surprisingly, the DYRK1A-mediated phosphorylation of α- and β-adaptins led to dissociation of the AP2 complex, and released only β-adaptin from the CCVs. AP180 was phosphorylated by DYRK1A also in the membrane-free fractions, but α- and β-adaptins were not. DYRK1A was detected in the isolated CCVs and was co-localized with clathrin in neurons from mouse brain sections and from primary cultured rat hippocampus. Previously, we proposed that DYRK1A inhibits the onset of clathrin-mediated endocytosis in neurons by phosphorylating dynamin 1, amphiphysin 1, and synaptojanin 1. Current results suggest that besides the inhibition, DYRK1A promotes the uncoating process of endocytosed CCVs.

  • mnb DYRK1A phosphorylation regulates the interactions of synaptojanin 1 with endocytic accessory proteins
    Biochemical and Biophysical Research Communications, 2006
    Co-Authors: Tatyana Adayev, Noriko Murakami, Mochou Chenhwang, Rong Wang, Yuwen Hwang
    Abstract:

    Abstract MNB/DYRK1A is a proline-directed serine/threonine kinase implicated in Down syndrome (DS). In an earlier screening, two proteins from adult rat brain, one 100 kDa and the other 140 kDa, were found to be prominently phosphorylated by the kinase. The 100-kDa protein was previously characterized as an isoform of dynamin 1. In this study, we identified the 140-kDa protein as synaptojanin 1 (SJ1). MNB/DYRK1A phosphorylates SJ1 at multiple sites and produces complex behaviors in binding to amphiphysin 1 and intersectin 1 (ITSN1). However, the phosphorylation has little effect on the phosphatidylinositol phosphatase activity of SJ1. These results suggest that MNB/DYRK1A is involved in regulating the recruitment activity but not the phosphatase activity of SJ1. Our findings may be especially important in the etiology of DS because MNB/DYRK1A, SJ1, and ITSN1 are all located at or near the region of human chromosome 21, which is postulated to be involved in the disease.

  • phosphorylation of amphiphysin i by minibrain kinase dual specificity tyrosine phosphorylation regulated kinase a kinase implicated in down syndrome
    Journal of Biological Chemistry, 2006
    Co-Authors: Noriko Murakami, Wen Xie, Mochou Chenhwang, Andrzej Wieraszko, Yuwen Hwang
    Abstract:

    Minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase (Mnb/DYRK1A) is a proline-directed serine/threonine kinase encoded in the Down syndrome critical region of human chromosome 21. This kinase has been shown to phosphorylate dynamin 1 and synaptojanin 1. Here we report that amphiphysin I (Amph I) is also a Mnb/DYRK1A substrate. This kinase phosphorylated native Amph I in rodent brains and recombinant human Amph I expressed in Escherichia coli. Serine 293 (Ser-293) was identified as the major site, whereas serine 295 and threonine 310 were found as minor kinase sites. In cultured cells, recombinant Amph I was phosphorylated at Ser-293 by endogenous kinase(s). Because mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) has been suggested to phosphorylate Amph I at Ser-293, our efforts addressed whether Ser-293 is phosphorylated in vivo by MAPK/ERK or by Mnb/DYRK1A. Overnight serum-withdrawal inactivated MAPK/ERK; nonetheless, Ser-293 was phosphorylated in Chinese hamster ovary and SY5Y cells. Epigallocatechin-3-gallate, a potent Mnb/DYRK1A inhibitor in vitro, apparently reduced the phosphorylation at Ser-293, whereas PD98059, a potent MAPK/ERK inhibitor, did not. High frequency stimulation of mouse hippocampal slices reduced the phosphorylation at Ser-293, albeit in the midst of MAPK/ERK activation. The endophilin binding in vitro was inhibited by phosphorylating Amph I with Mnb/DYRK1A. However, phosphorylation at Ser-293 did not appear to alter cellular distribution patterns of the protein. Our results suggest that Mnb/DYRK1A, not MAPK/ERK, is responsible for in vivo phosphorylation of Amph I at Ser-293 and that phosphorylation changes the recruitment of endophilin at the endocytic sites.