Papers by Kazuhito Tomizawa
The Japanese Biochemical Society/The Molecular Biology Society of Japan, Nov 2, 2015
Background Information. Cortactin contributes to growth cone morphogenesis by forming with dynami... more Background Information. Cortactin contributes to growth cone morphogenesis by forming with dynamin, ringshaped complexes that mechanically bundle and stabilise F-actin. However, the regulatory mechanism of cortactin action is poorly understood. Results. Immunofluorescence microscopy revealed that protein kinase C (PKC) α colocalises with cortactin at growth cone filopodia in SH-SY5Y neuroblastoma cells. PKC activation by phorbol 12-myristate 13-acetate causes cortactin phosphorylation, filopodial retraction and F-actin-bundle loss. Moreover, PKCα directly phosphorylates cortactin in vitro at S135/T145/S172, mitigating both cortactin's actin-binding and actin-crosslinking activity, whereas cellular expression of a phosphorylation-mimetic cortactin mutant hinders filopodial formation with a significant decrease of actin bundles. Conclusions. Our results indicate that PKC-mediated cortactin phosphorylation might be implicated in the maintenance of growth cone.
Molecular and Cellular Neuroscience, 2012
Although synaptotagmin I, which is a calcium (Ca 2+)-binding synaptic vesicle protein, may trigge... more Although synaptotagmin I, which is a calcium (Ca 2+)-binding synaptic vesicle protein, may trigger soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated synaptic vesicle exocytosis, the mechanisms underlying the interaction between these proteins remains controversial, especially with respect to the identity of the protein(s) in the SNARE complex that bind(s) to synaptotagmin and whether Ca 2+ is required for their highly effective binding. To address these questions, native proteins were solubilized, immunoprecipitated from rat brain extracts, and analyzed by immunoblotting. SNARE complexes comprising syntaxin 1, 25-kDa synaptosomal-associated protein (SNAP-25), and synaptobrevin 2 were coprecipitated with synaptotagmin I in the presence of ethylene glycol tetraacetic acid. The amount of coprecipitated proteins was significantly unaltered by the addition of Ca 2+ to the brain extract. To identify the component of the SNARE complex that bound to synaptotagmin, SNARE was coexpressed with synaptotagmin in HEK293 cells and immunoprecipitated. Syntaxin, but not SNAP-25 and synaptobrevin, bound to synaptotagmin in a Ca 2+-independent manner, and the binding was abolished in the presence of 1 M NaCl. Synaptotagmin contains 2 Ca 2+-binding domains (C 2 A, C 2 B). Mutating the positively charged lysine residues in the putative effector-binding region of the C 2 B domain, which are critical for transmitter release, markedly inhibited synaptotagmin-syntaxin binding, while similar mutations in the C 2 A domain had no effect on binding. Synaptotagmin-syntaxin binding was reduced by mutating multiple negatively charged glutamate residues in the amino-terminal half of the syntaxin SNARE motif. These results indicate that synaptotagmin I binds to syntaxin 1 electrostatically through its C 2 B domain effector region in a Ca 2+-independent 3 fashion, providing biochemical evidence that synaptotagmin I binds SNARE complexes before Ca 2+ influx into presynaptic nerve terminals.
Journal of Neurochemistry, Jun 30, 2005
Cdk5 (cyclin-dependent kinase 5) activity is dependent upon association with one of two neuron-sp... more Cdk5 (cyclin-dependent kinase 5) activity is dependent upon association with one of two neuron-specific activators, p35 or p39. Genetic deletion of Cdk5 causes perinatal lethality with severe defects in corticogenesis and neuronal positioning. p35-/mice are viable with milder histological abnormalities. Although substantial evidence implicates Cdk5 in synaptic plasticity, its role in learning and memory has not been evaluated using mutant mouse models. We report here that p35-/mice have deficiencies in spatial learning and memory. Close examination of hippocampal circuitry revealed subtle histological defects in CA1 pyramidal cells. Furthermore, p35-/mice exhibit impaired long-term depression and depotentiation of long-term potentiation in the Schaeffer collateral CA1 pathway. Moreover, the Cdk5-dependent phosphorylation state of protein phosphatase inhibitor-1 was increased in 4week-old mice due to increased levels of p39, which colocalized with inhibitor-1 and Cdk5 in the cytoplasm. These results demonstrate that p35-dependent Cdk5 activity is important to learning and synaptic plasticity. Deletion of p35 may shift the substrate specificity of Cdk5 due to compensatory expression of p39.
PubMed, Aug 1, 2018
Genetic variation in Cdk5 Regulatory Associated Protein 1-Like 1 (CDKAL1) is associated with the ... more Genetic variation in Cdk5 Regulatory Associated Protein 1-Like 1 (CDKAL1) is associated with the development of type 2 diabetes (T2D). Dysfunction of CDKAL1 impairs the translation of proinsulin, which leads to glucose intolerance. Eperisone, an antispasmodic agent, has been shown to ameliorate glucose intolerance in Cdkal1-deficient mice. We have launched a phase II clinical study to investigate the potential anti-diabetic effect of eperisone in T2D patients carrying risk or non-risk alleles of CDKAL1. The primary endpoint is the change of hemoglobin A1c (HbA1c) levels. We also examined whether the efficacy of eperisone in T2D patients is associated with CDKAL1 activity.
Neuroscience Research, 2009
s S79 on presynaptic membranes to organize active zones. However, laminin 2 does not localize at ... more s S79 on presynaptic membranes to organize active zones. However, laminin 2 does not localize at the brain synapses. We hypothesized that the presynaptic VGCC interacts with a new ligand to induce presynaptic differentiation in the brain. We purified VGCC interacting proteins by affinity pull-down from the synaptic membrane fraction of mouse brains, and identified the ligand as ATP synthase by mass spectrometry. The direct binding of the two was confirmed by immunoprecipitation using recombinant proteins. We detected the ATP synthase on postsynaptic membranes by immunoelectron microscopy. In cultured cerebellar granule cells, beads coated with recombinant ATP synthase induced aggregations of synapsin 1, SV2, and bassoon at the bead-neuron contact sites. These results suggest that VGCC interacts with cell surface ATP synthase to induce presynaptic differentiation in the brain. doi:10.1016/j.neures.2009.09.291 P1-b01 Unbiased expression screen for synaptogenic proteins based on fibroblast-neuron coculture Daisaku Yokomaku, Hideto Takahashi, Tabrez J. Siddiqui, Michael W. Linhoff, Ann Marie Craig Brain Res. Centre, University of British Columbia, Vancouver, Canada Formation and maturation of chemical synapses in the brain requires interactions between preand post-synaptic partners. Previous reports have uncovered some “synaptogenic” proteins. However, few studies have attempted global identification of synaptogenic proteins. To search for novel synaptogenic proteins, we created a set of full-length enriched cDNA expression libraries from rat brain. For co-culture assay, we transfected each sampling DNA pool into COS-7 cells, harvested them 24 h later, and then seeded them on hippocampal neuron coverslips. After 24 h of co-culture, coverslips were immunostained with synapsin I, PSD-95 and gephyrin. A cDNA pool was considered positive if it generated any COS cells with significant associated synapsin clusters unapposed to PSD-95 or gephyrin. We identified several novel synaptogenic proteins by this method. In conclusion, our novel screen using fibroblast-neuron co culture assay is a useful and powerful method for the identification of synaptogenic proteins. doi:10.1016/j.neures.2009.09.292 P1-b02 Phenotype analysis of syntaxin1B knockout mice Tomonori Fujiwara1, Masumi Sanada1, Takefumi Kofuji 2, Tatsuya Mishima1, Masami Kanai-Azuma3, Kimio Akagawa1 1 Dept. Cell Physiology, Kyorin University School of Medicine, Tokyo, Japan; 2 Radio Isotope Lab., Kyorin University School of Medicine, Tokyo, Japan; 3 Dept. Anatomy, Kyorin University School of Medicine, Tokyo, Japan Syntaxin1 has been thought to regulate the exocytosis of synaptic vesicles. In neurons, two types of syntaxin1 isoforms, HPC-1/syntaxin1A (STX1A) and syntaxin1B (STX1B), both of which are believed to have similar function in CNS, are expressed. Previously, we have reported that STX1A null mutant mice developed normally and glutamatergic or GABAergic synaptic transmission in vitro was normal, although the synaptic plasticity in vitro and in vivo was impaired (Fujiwara et al., 2006). In this study, we have generated STX1B gene knockout mice. STX1B heterozygous mutant mice exhibited seizure phenotype, but survived at least until 6 month. STX1B null mutant mice were born alive, but were dead within 2 weeks after birth unlike the case of STX1A null mutant mice. In addition, null mutant mice revealed morphological abnormalities in CNS. Differences of the phenotypes between null mutant mice of STX1A and STX1B will be discussed. doi:10.1016/j.neures.2009.09.293 P1-b03 Negatively charged amino acid residues of syntaxin 1 critical for synaptotagmin 1 binding Toshio Masumoto1, Tei-ich Nishiki1, Iori Omori1, Kazuhito Tomizawa2, Hideki Matsui1 1 Dept. Physiol, Okayama Univ., Okayama, Japan; 2 Dept. Molecular Physiol, Kumamoto Univ., Kumamoto, Japan Recently, we have proposed a model where synaptotagmin (syt) 1 binds a neural SNARE complex in presynaptic nerve terminals at resting state. To study the interaction between syt1 and SNARE, recombinant proteins expressed in HEK293 cells were immunoprecipitated with an anti-syt1 antibody. Among 3 kinds of SNAREs, syntaxin bound syt1 in the presence of EGTA, but not SNAP-25 and VAMP-2. A SNARE complex binding-defective syt1 mutant markedly reduced syntaxin binding, indicating that syntaxin mediates Ca2+-independent syt1/SNARE complex interaction. Fourteen negatively charged, conserved amino acid residues in the SNARE motif (9 Glu (E1-E9) and 5 Asp (D1-D5) residues) of syntaxin were replaced with Gln (Q) or Asn (N) in combination with an adjacent one. Three mutants (E1,2Q, E3,4Q, and E5Q/D1N) reduced their syt1 binding by 30-40%, indicating importance of multiple Glu and/or Asp of syntaxin for syt1 binding. Thus, before Ca2+ influx, syt1 could bind to the SNARE complex that is thought to assemble during priming steps. doi:10.1016/j.neures.2009.09.294 P1-b04 Synaptic integrations switched by neuromodulators: Their cellular…
eLife, Nov 14, 2019
Cholesterol is a major structural component of the plasma membrane (PM). The majority of PM chole... more Cholesterol is a major structural component of the plasma membrane (PM). The majority of PM cholesterol forms complexes with other PM lipids, making it inaccessible for intracellular transport. Transition of PM cholesterol between accessible and inaccessible pools maintains cellular homeostasis, but how cells monitor the accessibility of PM cholesterol remains unclear. We show that endoplasmic reticulum (ER)-anchored lipid transfer proteins, the GRAMD1s, sense and transport accessible PM cholesterol to the ER. GRAMD1s bind to one another and populate ER-PM contacts by sensing a transient expansion of the accessible pool of PM cholesterol via their GRAM domains. They then facilitate the transport of this cholesterol via their StART-like domains. Cells that lack all three GRAMD1s exhibit striking expansion of the accessible pool of PM cholesterol as a result of less efficient PM to ER transport of accessible cholesterol. Thus, GRAMD1s facilitate the movement of accessible PM cholesterol to the ER in order to counteract an acute increase of PM cholesterol, thereby activating non-vesicular cholesterol transport.
Nature Medicine, Sep 11, 2005
Biomolecules
SARS-CoV-2 infection alters cellular RNA content. Cellular RNAs are chemically modified and event... more SARS-CoV-2 infection alters cellular RNA content. Cellular RNAs are chemically modified and eventually degraded, depositing modified nucleosides into extracellular fluids such as serum and urine. Here we searched for COVID-19-specific changes in modified nucleoside levels contained in serum and urine of 308 COVID-19 patients using liquid chromatography-mass spectrometry (LC-MS). We found that two modified nucleosides, N6-threonylcarbamoyladenosine (t6A) and 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A), were elevated in serum and urine of COVID-19 patients. Moreover, these levels were associated with symptom severity and decreased upon recovery from COVID-19. In addition, the elevation of similarly modified nucleosides was observed regardless of COVID-19 variants. These findings illuminate specific modified RNA nucleosides in the extracellular fluids as biomarkers for COVID-19 infection and severity.
Communications Biology
In mammalian mitochondria, translation of the AUA codon is supported by 5-formylcytidine (f5C) mo... more In mammalian mitochondria, translation of the AUA codon is supported by 5-formylcytidine (f5C) modification in the mitochondrial methionine tRNA anticodon. The 5-formylation is initiated by NSUN3 methylase. Human NSUN3 mutations are associated with mitochondrial diseases. Here we show that Nsun3 is essential for embryonic development in mice with whole-body Nsun3 knockout embryos dying between E10.5 and E12.5. To determine the functions of NSUN3 in adult tissue, we generated heart-specific Nsun3 knockout (Nsun3HKO) mice. Nsun3HKO heart mitochondria were enlarged and contained fragmented cristae. Nsun3HKO resulted in enhanced heart contraction and age-associated mild heart enlargement. In the Nsun3HKO hearts, mitochondrial mRNAs that encode respiratory complex subunits were not down regulated, but the enzymatic activities of the respiratory complexes decreased, especially in older mice. Our study emphasizes that mitochondrial tRNA anticodon modification is essential for mammalian emb...
International Journal of Oncology, 2019
Dynamin copolymerizes with cortactin to form a ring-like complex that bundles and stabilizes acti... more Dynamin copolymerizes with cortactin to form a ring-like complex that bundles and stabilizes actin filaments. Actin bundle formation is crucial for generation of filopodia and lamellipodia, which guide migration, invasion, and metastasis of cancer cells. However, it is unknown how the dynamin-cortactin complex regulates actin bundle formation. The present study investigated phosphorylation of cortactin by cyclin-dependent kinase 5 (CDK5) and its effect on actin bundle formation by the dynamin-cortactin complex. CDK5 directly phosphorylated cortactin at T145/T219 in vitro. Phosphomimetic mutants in which one or both of these threonine residues was substituted by aspartate were used. The three phosphomimetic mutants (T145D, T219D and T145DT219D) had a decreased affinity for F-actin. Furthermore, electron microscopy demonstrated that these phosphomimetic mutants could not form a ring-like complex with dynamin 1. Consistently, the dynamin 1-phosphomimetic cortactin complexes exhibited d...
The Journal of Biochemistry, 2020
A fundamental aspect of mitochondria is that they possess DNA and protein translation machinery. ... more A fundamental aspect of mitochondria is that they possess DNA and protein translation machinery. Mitochondrial DNA encodes 22 tRNAs that translate mitochondrial mRNAs to 13 polypeptides of respiratory complexes. Various chemical modifications have been identified in mitochondrial tRNAs via complex enzymatic processes. A growing body of evidence has demonstrated that these modifications are essential for translation by regulating tRNA stability, structure and mRNA binding, and can be dynamically regulated by the metabolic environment. Importantly, the hypomodification of mitochondrial tRNA due to pathogenic mutations in mitochondrial tRNA genes or nuclear genes encoding modifying enzymes can result in life-threatening mitochondrial diseases in humans. Thus, the mitochondrial tRNA modification is a fundamental mechanism underlying the tight regulation of mitochondrial translation and is essential for life. In this review, we focus on recent findings on the physiological roles of 5-tau...
Naked mole-rats (NMRs) have a very low spontaneous carcinogenesis rate, which has prompted scient... more Naked mole-rats (NMRs) have a very low spontaneous carcinogenesis rate, which has prompted scientists to study their cancer resistance mechanisms in order to provide clues for human cancer prevention. Although cancer resistance in NMRs has been intensively investigated at the cellular level, it is still unknown how strongly resistant NMR individuals are to carcinogenesis and how NMR tissues respond to experimental carcinogenesis induction. Here, we show that NMRs exhibit extraordinary resistance against potent chemical carcinogenesis induction through a dampened inflammatory response. Although carcinogenic insults damaged skin cells of both NMRs and mice, NMR skin showed markedly lower immune cell infiltration and reduced induction of inflammatory genes. NMRs harbor loss-of-function mutations in receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) genes, which are essential for necroptosis, a type of necrotic cell death that activates strong infl...
eLife, 2019
Cholesterol is a major structural component of the plasma membrane (PM). The majority of PM chole... more Cholesterol is a major structural component of the plasma membrane (PM). The majority of PM cholesterol forms complexes with other PM lipids, making it inaccessible for intracellular transport. Transition of PM cholesterol between accessible and inaccessible pools maintains cellular homeostasis, but how cells monitor the accessibility of PM cholesterol remains unclear. We show that endoplasmic reticulum (ER)-anchored lipid transfer proteins, the GRAMD1s, sense and transport accessible PM cholesterol to the ER. GRAMD1s bind to one another and populate ER-PM contacts by sensing a transient expansion of the accessible pool of PM cholesterol via their GRAM domains. They then facilitate the transport of this cholesterol via their StART-like domains. Cells that lack all three GRAMD1s exhibit striking expansion of the accessible pool of PM cholesterol as a result of less efficient PM to ER transport of accessible cholesterol. Thus, GRAMD1s facilitate the movement of accessible PM cholester...
iScience, 2019
2-Methylthio-N 6-isopentenyl modification of adenosine (ms 2 i 6 A) is an evolutionally conserved... more 2-Methylthio-N 6-isopentenyl modification of adenosine (ms 2 i 6 A) is an evolutionally conserved modification found in mitochondrial (mt)-tRNAs. Cdk5 regulatory subunit-associated protein 1 (CDK5RAP1) specifically converts N6-isopentenyladenosine (i 6 A) to ms 2 i 6 A at position A37 of four mt-DNA-encoded tRNAs, and the modification regulates efficient mitochondrial translation and energy metabolism in mammals. Here, we report that the ms 2 conversion mediated by CDK5RAP1 in mt-tRNAs is required to sustain glioma-initiating cell (GIC)-related traits. CDK5RAP1 maintained the self-renewal capacity, undifferentiated state, and tumorigenic potential of GICs. This regulation was not related to the translational control of mt-proteins. CDK5RAP1 abrogated the antitumor effect of i 6 A by converting i 6 A to ms 2 i 6 A and protected GICs from excessive autophagy triggered by i 6 A. The elevated activity of CDK5RAP1 contributed to the amelioration of the tumor-suppressive effect of i 6 A and promoted GIC maintenance. This work demonstrates that CDK5RAP1 is crucial for the detoxification of endogenous i 6 A and that GICs readily utilize this mechanism for survival.
Endocrine Journal, 2019
CDK5 regulatory subunit associated protein 1-like 1 (CDKAL1) is a tRNA-modifying enzyme that cata... more CDK5 regulatory subunit associated protein 1-like 1 (CDKAL1) is a tRNA-modifying enzyme that catalyzes 2methylthiolation (ms 2) and has been implicated in the development of type 2 diabetes (T2D). CDKAL1-mediated ms 2 is important for efficient protein translation and regulates insulin biosynthesis in pancreatic cells. Interestingly, an association between T2D and release of growth hormone (GH) has been reported in humans. However, it is unknown whether CDKAL1 is important for hormone production in the pituitary gland. The present study investigated the role of CDKAL1 in GHproducing pituitary adenomas (GHPAs). CDKAL1 activity was suppressed in GHPAs, as evidenced by a decrease in ms 2 , compared with non-functioning pituitary adenomas (NFPAs), which do not produce specific hormones. Downregulation of Cdkal1 using small interfering and short hairpin RNAs increased the biosynthesis and secretion of GH in rat GH3 cells. Depletion of Cdkal1 increased the cytosolic calcium level via downregulation of DnaJ heat shock protein family (Hsp40) member C10 (Dnajc10), which is an endoplasmic reticulum protein related to calcium homeostasis. This stimulated transcription of GH via upregulation of Pit-1. Moreover, CDKAL1 activity was highly sensitive to proteostatic stress and was upregulated by suppression of this stress. Taken together, these results suggest that dysregulation of CDKAL1 is involved in the pathogenesis of GHPAs, and that modulation of the proteostatic stress response might control CDKAL1 activity and facilitate treatment of GHPAs.
ABSTRACTPost-transcriptional modifications in mitochondrial tRNAs (mt-tRNAs) play critical roles ... more ABSTRACTPost-transcriptional modifications in mitochondrial tRNAs (mt-tRNAs) play critical roles in mitochondrial protein synthesis, which produces respiratory chain complexes. In this study, we used mass spectrometric analysis to map 5-methylcytidine (m5C) at positions 48–50 in eight mouse and six human mt-tRNAs. We also confirmed the absence of m5C in mt-tRNAs isolated fromNsun2knockout (KO) mice, as well as fromNSUN2KO human culture cells. In addition, we successfully reconstituted m5C at positions 48–50 of mt-tRNAin vitrowith NSUN2 protein in the presence ofS-adenosylmethionine (SAM). Although NSUN2 is predominantly localized to the nucleus and introduces m5C into cytoplasmic tRNAs and mRNAs, structured illumination microscopy (SIM) clearly revealed NSUN2 foci inside mitochondria. These observations provide novel insights into the role of NSUN2 in the physiology and pathology of mitochondrial functions.
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Papers by Kazuhito Tomizawa