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Kenji Sugimoto

Publications and source records attributed to Kenji Sugimoto.

24 records · Page 2Linked to original sources
PubMed2002

G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis.

Covalent modification of histone tails is crucial for transcriptional regulation, mitotic chromosomal condensation, and heterochromatin formation. Histone H3 lysine 9 (H3-K9) methylation catalyzed by the Suv39h family proteins is essential for establishing the architecture of pericentric heterochromatin. We recently identified a mammalian histone methyltransferase (HMTase), G9a, which has strong HMTase activity towards H3-K9 in vitro. To investigate the in vivo functions of G9a, we generated G9a-deficient mice and embryonic stem (ES) cells. We found that H3-K9 methylation was drastically decreased in G9a-deficient embryos, which displayed severe growth retardation and early lethality. G9a-deficient ES cells also exhibited reduced H3-K9 methylation compared to wild-type cells, indicating that G9a is a dominant H3-K9 HMTase in vivo. Importantly, the loss of G9a abolished methylated H3-K9 mostly in euchromatic regions. Finally, G9a exerted a transcriptionally suppressive function that depended on its HMTase activity. Our results indicate that euchromatic H3-K9 methylation regulated by G9a is essential for early embryogenesis and is involved in the transcriptional repression of developmental genes.

Acetylation↗
PubMed2002

Aurora-B associated protein phosphatases as negative regulators of kinase activation.

The human serine/threonine kinase Aurora-B is structurally related to the protein kinase Ipl1p from S cerevisiae and aurora from Drosophila melanogaster, which are key regulators of mitosis. The present study shows that human Aurora-B is activated by okadaic acid and forms complexes with the protein serine/threonine phosphatase type 1 (PP1) or PP2A, but not with PP5. These data identified Aurora-B associated protein phosphatases as negative regulators of kinase activation. We then used a series of substrates based on a histone H3 phosphorylation site (residues 5-15) to determine the substrate specificity of human Aurora-B. We found that this enzyme is an arginine-directed kinase that can phosphorylate histone H3 at serines 10 and 28 in vitro, suggesting that human Aurora-B is a mitotic histone H3 kinase.

Animals↗
PubMed2002

A new fluorescent biosensor for inositol trisphosphate.

An intracellular second messenger d-myo-inositol-1,4,5-trisphosphate (IP3) is a key biological signaling molecule that controls the cellular Ca2+ concentration. We report the preparation and evaluation of a functionalized protein-based sensor for IP3 by exploring the selective IP3 binding properties of pleckstrin homology (PH) domain. Signal transduction is imparted to the protein by mutation of proximal residues to cysteine and then alkylation of the active site by various fluorophore derivatives. This creates functionalized proteins that show micromolar affinity for IP3, reasonably strong fluorescence emission, and wavelength changes in the fluorophore and selectivity higher than the original PH domain among different inositol phosphate derivatives.

Amino Acid Sequence↗
PubMed2002

The binding of lamin B receptor to chromatin is regulated by phosphorylation in the RS region.

Binding of lamin B receptor (LBR) to chromatin was studied by means of an in vitro assay system involving recombinant fragments of human LBR and Xenopus sperm chromatin. Glutathione-S-transferase (GST)-fused proteins including LBR fragments containing the N-terminal region (residues 1-53) and arginine-serine repeat-containing region (residues 54-89) bound to chromatin. The binding of GST-fusion proteins incorporating the N-terminal and arginine-serine repeat-containing regions to chromatin was suppressed by mild trypsinization of the chromatin and by pretreatment with a DNA solution. A new cell-free system for analyzing the cell cycle-dependent binding of a protein to chromatin was developed from recombinant proteins, a Xenopus egg cytosol fraction and sperm chromatin. The system was applied to analyse the binding of LBR to chromatin. It was shown that the binding of LBR fragments to chromatin was stimulated by phosphorylation in the arginine-serine repeat-containing region by a protein kinase(s) in a synthetic phase egg cytosol. However, the binding of LBR fragments was suppressed by phosphorylation at different residues in the same region by a kinase(s) in a mitotic phase cytosol. These results suggested that the cell cycle-dependent binding of LBR to chromatin is regulated by phosphorylation in the arginine-serine repeat-containing region by multiple kinases.

Animals↗
PubMed2002

Molecular dynamics of Aurora-A kinase in living mitotic cells simultaneously visualized with histone H3 and nuclear membrane protein importinalpha.

Aurora-A is known to be a mitotic kinase required for spindle assembly. We constructed a human stable cell-line in which Aurora-A, histone H3 and importinalpha were differentially expressed as fusions to green, cyan, and red fluorescent proteins (GFP, CFP and DsRed). In interphase cells, GFP-Aurora-A was localized in the centrosome. Its molecular behavior in living mitotic cells was extensively analyzed by an advanced timelapse image analyzing system. In G2 phase, duplicated centrosomal dots of Aurora-A separated and moved to the opposite poles, a process requiring 18 min. In prophase, the Aurora-A dots approached closer and the nuclear membrane of DsRed-importinalpha beneath them became thick and invaginated, resulting in a "dumb-bell" shaped nucleus with condensed chromatin. As the importinalpha membrane further shrank and disappeared, the condensed chromatin was excluded from the nucleus and the Aurora-A dots grew rapidly into a spindle-like structure. Congression of mitotic chromosomes continued for 20-50 min until they were properly aligned at the spindle equator and then the sister chromatids started to segregate, taking 4-6 min for them to reach the poles. An importinalpha membrane reappeared around the surface of chromatin 10 min after anaphase onset. Aurora-A gradually decreased in size in telophase and returned to the surface of the newly formed small sister nuclei. These observations showed that the morphological change of Aurora-A was cooperated with the breakdown and reformation of nuclear membrane. Immunostaining with anti-alpha or gamma-tubulin further indicated that Aurora-A was involved in the formation of mitotic spindle in metaphase as well as the subsequent chromosome movement in anaphase.

Aurora Kinases↗