Time filter

Source Type

Sakamoto S.,Tohoku University | Ishizaki T.,Tohoku University | Okawa K.,Drug Discovery Research Laboratories | Watanabe S.,Tohoku University | And 3 more authors.
Journal of Cell Science | Year: 2012

Regulation of the actin cytoskeleton is crucial for cell morphology and migration. mDia is an actin nucleator that produces unbranched actin filaments downstream of Rho. However, the mechanisms by which mDia activity is regulated in the cell remain unknown. We pulled down Liprin-α as an mDia-binding protein. The binding is mediated through the central region of Liprin-α and through the Nterminal Dia-inhibitory domain (DID) and dimerization domain (DD) of mDia. Liprin-α competes with Dia autoregulatory domain (DAD) for binding to DID, and binds preferably to the open form of mDia. Overexpression of a Liprin-α fragment containing the mDiabinding region decreases localization of mDia to the plasma membrane and attenuates the Rho-mDia-mediated formation of stress fibers in cultured cells. Conversely, depletion of Liprin-α by RNA interference (RNAi) increases the amount of mDia in the membrane fraction and enhances formation of actin stress fibers. Thus, Liprin-α negatively regulates the activity of mDia in the cell by displacing it from the plasma membrane through binding to the DID-DD region. © 2012.

Tanaka G.,Kyoto University | Nakase I.,Kyoto University | Fukuda Y.,Kyoto University | Masuda R.,Kyoto University | And 12 more authors.
Chemistry and Biology | Year: 2012

CXCR4 is a coreceptor of HIV-1 infection in host cells. Through a photocrosslinking study to identify receptors involved in internalization of oligoarginine cell-penetrating peptides (CPPs), we found that CXCR4 serves as a receptor that stimulates macropinocytic uptake of the arginine 12-mer peptide (R12) but not of the 8-mer. We also found that stimulating CXCR4 with its intrinsic ligands, stromal cell-derived factor 1α and HIV-1 envelope glycoprotein 120, induced macropinocytosis. R12 had activity to prevent viral infection for HIV-1IIIB, a subtype of HIV-1 that uses CXCR4 as a coreceptor for entry into susceptible cells, whereas the addition of a macropinocytosis inhibitor, dimethylamiloride, resulted in enhancement of viral infection. The present study shows that CXCR4 triggers macropinocytosis, which may have implications for the cellular uptake of oligoarginine CPPs and internalization of HIV. © 2012 Elsevier Ltd.

Kawasoe M.,Kyoto University | Yamamoto Y.,Kyoto University | Okawa K.,Drug Discovery Research Laboratories | Funato T.,Tohoku Fukushi University | And 9 more authors.
Experimental Hematology | Year: 2013

The elucidation of drug resistance mechanisms is important in the development of clinical therapies for the treatment of leukemia. To study the drug resistance mechanisms, protein expression profiles of 1-β-D-arabinofuranosylcytosine (AraC)-sensitive K562 (K562S) cells and AraC-resistant K562 (K562AC) cells were compared using two-dimensional fluorescence difference gel electrophoresis. In a comparison of protein expression profiles, 2073 protein spots were found to be altered, and 15 proteins of them were remarkably altered. These proteins were identified by mass spectrometry. The most differently expressed proteins were aldehyde dehydrogenase 1 family member A2 (ALDH1A2) and vimentin. Both proteins were verified using reverse transcriptase polymerase chain reaction and Western blot analysis. ALDH1A2 protein was found to be effective in AraC resistance. ALDH1A2 knock-down induced sensitivity to AraC treatment in K562AC cells, and ALDH1A2 overexpressed K562S cells acquired the AraC resistance. Furthermore, the findings also suggest that ALDH1A2 expression is increased after the appearance of AraC resistance in clinical cases. These results will be helpful in understanding the mechanism of AraC resistance. © 2013 ISEH - Society for Hematology and Stem Cells.

Orihashi K.,Kyoto University | Orihashi K.,Osaka City University | Tojo H.,Osaka University | Okawa K.,Drug Discovery Research Laboratories | And 3 more authors.
Biological Chemistry | Year: 2012

Mammalian carboxylesterase (CES) is well known as a biotransformation enzyme for prodrugs and xenobiotics. Here, we purified CES as a GPI-anchored protein (GPI-AP)- releasing factor (GPIase) that releases such protein from the cell surface. All five isoforms of CES showed this activity to various degrees. When the serine residue of the catalytic triad for esterase was replaced by alanine, esterase activity was completely disrupted, while full GPIase activity remained, suggesting that these two activities are exhibited via different mechanisms. CES6, a new class of mammalian CES, exhibited the highest GPIase activity and released specific GPI-APs from the cell surface after lipid raft fluidization. The released product contained a GPI component, indicating that GPI-AP was released by cleavage in GPI. These results revealed for the first time that CES recognizes and catalyzes macromolecule GPI-AP as well as small molecules. © 2012 by Walter de Gruyter · Berlin · Boston.

Ohsawa Y.,Kawasaki Medical School | Okada T.,Kawasaki Medical School | Nishimatsu S.-I.,Kawasaki Medical School | Ishizaki M.,Kumamoto University | And 10 more authors.
Laboratory Investigation | Year: 2012

Skeletal muscle expressing Pro104Leu mutant caveolin 3 (CAV3 P104L) in mouse becomes atrophied and serves as a model of autosomal dominant limb-girdle muscular dystrophy 1C. We previously found that caveolin 3-deficient muscles showed activated intramuscular transforming growth factor beta (TGF-Β) signals. However, the cellular mechanism by which loss of caveolin 3 leads to muscle atrophy is unknown. Recently, several small-molecule inhibitors of TGF-Β type I receptor (TΒRI) kinase have been developed as molecular-targeting drugs for cancer therapy by suppressing intracellular TGF-Β1,-Β2, and-Β3 signaling. Here, we show that a TΒRI kinase inhibitor, Ki26894, restores impaired myoblast differentiation in vitro caused by activin, myostatin, and TGF-Β1, as well as CAV3 P104L. Oral administration of Ki26894 increased muscle mass and strength in vivo in wild-type mice, and improved muscle atrophy and weakness in the CAV3 P104L mice. The inhibitor restored the number of satellite cells, the resident stem cells of adult skeletal muscle, with suppression of the increased phosphorylation of Smad2, an effector, and the upregulation of p21 (also known as Cdkn1a), a target gene of the TGF-Β family members in muscle. These data indicate that both TGF-Β-dependent reduction in satellite cells and impairment of myoblast differentiation contribute to the cellular mechanism underlying caveolin 3-deficient muscle atrophy. TΒRI kinase inhibitors could antagonize the activation of intramuscular anti-myogenic TGF-Β signals, thereby providing a novel therapeutic rationale for the alternative use of this type of anticancer drug in reversing muscle atrophy in various clinical settings. © 2012 USCAP, Inc All rights reserved.

Discover hidden collaborations