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Phosphorylation on TRPV4 Serine Residue 824 Enhances Its Association with PGM1

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American Journal of Molecular Biology 2016年第1期6卷 33-44页

作者： Sung Hwa Shin eun jeoung lee Sunghee Hyun Sang Sun Kang Department of Biology Education Chungbuk National University Cheongju Republic of Korea Department of Internal Medicine Chungbuk National University Cheongju Republic of Korea Department of Biomedical Laboratory Science Eulji University Daejeon Republic of Korea

The TRPV4 cation channel is expressed in a broad range of tissues and participates in the generation of a Ca2+ signal and/or depolarization of membrane potential. Here, human phosphoglucomutase- 1 (PGM1), an enzyme that converts glucose-6 phosphate to glucose-1 phosphate in the glycolysis pathway, as the first auxiliary protein of TRPV4 Ca2+ channels, is identified with yeast two hybrid system, coimmunoprecipitation, confocal microscopy, and GST pull-down assays. TRPV4 forms a complex with PGM1 through its C-terminal cytoplasmic domain. Because it is demonstrated that TRPV4 serine residue 824 (S824) is phosphorylated by serum/glucocorticoid regulated kinase 1, we elucidate the effect of TRPV4 S824 phosphorylation on TRPV association with PGM1. Even an inactivated mutant version of TRPV4, S824A, exhibited a decreased ability to bind PGM1, an activated phosphomimetic mutant version of TRPV4, S824D, exhibited enhanced binding to PGM1. Thus, formation of the TRPV4/PGM1 complex and localization of this complex to the plasma membrane appear to be regulated by the phosphorylation status of residue S824 in TRPV4. The newly identified interactor of TRPV4 may help the molecular pathways modulating transport activity or glucose metabolism, respectively.

关键词： Membrane Localization Phosphoglucomutase1 Phosphorylation Protein-Protein Interaction TRPV4

Ablation of TRPV4 in HepG2 with Its CRISPR/Cas9 Enhances Its Wound Healing

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American Journal of Molecular Biology 2020年第1期10卷 74-89页

作者： eun jeoung lee Sung Hwa Shin Sunghee Hyun Sang Sun Kang CdmoGen Cheongju Republic of Korea Department of Pre-Medicine School of Medicine Eulji University Daejeon Republic of Korea Department of Biology Education Chungbuk National University Cheongju Republic of Korea

TRPV4 activity modulates cell activities including receptor trafficking and transcriptional or translational regulations. We tested its CRISPR/Cas9 scissor efficacy in HepG2 (HEK293) cell noticed that it worked well in both cell lines to eliminate TRPV4 genome sequences. To confirm TRPV4 functions in the cell morphology maintenance and cell growth (beyond Ca2+ channel), we compared its wound healing, cell surface area, survival property and soft agar growth ability after deletion of TRPV4 gene in the cells with its CRISPR/Cas9 system. With these experiments, we confirmed that TRPV4 is required not only to function as Ca2+ channel but also to maintain its proper cell morphology as a corner stone protein on the cell adhesion junction.

关键词： TRPV4 Ca2+ Ion Channel E-Cadherin Tubulin Soft Agar Growth CRISPR/Cas9 Scissor System

SGK1 Inhibits ULK2 Autophagy Activity

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American Journal of Molecular Biology 2020年第1期10卷 12-28页

作者： Sung Hwa Shin eun jeoung lee Sunghee Hyun Sang Sun Kang Department of Biology Education Chungbuk National University Cheongju Korea Department of Biomedical Laboratory Science Eulji University Daejeon Korea

Serum- and glucocorticoid-induced kinase 1 (SGK1) is known to have consensus sequence of phosphorylation site R-x-R-x-x-(S/T)-Φ, where Φ is any hydrophobic amino acid and arginine residues are conserved at positions −5 and −3 relative to positions of Ser/Thr residues that are phosphorylated in the presence of SGK1. UNC-21-like kinase 2 (ULK2) also harbors putative SGK1 phosphorylation sites at both Ser507 (502RsRnsSG508) and Ser750 (745RtRttSV751) residues. Thus, the objective of this study was to determine whether Ser507 and Ser750 residues of ULK2 could be phosphorylation sites of SGK1 as one of its authentic substrate proteins. Using ULK2 507 and 750 serine residue un- or phosphorylation analog (S507AS750A or 507DS750D), we observed that modification of Ser507 or Ser750 residue was required to activate the kinase activity of ULK2 and sensitize ULK2 to stress or starvation while simultaneously enhancing its active state and autophagy characteristics, suggesting that phosphorylation at Ser750 or Ser507 residue could modulate its subcellular localization and protein interaction with AMPK1α to activate ULK2. We also observed that ULK2 autophagy activity was enhanced by GSK650394 (an SGK1 inhibitor) to compensate survival capacity through increasing its association with LC3 and phosphorylation. When SGK1 known to be associated with cell survival was inhibited by GSK650394, ULK2 autophagy pathway was activated to avoid cell death alternatively. Thus, our observations indicate that phosphorylation of ULK2 by SGK1 can regulate cell survival as an alternative modulation of ULK2 functions.

关键词： ULK-2 SGK1 Phosphorylation Autophagy Cell Survival Signal Transduction

The interaction between Fe65 and Tip60 is regulated by S-nitrosylation on 440 cystein residue of Fe65

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Advances in Biological Chemistry 2011年第3期1卷 109-118页

作者： eun jeoung lee Sung Hwa Shin Sunghee Hyun Jaesun Chun Sang Sun Kang 不详

The S-Nitrosylation of protein thiol groups by NO is a widely recognized protein modification. The treat-ment of cells with NOBF4 induces the S-nitrosylation of FE65. In this study, we present evidence showing that FE65 modified by NO (Nitric Oxide) via S-nitrosylation induces functional changes in the protein that inhibits the HAT activity of Tip60. The results of mutational analysis of FE65 demonstrated further that the cysteine residue of FE65 (Cys440) is critical to the process of S-nitrosylation. The mutation of the cysteine residue which completely ablated the S-nitrosylation of FE65 also lost its inhibitory effects on Tip60 HAT activity. Thus, our findings show, for the first time, that the novel regulation mechanism of Tip60 activity may operate via FE65 binding, which is enhanced by S-nitrosylation on the FE65 Cys440 residue. This study describes the interaction between FE65 and Tip60, which is enhanced by a posttransla-tional modification of FE65 (through S-nitrosylation) by NO, promoting the association of the FE65-Tip60 protein complex and inhibiting both the HAT activity of Tip60 and cell death.

关键词： S-Nitrosylation NO(Nitric Oxide) FE65 HAT activity Tip60