2015 Fiscal Year Research-status Report
Study on the mechanism of Girdin-mediated amino acid signaling and regulation of mTORC1 activity in cancer cells
| Project/Area Number |
15K21061
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| Research Institution | Nagoya University |
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Principal Investigator |
翁 良 名古屋大学, 医学(系)研究科(研究院), 特任助教 (20729280)
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| Project Period (FY) |
2015-04-01 – 2017-03-31
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| Keywords | Cell metabolism / mTOR / Girdin |
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| Outline of Annual Research Achievements |
In FY2015, we mainly focused on analyzing the interaction between Girdin and CD98/LAT1 and the effect of Girdin/CD98 on mTORC1 activation.
1. We purifed GST-fused CD98 cytoplasmic domain and Girdin-NT domain tagged with the His epitope using bacterial expression system, and in vitro binding assays confirmed direct interaction between CD98 and Girdin.
2. We overexpressed MAPKK dominant active form, which induced the activation of Erk, and found it increased the endogenous Girdin-CD98/LAT1 interaction. We also found treat cell with MAPKK inhibitor U0126 inhibits Girdin-CD98/LAT1 interaction. The data show that Erk-mediated Girdin phosphorylation is required for its interaction with CD98/LAT1.
3. We compared mTORC1 activity in control cells, Girdin knockdown cells and CD98 overexpression cells, and found Girdin regulates mTORC1 activity via CD98.
4. We checked the effect of Girdin knockdown and overexpression on cell surface CD98 by using a cell surface protein isolation kit (Pierce), which showed Girdin knockdown increased cell surface CD98, whereas Girdin overexpression decrease it.
5. We analyzed cytosolic and lysosomal amino acids by using an amino acids analyzer, and found Girdin and CD98 overexpression significantly decreased cytosolic lysosomal glutamine concentration, but has little effect on Leu or Arg concentration. Accordingly we get the conclusion Girdin and CD98 regulates mTORC1 activity through modulation cytosolic glutamine concentration.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
All of our experimental plans are underway according to our initial plan.
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| Strategy for Future Research Activity |
(1) Biochemical analysis of the interaction between Girdin and CD98/LAT1
(1-b) Roles of Erk-mediated Girdin phosphorylation in Girdin-CD98/LAT1 interaction
In 2016, we will evaluate the quality of phospho-Girdin (Ser233/237)-specific antibody.We will also use this antibody to perform immunofluorescence to test the colocalization of phosphorylated Girdin and CD98/LAT1.
(2) Roles of Girdin-CD98/LAT1 complex in the regulation of mTORC1 activation and cancer progression
(2-b) Relevance of the expression of Girdin and CD98/LAT1 and Girdin phosphorylation in human cancers
Considering previous studies that the mTORC1 signaling is critical for cell proliferation and tumorigenesis, we will see whether Girdin-CD98/LAT1 interaction is also involved in tumorigenesis. Our experiments in 2016 also include the investigation of the expression of Girdin, CD98/LAT1, and mTORC1 pathway in human breast cancer tissues.
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| Causes of Carryover |
今年度の実験計画が予想よりも進捗が早く、また一部の試薬を複数の実験で共有することが可能であったため、助成金の一部を次年度に繰り越すことを考案した。
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| Expenditure Plan for Carryover Budget |
次年度は今年度までに解明した細胞内代謝制御の機序について、動物実験を用いた検証を予定している。繰り越した助成金は昨年度と同様に生化学消耗品に用いる他、動物実験のための各種費用に充当する予定である。
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Research Products
(3 results)
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[Journal Article] Identification of Meflin as a Potential Marker for Mesenchymal Stromal Cells.2016
Author(s)
Maeda K, Enomoto A, Hara A, Asai N, Kobayashi T, Horinouchi A, Maruyama S, Ishikawa Y, Nishiyama T, Kiyoi H, Kato T, Ando K, Weng L, Mii S, Asai M, Mizutani Y, Watanabe O, Hirooka Y, Goto H, Takahashi M
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Journal Title
Sci Rep
Volume: 6
Pages: 22288
DOI
Peer Reviewed / Open Access
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