| 研究実績の概要 |
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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| 今後の研究の推進方策 |
(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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