Neogenin, Defined as a GD3-associated Molecule by Enzyme-mediated Activation of Radical Sources, Confers Malignant Properties via Intracytoplasmic Domain in Melanoma Cells.
To investigate mechanisms for increased malignant properties in malignant melanomas by ganglioside GD3, enzyme-mediated activation of radical sources and subsequent mass spectrometry were performed using an anti-GD3 antibody and GD3-positive (GD3+) and GD3-negative (GD3-) melanoma cell lines. Neogenin, defined as a GD3-neighbored molecule, was largely localized in lipid/rafts in GD3+ cells. Silencing of neogenin resulted in the reduction of cell growth and invasion activity. Physical association between GD3 and neogenin was demonstrated by immunoblotting of the immunoprecipitates with anti-neogenin antibody from GD3+ cell lysates. The intracytoplasmic domain of neogenin (Ne-ICD) was detected in GD3+ cells at higher levels than in GD3- cells when cells were treated by a proteasome inhibitor but not when simultaneously treated with a γ-secretase inhibitor. Exogenous GD3 also induced increased Ne-ICD in GD3- cells. Overexpression of Ne-ICD in GD3- cells resulted in the increased cell growth and invasion activity, suggesting that Ne-ICD plays a role as a transcriptional factor to drive malignant properties of melanomas after cleavage with γ-secretase. γ-Secretase was found in lipid/rafts in GD3+ cells. Accordingly, immunocyto-staining revealed that GD3, neogenin, and γ-secretase were co-localized at the leading edge of GD3+ cells. All these results suggested that GD3 recruits γ-secretase to lipid/rafts, allowing efficient cleavage of neogenin. ChIP-sequencing was performed to identify candidates of target genes of Ne-ICD. Some of them actually showed increased expression after expression of Ne-ICD, probably exerting malignant phenotypes of melanomas under GD3 expression.
From the Department of Biochemistry II, Nagoya University Graduate School of Medicine, Nagoya 466-0065.
Kei
K
Kaneko
Department of Life Biomedical Sciences, Chubu University College of Life and Health Sciences, Kasuigai, Aichi 487-8501.
Yuki
Y
Ohkawa
From the Department of Biochemistry II, Nagoya University Graduate School of Medicine, Nagoya 466-0065.
Noboru
N
Hashimoto
From the Department of Biochemistry II, Nagoya University Graduate School of Medicine, Nagoya 466-0065.
Yuhsuke
Y
Ohmi
Department of Biochemistry, Faculty of Medicine, Saitama Medical University, Moroyama-machi, Iruma-gun, Saitama 350-0495, and.
Norihiro
N
Kotani
Department of Biochemistry, Kochi University School of Medicine, Kochi 783-8505, Japan.
Koichi
K
Honke
From the Department of Biochemistry II, Nagoya University Graduate School of Medicine, Nagoya 466-0065.
Mitsutaka
M
Ogawa
From the Department of Biochemistry II, Nagoya University Graduate School of Medicine, Nagoya 466-0065.
Tetsuya
T
Okajima
Department of Life Biomedical Sciences, Chubu University College of Life and Health Sciences, Kasuigai, Aichi 487-8501.
Keiko
K
Furukawa
From the Department of Biochemistry II, Nagoya University Graduate School of Medicine, Nagoya 466-0065, Department of Life Biomedical Sciences, Chubu University College of Life and Health Sciences, Kasuigai, Aichi 487-8501, koichi@isc.chubu.ac.jp.
Koichi
K
Furukawa
D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D005732:Gangliosides / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008545:Melanoma; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*
D005732:Gangliosides; C000611758:NEO1 protein, human; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; C026226:ganglioside, GD3; D053829:Amyloid Precursor Protein Secretases
ganglioside; lipid raft; melanoma; signaling; transcription factor; γ-secretase