Neogenin, Defined as a GD3-associated Molecule by Enzyme-mediated Activation of Radical Sources, Confers Malignant Properties via Intracytoplasmic Domain in Melanoma Cells.

Abstract

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.

• Authors: Kei Kaneko, Yuki Ohkawa, Noboru Hashimoto, Yuhsuke Ohmi, Norihiro Kotani, Koichi Honke, Mitsutaka Ogawa, Tetsuya Okajima, Keiko Furukawa, Koichi Furukawa
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 291 Issue 32 Pg. 16630-43 (08 05 2016) ISSN: 1083-351X [Electronic] United States
• PMID: 27288875 (Publication Type: Journal Article)
• Copyright: © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Chemical References

• Gangliosides
• NEO1 protein, human
• Neoplasm Proteins
• Nerve Tissue Proteins
• Receptors, Cell Surface
• ganglioside, GD3
• Amyloid Precursor Protein Secretases

Topics

• Amyloid Precursor Protein Secretases (genetics, metabolism)
• Cell Line, Tumor
• Gangliosides (genetics, metabolism)
• Gene Expression Regulation, Neoplastic
• Humans
• Melanoma
• Membrane Microdomains (genetics, metabolism)
• Neoplasm Proteins (genetics, metabolism)
• Nerve Tissue Proteins (genetics, metabolism)
• Receptors, Cell Surface (genetics, metabolism)