Functional Activation of Src Family Kinase Yes Protein Is Essential for the Enhanced Malignant Properties of Human Melanoma Cells Expressing Ganglioside GD3

Kazunori Hamamura,Momoko Tsuji,Hiroshi Hotta,Yuki Ohkawa,Masataka Takahashi,Hidenobu Shibuya,Hideyuki Nakashima,Yoshio Yamauchi,Noboru Hashimoto,Hisashi Hattori,Minoru Ueda,Keiko Furukawa,Koichi Furukawa

doi:10.1074/jbc.m110.164798

Kazunori Hamamura, Momoko Tsuji + Show 11 more

Open Access

https://doi.org/10.1074/jbc.m110.164798

Copy DOI

Abstract

The possible roles of Src family kinases in the enhanced malignant properties of melanomas related to GD3 expression were analyzed. Among Src family kinases only Yes, not Fyn or Src, was functionally involved in the increased cell proliferation and invasion of GD3-expressing transfectant cells (GD3+). Yes was located upstream of p130Cas and paxillin and at an equivalent level to focal adhesion kinase. Yes underwent autophosphorylation even before serum treatment and showed stronger kinase activity in GD3+ cells than in GD3- cells following serum treatment. Coimmunoprecipitation experiments revealed that Yes bound to focal adhesion kinase or p130Cas more strongly in GD3+ cells than in GD3- cells. As a possible mechanism for the enhancing effects of GD3 on cellular phenotypes, it was shown that majority of Yes was localized in glycolipid-enriched microdomain/rafts in GD3+ cells even before serum treatment, whereas it was scarcely detected in glycolipid-enriched microdomain/rafts in GD3- cells. An in vitro kinase assay of Yes revealed that coexistence of GD3 with Yes in membranous environments enhances the kinase activity of GD3- cell-derived Yes toward enolase, p125, and Yes itself. Knockdown of GD3 synthase resulted in the alleviation of tumor phenotypes and reduced activation levels of Yes. Taken together, these results suggest a role of GD3 in the regulation of Src family kinases.

Highlights

Lation of glycosylation machineries [4]
Several members of Src family kinases were expressed in the melanoma cells, only Yes was functionally involved in the enhanced signaling that contributes to the malignant phenotypes of melanoma cells
Knockdown of Src, Fyn, Yes, p130Cas, Paxillin, and Focal adhesion kinase (FAK) by siRNA—Melanoma cells were plated at 70ϳ80% confluency in 3.5- or 6-cm cell culture dishes and were cultured overnight

Summary

EXPERIMENTAL PROCEDURES

Antibodies—Anti-phosphotyrosine mAb PY20, anti-FAK (mouse mAb IgG1), anti-paxillin (mouse mAb IgG1), anti-Yes (mouse mAb IgG1), anti-Fyn (mouse mAb IgG2b), anti-Lck (mouse mAb IgG2a), anti-Lyn (mouse mAb IgG1), and antiflotillin-1 (mouse mAb IgG1) were from BD Transduction Laboratories. Knockdown of Src, Fyn, Yes, p130Cas, Paxillin, and FAK by siRNA—Melanoma cells were plated at 70ϳ80% confluency in 3.5- or 6-cm cell culture dishes and were cultured overnight They were transiently transfected with siRNAs (100 nM) of the target gene or control in Opti-MEM I medium (Invitrogen) with Lipofectamine 2000TM (Invitrogen) following the manufacturer’s instructions. In Vitro Kinase Assay—The lysates prepared from cells treated with FCS were immunoprecipitated using anti-Yes antibody, and the immunocomplexes were washed three times with washing buffer (50 mM Tris-HCl (pH7.5), 150 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 1 mM EDTA) and twice with kinase buffer (30 mM HEPES (pH7.5), 10 mM MgCl2, 2 mM MnCl2, 10 ␮M Na3VO4). Statistical Analysis—Statistical significance of data were determined using Student’s t test

RESULTS

Enhanced Phosphorylation and Kinase Activity of Yes under

DISCUSSION