Ganglioside GD3 Enhances Adhesion Signals and Augments Malignant Properties of Melanoma Cells by Recruiting Integrins to Glycolipid-enriched Microdomains

Yoshio Yamauchi,Yuki Ohkawa,Yuhsuke Ohmi,Orie Tajima,Sayaka Miyazaki,Koichi Furukawa,Keiko Furukawa,Maiko Miyata,Mariko Kambe,Kazunori Hamamura

doi:10.1074/jbc.m109.087791

Yoshio Yamauchi, Yuki Ohkawa

Open Access

https://doi.org/10.1074/jbc.m109.087791

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Abstract

Ganglioside GD3 is widely expressed in human malignant melanoma cell lines and tumors. Previously, we reported that GD3+ cells show stronger tyrosine phosphorylation of focal adhesion kinase (FAK), p130(Cas), and paxillin when treated with fetal calf serum than GD3- cells. In this study, we analyzed the changes in the signals mediated by the interaction between integrins and extracellular matrices (ECM) to clarify how GD3 enhances cell signals in the vicinity of the cell membrane. An adhesion assay with a real time cell electronic sensing system revealed that GD3+ cells had stronger adhesion to all extracellular matrices examined. In particular, GD3+ cells attached more strongly to collagen type I and type IV than controls. Correspondingly, they showed stronger tyrosine phosphorylation of FAK and paxillin during adhesion to collagen type I. In the floating pattern of detergent extracts, a high level of integrin beta1 was found in glycolipid-enriched microdomain (GEM)/rafts in GD3+ cells before adhesion, whereas a smaller amount of integrin beta1 was detected in the GEM/rafts of controls. Some phosphorylated forms of FAK as well as total FAK were found in GEM/rafts during cell adhesion only in GD3+ cells. Another signal consisting of integrin-linked kinase/Akt was also activated during adhesion more strongly in GD3+ cells than in controls. In double stained GD3+ cells, GD3 and integrin beta1 co-localized at the focal adhesion with a punctate pattern. All these results suggested that integrins assembled and formed a cluster in GEM/rafts, leading to the enhanced signaling and malignant properties under GD3 expression.

Highlights

Performed [2], the mechanisms of regulation are not well understood
We demonstrated that adaptor molecules such as focal adhesion kinase (FAK),4 p130Cas, and paxillin undergo stronger tyrosine phosphorylation after treatment with fetal calf serum (FCS) in GD3ϩ cells than in control cells, and that they are involved in increased cell proliferation and invasion with GD3 expression [11, 12]
There are a number of studies indicating that integrin expression is up-regulated during malignant transformation, and integrin-mediated signals consisting of FAK, integrin-linked kinase (ILK), and ERK/MAPK, which determine cell fates such as cell growth and invasion, are activated in melanomas [16]

Summary

EXPERIMENTAL PROCEDURES

Cell Cultures—G5, G11, and G12 are GD3ϩ transfectant cells generated from a GD3Ϫ mutant of SK-MEL-28 N1 [9] by using cloned GD3 synthase cDNA [10]. Anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP) was purchased from Cell Signaling Technology (Beverly, MA). Phosphorylation site-specific anti-p130Cas antibodies (Tyr-165, Tyr-249, and Tyr-410) were purchased from Cell Signaling Technology. Anti-phospho-paxillin antibodies, p-Paxillin (Tyr-31 and Tyr-118) were purchased from Santa Cruz Biotechnology, and p-Paxillin (Tyr-118) was from Cell Signaling Technology. Anti-ILK antibodies were purchased from BD Transduction (mouse mAb), and from Cell Signaling Technology (rabbit anti-ILK1). Plates coated with 0.01% PLL (Sigma) in Petri dishes for 5 min at room temperature were washed with PBS and blocked with minimal essential medium/BSA as previously described [18]. GD3ϩ cell (G5) with 4 kinds of siRNA was examined by immunoblotting using an anti-ILK antibody (BD Biosciences). Cells were incubated with biotin-conjugated anti-human CD29 (integrin ␤1) or mAb R24 in PBS containing 0.5% BSA for 60 min at room.

RESULTS

G11 V4 V9

DISCUSSION