Activation of Src family kinase Yes induced by Shiga toxin binding to globotriaosyl ceramide (Gb3/CD77) in low density, detergent-insoluble microdomains

Shiga toxin (Stx) is an enterotoxin produced by Shigella dysenteriae seroty pe 1 and enterohemorrhagic Escherichia coli, which binds specifically to gl obotriao-sylceramide, Gb3, on the cell surface and causes cell death. We pr eviously demonstrated that Stx induced apoptosis in human renal tubular cel l line ACHN cells (Taguchi, T,, Uchida, H,, Kiyokawa, N., Mori, T,, Sate, N ,, Horie, H., Takeda, T and Fujimoto, J, (1998) Kidney Int. 53, 1681-1688), To study the early signal transduction after Stx addition, Gb3-enriched mi crodomains were prepared from ACHN cells by sucrose density gradient centri fugation of Triton X-100 lysate as buoyant, detergent-insoluble microdomain s (DIM), Gb3 was only recovered in DIM and was associated with Src family k inase Yes. Phosphorylation of tyrosine residues of proteins in the DIM frac tion increased by 10 min and returned to the resting level by 30 min after the addition of Stx, Since the kinase activity of Yes changed with the same kinetics, Yes was thought to be responsible for the hyperphosphorylation o bserved in DIM proteins. Unexpectedly, however, all of the Yes kinase activ ity was obtained in the high density, detergent-soluble fraction. Yes was a ssumed to be activated and show increased Triton X-100 solubility in the ea rly phase of retrograde endocytosis of Stx-Gb3 complex. Since Yes activatio n by the Stx addition was suppressed by filipin pretreatment, Gb3-enriched microdomains containing cholesterol were deeply involved in Stx signal tran sduction.