Activation of Src Family Kinase Yes Induced by Shiga Toxin Binding to Globotriaosyl Ceramide (Gb3/CD77) in Low Density, Detergent-insoluble Microdomains

Abstract

Shiga toxin (Stx) is an enterotoxin produced by Shigella dysenteriae serotype 1 and enterohemorrhagic Escherichia coli, which binds specifically to globotriaosylceramide, Gb3, on the cell surface and causes cell death. We previously demonstrated that Stx induced apoptosis in human renal tubular cell line ACHN cells (Taguchi, T., Uchida, H., Kiyokawa, N., Mori, T., Sato, 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 microdomains were prepared from ACHN cells by sucrose density gradient centrifugation of Triton X-100 lysate as buoyant, detergent-insoluble microdomains (DIM). Gb3 was only recovered in DIM and was associated with Src family kinase Yes. Phosphorylation of tyrosine residues of proteins in the DIM fraction 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 observed in DIM proteins. Unexpectedly, however, all of the Yes kinase activity was obtained in the high density, detergent-soluble fraction. Yes was assumed to be activated and show increased Triton X-100 solubility in the early phase of retrograde endocytosis of Stx-Gb3 complex. Since Yes activation by the Stx addition was suppressed by filipin pretreatment, Gb3-enriched microdomains containing cholesterol were deeply involved in Stx signal transduction. Shiga toxin (Stx)1 of Shigella dysenteriae serotype 1 and enterohemorrhagic Escherichia coli is one of the major cause of hemolytic uremic syndrome (HUS). Stx consists of an A subunit of 32 kDa associated with five B subunits of 7.5 kDa each. The A subunits act to remove the adenine base at position 4324 of 28 S rRNA and are responsible for inactivation of protein synthesis and toxicity (2). The A subunits lacking B subunits, however, do not show any toxicity because of their inability to bind to the cell surface receptor. The B subunits bind specifically to cell surface glycosphingolipid (GSL) receptors-Gb3, 2 also known as CD77 or blood group P k (3). Once Stx is internalized, protein synthesis is suppressed, leading to cell death.Cell death is widely known to take place through two distinctive processes, necrosis or apoptosis. In contradiction to Williams's report (4), a number of recent studies have clearly demonstrated that Stx induces apoptosis in several different cell types, including Burkitt's lymphoma cells (5), Vero cells (6), human renal tubular derived ACHN cells (1), and normal human renal tubular epithelial cells (7,8). Especially, the later two studies indicate the importance of apoptotic cell death as one mechanism of damage to renal epithelium in the pathogenesis of HUS. Although the B subunit has no inhibitory effect on protein synthesis, a series of studies indicates that it alone participate to transduce cell signaling and can induce apoptosis in some instances such as Burkitt's lymphoma (5, 9). These reports en...

The Src Family Kinase Yes Triggers Hyperosmotic Activation of the Epidermal Growth Factor Receptor and CD95

“…9. In line with an important role of Yes in the hyperosmotic activation of the CD95 system in hepatocytes may also be the recent observation that Shiga toxin-induced cell death in the human renal tubular cell line ACNH is accompanied by a Shiga toxin-triggered Yes activation (45).…”

Src Kinase Mediates the Regulation of Phospholipase C-γ Activity by Glycosphingolipids

“…Signal transduction of non-receptor tyrosine kinases has also been demonstrated in association with several glycosphingolipids. This includes the activation of c-Src by exogenous GM3 (25), the activation of Lyn by anti-ganglioside GD3 antibody (22), activation of Lck with exogenous GM1 (26), the activation of Lyn with anti-␣GalGD1b antibody (27), and the activation of Yes by the globotriaosylceramide binding toxin shiga toxin (23). The mechanism responsible for the regulation of non-receptor tyrosine kinases by glycosphingolipids is not understood.…”

Membrane cytosolic translocation of verotoxin A1 subunit in target cells

“…However, at 16 u C, protein synthesis inhibition remained steady at~50 % from 3 to 6 h of VT1 treatment and was not augmented by lactacystin proteosomal inhibition. This protein synthesis inhibition could result from signal transduction involving VTA or VT1 holotoxin, rather than from cytosolic translocation (Katagiri et al, 1999;Lauvrak et al, 2006;Mori et al, 2000), as VTB alone was incapable of reducing protein synthesis at 16 u C.…”