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|Title:||The Escherichia coli subtilase cytotoxin A subunit specifically cleaves cell-surface GRP78 protein and abolishes COOH-terminal-dependent signaling|
de Ridder, G.
|Citation:||Journal of Biological Chemistry, 2012; 287(39):32755-32769|
|Publisher:||Amer Soc Biochemistry Molecular Biology Inc|
|Rupa Ray, Gustaaf G. de Ridder, Jerry P. Eu, Adrienne W. Paton, James C. Paton and Salvatore V. Pizzo|
|Abstract:||GRP78, a molecular chaperone with critical endoplasmic reticulum functions, is aberrantly expressed on the surface of cancer cells, including prostate and melanoma. Here it functions as a pro-proliferative and anti-apoptotic signaling receptor via NH2-terminal domain ligation. Auto-antibodies to this domain may appear in cancer patient serum where they are a poor prognostic indicator. Conversely, GRP78 COOH-terminal domain ligation is pro-apoptotic and anti-proliferative. There is no method to disrupt cell-surface GRP78 without compromising the total GRP78 pool, making it difficult to study cell-surface GRP78 function. We studied six cell lines representing three cancer types. One cell line per group expresses high levels of cell-surface GRP78, and the other expresses low levels (human hepatoma: Hep3B and HepG2; human prostate cancer: PC3 and 1-LN; murine melanoma: B16F0 and B16F1). We investigated the effect of Escherichia coli subtilase cytoxin catalytic subunit (SubA) on GRP78. We report that SubA specifically cleaves cell-surface GRP78 on HepG2, 1-LN, and B16F1 cells without affecting intracellular GRP78. B16F0 cells (GRP78low) have lower amounts of cleaved cell-surface GRP78. SubA has no effect on Hep3B and PC3 cells. The predicted 28-kDa GRP78 COOH-terminal fragment is released into the culture medium by SubA treatment, and COOH-terminal domain signal transduction is abrogated, whereas pro-proliferative signaling mediated through NH2-terminal domain ligation is unaffected. These experiments clarify cell-surface GRP78 topology and demonstrate that the COOH-terminal domain is necessary for pro-apoptotic signal transduction occurring upon COOH-terminal antibody ligation. SubA is a powerful tool to specifically probe the functions of cell-surface GRP78.|
|Keywords:||Animals; Humans; Mice; Escherichia coli; Melanoma; Prostatic Neoplasms; Subtilisins; Escherichia coli Proteins; Heat-Shock Proteins; Receptors, G-Protein-Coupled; Antibodies, Neoplasm; Autoantibodies; Signal Transduction; Catalytic Domain; Male; Hep G2 Cells; Proteolysis|
|Rights:||© 2012 by The American Society for Biochemistry and Molecular Biology, Inc.|
|Appears in Collections:||Microbiology and Immunology publications|
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