The Escherichia coli subtilase cytotoxin A subunit specifically cleaves cell-surface GRP78 protein and abolishes COOH-terminal-dependent signaling.

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 NH(2)-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 (GRP78(low)) 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 NH(2)-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.

• Authors: Rupa Ray, Gustaaf G de Ridder, Jerry P Eu, Adrienne W Paton, James C Paton, Salvatore V Pizzo
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 287 Issue 39 Pg. 32755-69 (Sep 21 2012) ISSN: 1083-351X [Electronic] United States
• PMID: 22851173 (Publication Type: Journal Article)

Chemical References

• Antibodies, Neoplasm
• Autoantibodies
• Endoplasmic Reticulum Chaperone BiP
• Escherichia coli Proteins
• HSPA5 protein, human
• Heat-Shock Proteins
• Hspa5 protein, mouse
• Receptors, G-Protein-Coupled
• Subtilisins
• subtilase cytotoxin, E coli

Topics

• Animals
• Antibodies, Neoplasm (genetics, metabolism)
• Autoantibodies (genetics, metabolism)
• Catalytic Domain
• Endoplasmic Reticulum Chaperone BiP
• Escherichia coli (enzymology)
• Escherichia coli Proteins (pharmacology)
• Heat-Shock Proteins (genetics, metabolism)
• Hep G2 Cells
• Humans
• Male
• Melanoma (genetics, metabolism)
• Mice
• Prostatic Neoplasms (genetics, metabolism)
• Proteolysis (drug effects)
• Receptors, G-Protein-Coupled (genetics, metabolism)
• Signal Transduction (drug effects)
• Subtilisins (pharmacology)