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

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Ray, R; de Ridder, GG; Eu, JP; Paton, AW; Paton, JC; Pizzo, SV

Published Date

  • September 21, 2012

Published In

Volume / Issue

  • 287 / 39

Start / End Page

  • 32755 - 32769

PubMed ID

  • 22851173

Pubmed Central ID

  • PMC3463347

Electronic International Standard Serial Number (EISSN)

  • 1083-351X

Digital Object Identifier (DOI)

  • 10.1074/jbc.M112.399808


  • eng

Conference Location

  • United States