Processing of Proaugurin Is Required to Suppress Proliferation of Tumor Cell Lines

Mol Endocrinol. 2011 May;25(5):776-84. doi: 10.1210/me.2010-0389. Epub 2011 Mar 24.

Abstract

Augurin is a secretory molecule produced in pituitary, thyroid, and esophagus and implicated in a wide array of physiological processes, from ACTH release to tumor suppression. However, the specific proaugurin-derived peptides present in various cell types are not yet known. In order to shed light on the posttranslational modifications required for biological activity, we here describe the posttranslational processing of proaugurin in AtT-20 and Lovo cells and identify proaugurin-derived products generated by convertases. In vitro cleavage of proaugurin with proprotein convertases produced multiple peptides, including a major product with a mass of 9.7 kDa by mass spectrometry. Metabolic labeling of C-terminally tagged proaugurin in AtT-20 and AtT-20/PC2 cells resulted in a major 15-kDa tagged form on SDS-PAGE, which likely corresponds to the 9.7-kDa in vitro fragment, with the added tag, its linker, and posttranslational modification(s). The secretion of neither proaugurin nor this cleavage product was stimulated by forskolin, indicating its lack of storage in regulated secretory granules and lack of cleavage by PC2. Incubation of cells with the furin inhibitor nona-d-arginine resulted in impaired cleavage of proaugurin, whereas metalloprotease inhibitors did not affect proaugurin proteolysis. These data support the idea that proaugurin is cleaved by furin and secreted via the constitutive secretory pathway. Interestingly, proaugurin was sulfated during trafficking; sulfation was completely inhibited by brefeldin A. Proliferation assays with three different tumor cell lines demonstrated that only furin-cleaved proaugurin could suppress cell proliferation, suggesting that proteolytic cleavage is a posttranslational requirement for proaugurin to suppress cell proliferation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Line, Tumor
  • Cell Proliferation*
  • Down-Regulation*
  • Furin / metabolism
  • Humans
  • Mice
  • Molecular Sequence Data
  • Molecular Weight
  • Neoplasm Proteins / metabolism*
  • Proprotein Convertases / metabolism
  • Protein Precursors / metabolism*
  • Protein Processing, Post-Translational
  • Secretory Pathway
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Neoplasm Proteins
  • Protein Precursors
  • esophageal cancer related gene 4 protein, mouse
  • Proprotein Convertases
  • FURIN protein, human
  • Furin