Isolation and biochemical characterization of the human Dkk-1 homologue, a novel inhibitor of mammalian Wnt signaling

J Biol Chem. 1999 Jul 2;274(27):19465-72. doi: 10.1074/jbc.274.27.19465.

Abstract

In an effort to isolate novel growth factors, we identified a human protein, designated Sk, that co-eluted with Neuregulin during chromatographic separation of conditioned medium from the SK-LMS-1 human leiomyosarcoma cell line. Degenerate oligonucleotides based on amino-terminal sequence analysis of the purified protein were used to isolate the corresponding cDNA from a library generated from this cell line. Sk is a novel 266-amino acid protein that contains a signal peptide sequence and two cysteine-rich domains with no similarity to other known growth factors. A single major 2-kilobase transcript was expressed in several embryonic tissues. Transfection of mammalian cells demonstrated that the protein was secreted and expressed as a doublet of approximately 35 kDa. In vitro translation and endoglycosylase analysis indicated that this doublet, which was also observed in cells expressing the endogenous protein, arises from posttranslational modification. A search of the GenBankTM data base revealed a match of Sk with Dkk-1, which is a novel secreted protein required for head induction in amphibian embryos and a potent Wnt inhibitor. When coexpressed with Wnt-2 in NIH3T3 cells, human Sk/Dkk-1 caused reversion of Wnt-2 induced morphological alterations and inhibited the Wnt-2 induced increase in uncomplexed beta-catenin levels. These results provide biochemical evidence that human Sk/Dkk-1 antagonizes Wnt signaling upstream of its effect on beta-catenin regulation.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cloning, Molecular
  • Humans
  • Intercellular Signaling Peptides and Proteins
  • Mice
  • Molecular Sequence Data
  • Proteins / chemistry
  • Proteins / isolation & purification*
  • Proto-Oncogene Proteins / antagonists & inhibitors*
  • Signal Transduction*
  • Transfection
  • Wnt Proteins
  • Wnt2 Protein
  • Zebrafish Proteins*

Substances

  • DKK1 protein, human
  • Dkk1 protein, mouse
  • Intercellular Signaling Peptides and Proteins
  • Proteins
  • Proto-Oncogene Proteins
  • WNT2 protein, human
  • Wnt Proteins
  • Wnt2 Protein
  • Zebrafish Proteins
  • dkk1b protein, zebrafish

Associated data

  • GENBANK/AF127563