CELL BIOLOGY AND METABOLISM
Isolation and Biochemical Characterization of the Human Dkk-1 Homologue, a Novel Inhibitor of Mammalian Wnt Signaling*

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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 β-catenin levels. These results provide biochemical evidence that human Sk/Dkk-1 antagonizes Wnt signaling upstream of its effect on β-catenin regulation.

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This project was support in part by Grants CA71997 (to P. F.) and CA71672 (to S. A. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF127563.