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

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.

Differential expression of the invertase-encoding SUC genes in Saccharomyces cerevisiae.

Invertase (INV) is encoded in Saccharomyces cerevisiae by a family of genes, comprising SUC1-SUC5 and SUC7. Production of INV is highly variable, dependent on the strain and SUC gene present in the cell. The differences in INV production derive from the structure of the genes or are dependent on the genetic background of the strain. Centromeric plasmids (based on YCp50) carrying one of the SUC genes (except SUC7) were introduced into a strain (SEY2101) lacking SUC genes. The INV produced by the transformants was dependent on the individual SUC genes, and correlated with INV mRNA levels. Plasmids in which SUC2 had been placed under control of promoters from the other SUC genes, were used to transform SEY2101 cells. The amounts of INV produced by cells carrying hybrid SUC genes were in agreement with the levels expected if the promoter controlled the expression of the SUC2 structural region. It is suggested that the differences in expression are a function of the transcription efficiency of the different SUC gene promoters, based on the divergence of 5' sequences.