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.
Fedi
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Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine; New York, New York 10029, USA. pfedi@smtplink.mssm.edu
Bafico
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Nieto Soria
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Burgess
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Miki
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Bottaro
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Kraus
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Aaronson
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D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors*; D015398:Signal Transduction; D014162:Transfection; D051153:Wnt Proteins; D051154:Wnt2 Protein; D029961:Zebrafish Proteins
C501126:DKK1 protein, human; C501127:Dkk1 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; C495244:WNT2 protein, human; D051153:Wnt Proteins; D051154:Wnt2 Protein; D029961:Zebrafish Proteins; C501128:dkk1b protein, zebrafish