Identification of a Domain of Axin That Binds to the Serine/Threonine Protein Phosphatase 2A and a Self-binding Domain*
- From the Department of Genetics and Development, College of Physicians & Surgeons, Columbia University, New York, New York 10032
Abstract
Axin is a negative regulator of embryonic axis formation in vertebrates, which acts through a Wnt signal transduction pathway involving the serine/threonine kinase GSK-3 and β-catenin. Axin has been shown to have distinct binding sites for GSK-3 and β-catenin and to promote the phosphorylation of β-catenin and its consequent degradation. This provides an explanation for the ability of Axin to inhibit signaling through β-catenin. In addition, a more N-terminal region of Axin binds to adenomatous polyposis coli (APC), a tumor suppressor protein that also regulates levels of β-catenin. Here, we report the results of a yeast two-hybrid screen for proteins that interact with the C-terminal third of Axin, a region in which no binding sites for other proteins have previously been identified. We found that Axin can bind to the catalytic subunit of the serine/threonine protein phosphatase 2A through a domain between amino acids 632 and 836. This interaction was confirmed by in vitro binding studies as well as by co-immunoprecipitation of epitope-tagged proteins expressed in cultured cells. Our results suggest that protein phosphatase 2A might interact with the Axin·APC·GSK-3·β-catenin complex, where it could modulate the effect of GSK-3 on β-catenin or other proteins in the complex. We also identified a region of Axin that may allow it to form dimers or multimers. Through two-hybrid and co-immunoprecipitation studies, we demonstrated that the C-terminal 100 amino acids of Axin could bind to the same region as other Axin molecules.
Footnotes
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↵* This work was supported by a fellowship (to W. H.) from the National Kidney Foundation and by grants (to F. C.) from the National Institutes of Health.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) AF076192.
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↵‡ Present address: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115.
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↵§ To whom correspondence should be addressed: Dept. of Genetics and Development, College of Physicians & Surgeons, Columbia University, 701 W. 168th St., New York, NY 10032. Tel.: 212-305-6814; Fax: 212-923-2090, E-mail: fdc3{at}columbia.edu.
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↵2 The nucleotide sequence for the mouse PP2Ac gene has been deposited under accession numberAF076192.
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↵3 F. Fagotto, E.-H. Jho, L. Zeng, and F. Costantini, unpublished data.
- Abbreviations:
- kb
-
kilobase pair(s)
- APC
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adenomatous polyposis coli
- PP2A
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protein phosphatase 2A
- GST
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glutathione S-transferase
- RGS
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regulation of G-protein signaling
- CMV
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cytomegalovirus
- PAGE
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polyacrylamide gel electrophoresis
- HA
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hemagglutinin.
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- Received July 15, 1998.
- Revision received November 11, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
