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© The Rockefeller University Press, 0021-9525/1999//741 $5.00
The Journal of Cell Biology, Volume 145, Number 4, , 1999 741-756

Domains of Axin Involved in Protein–Protein Interactions, Wnt Pathway Inhibition, and Intracellular Localization

François Fagotto^*, Eek-hoon Jho, Li Zeng, Thomas Kurth^*, Thomas Joos^*, Christine Kaufmann^*, and Frank Costantini

^* Division of Cell Biology, Max-Planck Institute for Developmental Biology, 72076 Tübingen, Germany; and Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, New York 10032

Axin was identified as a regulator of embryonic axis induction in vertebrates that inhibits the Wnt signal transduction pathway. Epistasis experiments in frog embryos indicated that Axin functioned downstream of glycogen synthase kinase 3β (GSK3β) and upstream of β-catenin, and subsequent studies showed that Axin is part of a complex including these two proteins and adenomatous polyposis coli (APC). Here, we examine the role of different Axin domains in the effects on axis formation and β-catenin levels. We find that the regulators of G-protein signaling domain (major APC-binding site) and GSK3β-binding site are required, whereas the COOH-terminal sequences, including a protein phosphatase 2A binding site and the DIX domain, are not essential. Some forms of Axin lacking the β-catenin binding site can still interact indirectly with β-catenin and regulate β-catenin levels and axis formation. Thus in normal embryonic cells, interaction with APC and GSK3β is critical for the ability of Axin to regulate signaling via β-catenin. Myc-tagged Axin is localized in a characteristic pattern of intracellular spots as well as at the plasma membrane. NH₂-terminal sequences were required for targeting to either of these sites, whereas COOH-terminal sequences increased localization at the spots. Coexpression of hemagglutinin-tagged Dishevelled (Dsh) revealed strong colocalization with Axin, suggesting that Dsh can interact with the Axin/APC/GSK3/β-catenin complex, and may thus modulate its activity.

Key Words: β-catenin • glycogen synthase kinase 3β (GSK3β) • adenomatous polyposis coli (APC) • Dishevelled (Dsh) • dorsal axis formation

Abbreviations used in this paper: aa, amino acid; APC, adenomatous polyposis coli; coIP, coimmunoprecipitate/coimmunoprecipitation; DAPI, 4',6-diamidino-2-phenylindole; Dsh, Dishevelled; FL, full-length; GSK3β, glycogen synthase kinase 3β; HA, hemagglutinin; HA–Dsh, hemagglutinin-tagged Dishevelled; IF, immunofluorescence; IP, immunoprecipitation; Myc–Axin, Myc-tagged Axin; pAb, polyclonal antibody; PP2A, protein phosphatase 2A; RGS, regulators of G-protein signaling; VSV-G vesicular stomatitis virus glycoprotein, VSV–APC, VSV-G–epitope-tagged APC.

The current address of Li Zeng is Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115.

The current address of Thomas Joos is Department of Biochemistry, Naturwissenschaftliches und Medizinishces Institut, Markwiesenstrasse 55, 72770 Reutlingen, Germany.

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