Control of b-Catenin Stability : Reconstitution of the Cytoplasmic Steps of the Wnt Pathway in Xenopus Egg Extracts

mechanistic aspects of wnt signaling still remain obscure. Recent reviews (see, for example, Cadigan and Nusse, 1997; Brown and Moon, 1998; Wodarz and Nusse, 1998; Sokol, 1999) present models that differ significantly in describing how the signal is propagated Adrian Salic,† Ethan Lee,† Leslie Mayer, and Marc W. Kirschner* Department of Cell Biology Harvard Medical School Boston, Massachusetts 02115 through the pathway and what proteins and interactions are functionally important. An incomplete list of poorly understood aspects in wnt signaling includes: (1) how Summary wnt-bound frizzled activates dsh; (2) how dsh interacts with GSK3/APC/axin/b-catenin complex and stabilizes Regulation of b-catenin degradation by intracellular components of the wnt pathway was reconstituted in b-catenin; (3) how b-catenin is recognized by the degradation machinery; (4) how GBP is regulated during wnt cytoplasmic extracts of Xenopus eggs and embryos. The ubiquitin-dependent b-catenin degradation in exsignaling; (5) what role is played by the tumor suppressor APC; and (6) what function, if any, is performed by other tracts displays a biochemical requirement for axin, GSK3, and APC. Axin dramatically accelerates while proteins that bind to known components of the pathway. A notable obstacle to a better understanding of wnt dishevelled inhibits b-catenin turnover. Through another domain, dishevelled recruits GBP/Frat1 to the signaling has been the lack of a biochemically tractable, in vitro system for studying the kinetics and the molecuAPC-axin-GSK3 complex. Our results confirm and extend models in which inhibition of GSK3 has two synerlar associations between components of the pathway. Since wnt signaling is active in early Xenopus developgistic effects: (1) reduction of APC phosphorylation and loss of affinity for b-catenin and (2) reduction of ment, we have developed extracts from both Xenopus eggs and embryos to study the pathway biochemically. b-catenin phosphorylation and consequent loss of its affinity for the SCF ubiquitin ligase complex. DishevWe reconstituted signaling downstream of dsh and examined the regulation and requirements for b-catenin elled thus stabilizes b-catenin, which can dissociate from the APC/axin complex and participate in tranturnover, the mechanism by which dsh and GBP inhibit the degradation of b-catenin, and the role of APC-axinscriptional activation. b-catenin interactions.