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The mechanism of cross talk between the Wnt signaling and cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]) pathways was studied. Prostaglandin E(1) (PGE(1)), isoproterenol, and dibutyryl cAMP (Bt(2)cAMP), all of which activate PKA, increased the cytoplasmic and nuclear beta-catenin protein level, and these actions were suppressed by a PKA inhibitor and RNA interference for PKA. PGE(1) and Bt(2)cAMP also increased T-cell factor (Tcf)-dependent transcription through beta-catenin. Bt(2)cAMP suppressed degradation of beta-catenin at the protein level. Although PKA did not affect the formation of a complex between glycogen synthase kinase 3beta (GSK-3beta), beta-catenin, and Axin, phosphorylation of beta-catenin by PKA inhibited ubiquitination of beta-catenin in intact cells and in vitro. Ser675 was found to be a site for phosphorylation by PKA, and substitution of this serine residue with alanine in beta-catenin attenuated inhibition of the ubiquitination of beta-catenin by PKA, PKA-induced stabilization of beta-catenin, and PKA-dependent activation of Tcf. These results indicate that PKA inhibits the ubiquitination of beta-catenin by phosphorylating beta-catenin, thereby causing beta-catenin to accumulate and the Wnt signaling pathway to be activated.

The Wnt signaling pathway plays a pivotal role in vertebrate early development and morphogenesis. Duplin (axis duplication inhibitor) interacts with beta-catenin and prevents its binding to Tcf, thereby inhibiting downstream Wnt signaling. Here we show that Duplin is expressed predominantly from early- to mid-stage mouse embryogenesis, and we describe the generation of mice deficient in Duplin. Duplin(-/-) embryos manifest growth retardation from embryonic day 5.5 (E5.5) and developmental arrest accompanied by massive apoptosis at E7.5. The mutant embryos develop into an egg cylinder but do not form a primitive streak or mesoderm. Expression of beta-catenin target genes, including those for T (brachyury), Axin2, and cyclin D1, was not increased in Duplin(-/-) embryos, suggesting that the developmental defect is not simply attributable to upregulation of Wnt signaling caused by the lack of this inhibitor. These results suggest that Duplin plays an indispensable role, likely by a mechanism independent of inhibition of Wnt signaling, in mouse embryonic growth and differentiation at an early developmental stage.

Axam has been identified as a novel Axin-binding protein that inhibits the Wnt signaling pathway. We studied the molecular mechanism by which Axam stimulates the downregulation of beta-catenin. The C-terminal region of Axam has an amino acid sequence similar to that of the catalytic region of SENP1, a SUMO-specific protease (desumoylation enzyme). Indeed, Axam exhibited activity to remove SUMO from sumoylated proteins in vitro and in intact cells. The Axin-binding domain is located in the central region of Axam, which is different from the catalytic domain. Neither the Axin-binding domain nor the catalytic domain alone was sufficient for the downregulation of beta-catenin. An Axam fragment which contains both domains was able to decrease the level of beta-catenin. On substitution of Ser for Cys(547) in the catalytic domain, Axam lost its desumoylation activity. Further, this Axam mutant decreased the activity to downregulate beta-catenin. Although Axam strongly inhibited axis formation and expression of siamois, a Wnt-response gene, in Xenopus embryos, Axam(C547S) showed weak activities. These results demonstrate that Axam functions as a desumoylation enzyme to downregulate beta-catenin and suggest that sumoylation is involved in the regulation of the Wnt signaling pathway.