Axin forms a complex with adenomatous polyposis coli gene product (APC), glycogen synthase kinase-3b (GSK3b), and b-catenin through di erent binding sites and downregulates b-catenin. GSK-3b-dependent phosphorylation of APC-(1211-2075) which has the Axin-binding site was facilitated by Axin, but that of APC-(959-1338) which lacks the Axin-binding site was not. Axin-(298-506) or Axin-(298-832), which has the GSK-3b-and bcatenin-but not APC-binding sites, did not enhance GSK-3b-dependent phosphorylation of either APC-(1211-2075) or APC-(959-1338). Furthermore, b-catenin stimulated the phosphorylation of APC-(959-1338) and APC-(1211-2075) by GSK-3b in the presence of Axin. Consistent with these in vitro observations, expression of b-catenin or Axin in COS cells promoted an SDS gel band shift of APC. These results indicate that APC complexed with Axin is e ectively phosphorylated by GSK-3b and that b-catenin may modulate this phosphorylation. In addition, the heterodimeric form of protein phosphatase 2A (PP2A) directly bound to Axin, and PP2A complexed with Axin dephosphorylated APC phosphorylated by GSK-3b. Taken together, these results suggest that GSK-3b-dependent phosphorylation of APC can be modulated by b-catenin and PP2A complexed with Axin. Oncogene (2000) 19, 537 ± 545.

Human erythrocyte protein phosphatase 2A, which comprises a 34-kDa catalytic C subunit, a 63-kDa regulatory A subunit and a 74-kDa regulatory BQ (N N) subunit, was phosphorylated at serine residues of BQ in vitro by cAMP-dependent protein kinase (A-kinase). In the presence and absence of 0.5 W WM okadaic acid (OA), A-kinase gave maximal incorporation of 1.7 and 1.0 mol of phosphate per mol of BQ, respectively. The K m value of A-kinase for CABQ was 0.17 þ 0.01 W WM in the presence of OA. The major in vitro phosphorylation sites of BQ were identified as Ser-60, -75 and -573 in the presence of OA, and Ser-75 and -573 in the absence of OA. Phosphorylation of BQ did not dissociate BQ from CA, and stimulated the molecular activity of CABQ toward phosphorylated H1 and H2B histones, 3.8-and 1.4-fold, respectively, but not toward phosphorylase a.z 1998 Federation of European Biochemical Societies.

Based on amino acid sequence data of a 74-kDa regulatory subunit (B" or ~) of a human heterotrimeric protein phosphatase 2A, a cDNA encoding the subunit was isolated from a human cerebral cortex library. The cDNA had an open reading frame encoding an Mr 66 138 protein of 570 amino acids. Bacterial expression of the cDNA yielded a protein immunoreactive with antisera specific to the 74-kDa subunit. The predicted primary structure of the subunit had no similarity to already reported sequences of PP2A regulatory subunits including A, B, and PR72. Potential phosphorylation sites for protein kinases A and C, a bipartite motif of putative nuclear localization signal, an SH3 accessible proline-rich domain, and a unique PQ repeat were found in the sequence. The subunit mRNA of about 2.9 kb was ubiquitously expressed in rat tissues.

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with glycogen synthase kinase-3␤ (GSK-3␤), ␤-catenin, adenomatous polyposis coli (APC) gene product, and Dvl, and it regulates GSK-3␤-dependent phosphorylation in the complex and the stability of ␤-catenin. Using yeast two-hybrid screening, we found that regulatory subunits of protein phosphatase 2A, PR61␤ and -␥, interact with Axin. PR61␤ or -␥ formed a complex with Axin in intact cells, and their interaction was direct. The binding site of PR61␤ on Axin was different from those of GSK-3␤, ␤-catenin, APC, and Dvl. Although PR61␤ did not affect the stability of ␤-catenin, it inhibited Dvl-and ␤-catenin-dependent T cell factor activation in mammalian cells. Moreover, it suppressed ␤-catenin-induced axis formation and expression of siamois, a Wnt target gene, in Xenopus embryos, suggesting that PR61␤ acts either at the level of ␤-catenin or downstream of it. Taken together with the previous observations that PR61 interacts with APC and functions upstream of ␤-catenin, these results demonstrate that PR61 regulates the Wnt signaling pathway at various steps.

Background: Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase distributed in eukaryotes from yeast to human, and plays pivotal roles in diverse cellular functions such as metabolism, cell cycle progression, gene expression and development. PP2A holoenzyme is a heterodimer of a catalytic subunit C and a regulatory subunit A, or a heterotrimer of C, A and a variable regulatory subunit consisting of three families; B, B 0 , and PR72. Specific functions for each variable subunit are not well understood.