Ikuo Sakamoto

Axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin

The regulators of G protein signaling (RGS) domain of Axin, a negative regulator of the Wnt signaling pathway, made a complex with full-length adenomatous polyposis coli (APC) in COS, 293, and L cells but not with truncated APC in SW480 or DLD-1 cells. The RGS domain directly interacted with the region containing the 20-amino acid repeats but not with that containing the 15-amino acid repeats of APC, although both regions are known to bind to β-catenin. In the region containing seven 20-amino acid repeats, the region containing the latter five repeats bound to the RGS domain of Axin. Axin and β-catenin simultaneously interacted with APC. Furthermore, Axin stimulated the degradation of β-catenin in COS cells. Taken together with our recent observations that Axin directly interacts with glycogen synthase kinase-3β (GSK-3β) and β-catenin and that it promotes GSK-3β-dependent phosphorylation of β-catenin, these results suggest that Axin, APC, GSK-3β, and β-catenin make a tetrameric complex, resulting in the regulation of the stabilization of β-catenin.

Inhibition of the Wnt Signaling Pathway by Idax, a Novel Dvl-Binding Protein

ABSTRACT In attempting to clarify the roles of Dvl in the Wnt signaling pathway, we identified a novel protein which binds to the PDZ domain of Dvl and named it Idax (for inhibition of the Dvl and Axin complex). Idax and Axin competed with each other for the binding to Dvl. Immunocytochemical analyses showed that Idax was localized to the same place as Dvl in cells and that expression of Axin inhibited the colocalization of Dvl and Idax. Further, Wnt-induced accumulation of β-catenin and activation of T-cell factor in mammalian cells were suppressed by expression of Idax. Expression of Idax inXenopus embryos induced ventralization with a reduction in the expression of siamois, a Wnt-inducible gene. Idax inhibited Wnt- and Dvl- but not β-catenin-induced axis duplication. It is known that Dvl is a positive regulator in the Wnt signaling pathway and that the PDZ domain is important for this activity. Therefore, these results suggest that Idax functions as a negative regulator of the Wnt signaling pathway by directly binding to the PDZ domain of Dvl.

A Novel β-Catenin-binding Protein Inhibits β-Catenin-dependent Tcf Activation and Axis Formation

β-Catenin is efficiently phosphorylated by glycogen synthase kinase-3β in the Axin complex in the cytoplasm, resulting in the down-regulation. In response to Wnt, β-catenin is stabilized and translocated into the nucleus where it stimulates gene expression through Tcf/Lef. Here we report a novel protein, designated Duplin (for axis duplication inhibitor), which negatively regulates the function of β-catenin in the nucleus. Duplin was located in the nucleus. Duplin bound directly to the Armadillo repeats of β-catenin, thereby inhibiting the binding of Tcf to β-catenin. It did not affect the stability of β-catenin but inhibited Wnt- or β-catenin-dependent Tcf activation. Furthermore, expression of Duplin in Xenopus embryos inhibited the axis formation and β-catenin-dependent axis duplication, and prevented the β-catenins ability to rescue ventralizing phenotypes induced by ultraviolet light irradiation. Thus, Duplin is a nuclear protein that inhibits β-catenin signaling.

Stimulation of retinal pigment epithelial cell growth by neuropeptides in vitro

PURPOSE The proliferation of many cell types are regulated by cytokines and neuropeptides by autocrine and paracrine mechanisms. Retinal pigment epithelial (RPE) cells are also regulated by cytokines. But RPE cells are very close to the neural retina which has some neuropeptides. The present study was to investigate the effects of neuropeptides on the growth of RPE cells. METHODS RPE cells were obtained from the eyes of 11 day old chick embryos and cultured in Dulbeccos modified Eagles culture medium containing 10% fetal calf serum. The growth of RPE cells was evaluated by [3H]-thymidine uptake. RESULTS Substance P, beta-endorphin and calcitonin gene-related peptide markedly stimulated the growth of RPE cells. The effects of methionine-enkephalin, somatostatin and vasoactive intestinal peptide were intermediate. The strongest effects of substance P, beta-endorphin and calcitonin gene-related peptide were observed at 10(-6) to 10(-7) M. The stimulation of RPE cells with beta-endorphin was inhibited by naloxone, suggesting that the stimulation with beta-endorphin is mediated by an opioid receptor. beta-endorphin and substance P induced RPE cell growth stimulating activity. Leucine-enkephalin and neuropeptide Y did not affect the growth of RPE cells. CONCLUSIONS These results suggest that neuropeptides play an important role in the regulation of RPE cell growth.