δ-Catenin interacts with LEF-1 and negatively regulates its transcriptional activity.

δ-Catenin and ß-catenin belong to different subfamilies of armadillo proteins but share some common binding partners, such as E-cadherin. This is the first study that demonstrated a novel common binding partner for δ-catenin and ß-catenin, lymphoid enhancer factor-1 (LEF-1). We found that the N-terminus of δ-catenin (amino acids 85-325) bound to the middle region of LEF-1 unlike ß-catenin. Overexpressed δ-catenin entered the nucleus and inhibited LEF-1-mediated transcriptional activity in Bosc23 and DLD-1 cell lines. The current study provided novel insights that will provide a better understanding of the effects of δ-catenin on Wnt/LEF-1-mediated transcriptional activity.

GSK-3 phosphorylates delta-catenin and negatively regulates its stability via ubiquitination/proteosome-mediated proteolysis.

Delta-catenin was first identified because of its interaction with presenilin-1, and its aberrant expression has been reported in various human tumors and in patients with Cri-du-Chat syndrome, a form of mental retardation. However, the mechanism whereby delta-catenin is regulated in cells has not been fully elucidated. We investigated the possibility that glycogen-synthase kinase-3 (GSK-3) phosphorylates delta-catenin and thus affects its stability. Initially, we found that the level of delta-catenin was greater and the half-life of delta-catenin was longer in GSK-3beta(-/-) fibroblasts than those in GSK-3beta(+/+) fibroblasts. Furthermore, four different approaches designed to specifically inhibit GSK-3 activity, i.e. GSK-3-specific chemical inhibitors, Wnt-3a conditioned media, small interfering RNAs, and GSK-3alpha and -3beta kinase dead constructs, consistently showed that the levels of endogenous delta-catenin in CWR22Rv-1 prostate carcinoma cells and primary cortical neurons were increased by inhibiting GSK-3 activity. In addition, it was found that both GSK-3alpha and -3beta interact with and phosphorylate delta-catenin. The phosphorylation of DeltaC207-delta-catenin (lacking 207 C-terminal residues) and T1078A delta-catenin by GSK-3 was noticeably reduced compared with that of wild type delta-catenin, and the data from liquid chromatography-tandem mass spectrometry analyses suggest that the Thr(1078) residue of delta-catenin is one of the GSK-3 phosphorylation sites. Treatment with MG132 or ALLN, specific inhibitors of proteosome-dependent proteolysis, increased delta-catenin levels and caused an accumulation of ubiquitinated delta-catenin. It was also found that GSK-3 triggers the ubiquitination of delta-catenin. These results suggest that GSK-3 interacts with and phosphorylates delta-catenin and thereby negatively affects its stability by enabling its ubiquitination/proteosome-mediated proteolysis.

Beta-catenin can bind directly to CRM1 independently of adenomatous polyposis coli, which affects its nuclear localization and LEF-1/beta-catenin-dependent gene expression.

Nuclear beta-catenin affects the developmental process and progression of tumors. However, the precise mechanism for the nuclear export of beta-catenin is not completely understood. We found that beta-catenin can bind directly to CRM1 through its central armadillo (ARM) repeats region, independently of the adenomatous polyposis coli (APC) protein. CRM1 overexpression transports nuclear beta-catenin into the cytoplasm and decreases LEF-1/beta-catenin-dependent transcriptional activity, which is also affected by the co-overexpression of E-cadherin. CRM1 competed with E-cadherin and LEF-1 for binding to beta-catenin. beta-catenin could interact directly with APC through its essential sequences between amino acids 342 and 350. The site-directed beta-catenin mutant (NES2(-)), which could interact with CRM1, but not with APC, still retained its ability to export from the nucleus and its transactivational activity. This suggests that CRM1 can function as an efficient nuclear exporter for beta-catenin independently of APC. These results strongly suggest that the CRM1-mediated pathway is involved in the efficient transport of nuclear beta-catenin in the nucleus of cells.

Identification of E2F1 as a positive transcriptional regulator for delta-catenin.

delta-Catenin is upregulated in human carcinomas. However, little is known about the potential transcriptional factors that regulate delta-catenin expression in cancer. Using a human delta-catenin reporter system, we have screened several nuclear signaling modulators to test whether they can affect delta-catenin transcription. Among beta-catenin/LEF-1, Notch1, and E2F1, E2F1 dramatically increased delta-catenin-luciferase activities while beta-catenin/LEF-1 induced only a marginal increase. Rb suppressed the upregulation of delta-catenin-luciferase activities induced by E2F1 but did not interact with delta-catenin. RT-PCR and Western blot analyses in 4 different prostate cancer cell lines revealed that regulation of delta-catenin expression is controlled mainly at the transcriptional level. Interestingly, the effects of E2F1 on delta-catenin expression were observed only in human cancer cells expressing abundant endogenous delta-catenin. These studies identify E2F1 as a positive transcriptional regulator for delta-catenin, but further suggest the presence of strong negative regulator(s) for delta-catenin in prostate cancer cells with minimal endogenous delta-catenin expression.

Delta-catenin-induced dendritic morphogenesis. An essential role of p190RhoGEF interaction through Akt1-mediated phosphorylation.

Delta-catenin was first identified through its interaction with Presenilin-1 and has been implicated in the regulation of dendrogenesis and cognitive function. However, the molecular mechanisms by which delta-catenin promotes dendritic morphogenesis were unclear. In this study, we demonstrated delta-catenin interaction with p190RhoGEF, and the importance of Akt1-mediated phosphorylation at Thr-454 residue of delta-catenin in this interaction. We have also found that delta-catenin overexpression decreased the binding between p190RhoGEF and RhoA, and significantly lowered the levels of GTP-RhoA but not those of GTP-Rac1 and -Cdc42. Delta-catenin T454A, a defective form in p190RhoGEF binding, did not decrease the binding between p190RhoGEF and RhoA. Delta-catenin T454A also did not lower GTP-RhoA levels and failed to induce dendrite-like process formation in NIH 3T3 fibroblasts. Furthermore, delta-catenin T454A significantly reduced the length and number of mature mushroom shaped spines in primary hippocampal neurons. These results highlight signaling events in the regulation of delta-catenin-induced dendrogenesis and spine morphogenesis.

Overexpressed LEF-1 proteins display different nuclear localization patterns of beta-catenin in normal versus tumor cells.

Beta-catenin not only plays a role in cadherin-dependent cell adhesion, but also interacts with T-cell factor (TCF)/lymphoid enhancer factor-1 (LEF-1) to affect gene expression. In this report, we describe the effects of exogenous LEF-1 and of treatment with leptomycin B (LMB), a specific inhibitor of CRM1-medicated nuclear export, on the nuclear localization and export of beta-catenin. Normal epithelial cells overexpressing LEF-1 accumulate nuclear beta-catenin in a LEF-1 concentration-dependent manner. Nuclear beta-catenin, once imported from the cytoplasm, is rapidly removed from the nucleus. Treatment with LMB results in dramatic retention of nuclear beta-catenin in normal epithelial cells transfected with LEF-1, and this effect is intensified by treatment of N-Acetyl-leucyl-leucyl-norleucinal together with LMB. Colon carcinoma cells containing an adenomatous polyposis coli mutation retain significant amounts of LEF-1 induced nuclear beta-catenin considerably after the time-point when beta-catenin disappears from the nuclei of LEF-1 transfected normal epithelial cells. beta-Catenin binds directly to CRM1, and overexpression of CRM1 reduces nuclear beta-catenin-mediated transactivation function.

Presenilin-1 D257A and D385A mutants fail to cleave Notch in their endoproteolyzed forms, but only presenilin-1 D385A mutant can restore its gamma-secretase activity with the compensatory overexpression of normal C-terminal fragment.

The enzyme gamma-secretase is involved in the cleavage of several type I membrane proteins, such as Notch 1 and amyloid precursor protein. Presenilin-1 (PS-1) is one of the critical integral membrane protein components of the gamma-secretase complex and is processed endoproteolytically, generating N- and C-terminal fragments (NTF and CTF, respectively). PS-1 is also known to incorporate into a high molecular weight complex by binding to other gamma-secretase components such as Nicastrin, Aph-1, and Pen-2. Mutations on PS-1 can alter the effects of gamma-secretase on its many substrates to different extents. Here, we showed that PS-1 mutants have a different activity for Notch cleavage, which depended on the PS-1 mutation site. We demonstrated that defective PS-1 mutants located in CTF, i.e. D385A and C410Y, could restore their gamma-secretase activities with the compensatory overexpression of wild type CTF or of minimal deleted CTF (amino acids 349-467). However, the defective PS-1 D257A mutant could not restore their gamma-secretase activities with the compensatory overexpression of wild type NTF. In comparison, both D257A NTF and D385A CTF could abolish the gamma-secretase activity of wild type and pathogenic PS-1 mutants. We also showed that PS-1 NTF but not CTF forms strong high molecular weight aggregates in SDS-PAGE. Taken together, results have shown that NTF and CTF integrate differently into high molecular weight aggregates and that PS-1 Asp-257 and Asp-385 have different accessibilities in their unendoproteolyzed conformation.