␦-Catenin was first identified through its interaction withPresenilin-1 and has been implicated in the regulation of dendrogenesis and cognitive function. However, the molecular mechanisms by which ␦-catenin promotes dendritic morphogenesis were unclear. In this study, we demonstrated ␦-catenin interaction with p190RhoGEF, and the importance of Akt1-mediated phosphorylation at Thr-454 residue of ␦-catenin in this interaction. We have also found that ␦-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. ␦-Catenin T454A, a defective form in p190RhoGEF binding, did not decrease the binding between p190RhoGEF and RhoA. ␦-Catenin T454A also did not lower GTP-RhoA levels and failed to induce dendrite-like process formation in NIH 3T3 fibroblasts. Furthermore, ␦-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 ␦-catenininduced dendrogenesis and spine morphogenesis.␦-Catenin was first identified by yeast two-hybrid screening as a molecule that interacts with Presenilin-1 (PS-1), 3 which is the most prominently mutated gene in familial Alzheimer disease (FAD) patients (1, 2). The interaction of ␦-catenin with PS-1, along with its abundant expression in neurons, suggests that ␦-catenin has specialized neuronal functions (3, 4). Indeed, ␦-catenin-deficient mice showed severe learning deficits and abnormal synaptic plasticity, suggesting a special role of ␦-catenin at the synapse (5). Furthermore, the hemizygous loss of the chromosomal 5p15.2 region, which contains the human ␦-catenin gene, results in the severe mental retardation associated with Cri du Chat syndrome. This chromosomal abnormality may account for 1% of all mentally retarded individuals (6).Structural analysis indicated that ␦-catenin is a member of the p120-Catenin (hereafter, p120 ctn ) subfamily of armadillo proteins and has a DSWV sequence at the C terminus that binds to the PDZ (PSD-95/Disc-larg/ZO-1) domain-containing proteins (7). ␦-Catenin also contains SH3 binding domains at the N terminus (4, 8), a GKKKKKKK sequence (putative NLS) that can potentially promote lipid intermixing (9), and a proline-rich domain that is likely to be involved in the interaction with the actin-binding protein, Profilin (4). The presence of 10 Arm repeats in ␦-catenin suggests its potential participation in various protein-protein interactions. In addition to PS-1, the ␦-catenin-associated proteins identified thus far include E-cadherin (4), S-SCAM (7), p0071 (10), Densin-180 (11), PSD-95, Abl (8), Cortactin (12), sphingosine kinase (13), and Kaiso (14), suggesting its many possible roles in cells. Our previous reports demonstrated that the overexpression of ␦-catenin induces the branching of dendrite-like processes in both NIH 3T3 fibroblasts and primary hippocampal neurons (15). We have also reported that an E18 hippocampal neuron ...

␦-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 ␦-catenin is regulated in cells has not been fully elucidated. We investigated the possibility that glycogen-synthase kinase-3 (GSK-3) phosphorylates ␦-catenin and thus affects its stability. Initially, we found that the level of ␦-catenin was greater and the half-life of ␦-catenin was longer in GSK-3␤ fibroblasts than those in GSK-3␤؉/؉ 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-3␣ and -3␤ kinase dead constructs, consistently showed that the levels of endogenous ␦-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-3␣ and -3␤ interact with and phosphorylate ␦-catenin. The phosphorylation of ⌬C207-␦-catenin (lacking 207 C-terminal residues) and T1078A ␦-catenin by GSK-3 was noticeably reduced compared with that of wild type ␦-catenin, and the data from liquid chromatography-tandem mass spectrometry analyses suggest that the Thr 1078 residue of ␦-catenin is one of the GSK-3 phosphorylation sites. Treatment with MG132 or ALLN, specific inhibitors of proteosome-dependent proteolysis, increased ␦-catenin levels and caused an accumulation of ubiquitinated ␦-catenin. It was also found that GSK-3 triggers the ubiquitination of ␦-catenin. These results suggest that GSK-3 interacts with and phosphorylates ␦-catenin and thereby negatively affects its stability by enabling its ubiquitination/proteosome-mediated proteolysis.

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

The enzyme ␥-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 ␥-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 ␥-secretase components such as Nicastrin, Aph-1, and Pen-2. Mutations on PS-1 can alter the effects of ␥-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 ␥-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 ␥-secretase activities with the compensatory overexpression of wild type NTF. In comparison, both D257A NTF and D385A CTF could abolish the ␥-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.

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.