When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES) has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i) there are no publicly available cloning vectors harboring a 2A peptide gene and (ii) comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.

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␦-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 ...

Runx2 plays essential roles in bone formation and chondrocyte maturation. Akt promotes osteoblast differentiation induced by the bone morphogenetic proteins BMP2 and enhances the function and transcriptional activity of Runx2. However, the precise molecular mechanism underlying the relationship between Runx2 and Akt is not well understood. In this study, we examined the role of Akt in regulating Runx2 function. We found that Akt increases the stability of Runx2 protein. However, the level of Runx2 mRNA was not affected by Akt, and we did not find any evidence for direct modification of Runx2 by Akt. Instead, we found evidence that Akt induces the phosphorylation of the Smad ubiquitination regulatory factor Smurf2 and decreases the level of Smurf2 protein through ubiquitin/proteasome-mediated degradation of Smurf2. Akt also alleviates Smurf2-mediated suppression of Runx2 transcriptional activity. Taken together, our results suggest that Akt regulates osteoblast differentiation, at least in part, by enhancing the protein stability and transcriptional activity of Runx2 through regulation of ubiquitin/proteasome-mediated degradation of Smurf2.