A Highlights from MBoC Selection: Emi2 Inhibition of the Anaphase-promoting Complex/Cyclosome Absolutely Requires Emi2 Binding via the C-Terminal RL Tail

Emi2 (also called Erp1) inhibits the anaphase-promoting complex/cyclosome (APC/C) and thereby causes metaphase II arrest in unfertilized vertebrate eggs. Both the D-box and the zinc-binding region (ZBR) of Emi2 have been implicated in APC/C inhibition. However, it is not well known how Emi2 interacts with and hence inhibits the APC/C. Here we show that Emi2 binds the APC/C via the C-terminal tail, termed here the RL tail. When expressed in Xenopus oocytes and egg extracts, Emi2 lacking the RL tail fails to interact with and inhibit the APC/C. The RL tail itself can directly bind to the APC/C, and, when added to egg extracts, either an excess of RL tail peptides or anti-RL tail peptide antibody can dissociate endogenous Emi2 from the APC/C, thus allowing APC/C activation. Furthermore, and importantly, the RL tail–mediated binding apparently promotes the inhibitory interactions of the D-box and the ZBR (of Emi2) with the APC/C. Finally, Emi1, a somatic paralog of Emi2, also has a functionally similar RL tail. We propose that the RL tail of Emi1/Emi2 serves as a docking site for the APC/C, thereby promoting the interaction and inhibition of the APC/C by the D-box and the ZBR.     

Effects of Forced Expression of an NH2-terminal Truncated β-Catenin on Mouse Intestinal Epithelial Homeostasis

β-Catenin functions as a downstream component of the Wnt/Wingless signal transduction pathway and as an effector of cell–cell adhesion through its association with cadherins. To explore the in vivo effects of β-catenin on proliferation, cell fate specification, adhesion, and migration in a mammalian epithelium, a human NH₂-terminal truncation mutant (ΔN89β-catenin) was expressed in the 129/Sv embryonic stem cell–derived component of the small intestine of adult C57Bl/6–ROSA26↔ 129/Sv chimeric mice. ΔN89β-Catenin was chosen because mutants of this type are more stable than the wild-type protein, and phenocopy activation of the Wnt/Wingless signaling pathway in Xenopus and Drosophila. ΔN89β-Catenin had several effects. Cell division was stimulated fourfold in undifferentiated cells located in the proliferative compartment of the intestine (crypts of Lieberkühn). The proliferative response was not associated with any discernible changes in cell fate specification but was accompanied by a three- to fourfold increase in crypt apoptosis. There was a marked augmentation of E-cadherin at the adherens junctions and basolateral surfaces of 129/Sv (ΔN89β-catenin) intestinal epithelial cells and an accompanying slowing of cellular migration along crypt-villus units. 1–2% of 129/Sv (ΔN89β-catenin) villi exhibited an abnormal branched architecture. Forced expression of ΔN89β-catenin expression did not perturb the level or intracellular distribution of the tumor suppressor adenomatous polyposis coli (APC). The ability of ΔN89β-catenin to interact with normal cellular pools of APC and/or augmented pools of E-cadherin may have helped prevent the 129/Sv gut epithelium from undergoing neoplastic transformation during the 10-mo period that animals were studied. Together, these in vivo studies emphasize the importance of β-catenin in regulating normal adhesive and signaling functions within this epithelium.     

Dynamics of β-Catenin Interactions with APC Protein Regulate Epithelial Tubulogenesis

Epithelial tubulogenesis involves complex cell rearrangements that require control of both cell adhesion and migration, but the molecular mechanisms regulating these processes during tubule development are not well understood. Interactions of the cytoplasmic protein, β-catenin, with several molecular partners have been shown to be important for cell signaling and cell–cell adhesion. To examine if β-catenin has a role in tubulogenesis, we tested the effect of expressing NH₂-terminal deleted β-catenins in an MDCK epithelial cell model for tubulogenesis. After one day of treatment, hepatocyte growth factor/scatter factor (HGF/ SF)-stimulated MDCK cysts initiated tubulogenesis by forming many long cell extensions. Expression of NH₂-terminal deleted β-catenins inhibited formation of these cell extensions. Both ΔN90 β-catenin, which binds to α-catenin, and ΔN131 β-catenin, which does not bind to α-catenin, inhibited formation of cell extensions and tubule development, indicating that a function of β-catenin distinct from its role in cadherin-mediated cell–cell adhesion is important for tubulogenesis. In cell extensions from parental cysts, adenomatous polyposis coli (APC) protein was localized in linear arrays and in punctate clusters at the tips of extensions. Inhibition of cell extension formation correlated with the colocalization and accumulation of NH₂-terminal deleted β-catenin in APC protein clusters and the absence of linear arrays of APC protein. Continued HGF/ SF treatment of parental cell MDCK cysts resulted in cell proliferation and reorganization of cell extensions into multicellular tubules. Similar HGF/SF treatment of cysts derived from cells expressing NH₂-terminal deleted β-catenins resulted in cells that proliferated but formed cell aggregates (polyps) within the cyst rather than tubules. Our results demonstrate an unexpected role for β-catenin in cell migration and indicate that dynamic β-catenin–APC protein interactions are critical for regulating cell migration during epithelial tubulogenesis.     

NH2-terminal Deletion of β-Catenin Results in Stable Colocalization of Mutant β-Catenin with Adenomatous Polyposis Coli Protein and Altered MDCK Cell Adhesion

β-Catenin is essential for the function of cadherins, a family of Ca²⁺-dependent cell–cell adhesion molecules, by linking them to α-catenin and the actin cytoskeleton. β-Catenin also binds to adenomatous polyposis coli (APC) protein, a cytosolic protein that is the product of a tumor suppressor gene mutated in colorectal adenomas. We have expressed mutant β-catenins in MDCK epithelial cells to gain insights into the regulation of β-catenin distribution between cadherin and APC protein complexes and the functions of these complexes. Full-length β-catenin, β-catenin mutant proteins with NH₂-terminal deletions before (ΔN90) or after (ΔN131, ΔN151) the α-catenin binding site, or a mutant β-catenin with a COOH-terminal deletion (ΔC) were expressed in MDCK cells under the control of the tetracycline-repressible transactivator. All β-catenin mutant proteins form complexes and colocalize with E-cadherin at cell–cell contacts; ΔN90, but neither ΔN131 nor ΔN151, bind α-catenin. However, β-catenin mutant proteins containing NH₂-terminal deletions also colocalize prominently with APC protein in clusters at the tips of plasma membrane protrusions; in contrast, full-length and COOH-terminal– deleted β-catenin poorly colocalize with APC protein. NH₂-terminal deletions result in increased stability of β-catenin bound to APC protein and E-cadherin, compared with full-length β-catenin. At low density, MDCK cells expressing NH₂-terminal–deleted β-catenin mutants are dispersed, more fibroblastic in morphology, and less efficient in forming colonies than parental MDCK cells. These results show that the NH₂ terminus, but not the COOH terminus of β-catenin, regulates the dynamics of β-catenin binding to APC protein and E-cadherin. Changes in β-catenin binding to cadherin or APC protein, and the ensuing effects on cell morphology and adhesion, are independent of β-catenin binding to α-catenin. These results demonstrate that regulation of β-catenin binding to E-cadherin and APC protein is important in controlling epithelial cell adhesion.     

Functional interaction between peroxisome proliferator-activated receptors-alpha and Mef-2C on human carnitine palmitoyltransferase 1beta (CPT1beta) gene activation

Muscle-type carnitine palmitoyltransferase 1 (CPT1β) is considered to be the gene that controls fatty acid mitochondrial β-oxidation. A functional peroxisome proliferator-activated receptor (PPAR) responsive element (PPRE) and a myocite-specific (MEF2) site that binds MEF2A and MEF2C in the promoter of this gene had been previously identified. We investigated the roles of the PPRE and the MEF2 binding sites and the potential interaction between PPARα and MEF2C regulating the CPT1β gene promoter. Mutation analysis indicated that the MEF2 site contributed to the activation of the CPT1β promoter by PPAR in C2C12 cells. The reporter construct containing the PPRE and the MEF2C site was synergistically activated by co-expression of PPAR, retinoid X receptor (RXR) and MEF2C in non-muscle cells. Moreover, protein-binding assays demonstrated that MEF2C and PPAR specifically bound to one another in vitro. Also for the synergistic activation of the CPT1β gene promoter by MEF2C and PPARα-RXRα, a precise arrangement of its binding sites was essential.     

Impaired Cdc20 signaling promotes senescence in normal cells and apoptosis in non–small cell lung cancer cells

Cellular senescence is a form of irreversible growth arrest that cancer cells evade. The cell division cycle protein 20 homolog (Cdc20) is a positive regulator of cell division, but how its dysregulation may relate to senescence is unclear. Here, we find that Cdc20 mRNA and protein expression are downregulated in stress-induced premature senescent lung fibroblasts in a p53-dependent manner. Either Cdc20 downregulation or inhibition of anaphase-promoting complex/cyclosome (APC/C) is sufficient to induce premature senescence in lung fibroblasts, while APC/C activation inhibits stress-induced premature senescence. Mechanistically, we show both Cdc20 downregulation and APC/C inhibition induce premature senescence through glycogen synthase kinase (GSK)-3β–mediated phosphorylation and downregulation of securin expression. Interestingly, we determined Cdc20 expression is upregulated in human lung adenocarcinoma. We find that downregulation of Cdc20 in non–small cell lung cancer (NSCLC) cells is sufficient to inhibit cell proliferation and growth in soft agar and to promote apoptosis, but not senescence, in a manner dependent on downregulation of securin following GSK-3β-mediated securin phosphorylation. Similarly, we demonstrate securin expression is downregulated and cell viability is inhibited in NSCLC cells following inhibition of APC/C. Furthermore, we show chemotherapeutic drugs downregulate both Cdc20 and securin protein expression in NSCLC cells. Either Cdc20 downregulation by siRNA or APC/C inhibition sensitize, while securin overexpression inhibits, chemotherapeutic drug-induced NSCLC cell death. Together, our findings provide evidence that Cdc20/APC/C/securin-dependent signaling is a key regulator of cell survival, and its disruption promotes premature senescence in normal lung cells and induces apoptosis in lung cancer cells that have bypassed the senescence barrier.     

Paraoxonase 2 Serves a Proapopotic Function in Mouse and Human Cells in Response to the Pseudomonas aeruginosa Quorum-sensing Molecule N-(3-Oxododecanoyl)-homoserine Lactone

Pseudomonas aeruginosa use quorum-sensing molecules, including N-(3-oxododecanoyl)-homoserine lactone (C12), for intercellular communication. C12 activated apoptosis in mouse embryo fibroblasts (MEF) from both wild type (WT) and Bax/Bak double knock-out mice (WT MEF and DKO MEF that were responsive to C12, DKO_R MEF): nuclei fragmented; mitochondrial membrane potential (Δψ_mito) depolarized; Ca²⁺ was released from the endoplasmic reticulum (ER), increasing cytosolic [Ca²⁺] (Ca_cyto); and caspase 3/7 was activated. DKO_R MEF had been isolated from a nonclonal pool of DKO MEF that were non-responsive to C12 (DKO_NR MEF). RNAseq analysis, quantitative PCR, and Western blots showed that WT and DKO_R MEF both expressed genes associated with cancer, including paraoxonase 2 (PON2), whereas DKO_NR MEF expressed little PON2. Adenovirus-mediated expression of human PON2 in DKO_NR MEF rendered them responsive to C12: Δψ_mito depolarized, Ca_cyto increased, and caspase 3/7 activated. Human embryonic kidney 293T (HEK293T) cells expressed low levels of endogenous PON2, and these cells were also less responsive to C12. Overexpression of PON2, but not PON2-H114Q (no lactonase activity) in HEK293T cells caused them to become sensitive to C12. Because [C12] may reach high levels in biofilms in lungs of cystic fibrosis (CF) patients, PON2 lactonase activity may control Δψ_mito, Ca²⁺ release from the ER, and apoptosis in CF airway epithelia. Coupled with previous data, these results also indicate that PON2 uses its lactonase activity to prevent Bax- and Bak-dependent apoptosis in response to common proapoptotic drugs like doxorubicin and staurosporine, but activates Bax- and Bak-independent apoptosis in response to C12.     

Novel oxindole/benzofuran hybrids as potential dual CDK2/GSK-3β inhibitors targeting breast cancer: design,synthesis, biological evaluation,and in silico studies

The serine/threonine protein kinases CDK2 and GSK-3β are key oncotargets in breast cancer cell lines, therefore, in the present study three series of oxindole-benzofuran hybrids were designed and synthesised as dual CDK2/GSK-3β inhibitors targeting breast cancer (5a–g, 7a–h, and 13a–b). The N¹-unsubstituted oxindole derivatives, series 5, showed moderate to potent activity on both MCF-7 and T-47D breast cancer cell lines. Compounds 5d–f showed the most potent cytotoxic activity with IC₅₀ of 3.41, 3.45 and 2.27 μM, respectively, on MCF-7 and of 3.82, 4.53 and 7.80 μM, respectively, on T-47D cell lines, in comparison to the used reference standard (staurosporine) IC₅₀ of 4.81 and 4.34 μM, respectively. On the other hand, the N¹-substituted oxindole derivatives, series 7 and 13, showed moderate to weak cytotoxic activity on both breast cancer cell lines. CDK2 and GSK-3β enzyme inhibition assay of series 5 revealed that compounds 5d and 5f are showing potent dual CDK2/GSK-3β inhibitory activity with IC₅₀ of 37.77 and 52.75 nM, respectively, on CDK2 and 32.09 and 40.13 nM, respectively, on GSK-3β. The most potent compounds 5d–f caused cell cycle arrest in the G2/M phase in MCF-7 cells inducing cell apoptosis because of the CDK2/GSK-3β inhibition. Molecular docking studies showed that the newly synthesised N¹-unsubstituted oxindole hybrids have comparable binding patterns in both CDK2 and GSK-3β. The oxindole ring is accommodated in the hinge region interacting through hydrogen bonding with the backbone CO and NH of the key amino acids Glu81 and Leu83, respectively, in CDK2 and Asp133 and Val135, respectively, in GSK-3β. Whereas, in series 7 and 13, the N¹-substitutions on the oxindole nucleus hinder the compounds from achieving these key interactions with hinge region amino acids what rationalises their moderate to low anti-proliferative activity.