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CPEB1 regulates beta-catenin mRNA translation and cell migration in astrocytes.
ABSTRACT: A crucial step in directed cell migration is the recruitment of cytoskeletal regulatory and signaling proteins to the leading edge of the cell. One protein localized to the leading edge of a migrating astrocyte is beta-catenin. Using an in vitro wound-healing assay, we show that the localization of beta-catenin to the leading edge is dependent upon new protein synthesis at the time of wounding. We examined the mRNA encoding beta-catenin for potential regulatory elements and identified a conserved cytoplasmic polyadenylation element in the 3'-untranslated region (UTR). We now show that the CPE-binding protein (CPEB1) is expressed in astrocytes and that translation of beta-catenin mRNA is regulated by CPEB1. Further, expression of a mutant CPEB1 protein in astrocytes not only blocks beta-catenin protein localization, it also inhibits cell migration. These findings demonstrate a role for CPEB1-mediated protein synthesis in the localization of beta-catenin protein to the leading edge of migrating astrocytes and in regulating directed cell motility.
Project description:The Cytoplasmic Polyadenylation Element Binding proteins are RNA binding proteins involved in the translational regulation of mRNA. During cell cycle progression, CPEB1 is labeled for degradation by phosphorylation-dependent ubiquitination by the SCF(β-TrCP) ligase. The peptidyl-prolyl isomerase Pin1 plays a key role in CPEB1 degradation. Conditioned by the cell cycle stage, CPEB1 and Pin1 interactions occur in a phosphorylation-independent or -dependent manner. CPEB1 contains six potential phosphorylatable Pin1 binding sites. Using a set of biophysical techniques, we discovered that the pS210 site is unique, since it displays binding activity not only to the WW domain but also to the prolyl-isomerase domain of Pin1. The NMR structure of the Pin1 WW-CPEB1 pS210 (PDB ID: 2n1o) reveals that the pSerPro motif is bound in trans configuration through contacts with amino acids located in the first turn of the WW domain and the conserved tryptophan in the β3-strand. NMR relaxation analyses of Pin1 suggest that inter-domain flexibility is conferred by the modulation of the interaction with peptides containing the pS210 site, which is essential for degradation.
Project description:In mouse mammary epithelial cells, cytoplasmic polyadenylation element binding protein 1 (CPEB1) mediates the apical localization of ZO-1 mRNA, which encodes a critical tight junction component. In mice lacking CPEB1 and in cultured cells from which CPEB has been depleted, randomly distributed ZO-1 mRNA leads to the loss of cell polarity. We have investigated whether this diminution of polarity results in an epithelial-to-mesenchyme (EMT) transition and possible increased metastatic potential. Here, we show that CPEB1-depleted mammary epithelial cells alter their gene expression profile in a manner consistent with an EMT and also become motile, which are made particularly robust when cells are treated with transforming growth factor-?, an enhancer of EMT. CPEB1-depleted mammary cells become metastatic to the lung following injection into mouse fat pads while ectopically expressed CPEB1 prevents metastasis. Surprisingly, CPEB1 depletion causes some EMT/metastasis-related mRNAs to have shorter poly(A) tails while other mRNAs to have longer poly(A) tails. Matrix metalloproteinase 9 (MMP9) mRNA, which encodes a metastasis-promoting factor, undergoes poly(A) lengthening and enhanced translation upon CPEB reduction. Moreover, in human breast cancer cells that become progressively more metastatic, CPEB1 is reduced while MMP9 becomes more abundant. These data suggest that at least in part, CPEB1 regulation of MMP9 mRNA expression mediates metastasis of breast cancer cells.
Project description:Neuronal morphogenesis, the growth and arborization of neuronal processes, is an essential component of brain development. Two important but seemingly disparate components regulating neuronal morphology have previously been described. In the hippocampus, neurotrophins, particularly brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3), act to enhance cell growth and branching, while activity-induced branching was shown to be dependent upon intracellular beta-catenin. We now describe a molecular link between NT3 stimulation and beta-catenin increase in developing neurons and demonstrate that this process is required for the NT3-mediated increase in process branching. Here, we show that beta-catenin is rapidly increased specifically in growth cones following NT3 stimulation. This increase in beta-catenin is protein synthesis dependent and requires the activity of cytoplasmic polyadenylation element-binding protein-1 (CPEB1), an mRNA-binding protein that regulates mRNA translation. We find that CPEB1 protein binds beta-catenin mRNA in a CPE-dependent manner and that both localize to growth cones of developing hippocampal neurons. Both the NT3-mediated rapid increase in beta-catenin and process branching are abolished when CPEB1 function is inhibited. In addition, the NT3-mediated increase in beta-catenin in growth cones is dependent upon internal calcium and the activity of CaMKII (calcium/calmodulin-dependent kinase II). Together, these results suggest that CPEB1 regulates beta-catenin synthesis in neurons and may contribute to neuronal morphogenesis.
Project description:Cancer stem cells (CSCs) are a subpopulation of cells within tumors that are believed to possess pluripotent properties and thought to be responsible for tumor initiation, progression, relapse and metastasis. Cytoplasmic polyadenylation element-binding protein 1 (CPEB1), a sequence-specific RNA-binding protein that regulates mRNA polyadenylation and translation, has been linked to cancer progression and metastasis. However, the involvement of CPEB1 in hepatocellular carcinoma (HCC) remains unclear. In this study, we have demonstrated that CPEB1 directly regulates sirtuin 1 (SIRT1) mRNA to mediate cancer stemness in HCC. Cancer stemness was analyzed by self-renewal ability, chemoresistance, metastasis, expression of stemness-related genes and CSC marker-positive cell populations. The results indicate that CPEB1 is downregulated in HCC. Overexpression of CPEB1 dramatically reduced HCC cell stemness, whereas silencing CPEB1 enhances it. Using site-directed mutagenesis, a luciferase reporter assay, and immunoprecipitation, we found that CPEB1 could directly target the 3'-UTR of SIRT1, control poly(A) tail length and suppress its translation to mediate cancer stemness in vitro and in vivo. Overall, our findings suggest that the negative regulation between CPEB1 and SIRT1 contributes to the suppression of cancer stemness in HCC. CPEB1 may have potential as a therapeutic target in HCC.
Project description:Wnt5a is a representative ligand that activates the Wnt/beta-catenin-independent pathway, resulting in the regulation of cell adhesion, migration, and polarity, but its molecular mechanism is poorly understood. This report shows that Dishevelled (Dvl) binds to adenomatous polyposis coli (APC) gene product, and this binding is enhanced by Wnt5a. Dvl was involved in the stabilization of the plus end dynamics of microtubules as well as APC. Frizzled2 (Fz2) was present with Wnt5a at the leading edge of migrating cells and formed a complex with APC through Dvl. Fz2 also interacted with integrins at the leading edge, and Dvl and APC associated with and activated focal adhesion kinase and paxillin. The binding of APC to Dvl enhanced the localization of paxillin to the leading edge and was involved in Wnt5a-dependent focal adhesion turnover. Furthermore, this new Wnt5a signalling pathway was important for the epithelial morphogenesis in the three-dimensional culture. These results suggest that the functional and physical interaction of Dvl and APC is involved in Wnt5a/Fz2-dependent focal adhesion dynamics during cell migration and epithelial morphogenesis.
Project description:Inversin is a ciliary protein that critically regulates developmental processes and tissue homeostasis in vertebrates, partly through the degradation of Dishevelled (Dvl) proteins to coordinate Wnt signaling in planar cell polarity (PCP). Here, we investigated the role of Inversin in coordinating cell migration, which highly depends on polarity processes at the single-cell level, including the spatial and temporal organization of the cytoskeleton as well as expression and cellular localization of proteins in leading edge formation of migrating cells. Using cultures of mouse embryonic fibroblasts (MEFs) derived from inv(-/-) and inv(+/+) animals, we confirmed that both inv(-/-) and inv(+/+) MEFs form primary cilia, and that Inversin localizes to the primary cilium in inv(+/+) MEFs. In wound healing assays, inv(-/-) MEFs were severely compromised in their migratory ability and exhibited cytoskeletal rearrangements, including distorted lamellipodia formation and cilia orientation. Transcriptome analysis revealed dysregulation of Wnt signaling and of pathways regulating actin organization and focal adhesions in inv(-/-) MEFs as compared to inv(+/+) MEFs. Further, Dvl-1 and Dvl-3 localized to MEF primary cilia, and ?-catenin/Wnt signaling was elevated in inv(-/-) MEFs, which moreover showed reduced ciliary localization of Dvl-3. Finally, inv(-/-) MEFs displayed dramatically altered activity and localization of RhoA, Rac1, and Cdc42 GTPases, and aberrant expression and targeting of the Na(+)/H(+) exchanger NHE1 and ezrin/radixin/moesin (ERM) proteins to the edge of cells facing the wound. Phosphorylation of ?-catenin at the ciliary base and formation of well-defined lamellipodia with localization and activation of ERM to the leading edge of migrating cells were restored in inv(-/-) MEFs expressing Inv-GFP. Collectively, our findings point to the significance of Inversin in controlling cell migration processes, at least in part through transcriptional regulation of genes involved in Wnt signaling and pathways that control cytoskeletal organization and ion transport.
Project description:Wnt signaling regulates beta-catenin-mediated gene transcription and planar cell polarity (PCP). The Wnt co-receptor, Lrp6, is required for signaling along the beta-catenin arm. We show that Lrp6 downregulation (by morpholino injection) or overexpression in Xenopus embryos disrupts convergent extension, a hallmark feature of Wnt/PCP components. In embryos with decreased Lrp6 levels, cells of the dorsal marginal zone (DMZ), which undergoes extensive cellular rearrangements during gastrulation, exhibit decreased length:width ratios, decreased migration, and increased numbers of transient cytoplasmic protrusions. We show that Lrp6 opposes Wnt11 activity and localizes to the posterior edge of migrating DMZ cells and that Lrp6 downregulation enhances cortical and nuclear localization of Dsh and phospho-JNK, respectively. Taken together, these data suggest that Lrp6 inhibits Wnt/PCP signaling. Finally, we identify the region of the Lrp6 protein with Wnt/PCP activity to a stretch of 36 amino acids, distinct from regions required for Wnt/beta-catenin signaling. We propose a model in which Lrp6 plays a critical role in the switch from Wnt/PCP to Wnt/beta-catenin signaling.
Project description:Fragile X syndrome (FXS), the most common cause of inherited mental retardation and autism, is caused by transcriptional silencing of FMR1, which encodes the translational repressor fragile X mental retardation protein (FMRP). FMRP and cytoplasmic polyadenylation element-binding protein (CPEB), an activator of translation, are present in neuronal dendrites, are predicted to bind many of the same mRNAs and may mediate a translational homeostasis that, when imbalanced, results in FXS. Consistent with this possibility, Fmr1(-/y); Cpeb1(-/-) double-knockout mice displayed amelioration of biochemical, morphological, electrophysiological and behavioral phenotypes associated with FXS. Acute depletion of CPEB1 in the hippocampus of adult Fmr1(-/y) mice rescued working memory deficits, demonstrating reversal of this FXS phenotype. Finally, we find that FMRP and CPEB1 balance translation at the level of polypeptide elongation. Our results suggest that disruption of translational homeostasis is causal for FXS and that the maintenance of this homeostasis by FMRP and CPEB1 is necessary for normal neurologic function.
Project description:Host and virus interactions occurring at the post-transcriptional level are critical for infection but remain poorly understood. Here, we performed comprehensive transcriptome-wide analyses revealing that human cytomegalovirus (HCMV) infection results in widespread alternative splicing (AS), shortening of 3' untranslated regions (3' UTRs) and lengthening of poly(A)-tails in host gene transcripts. We found that the host RNA-binding protein CPEB1 was highly induced after infection, and ectopic expression of CPEB1 in noninfected cells recapitulated infection-related post-transcriptional changes. CPEB1 was also required for poly(A)-tail lengthening of viral RNAs important for productive infection. Strikingly, depletion of CPEB1 reversed infection-related cytopathology and post-transcriptional changes, and decreased productive HCMV titers. Host RNA processing was also altered in herpes simplex virus-2 (HSV-2)-infected cells, thereby indicating that this phenomenon might be a common occurrence during herpesvirus infections. We anticipate that our work may serve as a starting point for therapeutic targeting of host RNA-binding proteins in herpesvirus infections.
Project description:The cytoplasmic element binding protein 1 (CPEB1) regulates many important biological processes ranging from cell cycle control to learning and memory formation, by controlling mRNA translation efficiency via 3' untranslated regions (3'UTR). In the present study, we show that CPEB1 is significantly downregulated in human Glioblastoma Multiforme (GBM) tissues and that the restoration of its expression impairs glioma cell lines growth. We demonstrate that CPEB1 promotes the expression of the cell cycle inhibitor p27(Kip1) by specifically targeting its 3'UTR, and competes with miR-221/222 binding at an overlapping site in the 3'UTR, thus impairing miR-221/222 inhibitory activity. Upon binding to p27(Kip1) 3'UTR, CPEB1 promotes elongation of poly-A tail and the subsequent translation of p27(Kip1) mRNA. This leads to higher levels of p27(Kip1) in the cell, in turn significantly inhibiting cell proliferation, and confers to CPEB1 a potential value as a tumor suppressor in Glioblastoma.