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Role of cdc2 kinase phosphorylation and conserved N-terminal proteolysis motifs in cytoplasmic polyadenylation-element-binding protein (CPEB) complex dissociation and degradation.

ABSTRACT: Cytoplasmic polyadenylation-element-binding protein (CPEB) is a well-characterized and important regulator of translation of maternal mRNA in early development in organisms ranging from worms, flies and clams to frogs and mice. Previous studies provided evidence that clam and Xenopus CPEB are hyperphosphorylated at germinal vesicle breakdown (GVBD) by cdc2 kinase, and degraded shortly after. To examine the conserved features of CPEB that mediate its modification during meiotic maturation, we microinjected mRNA encoding wild-type and mutated clam CPEB into Xenopus oocytes that were subsequently allowed to mature with progesterone. We observed that (i) ectopically expressed clam CPEB is phosphorylated at GVBD and subsequently degraded, mirroring the fate of the endogenous Xenopus CPEB protein, (ii) mutation of nine Ser/Thr Pro-directed kinase sites prevents phosphorylation and degradation and (iii) deletion of the PEST box, and to a lesser extent of the putative cyclin destruction box, generates a stable and phosphorylated version of CPEB. We conclude that phosphorylation of both consensus and non-consensus sites by cdc2 kinase targets clam CPEB for PEST-mediated destruction. We also show that phosphorylation of CPEB mediates its dissociation from ribonucleoprotein complexes, prior to degradation. Our findings reinforce results obtained in Xenopus, and have implications for CPEB from other invertebrates including Drosophila, Caenorhabditis elegans and Aplysia, which lack PEST boxes.

SUBMITTER: Thom G

PROVIDER: S-EPMC1223136 | biostudies-other | 2003 Feb

REPOSITORIES: biostudies-other

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Project description:ObjectiveObesity represents a growing health problem that is reaching pandemic dimensions and lacks effective cures, thus highlighting an urgent need for better mechanistic understanding and new therapeutic strategies. Unlike transcription, the function of translation in obesity has hardly been investigated. Here, we fill this knowledge gap by pinpointing a crucial function for gene regulation at the step of translation in diet-induced obesity.MethodsWe performed studies with human adipose tissue, high-fat-diet-induced obese mice and rats, CPEB4-knockout mice, and adipocyte lines. Cells were transfected with small-interfering RNAs that knockdown CPEB4. Transcriptome-wide identification and validation of CPEB4 targets in adipocytes were obtained by RNA-protein coimmunoprecipitation and high-throughput sequencing. The effect of CPEB4 depletion on high-fat-diet-induced dysbiosis was determined by 16S ribosomal-RNA gene sequencing and microbiome bioinformatics.ResultsWe show that cytoplasmic polyadenylation element-binding protein 4 (CPEB4), which controls the translation of specific mRNAs by modulating their poly(A) tails, is highly expressed in visceral fat of obese but not lean humans and rodents (mice and rats), where it orchestrates an essential post-transcriptional reprogramming for aggravation of high-fat-diet-induced obesity. Mechanistically, CPEB4 overexpression in obese adipocytes activates the translation of factors essential for adipose tissue expansion (Cebpb, Stat5a) and adipocyte-intrinsic immune-like potential (Ccl2, Tlr4), as demonstrated by RNA-immunoprecipitation and high-throughput sequencing and experimentally validated in vivo. Consistently blocking CPEB4 production in knockout mice protects against diet-induced body weight gain and reduces adipose tissue enlargement and inflammation. In addition, the depletion of CPEB4 specifically in obese adipocytes using short hairpin RNAs decreases cell differentiation, lipid accumulation, and the proinflammatory and migratory capacity of macrophages. The absence of CPEB4 also attenuates high-fat diet-induced dysbiosis, shaping the microbiome composition toward a more beneficial profile, as shown by microbiome bioinformatics analysis.ConclusionOur study identifies CPEB4 as a driver and therapeutic target to combat obesity.

Project description:BackgroundOver-expression of cyclooxygenase (COX)-2 promotes breast cancer progression by multiple mechanisms, including induction of stem-like cells (SLC). Combined gene expression and microRNA microarray analyses of empty vector vs COX-2- transfected COX-2 low MCF7 breast cancer cell line identified two COX-2-upregulated microRNAs, miR-526b and miR-655, both found to be oncogenic and SLC-promoting. Cytoplasmic Polyadenylation Element-Binding Protein 2 (CPEB2) was the single common target of both microRNAs, the functions of which remain controversial. CPEB2 has multiple isoforms (A-F), and paradoxically, a high B/A ratio was reported to impart anoikis-resistance and metastatic phenotype in triple- negative breast cancer cells. We tested whether CPEB2 is a tumor suppressor in mammary epithelial cells.MethodsWe knocked-out CPEB2 in the non-tumorigenic mammary epithelial cell line MCF10A by CRISPR/Cas9-double nickase approach, and knocked-down CPEB2 with siRNAs in the poorly malignant MCF7 cell line, both lines being high CPEB2-expressing. The resultant phenotypes for oncogenity were tested in vitro for both lines and in vivo for CPEB2KO cells. Finally, CPEB2 expression was compared between human breast cancer and non-tumor breast tissues.ResultsCPEB2 (isoform A) expression was inversely correlated with COX-2 or the above microRNAs in COX-2-divergent breast cancer cell lines. CPEB2KO MCF10A cells exhibited oncogenic properties including increased proliferation, migration, invasion, EMT (decreased E-Cadherin, increased Vimentin, N-Cadherin, SNAI1, and ZEB1) and SLC phenotype (increased tumorsphere formation and SLC marker-expression). Tumor-suppressor p53 protein was shown to be a novel translationally-regulated target of CPEB2, validated with polysome profiling. CPEB2KO, but not wild-type cells produced lung colonies upon intravenous injection and subcutaneous tumors and spontaneous lung metastases upon implantation at mammary sites in NOD/SCID/IL2Rϒ-null mice, identified with HLA immunostaining. Similarly, siRNA-mediated CPEB2 knockdown in MCF7 cells promoted oncogenic properties in vitro. Human breast cancer tissues (n = 105) revealed a lower mRNA expression for CPEB2 isoform A and also a lower A/B isoform ratio than in non-tumour breast tissues (n = 20), suggesting that CPEB2A accounts for the tumor-suppressor functions of CPEB2.ConclusionsCPEB2, presumably the isoform A, plays a key role in suppressing tumorigenesis in mammary epithelial cells by repressing EMT, migration, invasion, proliferation and SLC phenotype, via multiple targets, including a newly-identified translational target p53.

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Role of cdc2 kinase phosphorylation and conserved N-terminal proteolysis motifs in cytoplasmic polyadenylation-element-binding protein (CPEB) complex dissociation and degradation.

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