Impaired plasma membrane localization of ubiquitin ligase complex underlies 3-M syndrome development
Pu Wang, … , Scott E. Parnell, Yue Xiong
Pu Wang, … , Scott E. Parnell, Yue Xiong
Published July 25, 2019
Citation Information: J Clin Invest. 2019;129(10):4393-4407. https://doi.org/10.1172/JCI129107.
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Research Article Development Genetics

Impaired plasma membrane localization of ubiquitin ligase complex underlies 3-M syndrome development

Abstract

3-M primordial dwarfism is an inherited disease characterized by severe pre- and postnatal growth retardation and by mutually exclusive mutations in 3 genes, CUL7, OBSL1, and CCDC8. The mechanism underlying 3-M dwarfism is not clear. We showed here that CCDC8, derived from a retrotransposon Gag protein in placental mammals, exclusively localized on the plasma membrane and was phosphorylated by CK2 and GSK3. Phosphorylation of CCDC8 resulted in its binding first with OBSL1, and then CUL7, leading to the membrane assembly of the 3-M E3 ubiquitin ligase complex. We identified LL5β, a plasma membrane protein that regulates cell migration, as a substrate of 3-M ligase. Wnt inhibition of CCDC8 phosphorylation or patient-derived mutations in 3-M genes disrupted membrane localization of the 3-M complex and accumulated LL5β. Deletion of Ccdc8 in mice impaired trophoblast migration and placental development, resulting in intrauterine growth restriction and perinatal lethality. These results identified a mechanism regulating cell migration and placental development that underlies the development of 3-M dwarfism.

Authors

Pu Wang, Feng Yan, Zhijun Li, Yanbao Yu, Scott E. Parnell, Yue Xiong

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Figure 6

CCDC8 regulates ECM remodeling and cell migration.

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CCDC8 regulates ECM remodeling and cell migration. (A) U2OS cells stably...
(A) U2OS cells stably expressing shRNA targeting CCDC8 were subjected to transwell cell migration or invasion assay. Data are represented as mean ± SEM from 3 replicates. Significance was determined by 1-way ANOVA and Dunnett’s multiple-comparisons test. **Adjusted P < 0.01, ***adjusted P < 0.001. (B) U2OS cells stably expressing shRNA targeting CCDC8 were cultured on cover glasses precoated with fluorescent gelatin for 8 hours, followed by microscopic examination showing gelatin degradation. Scale bars: 10 μm. (C) Quantification of relative degradation area in B. Fifty cells in each group were chosen randomly. Dots indicate the percentage of area of individual cells being degraded. Lines indicate mean ± SEM. Significance was determined by 1-way ANOVA and Dunnett’s multiple-comparisons test. **Adjusted P < 0.01, ****adjusted P < 0.0001. (D) MEFs of indicated genotypes were subjected to transwell cell migration or invasion assay. Data are represented as mean ± SEM from 3 replicates. Significance was determined by Student’s t test. *P < 0.05, **P < 0.01. (E) RT-qPCR showing Ccdc8 and LL5β mRNA expression in mouse trophoblast stem cells (TSCs) and in vitro–differentiated trophoblast giant cells (TGCs) or syncytiotrophoblast layer II (SynT-II) cells. Cdx2, Prl3d1, and SynB are markers for TSCs, TGCs, and SynT-II, respectively. FGF, fibroblast growth factor; RA, retinoic acid; CHIR, GSK3 inhibitor. (F) TSCs or in vitro–differentiated SynT-II cells were subjected to transwell cell migration or invasion assay. Data are represented as mean ± SEM from 3 replicates. Significance was determined by 1-way ANOVA and Dunnett’s multiple-comparisons test. **Adjusted P < 0.01. (G) LL5β in wild-type or Ccdc8-knockout SynT-II cells was determined by direct Western blotting analysis.