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 7

3-M syndrome patient–derived mutations disrupt the assembly of the 3-M complex on the plasma membrane and the regulation of LL5β.

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3-M syndrome patient–derived mutations disrupt the assembly of the 3-M c...
(A) Schematic representation of domains, binding proteins, and 3-M patient–derived mutations in CUL7. (B) mKate-fused wild-type or mutant CCDC8 was coexpressed with CUL7-mTagBFP and OBSL1-EGFP in U2OS cells, followed by confocal microscopic analyses of protein localization. Scale bars: 10 μm. (C) FLAG-CUL7 and HA-OBSL1 were coexpressed in HEK293 cells. CUL7-OBSL1 association was determined by IP and Western analysis. (D) U2OS cells with CCDC8-21×FLAG and 16×MYC-CUL7 double knockin (DKI, see Figure 2 for details) were subjected to CRISPR-mediated genome editing to introduce a homozygous L1588P mutation. The binding between endogenous CUL7 and CCDC8 was determined by IP and Western. (E) Total cell lysates derived from parental, DKI, and DKI/L1588P U2OS cells were fractionated by centrifugation, and total lysate (Lys), cytoplasm (Cyto), and membrane (Mem) fractions were analyzed by Western blotting. (F) The steady-state level of LL5β protein was determined by direct Western blot in parental, DKI, and DKI/L1588P U2OS cells. (G) Schematic representation of the function and regulation of 3-M E3 ubiquitin ligase. MT, microtubule.