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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

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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 2

CCDC8 localizes on and recruits OBSL1 and CUL7 to the plasma membrane.

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CCDC8 localizes on and recruits OBSL1 and CUL7 to the plasma membrane.
(...
(A) Schematic representation of the domain structure and knockin epitope tagging of endogenous CUL7 and CCDC8 to generate 16×MYC-CUL7 and CCDC8-21×FLAG double-knockin (DKI) U2OS cells. (B) Validation of DKI U2OS cells. Cell lysates from DKI and parental U2OS cells were subjected to anti-FLAG immunoprecipitation (IP) and Western blot. (C) Immunostaining of DKI and parental U2OS cells with antibodies specific for MYC, FLAG, or plasma membrane marker Na+/K+-ATPase demonstrates the colocalization of endogenous (endo.) CCDC8 and CUL7 on the plasma membrane. Scale bars: 10 μm. (D) U2OS cells transfected with shRNA targeting CCDC8 were fractionated by centrifugation, and total lysate (Lys), cytoplasm (Cyto), and membrane (Mem) fractions were analyzed by Western blot with antibodies recognizing the indicated proteins. CUL7 and CUL9 were recognized by the same monoclonal antibody. (E) Induced overexpression of CCDC8-mKate localized CUL7-mTagBFP and OBSL1-EGFP onto the plasma membrane. Dox, doxycycline. Scale bars: 10 μm.

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