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 October 1, 2019; First 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 5

3-M E3 ligase ubiquitinates and degrades plasma membrane protein LL5β.

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3-M E3 ligase ubiquitinates and degrades plasma membrane protein LL5β. (...
(A) Schematic representation of the strategy for identifying proteins interacting with the 3-M complex. Endogenous CCDC8, FBXW8, CUL7, CUL9, and OBSL1 were tagged with 3×FLAG tag by CRISPR in U2OS cells. Individual protein complexes were purified using FLAG antibody and analyzed by mass spectrometry. (B) Validation of LL5β as a 3-M–interacting protein. Endogenous 3×FLAG-tagged CCDC8, FBXW8, CUL7, CUL9, or OBSL1 protein was immunoprecipitated with FLAG antibody, followed by Western blotting (IB) analyses. (C and D) U2OS cells were transfected with plasmid expressing indicated proteins (C) or infected with shRNA lentivirus targeting indicated proteins (D). The ubiquitylation of overexpressed (C) or endogenous (D) LL5β ubiquitylation was determined by IP and Western blotting analyses. (E) U2OS cells were infected with lentivirus expressing shRNA targeting CUL7 or CCDC8, followed by direct Western blotting to detect indicated proteins. The quantitation of relative abundance of LL5β is shown. (F) U2OS cells with endogenous 3×FLAG-OBSL1 tagged by CRISPR were transfected with siRNA targeting OBSL1, followed by direct Western blotting to detect indicated proteins. (G and H) U2OS cells were treated with GSK3 or CK2 inhibitors (G) or Wnt3a (H), followed by direct Western blotting to detect indicated proteins. (I) CUL7 ubiquitinated LL5β in vitro. Indicated proteins were individually immunopurified from 293F cells and eluted using antigenic peptide. Substrate LL5β was incubated with a mixture of purified proteins as indicated in the presence of UBE1, UBE2D3, and HA-ubiquitin (HA-Ub) with or without ATP. After the reaction, LL5β was denatured by boiling in 1% SDS and immunoprecipitated, followed by SDS-PAGE and Western blot.