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 3

CCDC8 is phosphorylated by CK2 and GSK3.

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CCDC8 is phosphorylated by CK2 and GSK3. (A) Schematic representation of...
(A) Schematic representation of the domain structure and reported serine phosphorylation sites on the CCDC8 protein. The sequences adjacent to the putative phosphorylated serines and their predicted kinases are shown. (B) U2OS and HEK293 cells were treated with kinase inhibitors targeting GSK3 (by CHIR-98012), CK2 (by CX4945), CDK1 (by RO-3306), and PKC (by sotrastaurin), and lysates were separated by either 10% conventional SDS-PAGE or 6% Zn Phos-tag PAGE, followed by Western blotting using an antibody specific for CCDC8. (C) U2OS cells with endogenous CCDC8 3×FLAG-tagged by CRISPR were treated with GSK3 or CK2 inhibitors. Endogenous CCDC8 was precipitated by anti-FLAG antibody, followed by Western blot with anti-CCDC8, anti–phosphorylated serine 142 (α–p-Ser142), or anti–phosphorylated serine 146 (α–p-Ser146) antibodies. (D) Purified recombinant CCDC8 proteins were incubated with or without recombinant CK2 in kinase buffer containing ATP-γ-S, followed by treatment with p-nitrobenzyl mesylate to alkylate thiophosphates and form thiophosphate ester epitopes. In vitro phosphorylation of CCDC8 by CK2 was detected by Western blot with an antibody recognizing the thiophosphate ester. (E) Recombinant CCDC8 proteins were immobilized on magnetic beads, incubated first with CK2 in buffers containing ATP, and then with GSK3 in buffers containing ATP-γ-S. The phosphorylation by GSK3 was detected by alkylation with p-nitrobenzyl mesylate and Western blot with an antibody recognizing the thiophosphate ester.