Related Experiment Video

Updated: Apr 11, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

6.2K

CDK/mTOR-dependent phosphorylation of UBE2H restrains its charging with ubiquitin and regulates CTLH-dependent

Yingqian Chen, Valentina Rossio, Joao A Paulo

    Biorxiv : the Preprint Server for Biology
    |April 10, 2026
    PubMed

    Insights

    Cell cycle and nutrient signals regulate the C-terminal to LisH (CTLH) E3 ligase complex activity by controlling UBE2H enzyme phosphorylation. This phosphorylation limits ubiquitin charging, impacting CTLH substrate degradation and cellular processes.

    Area of Science:

    • Molecular Biology
    • Cell Biology
    • Biochemistry

    Background:

    • The C-terminal to LisH (CTLH) complex is a multi-subunit E3 ubiquitin ligase with crucial biological roles.
    • Understanding the regulatory mechanisms controlling CTLH complex ubiquitylation activity is essential.

    Purpose of the Study:

    • To investigate how the catalytic activity of the CTLH E3 ligase complex is regulated.
    • To identify key factors that modulate CTLH ubiquitylation capacity.

    Main Methods:

    • Phosphoproteomic analysis of the E2 enzyme UBE2H.
    • Biochemical assays to assess UBE2H ubiquitin charging.
    • Cellular assays to evaluate CTLH-mediated substrate degradation and proliferation.
    • Identification of CTLH substrates and recognition motifs.

    Main Results:

    • CDK and mTOR-dependent phosphorylation of UBE2H at N-terminal serine residues (S3/S5) inhibits its ubiquitin-charging capacity.
    • Mitotic CDKs inactivate UBE2H during mitosis, while mTOR restrains charging during interphase, linking CTLH activity to nutrient status.
    • Preventing UBE2H phosphorylation enhances CTLH activity, substrate degradation, and causes proliferation and mitotic defects.
    • NEK9 and AAMP identified as novel CTLH substrates, with a DR-like C-degron recognized by MKLN1.

    Conclusions:

    • Regulation of the E2 enzyme UBE2H by cell cycle (CDK) and nutrient (mTOR) signaling pathways dynamically controls CTLH E3 ligase activity.
    • This regulatory mechanism ensures that CTLH-dependent ubiquitylation is coupled to specific cellular states and nutrient availability.
    • The findings provide new insights into the intricate regulation of ubiquitin ligase complexes and their substrates.

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