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Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Published on: May 10, 2015

USP2 is an SKP2 deubiquitylase that stabilizes both SKP2 and its substrates

Fengwu Zhang¹, Yongchao Zhao², Yi Sun³

¹Cancer Institute of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China.

The Journal of Biological Chemistry

|August 23, 2021

Abstract:

The stability of a protein is regulated by a balance between its ubiquitylation and deubiquitylation. S-phase kinase-associated protein 2 (SKP2) is an oncogenic F-box protein that recognizes tumor suppressor substrates for targeted ubiquitylation by the E3 ligase SKP1-Cullin1-F-box and degradation by proteasome. SKP2 is itself ubiquitylated by the E3 ligases APC/C^CDH1 and SCF^FBXW2, and deubiquitylated by deubiquitylases (DUBs) USP10 and USP13. Given the biological significance of SKP2, it is likely that the other E3s or DUBs may also regulate its stability. Here, we report the identification and characterization of USP2 as a new DUB. We first screened a panel of DUBs and found that both USP2 and USP21 bound to endogenous SKP2, but only USP2 deubiquitylated and stabilized SKP2 protein. USP2 inactivation via siRNA knockdown or small-molecule inhibitor treatment remarkably shortened SKP2 protein half-life by enhancing its ubiquitylation and subsequent degradation. Unexpectedly, USP2-stabilized SKP2 did not destabilize its substrates p21 and p27. Mechanistically, USP2 bound to SKP2 via the leucine-rich repeat substrate-binding domain on SKP2 to disrupt the SKP2-substrate binding, leading to stabilization of both SKP2 and these substrates. Biologically, growth suppression induced by USP2 knockdown or USP2 inhibitor is partially mediated via modulation of SKP2 and its substrates. Our study revealed a new mechanism of the cross-talk among the E3-DUB substrates and its potential implication in targeting the USP2-SKP2 axis for cancer therapy.

Insights

USP2 deubiquitylase stabilizes oncogenic SKP2 protein, impacting cancer therapy. USP2 inactivation shortens SKP2 half-life, affecting its substrates p21 and p27, revealing a new therapeutic target.

Area of Science:

Biochemistry
Molecular Biology
Cancer Research

Background:

Protein stability is crucial, regulated by ubiquitylation and deubiquitylation.
S-phase kinase-associated protein 2 (SKP2) is an oncogenic F-box protein targeting tumor suppressors for degradation.
SKP2 stability is regulated by E3 ligases and deubiquitylases (DUBs) like USP10 and USP13.

Purpose of the Study:

To identify and characterize novel DUBs regulating SKP2 stability.
To elucidate the mechanism by which USP2 affects SKP2 and its substrates.
To explore the therapeutic potential of targeting the USP2-SKP2 axis in cancer.

Main Methods:

Screening of DUBs for interaction with endogenous SKP2.
siRNA knockdown and small-molecule inhibitor treatment to inactivate USP2.
Analysis of SKP2 protein half-life, ubiquitylation, and degradation.
Investigation of SKP2-substrate binding disruption via leucine-rich repeat domain interaction.
Assessment of biological effects of USP2 modulation on cell growth.

Main Results:

USP2 and USP21 bind to SKP2; USP2 deubiquitylates and stabilizes SKP2.
USP2 inactivation significantly reduces SKP2 half-life by increasing ubiquitylation and degradation.
USP2-mediated stabilization of SKP2 did not destabilize its substrates p21 and p27.
USP2 disrupts SKP2-substrate binding through its interaction with SKP2's leucine-rich repeat domain.
USP2 knockdown or inhibition causes growth suppression partially via SKP2 and its substrates.

Conclusions:

USP2 is a novel DUB that stabilizes SKP2 protein.
USP2 stabilizes SKP2 by disrupting its interaction with substrates, leading to stabilization of both.
Targeting the USP2-SKP2 axis presents a potential therapeutic strategy for cancer treatment.

Read full analysis

Keywords:

SKP1-Cullin1-F-box E3 (SCF E3)SKP2 USP2 deubiquitylation ubiquitylation

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