Covalent Modification of the NF-κB Essential Modulator (NEMO) by a Chemical Compound Can Regulate Its Ubiquitin Binding Properties in Vitro

Christopher Hooper,Shawn S Jackson,Emma E Coughlin,Joshua J Coon,Shigeki Miyamoto

doi:10.1074/jbc.m114.582478

Christopher Hooper, Shawn S Jackson + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m114.582478

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Abstract

Post-translational modification by ubiquitin plays important roles in multiple physiological and pathological processes. Ubiquitin-binding proteins play a critical role in recognizing and relaying polyubiquitin-based signaling. NEMO (NF-κB Essential Modulator) is a central player in canonical NF-κB signaling whose major function is to bind to Lys-63- and/or M1- (or linear) linked polyubiquitin chains generated in response to cell stimulation. Here we show that Withaferin A (WA), a steroidal lactone, causes a change in NEMO's interaction with specific types of polyubiquitin chains in vitro. WA induces full-length recombinant NEMO to bind to long Lys-48-linked polyubiquitin chains but not tetra-ubiquitin species. Significantly, the UBAN (ubiquitin binding in ABIN and NEMO) domain, essential for the ability of NEMO to bind M1/Lys-63-linked polyubiquitin, is dispensable for the WA-induced gain-of-function activity. Mass spectrometric analysis demonstrated that WA covalently modifies NEMO on a cysteine residue within the C-terminal zinc finger (ZF) domain. Point mutations to the ZF can reverse the WA-induced Lys-48-polyubiquitin binding phenotype. Our study demonstrates the feasibility of directly altering the ubiquitin interaction properties of an ubiquitin-binding protein by a chemical compound, thereby shedding light on a novel drug class to potentially alter polyubiquitin-based cellular processes.

Highlights

NEMO is a known ubiquitin-binding protein that functions as a key regulator of NF-␬B activation
In this study we show that a chemical compound termed Withaferin A (WA), a steroidal lactone, can covalently modify NEMO to induce a gain-of-function phenotype to bind Lys-48-linked polyubiquitin chains in vitro and in vivo
We found that GST-NEMO strongly prefers Lys-63-linked polyubiquitin chains to Lys-48linked chains with further preference for longer polyubiquitin chains (Fig. 1B)

Summary

Background

NEMO is a known ubiquitin-binding protein that functions as a key regulator of NF-␬B activation. Results: WA is able to covalently modify NEMO to induce a gain-of-function binding to long Lys-48-linked polyubiquitin chains. Ubiquitin-binding proteins play a critical role in recognizing and relaying polyubiquitin-based signaling. NEMO (NF-␬B Essential Modulator) is a central player in canonical NF-␬B signaling whose major function is to bind to Lys-63- and/or M1- (or linear) linked polyubiquitin chains generated in response to cell stimulation. WA induces full-length recombinant NEMO to bind to long Lys-48-linked polyubiquitin chains but not tetra-ubiquitin species. The UBAN (ubiquitin binding in ABIN and NEMO) domain, essential for the ability of NEMO to bind M1/Lys-63-linked polyubiquitin, is dispensable for the WA-induced gain-of-function activity. Our study demonstrates the feasibility of directly altering the ubiquitin interaction properties of an ubiquitin-binding protein by a chemical compound, thereby shedding light on a novel drug class to potentially alter polyubiquitin-based cellular processes. While its initial discovery characterized ubiquitin as a degradative signal, recent studies have shown a plethora of non-degradative signaling functions

To whom correspondence should be addressed

EXPERIMENTAL PROCEDURES

RESULTS

D GST-NEMO PH006594 S956066 T272299 PH004604 T194999

DISCUSSION