Covalent Modification of the NF-{kappa}B Essential Modulator (NEMO) by a Chemical Compound can Regulate its Ubiquitin Binding Properties In Vitro [Signal Transduction]

October 8th, 2014 by Hooper, C., Jackson, S. S., Coughlin, E. E., Coon, J. J., Miyamoto, S.

Posttranslational 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 K63- 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 K48-linked polyubiquitin chains but not tetra-ubiquitin species. Significantly, the UBAN (ubiquitin binding in ABIN and NEMO) domain, essential for NEMO′s ability to bind M1/K63-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 K48-polyubiquitin binding phenotype. Our study demonstrates the feasibility of directly altering the ubiquitin interaction properties of a ubiquitin-binding protein by a chemical compound, thereby shedding light on a novel drug class to potentially alter polyubiquitin-based cellular processes.
  • Posted in Journal of Biological Chemistry, Publications
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