Substrate Modification with Lysine 63-linked Ubiquitin Chains through the UBC13-UEV1A Ubiquitin-conjugating Enzyme

Donald G. Payan,Jianing Huang,GuoQiang Dong,Matthew D. Petroski,Sarkiz Daniel-Issakani,Xiulan Zhou

doi:10.1074/jbc.m703911200

Abstract

Protein modification with lysine 63-linked ubiquitin chains has been implicated in the non-proteolytic regulation of signaling pathways. To understand the molecular mechanisms underlying this process, we have developed an in vitro system to examine the activity of the ubiquitin-conjugating enzyme UBC13-UEV1A with TRAF6 in which TRAF6 serves as both a ubiquitin ligase and substrate for modification. Although TRAF6 potently stimulates the activity of UBC13-UEV1A to synthesize ubiquitin chains, it is not appreciably ubiquitinated. We have determined that the presentation of Lys(63) of ubiquitin by UEV1A suppresses TRAF6 modification. Based on our observations, we propose that the modification of proteins with Lys(63)-linked ubiquitin chains occurs through a UEV1A-independent substrate modification and UEV1A-dependent Lys(63)-linked ubiquitin chain synthesis mechanism.

Highlights

Chains of specific ubiquitin-ubiquitin linkages result in distinct protein fates
Fractionation experiments initially identified UBC13 as a TRAF6 interacting protein and a variety of cell-based and in vitro experiments suggest that TRAF6 modification with Lys63-linked ubiquitin chains has a non-proteolytic function in NF-␬B activation through the kinase TAK1 [15,16,17]
We examine how UBC13-UEV1A functions with this ubiquitin ligase to promote TRAF6 auto-ubiquitination and uncover that the activity provided by UEV1A primarily promotes Lys63-linked ubiquitin chain synthesis that effectively reduces TRAF6 modification

Summary

Protein Expression and Purification

Ubiquitin-activating Enzyme (E1)—Human E1 containing an N-terminal hexahistidine tag was expressed in Sf9 insect cells from recombinant baculovirus and purified from lysates by NiNTA chromatography, followed by Mono Q-Sepharose separation. Lys63-linked Substrate Ubiquitination by UBC13-UEV1A mM Tris-HCl, pH 8.0, at 25 °C, 100 mM NaCl, 0.5 mM EDTA, 0.5 mM Tris(2-carboxyethyl)phosphine, and 10% glycerol. Protein concentration was measured using Bio-Rad Protein Assay reagent using ␥-globulin as a standard and purity was assessed by SDS-PAGE followed by Coomassie staining (see Fig. 1A, lane 5). FLAG-ubiquitin (ubiquitin containing an N-terminal FLAG epitope) was expressed in E. coli and purified by acid elution from anti-FLAG-Sepharose. Protein concentration was measured using Bio-Rad Protein Assay reagent as per the manufacturer’s instructions and verified by SDS-PAGE and Coomassie staining

Ubiquitination Reactions

RESULTS