Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade.
Ubiquitination of proteins involves the concerted action of the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes and E3 ubiquitin-protein ligases. It has been proposed that E3s function as 'docking proteins', specifically binding substrate proteins and specific E2s, and that ubiquitin is then transferred directly from E2s to substrates. We show here that formation of a ubiquitin thioester on E6-AP, an E3 involved in the human papillomavirus E6-induced ubiquitination of p53 (refs 6-10), is an intermediate step in E6-AP-dependent ubiquitination. The order of ubiquitin transfer is from E1 to E2, from E2 to E6-AP, and finally from E6-AP to a substrate. This cascade of ubiquitin thioester complexes suggests that E3s have a defined enzymatic activity and do not function simply as docking proteins. The cysteine residue of E6-AP responsible for ubiquitin thioester formation was mapped to a region that is highly conserved among several proteins of unknown function, suggesting that these proteins share the ability to form thioesters with ubiquitin.
Scheffner
M
M
Deutsches Krebsforschungszentrum, Angewandte Tumorvirologie, Heidelberg, Germany.
Nuber
U
U
Huibregtse
J M
JM
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D003545:Cysteine / Q000378:metabolism; D004952:Esters / Q000378:metabolism; D008025:Ligases / Q000378:metabolism*; D008969:Molecular Sequence Data; D027383:Papillomaviridae / Q000378:metabolism; D011994:Recombinant Proteins; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D004952:Esters; D011994:Recombinant Proteins; D014452:Ubiquitins; D014764:Viral Proteins; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D003545:Cysteine