Monoubiquitination empowers ubiquitin chain elongation.

Ubiquitination often generates lysine 48-linked polyubiquitin chains that signal proteolytic destruction of the protein target. A significant subset of ubiquitination proceeds by a priming/extending mechanism, in which a substrate is first monoubiquitinated with a priming E2-conjugating enzyme or a set of E3 ARIH/E2 enzymes specific for priming. This is then ...
followed by ubiquitin (Ub) chain extension catalyzed by an E2 enzyme capable of elongation. This report provides further insights into the priming/extending mechanism. We employed reconstituted ubiquitination systems of substrates CK1? (casein kinase 1?) and ?-catenin by Cullin-RING E3 Ub ligases (CRLs) CRL4CRBN and CRL1?TrCP, respectively, in the presence of priming E2 UbcH5c and elongating E2 Cdc34b (cell division cycle 34b). We have established a new "apyrase chase" strategy that uncouples priming from chain elongation, which allows accurate measurement of the decay rates of the ubiquitinated substrate with a defined chain length. Our work has revealed highly robust turnover of monoubiquitinated ?-catenin that empowers efficient polyubiquitination. The results of competition experiments suggest that the interactions between the ubiquitinated ?-catenin and CRL1?TrCP are highly dynamic. Moreover, ubiquitination of the Ub-modified ?-catenin appeared more resistant to inhibition by competitors than the unmodified substrate, suggesting tighter binding with CRL1?TrCP. These findings support a role for conjugated Ub in enhancing interactions with E3.
Mesh Terms:
Ubiquitin, Ubiquitin-Conjugating Enzymes, Ubiquitin-Protein Ligases, Ubiquitination, beta Catenin, beta-Transducin Repeat-Containing Proteins
J Biol Chem
Date: Mar. 01, 2024
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