Proximity-induced activation of human Cdc34 through heterologous dimerization

Abstract

Cdc34 is an E2-conjugating enzyme required for catalyzing the polyubiquitination reaction mediated by the Skp1⅐CUL1⅐F-box (SCF) protein E3 ubiquitin (Ub) ligase. Here, we show that the activity of human Cdc34 in the Ub-Ub ligation reaction was enhanced dramatically by SCF's core Ub ligase module, composed of a heterodimeric complex formed by the ROC1 RING finger protein and the CUL1 C terminus that contains a Nedd8 moiety covalently conjugated at K720. Unexpectedly, we found that N-terminal fusion of a GST moiety to human Cdc34 generated dimeric GSTCdc34 that was constitutively active in supporting the assembly of K48-linked polyUb chains independently of SCF. Furthermore, fusion of a FK506-binding protein (FKBP) to the N terminus of human Cdc34 yielded FKBP-Cdc34 that was induced to form a dimer upon treatment with the chemical inducer AP20187. The AP20187-induced dimeric form of FKBP-Cdc34 was substantially more active than the monomer in catalyzing Ub-Ub ligation. Thus, juxtaposition of human Cdc34 activates its catalytic capability, suggesting that the SCF-mediated polyubiquitination reaction may require the conversion of Cdc34 from an inactive monomer to a highly active dimeric form.SCF ͉ ubiquitination U biquitin (Ub)-dependent proteolysis is a remarkably intricate biochemical process in which the 26S proteasome recognizes and subsequently degrades a target protein tagged with lysine-48-linked polyUb chains, generated through a modification reaction known as ubiquitination. Ubiquitination requires activation of monomeric Ub through formation of an initial thiol-ester bond with the E1 activating enzyme followed by transfer of the thiol-ester-linked Ub to an E2 conjugating enzyme (1). The enzymatic generation of signaling Ub chains attached to a substrate is mediated typically by an E3 Ub ligase, which contains either a HECT domain or a RING finger motif and functions by recognizing both the substrate and an E2 (2).The Skp1⅐CUL1⅐F-box protein⅐ROC1 (SCF) complex is a prototype of cullin (CUL)-based RING E3 ligases that are defined by two signature components: a CUL-family molecule and its RING finger partner ROC1͞Rbx1͞Hrt1 (reviewed in ref.3). SCF utilizes the CUL1 scaffold to anchor a substratetargeting module (comprised of the Skp1⅐F-box protein heterodimer) and to tether ROC1 (via the CUL domain), respectively (4 -6). As a result of this multisubunit interaction, an F-box protein (member of a large family of substrate-targeting molecules) is placed in proximity to ROC1, which functions to recruit an E2 conjugating enzyme (7-9), thereby initiating the ubiquitination of a bound substrate. As revealed by a large body of genetic and biochemical experiments, the SCF and other CUL-based RING E3 ligases are activated by conjugation of Nedd8 to a conserved lysine residue within a CUL protein (a process termed neddylation) (3), which mechanistically parallels ubiquitination.In budding yeast, the activity of SCF requires Cdc34, a class II E2 Ub-conjugating enzyme that catalyzes the formation of K48-linked polyUb cha...

Differential Ubiquitin Binding by the Acidic Loops of Ube2g1 and Ube2r1 Enzymes Distinguishes Their Lys-48-ubiquitylation Activities

“…3). This result is consistent with the previous report that the dimerization of Ube2r1 increases its Lys-48-ubiquitylation activity (25). On the other hand, with Ube2g1 the size distribution of Lys-48 polyubiquitin products did not depend on the E2 concentration, and thus catalysis by Ube2g1 likely involves a reaction between the E2ϳUB thioester and an acceptor ubiquitin that is not associated with a second E2ϳUB molecule.…”

Characterization of a Novel Ubiquitin-Conjugating Enzyme That Regulates β1,4-Galactosyltransferase-1 in Embryonic Stem Cells

“…Interestingly, the 120-kDa dimer of GTAP is the only form that binds Ub through thiolester activation. As a prerequisite for Ub activation, this type of self-association has been described previously in other E2 enzymes [27,28]. Taken together, these results suggest that a homodimer of GTAP may represent the biologically active form of the enzyme.…”

SCF E3-Mediated Autoubiquitination Negatively Regulates Activity of Cdc34 E2 but Plays a Nonessential Role in the Catalytic Cycle In Vitro and In Vivo

“…On the one hand, the C-terminal fragment of Cdc34 implicated in SCF binding (32) has also been shown to play a role in Cdc34 oligomerization (36). Dimerization of human Cdc34 via fusion to glutathione S-transferase or FK506-binding protein mimics the requirement for SCF in Cdc34 activation in vitro (13), suggesting that SCF could activate Cdc34 by promoting its oligomerization. A further link between Cdc34 oligomerization and function is suggested by the finding that charging of the Cdc34 active site with ubiquitin, a prerequisite for a functional interaction with SCF (7), allows detection of the Cdc34-Cdc34 interaction in yeast extracts (45 oligomerization has been proposed to be independent of SCF, based on the observation that the Cdc34-Cdc34 interaction could be detected in SCF mutant strains defective in Cdc34 binding (45).…”