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

Abstract

One of the several still unexplained aspects of the mechanism by which the Cdc34/SCF RING-type ubiquitin ligases work is the marked stimulation of Cdc34 autoubiquitination, a phenomenon of unknown mechanism and significance. In in vitro experiments with single-lysine-containing Cdc34 mutant proteins of Saccharomyces cerevisiae, we found that the SCF-mediated stimulation of autoubiquitination is limited to specific N-terminal lysines modified via an intermolecular mechanism. In a striking contrast, SCF quenches autoubiquitination of C-terminal lysines catalyzed in an intramolecular manner. Unlike autoubiquitination of the C-terminal lysines, which has no functional consequence, autoubiquitination of the N-terminal lysines inhibits Cdc34. This autoinhibitory mechanism plays a nonessential role in the catalytic cycle, as the lysineless K0 Cdc34 ⌬C is indistinguishable from Cdc34 ⌬C in ubiquitination of the prototype SCF Cdc4 substrate Sic1 in vitro, and replacement of the CDC34 gene with either the K0 cdc34 ⌬C or the cdc34 ⌬C allele in yeast has no cell cycle phenotype. We discuss the implications of these findings for the mechanism of Cdc34 function with SCF.Posttranslational modification of proteins with ubiquitin has emerged as a major regulatory mechanism in eukaryotic cells, with both proteolysis-related and non-proteolysis-related functions (14,19,34,35). The variety of mechanisms of substrate recognition by specific ubiquitin ligases well reflects the large number of proteins targeted for ubiquitination. However, once a substrate is selected, its ubiquitination is catalyzed via a conserved cascade of ubiquitin transfer reactions.The first two steps of the ubiquitin transfer cascade do not involve a specific substrate protein and appear to be facilitated in a constitutive manner. In the first step, E1, called a ubiquitin-activating enzyme, forms a ubiquitin adenylate and subsequently a high-energy thiol ester bond with a carboxyl group of ubiquitin, thus activating it for nucleophilic attack. In the second step, the activated ubiquitin is trans-esterified to the active-site cysteine of one of several E2 ubiquitin-conjugating enzymes. The diversity in the ubiquitin transfer cascade begins when the ubiquitin-charged E2 is recruited to the proximity of a substrate through an interaction with a specific E3, which can be, but is not always, an enzyme. In the case of HECT (homology to E6-AP C terminus)-type E3s, E2 transfers ubiquitin to the catalytic site of E3 and E3 catalyzes ubiquitination. In contrast, in the case of RING (really interesting new gene)-type E3s, which do not have their own catalytic sites, E2 is directly responsible for ubiquitination of the E3-bound substrate. It is still unknown why such a difference evolved and why the enzymatic activity of HECT-type E3s is required if RING-type E3s can depend on the activity of E2 without having their own catalytic sites.

Rhes, a Physiologic Regulator of Sumoylation, Enhances Cross-sumoylation between the Basic Sumoylation Enzymes E1 and Ubc9

“…Without SCF, Cdc34 is autoubiquitinated via intramolecular modification with no effect on enzyme function. However, when SCF is present, Cdc34 transfers ubiquitin to separate Cdc34 molecules via an intermolecular reaction, which inhibits their activity (12). We find that Ubc9 is incapable of either reaction and depends on E1 for sumoylation.…”

“…Sumoylation of E1 at multiple lysines has previously been reported (6). To determine whether E1 is modified through intramolecular autosumoylation, similar to ubiquitin E1 and E2 (12), or whether it is dependent on transfer of SUMO from Ubc9, we performed in vitro sumoylation assays in the presence and absence of Ubc9 and with the catalytically inactive Ubc9-C93A (Fig. 2D).…”

UBC9 Autosumoylation Negatively Regulates Sumoylation of Septins in Saccharomyces cerevisiae

“…For example, Cdc34, an ubiquitin E2 enzyme, is autoubiquitinated in the presence of SCF (Skp1, Cdc53/Cull, and F-box protein), inhibiting its ubiquitin conjugating activity to transfer ubiquitin to separate Cdc34 molecules (49). Knipscheer et al (26) recently reported that mammalian Ubc9 autosumoylation regulates its target discrimination.…”

Synthesis of Free and Proliferating Cell Nuclear Antigen-bound Polyubiquitin Chains by the RING E3 Ubiquitin Ligase Rad5

“…There is evidence that RING E3s like Rad5 may play a more active role in ubiquitination than simply bringing the substrate into close proximity with the E2ϳUb. Several RING E3s have been shown to stimulate the synthesis of unanchored polyUb chains or autoubiquitination of their cognate E2s in the absence of substrates (22)(23)(24). This stimulation may be related to the ability of RING E3s to enhance reactivity of the E2ϳUb thiolester bond through allosteric effects (25,26).…”