An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme

In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.     

Coordinating developmental signaling: novel roles for the Hippo pathway

Genetic and biochemical studies have defined the Hippo pathway as a central mediator of developmental and pathogenic signals. By directing intracellular signaling events, the Hippo pathway fine-tunes cell proliferation, cell death, and cell-fate decisions, and coordinates these cues to specify animal organ size. Recent studies have revealed that Hippo pathway-mediated processes are interconnected with those of other key signaling cascades, such as those mediated by TGF-β and Wnt growth factors. Moreover, several reports have described a role for cell contact-mediated polarity proteins in Hippo pathway regulation. Emerging details suggest that crosstalk between these signals drives fundamental developmental processes, and deregulated intercellular communication influences disease progression, such as cancer. We review recent data with a focus on how the Hippo pathway integrates its activity with other signaling pathways.     

Cdc34 self-association is facilitated by ubiquitin thiolester formation and is required for its catalytic activity

Using a coimmunoprecipitation strategy, we showed that the Cdc34 ubiquitin (Ub)-conjugating enzyme from Saccharomyces cerevisiae self-associates in cell lysates, thereby indicating an in vivo interaction. The ability of Cdc34 to interact with itself is not dependent on its association with the ubiquitin ligase Skp1-Cdc53/Cul1-Hrt1-F-box complex. Rather, this interaction depends upon the integrity of the Cdc34-Ub thiolester. Furthermore, several principal determinants within the Cdc34 catalytic domain, including the active-site cysteine, amino acid residues S73 and S97, and its catalytic domain insertion, also play a role in self-association. Mutational studies have shown that these determinants are functionally important in vivo and operate at the levels of both Cdc34-Ub thiolester formation and Cdc34-mediated multi-Ub chain assembly. These determinants are spatially situated in a region that is close to the active site, corresponding closely to the previously identified E2-Ub interface. These observations indicate that the formation of the Cdc34-Ub thiolester is important for Cdc34 self-association and that the interaction of Cdc34-Ub thiolesters is in turn a prerequisite for both multi-Ub chain assembly and Cdc34's essential function(s). A conclusion from these findings is that the placement of ubiquitin on the Cdc34 surface is a structurally important feature of Cdc34's function.