Control of APC/C-dependent ubiquitin chain elongation by reversible phosphorylation.

Most metazoan E3 ligases contain a signature RING domain that promotes the transfer of ubiquitin from the active site of E2 conjugating enzymes to lysine residues in substrates. Although these RING-E3s depend on E2 enzymes for catalysis, how they turn on their E2s at the right time and place remains poorly understood. Here we report a phosphorylation-dependent mechanism that ensures timely activation of the E2 Ube2S by its RING-E3, the anaphase-promoting complex (APC/C); while phosphorylation of a specific serine residue in the APC/C coactivator Cdc20 prevents delivery of Ube2S to the APC/C, removal of this mark by PP2A(B56) allows Ube2S to bind the APC/C and catalyze ubiquitin chain elongation. PP2A(B56) also stabilizes kinetochore-microtubule attachments to shut off the spindle checkpoint, suggesting that cells regulate the E2-E3 interplay to coordinate ubiquitination with critical events during cell division.

Microtubule-dependent regulation of mitotic protein degradation.

Accurate cell division depends on tightly regulated ubiquitylation events catalyzed by the anaphase-promoting complex (APC/C). Among its many substrates, the APC/C triggers the degradation of proteins that stabilize the mitotic spindle, and loss or accumulation of such spindle assembly factors can result in aneuploidy and cancer. Although critical for cell division, it has remained poorly understood how the timing of spindle assembly factor degradation is established during mitosis. Here, we report that active spindle assembly factors are protected from APC/C-dependent degradation by microtubules. In contrast, those molecules that are not bound to microtubules are highly susceptible to proteolysis and turned over immediately after APC/C activation. The correct timing of spindle assembly factor degradation, as achieved by this regulatory circuit, is required for accurate spindle structure and function. We propose that the localized stabilization of APC/C substrates provides a mechanism for the selective disposal of cell-cycle regulators that have fulfilled their mitotic roles.

Dynamic regulation of ubiquitin-dependent cell cycle control.

Recent work revealed that cullin-RING ligases and the anaphase-promoting complex, two classes of ubiquitin ligases that are essential for cell division in all eukaryotes, are regulated in a highly dynamic manner. Here, we describe mechanisms that establish the dynamic regulation of these crucial ubiquitylation enzymes and discuss the functional consequences for cell division control.

Antibiotic interactions that select against resistance.

Multidrug combinations are increasingly important in combating the spread of antibiotic-resistance in bacterial pathogens. On a broader scale, such combinations are also important in understanding microbial ecology and evolution. Although the effects of multidrug combinations on bacterial growth have been studied extensively, relatively little is known about their impact on the differential selection between sensitive and resistant bacterial populations. Normally, the presence of a drug confers an advantage on its resistant mutants in competition with the sensitive wild-type population. Here we show, by using a direct competition assay between doxycycline-resistant and doxycycline-sensitive Escherichia coli, that this differential selection can be inverted in a hyper-antagonistic class of drug combinations. Used in such a combination, a drug can render the combined treatment selective against the drug's own resistance allele. Further, this inversion of selection seems largely insensitive to the underlying resistance mechanism and occurs, at sublethal concentrations, while maintaining inhibition of the wild type. These seemingly paradoxical results can be rationalized in terms of a simple geometric argument. Our findings demonstrate a previously unappreciated feature of the fitness landscape for the evolution of resistance and point to a trade-off between the effect of drug interactions on absolute potency and the relative competitive selection that they impose on emerging resistant populations.