Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1.

Progress through mitosis is controlled by the sequential destruction of key regulators including the mitotic cyclins and securin, an inhibitor of anaphase whose destruction is required for sister chromatid separation. Here we have used live cell imaging to determine the exact time when human securin is degraded in mitosis. We show that the timing of securin destruction is set by the spindle checkpoint; securin destruction begins at metaphase once the checkpoint is satisfied. Furthermore, reimposing the checkpoint rapidly inactivates securin destruction. Thus, securin and cyclin B1 destruction have very similar properties. Moreover, we find that both cyclin B1 and securin have to be degraded before sister chromatids can separate. A mutant form of securin that lacks its destruction box (D-box) is still degraded in mitosis, but now this is in anaphase. This destruction requires a KEN box in the NH2 terminus of securin and may indicate the time in mitosis when ubiquitination switches from APCCdc20 to APCCdh1. Lastly, a D-box mutant of securin that cannot be degraded in metaphase inhibits sister chromatid separation, generating a cut phenotype where one cell can inherit both copies of the genome. Thus, defects in securin destruction alter chromosome segregation and may be relevant to the development of aneuploidy in cancer.

APC/C and SCF: controlling each other and the cell cycle.

Regulated protein degradation has emerged as a key recurring theme in multiple aspects of cell-cycle regulation. Importantly, the irreversible nature of proteolysis makes it an invaluable complement to the intrinsically reversible regulation through phosphorylation and other post-translational modifications. Consequently, ubiquitin-protein ligases, the protagonists of regulated protein destruction, have gained prominence that compares to that of the cyclin-dependent kinases (Cdks) in driving the eukaryotic cell-cycle clock. This review will focus on the two main players, the related ubiquitin-protein ligases APC/C and SCF, and how they control cell-cycle progression. I will also try to delineate the regulation and interplay of these destruction mechanisms, which are intricately connected to the kinase network as well as to extrinsic signals. Moreover, cell-cycle ubiquitin-protein ligases are themselves subject to proteolytic control in cis as well as in trans. Finally, a careful comparison of the functions and regulation of APC/C and SCF shows that, in certain aspects, their logic of action is fundamentally different.

TPR subunits of the anaphase-promoting complex mediate binding to the activator protein CDH1.

BACKGROUND: Chromosome segregation and mitotic exit depend on activation of the anaphase-promoting complex (APC) by the substrate adaptor proteins CDC20 and CDH1. The APC is a ubiquitin ligase composed of at least 11 subunits. The interaction of APC2 and APC11 with E2 enzymes is sufficient for ubiquitination reactions, but the functions of most other subunits are unknown. RESULTS: We have biochemically characterized subcomplexes of the human APC. One subcomplex, containing APC2/11, APC1, APC4, and APC5, can assemble multiubiquitin chains but is unable to bind CDH1 and to ubiquitinate substrates. The other subcomplex contains all known APC subunits except APC2/11. This subcomplex can recruit CDH1 but fails to support any ubiquitination reaction. In vitro, the C termini of CDC20 and CDH1 bind to the closely related TPR subunits APC3 and APC7. Homology modeling predicts that these proteins are similar in structure to the peroxisomal import receptor PEX5, which binds cargo proteins via their C termini. APC activation by CDH1 depends on a conserved C-terminal motif that is also found in CDC20 and APC10. CONCLUSIONS: APC1, APC4, and APC5 may connect APC2/11 with TPR subunits. TPR domains in APC3 and APC7 recruit CDH1 to the APC and may thereby bring substrates into close proximity of APC2/11 and E2 enzymes. In analogy to PEX5, the different TPR subunits of the APC might function as receptors that interact with the C termini of regulatory proteins such as CDH1, CDC20, and APC10.

APC activators caught by their tails?

The complexity of the Anaphase-Promoting Complex (APC), the major ubiquitin ligase in mitotic control, has been puzzling investigators ever since its discovery. Recent biochemical and genetic studies have now provided insights not only into the architecture of the complex, but also into how activators are recruited to the APC. In this article, we discuss the implications of these findings on our current understanding of APC activation.

The WD40 propeller domain of Cdh1 functions as a destruction box receptor for APC/C substrates.

Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 and Cdh1 leads to ubiquitin-dependent degradation of securin and cyclin B and thereby promotes the initiation of anaphase and exit from mitosis. Cyclin B and securin ubiquitination depend on a destruction box (D box) sequence in these proteins, but how APC/C bound to Cdc20 or Cdh1 recognizes the D box is poorly understood. By using site-specific photocrosslinking in combination with mutational analyses, we show that the D box directly interacts with an evolutionarily conserved surface on the predicted WD40 propeller structure of Cdh1 and that this interaction is essential for processive substrate ubiquitination. We further show that Cdh1 specifically crosslinks to the APC/C subunit Cdc27 and that Cdh1 binding to APC/C depends on the presence of Cdc27. Our data imply that APC/C is activated by the association of Cdh1 with Cdc27, which enables APC/C to recognize the D box of substrates via Cdh1's propeller domain.

Early mitotic degradation of the homeoprotein HOXC10 is potentially linked to cell cycle progression.

Hox proteins are transcription factors involved in controlling axial patterning, leukaemias and hereditary malformations. Here, we show that HOXC10 oscillates in abundance during the cell cycle, being targeted for degradation early in mitosis by the ubiquitin-dependent proteasome pathway. Among abdominal-B subfamily members, the mitotic proteolysis of HOXC10 appears unique, since the levels of the paralogous HOXD10 and the related homeoprotein HOXC13 are constant throughout the cell cycle. When two destruction box motifs (D-box) are mutated, HOXC10 is stabilized and cells accumulate in metaphase. HOXC10 appears to be a new prometaphase target of the anaphase-promoting complex (APC), since its degradation coincides with cyclin A destruction and is suppressed by expression of a dominant-negative form of UbcH10, an APC-associated ubiquitin-conjugating enzyme. Moreover, HOXC10 co-immunoprecipitates the APC subunit CDC27, and its in vitro degradation is reduced in APC-depleted extracts or by competition with the APC substrate cyclin A. These data imply that HOXC10 is a homeoprotein with the potential to influence mitotic progression, and might provide a link between developmental regulation and cell cycle control.