Control of mitotic and meiotic transitions through regulated phosphorylation of Emi1 and Emi2

Foremost among ubiquitin ligases involved in cell cycle control is the Anaphase Promoting Complex or Cyclosome (APC/C). The APC/C is responsible for the ubiquitination of A- and B-type cyclins. It becomes active during mitosis, is essential for the metaphase-anaphase transition and mitotic exit, and remains active during G1 phase of the following cell cycle to restrain premature cyclin accumulation. The cell cycle can be simply considered in terms of alternating phases of high and low APC/C activity, which dictate periods of low and high Cdk activity, respectively. How is APC/C activity turned on and off? During recent years our laboratory has characterized vertebrate Emi1, an APC/C inhibitor that appears during late G1 phase and is destroyed during early mitosis. Emi1 accumulation is required for APC/C inactivation at the G1-S transition, and Emi1 destruction is required for mitotic APC/C activation. Thus, the presence of Emi1 defines an interval of APC/C inactivity and cyclin stability.; Emi1 destruction occurs when a previously unknown kinase phosphorylates a specific motif within Emi1 which is then recognized by the SCFbetaTrCP ubiquitin ligase for ubiquitination. Using a variety of biochemical and genetic techniques in Xenopus egg extracts and in cultured human cells, we demonstrate that the Polo-like kinase Plk1 is responsible for Emi1 destruction. We also explain the previously reported role of Cdk phosphorylation in Emi1 destruction by showing that phosphorylation by cyclin B/Cdk1 "primes" Emil for interaction with Plk1.; We also characterize Emi2, a previously unstudied Emi1 homologue, as an APC/C inhibitor that is similarly destroyed in mitosis through Plk1 phosphorylation and ubiquitination by the SCFbetaTrCP complex. Importantly, we demonstrate that Emi2 is protected from destruction during meiosis II, and that mimicking fertilization through addition of calcium to metaphase II-arrested egg extracts stimulates Emi2 destruction, which is critical for APC/C activation and completion of meiosis. We show that the calcium/calmodulin-dependent kinase II (CaMKII) phosphorylates Emi2 to generate a single, high-affinity Plk1-binding site, resulting in extremely rapid Emi2 destruction and meiotic exit. These results exemplify how related proteins and signaling pathways can be specifically engineered to respond to distinct inputs for unique biological purposes.