FIGURE 2.

Effect of protein kinases on Emi1-mediated inhibition of APC/C in a purified system. A, inactivation of Emi1 by purified cyclin B/CDK1. In the first incubation, 3 nm full-length recombinant Emi1 in complex with Skp1 (Skp1/Emi1) was treated by a phosphorylation mixture with purified cyclin B/CDK1 or Plk1, and 10 μm staurosporine where indicated. Following this incubation, phosphorylation was inhibited by addition of staurosporine (where not previously added), and a second incubation was carried out in the presence of a ubiquitylation mixture, purified mitotic APC/C, recombinant purified Cdc20, and ¹²⁵I-cyclin B as substrate. B, inactivation of the GST-tagged C-terminal 150-amino acid fragment of Emi1 by purified cyclin B/CDK1. As in A, 3 nm Skp1/Emi1 or the GST-tagged C-terminal 150 amino acids of Emi1 (GST-Emi1CT) were preincubated with the phosphorylation mixture, and purified cyclin B/CDK1 and staurosporine, where indicated, followed by a second incubation in which the ubiquitylation mixture and ¹²⁵I-cyclin B were added, along with purified mitotic APC/C, recombinant Cdc20, and staurosporine, as indicated. C, effect of cyclin B/CDK1 on the inhibition of APC/C^Cdh1 by Emi1. A two-step phosphorylation and ubiquitylation assay as described above. Where indicated, GST-Emi1CT and cyclin B/CDK were added in the first incubation, and purified S-phase APC/C with recombinant Cdh1 was added for the second incubation. Staurosporine was added in all cases following the first incubation. D, inactivation of Emi1 by cyclin B/CDK1 and cyclin A/CDK2. GST-tagged Emi1CT was incubated in the presence of buffer, purified cyclin B/CDK1, or purified cyclin A/CDK2, and then purified on glutathione beads, quantified, and assayed for inhibition of cyclin B ubiquitylation by APC/C^Cdc20 at 10 nm concentration of each Emi1CT preparation.