Figure 4.

Prephosphorylation of Xic1 on Thr-205 by cyclin E/Cdk2 bypasses the nuclear requirement for Xic1 destruction. (A) Prephosphorylated Xic1 is destroyed even in the absence of nuclei. GST-Xic1 was incubated with baculovirus-purified cyclin E/Cdk2 in the presence (lower tier) or absence of ATP (upper tier) for 30 min. Destruction assays were conducted plus or minus sperm and MG132 as indicated. Reactions were processed as in Fig. 1A except that membranes were immunoblotted with anti-Xic1 antibody. (B) Cyclin E/Cdk2 phosphorylates Xic1 on Thr-205. Phosphorylated (III and IV) and unphosphorylated (I and II) GST-Xic1 were prepared by kinase reactions with or without ATP (see Materials and Methods). Samples were digested with trypsin and analyzed by matrix-assisted laser desorption ionization–time of flight MS.