Figure 7

Effects of Speedy/Ringo A2 binding on the phosphorylation and dephosphorylation of Cdk2 on Thr-160. (A) Speedy/Ringo A2 hinders the phosphorylation of Cdk2 on Thr-160 by budding yeast Cak1p. GST-Cdk2^D145N (1 μg) was incubated with increasing amounts (0, 1, 2.5, 5, and 10 μg) of GST-Speedy/Ringo A2 or GST prior to phosphorylation by Cak1p (100 ng). The reactions were terminated and phosphorylated proteins were resolved by SDS-PAGE and detected by autoradiography. (B) Speedy/Ringo A2 binding did not stimulate Thr-160 phosphorylation by mammalian CAK. GST-Cdk2^D145N (1 μg; lanes 1, 4, 5) was preincubated with cyclin A (1 μg; lanes 2 and 4) or GST-Speedy/Ringo A2 (4 μg; lanes 3 and 5) before phosphorylation by CAK. Samples were processed as described in Methods. (C) Speedy/Ringo A2 hinders the dephosphorylation of Cdk2 on Thr-160 by PP2Cα. [³²P-T160]GST-Cdk2^D145N was preincubated with cyclin A (lanes 2 and 4), GST-Speedy/Ringo A2 (lanes 6 and 8), or buffer alone (lanes 1, 3, 5, 7) prior to addition of recombinant human PP2Cα (lanes 3, 4, 7, 8) or buffer (lanes 1, 2, 5, 6). [³²P-T160]GST-Cdk2^D145N was detected by autoradiography.