(
A) Purified NRD 1–203 without (left) and with (right) Cdk2-CycA phosphorylation. The construct has one highly conserved and strong Cdk consensus site ({S/T}Px{K/R}) at S4 and a less conserved weak consensus site ({S/T}P) at S35. The numbers in parentheses indicate the concluded number of added phosphoryl groups (+80 Da). (
B) Purified CSR-TAD 526–748 without (left) and with Plk1 (middle) and Cdk2-CycA (right) phosphorylation. The human construct contains 34 potential serine phosphorylation sites for Plk1, which lacks a strong consensus. The CSR-TAD contains three strong (T585, T596, S657) and six weak (T595, T612, S623, T647, S678, S689) Cdk consensus sites. Four of these total consensus sites (T585, T596, S623, and T647) are conserved in all five of the orthologs used in the sequence alignment in
Figure 2A. (
C) Purified TAD 696–748 without (left) and with Plk1 (right) phosphorylation. The TAD construct has only five serines, and we observe quantitative phosphorylation on four sites. (
D,E) Plk1 phosphorylation of a S715A mutant TAD results in three additional phosphoryl groups, while Plk1 phosphorylation of a S702A/S724A/S741A mutant TAD results in one additional phosphoryl group. From these and similar mutagenesis experiments, we conclude that Plk1 phosphorylated the TAD on S702, S715, S724, and S741.