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. 2019 May 28;8:e46131. doi: 10.7554/eLife.46131

Figure 4. Plk1 phosphorylation of Ser715 inhibits NRD-TAD association.

(A) ITC binding affinities of NRD and TAD-containing FoxM1 fragments following Cdk phosphorylation as indicated. (B) ITC binding affinity of NRD (1–203) for WT TAD (696-748) and TAD containing mutations at Plk1 sites. Measurements were made with and without Plk1 phosphorylation. (C) Ser715 is near the interface in the NMR structure of the NRD-TAD complex. Human amino acid numbering is used. (D) Luciferase reporter expression from the 6DB promoter as in Figure 3C. Only experiments in which significant differences in the relative luminescence between expression of FoxM1 alone (control, black) or co-expression with Plk1 (purple) are indicated with asterisks (*p<0.05, using two-tailed student’s t-test).

Figure 4.

Figure 4—figure supplement 1. Electrospray mass spectrometry characterization of kinase reactions.

Figure 4—figure supplement 1.

(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.
Figure 4—figure supplement 2. Phosphorylation dependent CSR association with the Plk1 polobox domain.

Figure 4—figure supplement 2.

ITC measurements of FoxM1 CSR (residues 573–635) titrated into Plk1 polobox (residues 345–603). Left is without CSR phosphorylation and right is CSR treated with Cdk2-CycA.