Fig. 1: Phosphorylation of PRC1 by CDK1 and PLK1.

A Schematic of the PRC1 sequence with domains and phosphorylation sites indicated, amino acid numbers refer to PRC1 isoform 2 which is used in all experiments. B Phos-tag gel of 5 µM PRC1-SNAP phosphorylated by 170 nM CDK1/cyclin B/CKS1 or 1 µM MBP-PLK1 for the indicated times, or incubated with 5 µM lambda protein phosphatase (λ-PP) for 30 min as negative control. n = 5 independent experiments. C Mass spectroscopic quantification of the degree of phosphorylation of PRC1 residues threonine 470, 481 and serine 513 (T470, T481, S513) by 170 nM CDK1/cyclin B/CKS1 and of PRC1 residues threonine 578 and 602 (T578, T602) by 1 µM MBP-PLK1 as a function of time. Data are presented as mean and errors bars are standard deviation. Lines are hyperbolic fits. n = 5 independent experiments for residues T470, T481, T578 and T602, and n = 2 independent experiments for residue S513. D, E Analytical size exclusion chromatography: Coomassie-stained SDS gels of PRC1 and kinases eluted from a gel filtration column at the indicated volumes. D 5 µM PRC1-SNAP or 5 µM CDK1/cyclin B/CKS1 or both mixed together in the presence of ATP were separated. E 5 µM PRC1 or 5 µM MBP-PLK1 or both mixed together either in the presence or absence of ATP, as indicated, were separated. The shift of a fraction of PRC1 and MBP-PLK1 in the presence of ATP to a smaller elution volume indicates binding of PLK1-phosphorylated PRC1 to MBP-PLK1. For D and E, n = 3 independent experiments. Source data are provided as a Source Data file.