Figure 3. CDK1-mediated phosphorylation at Ser1375 of RSF1 is necessary for PLK1 binding and recruitment to kinetochores.

(a) In vitro kinase assay: recombinant GST-RSF1-C2 or N3 proteins purified in the baculovirus system were incubated with the cyclin B1–CDK1 complex at 30 °C for 30 min in the presence of γ³²P-ATP. Incorporation of ³²P into RSF1 protein was visualized by autoradiography. Coomassie blue staining demonstrates equal protein loading. The asterisk indicates the hyper-phosphorylated form of RSF1. (b) RSF1 KO cells were transfected with the RSF1-C1-V5 plasmid and treated with paclitaxel for 16 h before harvest. Cells were pre-treated with MG132 for 1 h, and with the CDK1 inhibitor Ro3306 for 15 min. Co-immunoprecipitation experiments were carried out with an anti-V5 antibody. (c) Alignment of vertebrate RSF1 with the conserved CDK1 consensus motif (upper panel) and in vitro kinase assay of phosphorylation mutants of RSF1-C1. RSF1 KO HeLa cells transfected with the indicated plasmids were treated with paclitaxel for 16 h. RSF1-C1 and RSF1-C1^S1375A proteins were immunopurified using anti-V5 antibody and subjected to in vitro kinase assays (bottom panel). (d) The RSF1-C1^S1375A or RSF1-C1^S1375D plasmids were transfected into RSF1 KO cells, and mitotic lysates were immunoprecipitated with anti-V5 antibody, followed by immunoblotting. (e) RSF1 KO cells were introduced with indicated RSF1 constructs and treated with nocodazole for 4 h. Floating mitotic cells were subjected to chromosome spread immunostaining. Mitotic cells were analysed by immunofluorescence staining: PLK1 (green), RSF1 (red) or 4,6-diamidino-2-phenylindole (blue). Scale bar, 5 μm. See full blots Supplementary Fig. 8.