The subcellular localization of each protein was determined at least three times

n. Thus, binding to Plk1 contributes to H3T3 phosphorylation by Haspin. Plk1 inhibition had little effect on the residual H3T3ph produced by myc-DMXB-A site Haspin T128A, consistent with the idea that T128 phosphorylation promotes binding and phosphorylation of Haspin by Plk1. To determine if direct phosphorylation of Haspin by Plk1 regulates Haspin action, we mutated seven sites in Haspin that match the Plk1 consensus motif: four residues known to be phosphorylated in mitosis , and additional three in regions not covered by our previous mass spectrometric analysis. The myc-Haspin 7A mutant retained the S-pT-P epitope and binding to GST-PBD. However, like myc-Haspin T128A, it was partially defective in its ability to support H3T3ph in Haspin RNAi-rescue experiments. Therefore, we conclude that phosphorylation of Haspin by Plk1 is required for full generation of H3T3ph in mitosis. Role of Plk1 and Aurora B pathways of Haspin regulation We were interested in the relationship between previously defined Aurora B-dependent mechanisms that regulate H3T3ph, and the Plk1-dependent pathway defined here. Co-treatment with inhibitors of Aurora B and Plk1 essentially abolished detectable H3T3ph in nocodazole-arrested prometaphase-like cells, suggesting that these are two major pathways responsible for Haspin activity toward H3T3. It appears that Aurora B and Plk1-mediated Haspin phosphorylation can act at least partly independently: co-inhibition of Aurora B and Plk1 causes a greater decrease in the apparent molecular weight of Haspin in mitosis than inhibition of either kinase alone, and the Haspin T128A mutant remains moderately sensitive to Aurora B inhibition despite its near-complete loss of Plk1-dependent phosphorylation. Furthermore, weak but detectable H3T3ph is present in early prophase cells even when Aurora B is inhibited, while H3T3ph is delayed but partly recovers by late prometaphase when Plk1 is inhibited. Nevertheless, generation of H3T3ph is strongly influenced by Aurora B activity, even in prophase cells, and H3T3ph is partly 276 EMBO reports 2014 The Authors Linli Zhou et al Plk1 triggers histone phosphorylation by Haspin EMBO reports A B C D E F 2014 The Authors EMBO reports 277 EMBO reports Plk1 triggers histone phosphorylation by Haspin Linli Zhou et al A B D C reduced even at centromeres in Plk1-inhibited prometaphase cells. These patterns of inhibition suggest that the Cyclin B-Cdk1-primed phosphorylation of Haspin by Plk1 could enhance subsequent triggering of Aurora B-H3T3ph feedback loops. Indeed, instigation of the Plk1 pathway by Cdk activity early in mitosis may serve to activate a population of Haspin, generating low H3T3ph on chromatin. Recruitment of the CPC by H3T3ph would then bring Aurora B into proximity with Haspin to trigger the feedback loops that drive full H3T3ph and CPC localization in mitosis . Consistent with this, Plk1 inhibition or T128A mutation decreases the accumulation of Aurora B at PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19812126 centromeres in prometaphase, and Plk1 inhibition delays activation of Aurora B in inhibitor washout experiments, as measured by phosphorylation of the Aurora B substrate CENP-A S7. Role of multiple contributions to Haspin regulation It is likely that multiple inputs allow H3T3ph and CPC recruitment to be controlled in time, space and extent during the cell cycle. Phosphorylation of Haspin T128 by the master mitotic regulator Cyclin B-Cdk1 restricts H3T3ph to mitosis. The additional need for recognition of the S-pT128-P motif by Pl