The mitosis-specific phosphorylation of histone H3 at Thr3 (H3T3ph) plays an 
important role in chromosome segregation by recruiting Aurora B. H3T3 
phosphorylation is catalyzed by Haspin, an atypical protein kinase whose kinase 
domain is intrinsically active without phosphorylation at the activation loop. 
Here, we report the molecular basis for Haspin inhibition during interphase and 
its reactivation in M phase. We identify a conserved basic segment that 
autoinhibits Haspin during interphase. This autoinhibition is neutralized when 
Cdk1 phosphorylates the N terminus of Haspin in order to recruit Polo-like 
kinase (Plk1/Plx1), which, in turn, further phosphorylates multiple sites at the 
Haspin N terminus. Although Plx1, and not Aurora B, is critical for H3T3 
phosphorylation in Xenopus egg extracts, Plk1 and Aurora B both promote this 
modification in human cells. Thus, M phase-specific H3T3 phosphorylation is 
governed by the combinatorial action of mitotic kinases that neutralizes Haspin 
autoinhibition through a mechanism dependent on multisite phosphorylation.