Acknowledgments The authors thank Partners

Acknowledgments The authors thank Partners Healthcare for financial support. This work was also supported in part by NIH Grant R01CA122608 to J.M.G.H.
Introduction The phosphorylation of histone H3 is recognized as a hallmark of mitosis. Histone H3 phosphorylation at Thr3 (H3T3ph) acts as a mitotic ligand for Survivin (Kelly et al., 2010, Wang et al., 2010, Yamagishi et al., 2010), a subunit of the chromosomal passenger complex (CPC), which plays multiple essential roles during mitosis and meiosis (Carmena et al., 2012b). H3T3ph promotes CPC localization on mitotic chromatin, particularly at the centromere. Enrichment of the CPC on chir99021 locally activates its kinase subunit Aurora B by promoting autophosphorylation, leading to the downstream phosphorylation of a variety of substrates (De Antoni et al., 2012, Funabiki and Wynne, 2013, Kelly et al., 2010, Wang et al., 2012). Although the dephosphorylation of H3T3ph at the exit from M phase is required for proper chromosome decondensation and nuclear envelope formation (Kelly et al., 2010), the molecular mechanisms that limit H3T3ph to M phase remain unclear. Mitotic H3T3 phosphorylation is catalyzed by Haspin (Dai et al., 2005), which is an atypical protein kinase in several regards. For example, in most kinases, the highly conserved DFG (Asp-Phe-Gly) motif anchors the N-terminal portion of the activation segment and coordinates catalytic magnesium (Nolen et al., 2004), but, in Haspin, it is changed into DYT (Asp-Tyr-Thr). Crystal structure analysis of the Haspin kinase domain revealed that it exhibits an intrinsically active conformation in the absence of a phosphorylated activation loop, assisted by several unique insertions at its N- and C-terminal lobes (Eswaran et al., 2009, Villa et al., 2009). How can H3T3 phosphorylation be limited to M phase if the Haspin kinase domain is intrinsically active? Here, we reveal that Haspin activity is autoinhibited during interphase by a conserved basic segment adjacent to its kinase domain, and that the multisite phosphorylation of its N-terminal region by Cdk1 and Polo-like kinase (Plx1 in Xenopus egg extracts or Plk1 in human cells) in M phase neutralizes its autoinhibition.
Results
Discussion In this study, we revealed the mechanistic basis for cell-cycle-dependent activation of the H3T3 kinase Haspin. In interphase, Haspin is autoinhibited by the HBIS, which interacts with its kinase domain. When Cdk1 is activated upon entry into M phase and phosphorylates a specific priming site (T206 in Xenopus or T128 in humans) at the N terminus of Haspin, Plk1 docks on this priming phosphorylation site and subsequently phosphorylates multiple residues on the N terminus of Haspin, a cascade of events which neutralizes the action of HBIS and stimulates the kinase activity of Haspin (Figure 7E).
Experimental Procedures
Acknowledgments
Introduction Asymmetric cell division is a fundamental process in stem cell biology and development. The appropriate balance between symmetric and asymmetric divisions is important to expand and maintain the stem cell pools and to generate progenitors for differentiation. Disruption of this balance may lead to cancer (Gonzalez, 2007, Knoblich, 2010, Wodarz and Näthke, 2007). One essential step for asymmetric division is the proper distribution of polarization factors within the cell; this drives the unequal partition of cellular components. Moreover, the establishment of a correct cell division axis also relies on cell polarity cues, and is connected to the orientation of the mitotic spindle (Siller and Doe, 2009, Gönczy, 2008). Regulated polarization and spindle orientation are crucial in both unicellular and multicellular organisms, where they are involved in fundamental processes such as epithelial polarization, asymmetric cell division, morphogenesis, organogenesis, and development (Clevers, 2005, Quyn et al., 2010, Gray et al., 2010, Tanos and Rodriguez-Boulan, 2008).