A Positive Feedback Loop Involving Haspin and Aurora B Promotes CPC Accumulation at Centromeres in Mitosis

Abstract

Summary Haspin phosphorylates histone H3 at Thr-3 (H3T3ph) during mitosis [1, 2], providing a chromatin binding site for the chromosomal passenger complex (CPC) at centromeres to regulate chromosome segregation [3–5]. H3T3ph becomes increasingly focused at inner centromeres during prometaphase [1, 2], but little is known about how its level or location and the consequent chromosomal localization of the CPC are regulated. In addition, CPC binding to Shugoshin proteins contributes to centromeric Aurora B localization [5, 6]. Recruitment of the Shugoshins to centromeres requires the phosphorylation of Histone H2A at T120 (H2AT120ph) by the kinetochore kinase Bub1 [7], but the molecular basis for the collaboration of this pathway with H3T3ph has been unclear. Here, we show that Aurora B phosphorylates Haspin to promote generation of H3T3ph, and that Aurora B kinase activity is required for normal chromosomal localization of the CPC, indicating an intimate linkage between Aurora B and Haspin functions in mitosis. We propose that Aurora B activity triggers a CPC-Haspin-H3T3ph feedback loop that promotes generation of H3T3ph on chromatin. We also provide evidence that the Bub1-Shugoshin-CPC pathway supplies a signal that boosts the CPC-Haspin-H3T3ph feedback loop specifically at centromeres to produce the well-known accumulation of the CPC in these regions.

The Mitotic Protein Kinase Haspin and Its Inhibitors

“…This phosphorylation (H3T3ph) was demonstrated both in vitro and in several cell lines by immunofluorescence, using histone H3 Thr3 phospho-specific antibodies ( Figure 5 upper panel). Haspin depletion by siRNA eliminates H3T3 phosphorylation in mitotic cells, and ectopic overexpression of Haspin leads to abnormal H3T3 phosphorylation levels in interphase cells confirming that H3T3ph is specific of Haspin activity [7,14,15]. H3 is phosphorylated on Thr3 through most of mitosis.…”

“…A second parallel pathway involving histone H2A phosphorylation on Thr120 by Bub1 creates a binding site for Shugoshin, a protein involved in the protection of centromeric cohesion. Shugoshin binds directly to Survivin in yeast and to Borealin CPC member in human in a comparable manner [14,43,44].…”

“…The phosphorylation decreases rapidly at anaphase and can still be observed on telomeres present in the vicinity of Aurora B activity area on the midzone. It is no longer detected on chromosomes, when cells are in late telophase ( Figure 5 upper panel) [6,7,13,14].…”

“…Histone H3 phosphorylated on threonine 3 is directly recognized by the conserved BIR domain of Survivin, a member of the chromosomal passenger complex (CPC) [14,19,39], thus anchoring the CPC at centromeres. The CPC is a complex of four subunits, Survivin, Borealin, INCENP, and the Aurora B kinase.…”

“…Aurora B is an essential kinase, which regulates mitotic progression, including spindle assembly checkpoint, condensation, and chromosomal biorientation and cytokinesis [40][41][42]. Additionally, Aurora B phosphorylates Haspin N-terminus on several sites allowing its over-activation and creating a positive feedback loop triggering the accumulation of CPC at the centromeres [14] (Figure 4 lower panel).…”