Mechanism of Aurora B Activation by INCENP and Inhibition by Hesperadin

Abstract

Aurora family serine/threonine kinases control mitotic progression, and their deregulation is implicated in tumorigenesis. Aurora A and Aurora B, the best-characterized members of mammalian Aurora kinases, are approximately 60% identical but bind to unrelated activating subunits. The structure of the complex of Aurora A with the TPX2 activator has been reported previously. Here, we report the crystal structure of Aurora B in complex with the IN-box segment of the inner centromere protein (INCENP) activator and with the small molecule inhibitor Hesperadin. The Aurora B:INCENP complex is remarkably different from the Aurora A:TPX2 complex. INCENP forms a crown around the small lobe of Aurora B and induces the active conformation of the T loop allosterically. The structure represents an intermediate state of activation of Aurora B in which the Aurora B C-terminal segment stabilizes an open conformation of the catalytic cleft, and a critical ion pair in the kinase active site is impaired. Phosphorylation of two serines in the carboxyl terminus of INCENP generates the fully active kinase.

Centromere Targeting of the Chromosomal Passenger Complex Requires a Ternary Subcomplex of Borealin, Survivin, and the N-Terminal Domain of INCENP

“…In contrast, the interaction between Borealin and Survivin was not affected by Zn 2ϩ , as noted previously (Gassmann et al, 2004). These data confirm and extend previously reported interactions between the CPC proteins (Adams et al, 2000;Bishop and Schumacher, 2002;Honda et al, 2003;Gassmann et al, 2004;Sessa et al, 2005). While Aurora B associates with the C terminus of INCENP via the IN-box, Survivin and Borealin interact with the N-terminal 58 amino acids of INCENP and bind to each other.…”

“…None of the constructs coprecipitated the centromeric protein CENP-A (lanes 1-7, second row from bottom) or the kinetochore protein Hec1 (lanes 1-7, bottom row). Together, these results support data on the interaction between the C terminus of INCENP containing the IN-box motif and Aurora B (Bishop and Schumacher, 2002;Bolton et al, 2002;Honda et al, 2003;Yasui et al, 2004;Sessa et al, 2005) and show that a ternary complex between Borealin, Survivin, and the first 58 amino acids of INCENP (INCENP 1-58 ) exists in vivo.…”

“…We suggest that the requirement for Aurora B is indirect, and results from the instability of fulllength INCENP in the absence of Aurora B. That GFP-INCENP 1-58 is stable in the absence of Aurora B suggests that the region that confers instability to INCENP must lie within the central coiled-coil domain or the C terminus of the protein, the region that interacts with the Aurora B kinase (Adams et al, 2000;Bishop and Schumacher, 2002;Bolton et al, 2002;Honda et al, 2003;Yasui et al, 2004;Sessa et al, 2005). Furthermore, the RNAi-rescue results with GFP-INCENP show that expression of the first 58 amino acids of INCENP rescues the protein levels of Survivin and Borealin in the transfected cells.…”

“…First, the C-terminal IN-box, which has previously been shown to function as an effector module in the binding and activation of Aurora B (Adams et al, 2000;Bishop and Schumacher, 2002;Honda et al, 2003;Yasui et al, 2004;Sessa et al, 2005); and second, a functional module required for centromere targeting associated with the N terminus of the INCENP protein ( Figure 10A). In the future, it will be important to elucidate the precise stoichiometry of the targeting subcomplex identified in this study and to analyze potential Aurora B-independent functions of this ternary module.…”

“…While it has been well established that an interaction of Aurora B with the IN-box, a highly conserved motif within the C terminus of INCENP, is required for full activation of the kinase, a previous analysis of chicken INCENP indicated that the N terminus of the protein might be involved in targeting the CPC to the centromere (Ainsztein et al, 1998;Adams et al, 2000;Bishop and Schumacher, 2002;Bolton et al, 2002;Honda et al, 2003;Yasui et al, 2004;Sessa et al, 2005). However, the molecular basis for this observation and the involvement of the other subunits of the CPC in the localization of the complex is poorly understood.…”