Aurora kinase inhibitors reveal mechanisms of HURP in nucleation of centrosomal and kinetochore microtubules

Abstract

The overexpression of Aurora kinases in multiple tumors makes these kinases appealing targets for the development of anticancer therapies. This study identified two small molecules with a furanopyrimidine core, IBPR001 and IBPR002, that target Aurora kinases and induce a DFG conformation change at the ATP site of Aurora A. Our results demonstrate the high potency of the IBPR compounds in reducing tumorigenesis in a colorectal cancer xenograft model in athymic nude mice. Human hepatoma up-regulated protein (HURP) is a substrate of Aurora kinase A, which plays a crucial role in the stabilization of kinetochore fibers. This study used the IBPR compounds as well as MLN8237, a proven Aurora A inhibitor, as chemical probes to investigate the molecular role of HURP in mitotic spindle formation. These compounds effectively eliminated HURP phosphorylation, thereby revealing the coexistence and continuous cycling of HURP between unphosphorylated and phosphorylated forms that are associated, respectively, with microtubules emanating from centrosomes and kinetochores. Furthermore, these compounds demonstrate a spatial hierarchical preference for HURP in the attachment of microtubules extending from the mother to the daughter centrosome. The finding of inequality in the centrosomal microtubules revealed by these small molecules provides a versatile tool for the discovery of new cell-division molecules for the development of antitumor drugs.

A water-mediated allosteric network governs activation of Aurora kinase A

“…The larger response to both ligands together is consistent with a shifting equilibrium, and provides a lower bound on the scale of the movement between the underlying structural states. These results are most consistent with apo AurA adopting a DFG-Out conformation observed in structures of the protein bound to an inhibitory nanobody 35 and the inhibitor VX-680 37 , in which the tip of the activation loop moves ~1 nanometer from its position in the active state. In this DFG-Out state the N-terminal β-strand of the activation loop is shifted in register but remains clamped to the catalytic loop (Figure 2d), restricting movement of the loop compared to other DFG-Out states.…”

A dynamic mechanism for allosteric activation of Aurora kinase A by activation loop phosphorylation

“…In both cases, a broad distribution centered at ~30 angstroms is observed for apo AurA, indicating that the activation loop is highly flexible under these conditions. This is consistent with adoption of the DFG-Out state, in which the C-terminal half of the activation loop lacks contacts with the rest of the kinase domain, and is typically disordered in x-ray structures 29–31 (Figure 2f). The addition of both ADP and Tpx2 together yields the longest distances (~55 angstroms) with the narrowest distributions, indicative of a well-defined structure consistent with the DFG-In state, in which the segment of the loop containing the labeling site is anchored to the C-terminal lobe of the kinase on both sides by backbone hydrogen bonds 22 (Figure 2f).…”

Centrosomes control kinetochore-fiber plus-end dynamics via HURP to ensure symmetric divisions

“…We next tested if endogenous HURP localization was under the control of the centrosomal protein kinase Aurora A, which has been proposed to regulate HURP based on HURP overexpression experiments (Wong et al, 2008;Wu et al, 2013;Yu et al, 2005).…”

“…K-fiber plus-end dynamics are set by kinetochores themselves, microtubuledepolymerases such as Kif2a, MCAK or Kif18A Ganem et al, 2005;Mayr et al, 2007;Wordeman et al, 2007) and plus-end binding proteins, such as CLASP, ch-TOG or HURP (Barr and Gergely, 2008;Maiato et al, 2003;Sillje et al, 2006). Of particular interest is the k-fiber stabilizing HURP-protein, since it is specifically enriched on a k-fiber section proximal to kinetochores; the mechanisms governing its function and localization are, however, unclear (Koffa et al, 2006;Sillje et al, 2006;Wong and Fang, 2006;Wong et al, 2008;Wu et al, 2013). Finally, k-fiber dynamics are also affected by k-fiber length, which is regulated by microtubule motors such as HSET, Kid, and Kif15 (Cai et al, 2009;Mayr et al, 2007;Sturgill and Ohi, 2013;Tokai-Nishizumi et al, 2005).…”