Structural Basis of Aurora-A Activation by TPX2 at the Mitotic Spindle

Abstract

Aurora-A is an oncogenic kinase essential for mitotic spindle assembly. It is activated by phosphorylation and by the microtubule-associated protein TPX2, which also localizes the kinase to spindle microtubules. We have uncovered the molecular mechanism of Aurora-A activation by determining crystal structures of its phosphorylated form both with and without a 43 residue long domain of TPX2 that we identified as fully functional for kinase activation and protection from dephosphorylation. In the absence of TPX2, the Aurora-A activation segment is in an inactive conformation, with the crucial phosphothreonine exposed and accessible for deactivation. Binding of TPX2 triggers no global conformational changes in the kinase but pulls on the activation segment, swinging the phosphothreonine into a buried position and locking the active conformation. The recognition between Aurora-A and TPX2 resembles that between the cAPK catalytic core and its flanking regions, suggesting this molecular mechanism may be a recurring theme in kinase regulation.

Phosphorylation of p53 Is Regulated by TPX2-Aurora A in Xenopus Oocytes

“…Previous work has suggested that Thr-295 is readily accessible to PP1 in interphase. Upon binding to TPX2 at nuclear envelope breakdown, biochemical and structural studies indicate that the T-loop is shielded from dephosphorylation by PP1 (11,14). TPX2 binding thus permits net autophosphorylation at Thr-295 and autoactivation of Aurora A, and TPX2 also targets the complex to polar microtubules (12,13).…”

“…Evidence from several laboratories indicates that activation occurs as a result of phosphorylation of a threonine residue in the T-loop of the kinase (4,9,10). Purification of Aurora A-activating activity from M phase Xenopus egg extracts led to an apparent activation mechanism in which autophosphorylation at the T-loop is stimulated by binding of the targeting protein for Xklp2 (TPX2) (11)(12)(13)(14). On the other hand, it has been shown that Aurora A activity can be inhibited by interaction with several proteins, including PP1 (protein phosphatase 1), AIP (Aurora A kinase-interacting protein), and, more recently, p53 (9,(15)(16)(17).…”

Protein kinases controlling the onset of mitosis

“…This would be consistent with observations made in vitro, in that close proximity of AurA molecules facilitates autophosphorylation and kinase activation. Structural studies where AurA and TPX2 were co-crystallized, confirmed the key role played by TPX2 in maintaining the phosphorylated T 288 residue in a buried position where it is protected from dephosphorylation, thus locking AurA in an active conformation [141]. The catalytic domain of AurA contains two R/K-V-X-F motifs that have been implicated in binding protein phosphatase 1 (PP1) in a functional manner [142].…”

Characterization of the TPX2 Domains Involved in Microtubule Nucleation and Spindle Assembly inXenopusEgg Extracts

“…A direct interaction of TPX2 with the mitotic kinase Aurora A has recently been described and seems to be required for the targeting of the kinase to spindle microtubules . This interaction is particularly interesting because it leads to the activation of the kinase (Bayliss et al, 2003;Eyers et al, 2003;Tsai et al, 2003). Finally a complex functional interaction between TPX2 and the dynein-dynactin complex leads to spindle pole localization of TPX2 and the targeting of Xklp2 (Wittmann et al, 2000).…”