A Novel Mechanism for Activation of the Protein Kinase Aurora A

Abstract

Segregation of chromosomes during mitosis requires interplay between several classes of protein on the spindle, including protein kinases, protein phosphatases, and microtubule binding motor proteins [1-4]. Aurora A is an oncogenic cell cycle-regulated protein kinase that is subject to phosphorylation-dependent activation [5-11]. Aurora A localization to the mitotic spindle depends on the motor binding protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2), but the protein(s) involved in Aurora A activation are unknown [11-13]. Here, we purify an activator of Aurora A from Xenopus eggs and identify it as TPX2. Remarkably, Aurora A that has been fully deactivated by Protein Phosphatase 2A (PP2A) becomes phosphorylated and reactivated by recombinant TPX2 in an ATP-dependent manner. Increased phosphorylation and activation of Aurora A requires its own kinase activity, suggesting that TPX2 stimulates autophosphorylation and autoactivation of the enzyme. Consistently, wild-type Aurora A, but not a kinase inactive mutant, becomes autophosphorylated on the regulatory T loop residue (Thr 295) after TPX2 treatment. Active Aurora A from bacteria is further activated at least 7-fold by recombinant TPX2, and TPX2 also impairs the ability of protein phosphatases to inactivate Aurora A in vitro. This concerted mechanism of stimulation of activation and inhibition of deactivation implies that TPX2 is the likely regulator of Aurora A activity at the mitotic spindle and may explain why loss of TPX2 in model systems perturbs spindle assembly [14-16]. Our finding that a known binding protein, and not a conventional protein kinase, is the relevant activator for Aurora A suggests a biochemical model in which the dynamic localization of TPX2 on mitotic structures directly modulates the activity of Aurora A for spindle assembly.

VX-680 Inhibits Aurora A and Aurora B Kinase Activity in Human Cells

“…2 and 3) and MLN8054, 19 demonstrates that it too is due to an autophosphorylation mechanism, as previously inferred from biochemical experiments with the Xenopus enzyme. 5,29,34 Interestingly, we found that pThr288 (active) Aurora A was specifically targeted to the centrosome during mitosis (Fig. 5D); this location is therefore a cytological marker of the activated kinase, and is likely to be enriched with Aurora A substrates that control spindle assembly.…”

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

“…Thus, it was important to inhibit Aurora A kinase activity more specifically. Previous work has demonstrated that TPX2 is an activator of Aurora A present in Meta II oocytes that does not activate Aurora B (11). Therefore, we specifically impaired Aurora A activity by ablation of TPX2 with antisense oligonucleotides.…”

“…TA, transactivation domain; DNABD, DNA binding domain; OD, oligomerization domain. from this laboratory had identified TPX2 as an activator of Aurora A present in Meta II (cytostatic factor-arrested) Xenopus egg extracts (11), and the binding site for TPX2 on Aurora A has been well characterized both at the structural level and by mutagenesis (12-14, 43, 44). Our data here show that the level of TPX2 markedly increases during maturation (Fig.…”

“…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).…”

PDZ-Binding Kinase/T-LAK Cell-Originated Protein Kinase, a Putative Cancer/Testis Antigen with an Oncogenic Activity in Breast Cancer

“…In this study, we used okadaic acid, which is known to induce premature mitosis, as a stimulus for activation of PBK/TOPK. Interestingly, previous reports indicated that okadaic acid induced Ser 10 phosphorylation of histone H3 through inhibition of protein phosphatases (28,29). For example, Aurora-A is deactivated by protein phosphatase 2A (PP2A), but to be reactivated by its autophosphorylation through binding with TPX2 (targeting protein for Xenopus kinesin-like protein 2) protein that impairs the activity of PP2A (29).…”