Activation of Aurora-A kinase by protein phosphatase inhibitor-2, a bifunctional signaling protein - PubMed (original) (raw)

Activation of Aurora-A kinase by protein phosphatase inhibitor-2, a bifunctional signaling protein

David L Satinover et al. Proc Natl Acad Sci U S A. 2004.

Abstract

Aurora-A kinase is necessary for centrosome maturation, for assembly and maintenance of a bipolar spindle, and for proper chromosome segregation during cell division. Aurora-A is an oncogene that is overexpressed in multiple human cancers. Regulation of kinase activity apparently depends on phosphorylation of Thr-288 in the T-loop. In addition, interactions with targeting protein for Xenopus kinesin-like protein 2 (TPX2) allosterically activate Aurora-A. The Thr-288 phosphorylation is reversed by type-1 protein phosphatase (PP1). Mutations in the yeast Aurora, Ipl1, are suppressed by overexpression of Glc8, the yeast homolog of phosphatase inhibitor-2 (I-2). In this study, we show that human I-2 directly and specifically stimulated recombinant human Aurora-A activity in vitro. The I-2 increase in kinase activity was not simply due to inhibition of PP1 because it was not mimicked by other phosphatase inhibitors. Furthermore, activation of Aurora-A was unaffected by deletion of the I-2 N-terminal PP1 binding motif but was eliminated by deletion of the I-2 C-terminal domain. Aurora-A and I-2 were recovered together from mitotic HeLa cells. Kinase activation by I-2 and TPX2 was not additive and occurred without a corresponding increase in T-loop phosphorylation. These results suggest that both I-2 and TPX2 function as allosteric activators of Aurora-A. This implies that I-2 is a bifunctional signaling protein with separate domains to inhibit PP1 and directly stimulate Aurora-A kinase.

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Figures

Fig. 2.

Fig. 2.

I-2 Stimulation of Aurora-A Kinase. (A) Time course of Aurora-A kinase activity with added I-2 (▴) or without (•). (B) Dose dependence of I-2 activation of Aurora-A using different kinase concentrations (key in Inset). The EC50 of I-2 with Aurora-A was 5 μM. Activity was normalized to untreated Aurora-A. (C) Recombinant I-2 (▪), I-2 deleted of the primary PP1 binding motif (▴, residues 14–197), or a C-terminal truncated I-2 (•, residues 0–118) were titrated into an Aurora-A kinase reaction as described under Materials and Methods.(D) Effects of I-2 on activity of various Aurora-A kinase mutants. Kinase assays were performed as described under Materials and Methods with MBP as substrate. The identity of Aurora-A is indicated by WT or the site of the point mutations. Parallel reactions without and with I-2 (–/+) were analyzed by phosphorimaging (top gel, [32P]MBP). Amount of MBP substrate is shown by Coomassie stain (middle gel, MBP). Loading control for the amount of Aurora-A kinase in the assay shown is presented (bottom gel, α-Aurora-A). Results are representative of three independent experiments.

Fig. 1.

Fig. 1.

Regulation of Aurora kinase activity by PP1 and I-2. (A) Recombinant Aurora-A was incubated ± PP1 for 45 min, then ± I-2 for 10 min. Assays containing 300 nM Aurora-A were performed for 10 min after addition of 100 μM radiolabeled ATP, as described in Materials and Methods. Samples were analyzed for (top to bottom) Aurora-A phosphorylation (α-Thr-288), total Aurora-A protein (α-Aurora-A), kinase activity by PhosphorImager ([32P]MBP), and the amount of MBP by Coomassie stain. Results are representative of at least three independent experiments. (B) Control reactions showing no kinase activity in the absence of Aurora-A. Kinase activity of I-2 stimulated Aurora-A is shown in lane 1 as a positive control. Assays with I-2 alone (lane 2), TPX2 alone (lane 3), or PP1 alone (lane 4) showed no 32P incorporation into MBP above background levels.

Fig. 3.

Fig. 3.

Association of endogenous I-2 and Aurora-A in mitotic HeLa cells. (A) I-2 was immunoprecipitated from mitotic HeLa cell extracts, and the recovered complexes were immunoblotted for I-2, Aurora-A, and PP1δ (top to bottom). The lane labeled L shows a 2% aliquot of the cell lysate, lane C is a control immunoprecipitate without antibody, and the I-2 lane is the specific anti-I-2 immunoprecipitate. (B) CNBr-activated Sepharose beads coupled with Aurora-A (Aur) or BSA as a negative control were used in a pull-down assay with extracts of mitotic HeLa cells. Beads were collected, and the unbound supernatant fraction (Sup) was reserved. The beads were then washed by centrifugation, and proteins on the pelleted beads (pellet) were analyzed by immunoblotting for α-I-2 (upper) and α-PP1 (lower).

Fig. 4.

Fig. 4.

Aurora-A activation by TPX2 and I-2. (A) Stimulation of lambda phosphatase (λ PPase) dephosphorylated Aurora-A does not fully reactivate the kinase. Lambda phosphatase was used to dephosphorylate recombinant Aurora-A. After addition of sodium vanadate to inactivate the lambda phosphatase, both recombinant Aurora-A kinase (400 nM) and dephosphorylated Aurora-A (400 nM) were preincubated for 10 min with either I-2 (27 μM) or TPX2 (2 μM), as indicated. Samples were assayed for kinase activity, and the specific activity (pmol of PO4 per μg of Aur-A per min) was calculated by PhosphorImager analysis of [32P]MBP relative to an internal standard. Numbers in parentheses in the graph represent fold activation of kinase activity normalized to recombinant Aurora-A kinase. The results show that lambda phosphatase treatment reduced kinase activity 25-fold (1.00 vs. 0.04). Subsequent activation by TPX2 or I-2 failed to restore activity to original levels. (B) I-2 does not restore T-loop phosphorylation at Thr-288 after lambda phosphatase treatment. Products of the kinase assay were analyzed by immunoblotting with T-loop phosphorylation site-specific antibody (α-Thr-288) and an Aurora-A antibody (α-Aurora-A) to show equal loading. Treatment with lambda phosphatase removed >95% of Thr-288 phosphorylation. Activation by TPX2 promoted a slight increase in Aurora-A Thr-288 phosphorylation but did not restore to original levels. No increase in Thr-288 phosphorylation was detected by I-2 addition either before or after lambda phosphatase treatment.

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