Activation of Aurora-A kinase by protein partner binding and phosphorylation are independent and synergistic

Abstract

Protein kinases are activated by phosphorylation and by the binding of activator proteins. The interplay of these two factors is incompletely understood. We applied energetic analysis to this question and characterized the activation process of the serine/threonine kinase Aurora-A by phosphorylation and by its protein partner, targeting protein for Xenopus kinesin-like protein 2 (TPX2). We discovered that these two activators act synergistically and without a predefined order: each can individually increase the activity of Aurora-A, and the effect of both bound together is the exact sum of their individual contributions to catalysis. Unexpectedly, the unphosphorylated enzyme has catalytic activity that is increased 15-fold by the binding of TPX2 alone. The energetic contribution of phosphorylation to catalysis is 2-fold greater than that of TPX2 binding, which is independent of the phosphorylation state of the enzyme. Based on this analysis, we propose a revised, fluid model of Aurora-A activation in which the first step is a reduction in the mobility of the activation loop by either TPX2 binding or phosphorylation. Furthermore, our results suggest that unphosphorylated Aurora-A bound to the mitotic spindle by TPX2 is catalytically active and that the phosphorylation state of Aurora-A is an inaccurate surrogate for its activity. Extending this form of analysis will allow us to compare quantitatively the effects of the whole network of kinase-activating partners. Comparison with other kinases showed that kinetic characterization detects those kinases whose activation loops undergo a rearrangement upon phosphorylation and thus whose unphosphorylated state offers a distinct target for the development of Type II inhibitors.

FIGURE 1.

Reaction scheme and free energy diagrams for Aurora-A catalysis. a, reaction scheme for a kinase following a random sequential mechanism: first one substrate binds (either A or B), then the other binds to form a triple complex, which reacts to form products P and Q. b, free energy diagram for the reaction scheme in a. ΔG_iA = RT ln K_ia, ΔG_mA = RT ln K_mA, and ΔG_cat ∝ RT ln k_cat. E·A·B^‡ denotes the high energy transition state of the phosphotransfer reaction. The relative positions of E·A and E·B on the reaction coordinate are arbitrary. Relative positions on the y axis have been drawn assuming saturating quantities of A and limiting quantities of B. These positions are arbitrary, and changing the concentration of reactants will only affect the position on the y axis and not the nomenclature or conclusions of this figure. c, simplified free energy diagram for the case where [B] ≪ K_mB. In this case, ΔG_cat encompasses the energetics of forming the triple complex and forming the high energy transition state and is proportional to a pseudo-k_cat as shown in the main text. Our assay conditions for Aurora-A are an example of this special case, and K_m and k_cat measurements presented in this study refer to the quantities shown in this figure.

FIGURE 2.

Normalized activity of Aurora-A and mutants. a, K_m for ATP determined for enzyme alone. b, TPX2 titration in 2 mm ATP (1 mm ATP for wild type). c, K_m for ATP determined in the presence of 20 μm TPX2 (5 μm TPX2 for wild type). Red solid line, wild type; red dashed line, T287A; blue solid line, T288A; blue dashed line, T287A/T288A. Red graphs are plotted on the left y axis, and blue graphs are plotted on the right y axis. All graphs are normalized for enzyme concentration. Error bars show S.E. of reaction rates (i.e. S.E. of the slope determined by linear regression of reaction time courses with each time course carried out in duplicate and replicate data fitted together).

FIGURE 3.

Two parallel paths of Aurora-A activation give rise to four different states of activity. a, the four different states of Aurora-A are shown schematically, each labeled with a roman numeral (i–iv), and are distributed on a vertical scale to indicate the catalytic activity relative to the lowest activity state. Numbers in bold (1–4) label each step for reference but have no numerical order or meaning. Kinetic constants indicate the approximate -fold change in the constant upon completion of the step. Each step is labeled with the associated -fold change in kinetic parameters. This was calculated in two different ways: the effect of phosphorylation in as close to physiological circumstances as possible (WT compared with T288A) and in brackets the effect of phosphorylation on Thr-288 alone (T287A compared with T287A/T288A). A fifth, dashed arrow connects the two states of intermediate activity. b, model of Aurora-A activation through stabilization of the activation loop (shown as a dashed line progressing to a solid line) and electrostatic communication between the phosphorylated activation loop and the catalytic center (shown as a double headed arrow).

FIGURE 4.

Activation loops of kinases for which activation process has been characterized by kinetic studies. The crystal structures of activation loops are shown from DFG to APE sequence motifs, which mark the beginning and end, respectively, of the kinase activation loop. a, Aurora-A; b, PKA; c, p38α; d, CDK2; e, v-Fes, the human viral homologue to avian v-Fps; f, ERK2. Residues whose phosphorylation affects kinase activity are shown in stick representation. The phosphate groups of the Thr(P) residues (Tyr(P) for v-Fes) superpose in the fully active kinases when the different molecules are aligned across the kinase domain. Red, phosphorylated kinase; dark blue, unphosphorylated kinase; orange, phosphorylated Aurora-A bound to TPX2 (a only)/phosphorylated CDK2 bound to cyclin A (d only); cyan, unphosphorylated CDK2 bound to cyclin A (d only). Protein Data Bank codes selected are 1OL5, 1OL6, and 1OL7 (Aurora-A); 1BKX and 2UZW (PKA); 3PY3 and 1OUK (p38α); 1B38, 1B39, 1JST, and 1FIN (CDK2); 3CBL and 3CD3 (v-Fes); and 1ERK and 2ERK (ERK2). The activation loop is not visible in the unphosphorylated Aurora-A structure and is assumed to be disordered.