Microtubules Accelerate the Kinase Activity of Aurora-B by a Reduction in Dimensionality

Abstract

Aurora-B is the kinase subunit of the Chromosome Passenger Complex (CPC), a key regulator of mitotic progression that corrects improper kinetochore attachments and establishes the spindle midzone. Recent work has demonstrated that the CPC is a microtubule-associated protein complex and that microtubules are able to activate the CPC by contributing to Aurora-B auto-phosphorylation in trans. Aurora-B activation is thought to occur when the local concentration of Aurora-B is high, as occurs when Aurora-B is enriched at centromeres. It is not clear, however, whether distributed binding to large structures such as microtubules would increase the local concentration of Aurora-B. Here we show that microtubules accelerate the kinase activity of Aurora-B by a “reduction in dimensionality.” We find that microtubules increase the kinase activity of Aurora-B toward microtubule-associated substrates while reducing the phosphorylation levels of substrates not associated to microtubules. Using the single molecule assay for microtubule-associated proteins, we show that a minimal CPC construct binds to microtubules and diffuses in a one-dimensional (1D) random walk. The binding of Aurora-B to microtubules is salt-dependent and requires the C-terminal tails of tubulin, indicating that the interaction is electrostatic. We show that the rate of Aurora-B auto-activation is faster with increasing concentrations of microtubules. Finally, we demonstrate that microtubules lose their ability to stimulate Aurora-B when their C-terminal tails are removed by proteolysis. We propose a model in which microtubules act as scaffolds for the enzymatic activity of Aurora-B. The scaffolding activity of microtubules enables rapid Aurora-B activation and efficient phosphorylation of microtubule-associated substrates.

Microtubule-associated proteins control the kinetics of microtubule nucleation

“…Paclitaxel-stabilized microtubules were prepared as described previously 69 . A small sample of the microtubule preparation was incubated on ice for 30 min and the polymerized tubulin concentration was determined spectrophotometrically using an extinction coefficient of ε = 115,000 M −1 cm −1 .…”

A mathematical model of Aurora B activity in prophase and metaphase

“…In these cases, the level of activity of Aurora B was approximately spatially homogeneous at all times. It is known that Aurora B binds to microtubules and diffuses along them [93,94] . This would have the effect of lowering the effective diffusion coefficient D in our model.…”

“…Table 1 . 5 demonstrates that a low diffusion coefficient, for example due to binding to microtubules [93,94] , can result in a gradient of Aurora B activity emanating from centromeres. This gradient can eventually disappear, in the case of a bistable switch, or be maintained as the system approaches equilibrium, in the case of a sigmoidal response.…”

“…In this case, a simpler model that includes soluble and bound fractions of Aurora B but no spatial variation may be more appropriate. However, if Aurora B binding to microtubules does significantly lower the effective diffusion coefficient [93,94] then our model in Section 2.2 would be more appropriate in order to capture the spatial variation in Aurora B activation. The modelling in Section 2 assumes that the rates of reactions for bound components and soluble components are the same.…”

“…Future modelling may explicitly include microtubules, which have been shown to be important for the activation of Aurora B [93,94] . Association of Aurora B with microtubules was not explicitly included in our model.…”