Noujaim M et al. (2014), Microtubules accelerate the kinase activity of ...

XB-ART-53389

PLoS One 2014 Feb 03;92:e86786. doi: 10.1371/journal.pone.0086786.

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Microtubules accelerate the kinase activity of Aurora-B by a reduction in dimensionality.

Noujaim M , Bechstedt S , Wieczorek M , Brouhard GJ .

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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.

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	Figure 2. The CCA diffuses on microtubules via electrostatic interactions.(A) Still image (top) and kymographs (bottom) of the CCA-GFP (green) interacting with microtubules (red). The back-and-forth movements of a 1D random walk are evident. (i) A color-combined kymograph of CCA-GFP diffusion. (ii) An inverted grayscale image of the CCA-GFP signal shown in (i). (B) Plot of the mean squared displacement, against time for the CCA-GFP. A linear relationship is observed, and slope of the fit (blue line), , is related to the diffusion coefficient by . (C) Histogram of the duration of CCA-GFP binding events. An exponential decay fit (blue line) gives a mean duration of = 1.5 s. (D) Plot of the intensity of the CCA-GFP on the microtubule lattice against the concentration of the CCA-GFP. The data is well described by a conventional binding isotherm (blue line). (E) Box plot of the intensity of the CCA-GFP on microtubules at four different salt concentrations. (F) Box plot of the intensity of the CCA-GFP on microtubules for paclitaxel-stabilized microtubules and subtilisin-digested microtubules (labeled).
	Figure 3. Microtubules accelerate Aurora-B activation by a reduction in dimensionality.(A) Schematic of a model for Aurora-B auto-activation. An active kinase (, bright red) binds to an inactive kinase (, dim red) with an equilibrium constant, , to form a complex, . The kinase reaction occurs with a catalytic constant, , producing two active kinases (right). (B) Images of P radiograms showing radioactive bands corresponding to phosphorylated INBox motif. The images correspond to 4 microtubule concentrations (labeled at left). The lanes in the radiogram correspond to different time points (labeled at bottom). At higher microtubule concentrations, the radioactive bands become more intense at earlier time points. (C) Plot of the INBox motif band intensity against time for 4 microtubule concentrations. (D) Plot of the mean first passage time, , against the size of the confining space in which the molecules diffuse, . The plot shows both 3D diffusion (red hashed line) and 1D diffusion (green solid line) using the diffusion coefficients for Aurora-B (3D case, estimated; 1D case, measured). 1D diffusion is significantly faster when the molecules diffuse within spaces 2 µm in radius.
	Figure 4. Disruption of Microtubule binding releases Aurora-B to soluble substrates.(A) Bar graph showing the normalized Histone H3 band intensity in P radiograms under three conditions: no microtubules (Buffer), paclitaxel-stabilized microtubules (MTs), and subtilisin-digested microtubule (Subtilisin). (B) Bar graph showing the normalized MCAKAAA band intensity in P radiograms under three conditions: no microtubules (Buffer), paclitaxel-stabilized microtubules (MTs), and subtilisin-digested microtubule (Subtilisin). (C) Bar graph showing the normalized INBox motif band intensity in P radiograms under three conditions: no microtubules (Buffer), paclitaxel-stabilized microtubules (MTs), and subtilisin-digested microtubule (Subtilisin). Note that the Histone H3, MCAKAAA, and INBox band intensities were normalized independently, so the data cannot be used to compare the absolute levels of phosphorylation across the different substrates.
	Figure 1. Microtubules sequester Aurora-B activity toward microtubule-associated substrates.(A) Bar graph showing the normalized Histone H3 band intensity in P radiograms at 3 different microtubule (MT) concentrations. (B) Image of a P radiogram showing radioactive bands corresponding to phosphorylated INBox motif (labeled) and Histone H3 (labeled) at 2 different microtubule concentrations. (C) Bar graph showing the normalized INBox band intensity in P radiograms at 3 different microtubule (MT) concentrations. (D) Image of an SDS-PAGE gel showing bands corresponding to Aurora-B-GFP (labeled), tubulin (labeled), and Histone H3 (labeled) at 2 different microtubule concentrations. The band for the INBox construct, at 52 kDa, is masked by the tubulin band. (E) Bar graph showing the normalized MCAKAAA band intensity in P radiograms at 3 different microtubule (MT) concentrations. (F) Image of a P radiogram showing radioactive bands corresponding to phosphorylated MCAKAAA (labeled) and the INBox motif (labeled) at 2 different microtubule concentrations.

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