Microtubules accelerate the kinase activity of Aurora-B by a reduction in dimensionality.
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.
Department of Biology, McGill University, Montréal, Québec, Canada.
Michael
M
Noujaim
Department of Biology, McGill University, Montréal, Québec, Canada.
Susanne
S
Bechstedt
Department of Biology, McGill University, Montréal, Québec, Canada.
Michal
M
Wieczorek
Department of Biology, McGill University, Montréal, Québec, Canada.
Gary J
GJ
Brouhard
D000818:Animals; D064107:Aurora Kinase B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002503:Centromere / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007700:Kinetics; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D029867:Xenopus Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism
D002868:Chromosomal Proteins, Non-Histone; C506222:INCENP protein, Xenopus; D029867:Xenopus Proteins; D064107:Aurora Kinase B