Abstract
To understand how the chromosomal passenger complex ensures chromosomal stability, it is crucial to identify its substrates and to find ways to specifically inhibit the enzymatic core of the complex, Aurora B. We therefore developed a chemical genetic approach to selectively inhibit human Aurora B. By mutating the gatekeeper residue Leu-154 in the kinase active site, the ATP-binding pocket was enlarged, but kinase function was severely disrupted. A unique second site suppressor mutation was identified that rescued kinase activity in the Leu-154 mutant and allowed the accommodation of bulky N(6)-substituted adenine analogs. Using this analog-sensitive Aurora B kinase, we found that retention of the chromosomal passenger complex at the centromere depends on Aurora B kinase activity. Furthermore, analog-sensitive Aurora B was able to use bulky ATPγS analogs and could thiophosphorylate multiple proteins in cell extracts. Utilizing an unbiased approach for kinase substrate mapping, we identified several novel substrates of Aurora B, including the nucleosomal-binding protein HMGN2. We confirmed that HMGN2 is a bona fide Aurora B substrate in vivo and show that its dynamic association to chromatin is controlled by Aurora B.
Highlights
Faithful chromosome segregation requires that the duplicated sister chromatids bi-orient on the mitotic spindle and that anaphase onset does not start before this is accom
INCENP, borealin, and survivin, and the binding of Aurora B to the C-terminal IN-box of INCENP is required for full Aurora B kinase activity [17,18,19]
HMGN2 Is a Mitotic Substrate of Aurora B—To further validate our approach, we asked whether the high mobility group nucleosomal binding protein 2 (HMGN2) we identified as a potential Aurora B substrate (Table II and Fig. 5B) could be validated as a bona fide in vivo substrate of the kinase
Summary
Chemical genetics refers to a strategy where a kinase is genetically engineered to render it capable of utilizing nonnatural ATP analogs to be preferentially utilized as substrates and to be sensitive to unique inhibition by cellpermeable ATP analogs [6, 7] This so-called analog-sensitive kinase harbors a specific mutation in the ATP-binding pocket that changes a bulky amino acid (i.e. methionine, leucine, phenylalanine, or threonine) into a small amino acid (glycine or alanine). We show that the active Aurora B is capable of using bulky ATP␥S analogs to thiophosphorylate multiple proteins in complex cell extracts, including a number of known Aurora B substrates Because this approach is not biased with respect to known consensus sites or for particular functional categories of putative substrates, it is useful for identifying novel direct substrates. We found a number of potential novel Aurora B phosphorylation sites on previously reported substrates, as well as novel substrates of the kinase including the nucleosomal-binding protein HMGN2
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