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Novel Binding of the Mitotic Regulator TPX2 (Target Protein for Xenopus Kinesin-like Protein 2) to Importin-α*

Astrid V Giesecke,M. Stewart

Published 2010 in Journal of Biological Chemistry

ABSTRACT

Several aspects of mitotic spindle assembly are orchestrated by the Ran GTPase through its modulation of the interaction between spindle assembly factors and importin-α. One such factor is TPX2 that promotes microtubule assembly in the vicinity of chromosomes. TPX2 is inhibited when bound to importin-α, which occurs when the latter is bound to importin-β. The importin-α:β interaction is disrupted by the high RanGTP concentration near the chromosomes, releasing TPX2. In more distal regions, where Ran is predominantly GDP-bound, TPX2 remains bound to importin-α and so is inhibited. Here we use a combination of structural and biochemical methods to define the basis for TPX2 binding to importin-α. A 2.2 Å resolution crystal structure shows that the primary nuclear localization signal (284KRKH287) of TPX2, which has been shown to be crucial for inhibition, binds to the minor NLS-binding site on importin-α. This atypical interaction pattern was confirmed using complementary binding studies that employed importin-α variants in which binding to either the major or minor NLS-binding site was impaired, together with competition assays using the SV40 monopartite NLS that binds primarily to the major site. The different way in which TPX2 binds to importin-α could account for much of the selectivity necessary during mitosis because this would reduce the competition for binding to importin-α from other NLS-containing proteins.

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