期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 285, 期 23, 页码 17628-17635出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M110.102343
关键词
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资金
- Wellcome Trust
- Medical Research Council [MC_U105178939] Funding Source: researchfish
- MRC [MC_U105178939] Funding Source: UKRI
Several aspects of mitotic spindle assembly are orchestrated by the Ran GTPase through its modulation of the interaction between spindle assembly factors and importin-alpha. One such factor is TPX2 that promotes microtubule assembly in the vicinity of chromosomes. TPX2 is inhibited when bound to importin-alpha, which occurs when the latter is bound to importin-beta. The importin-alpha:beta 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-alpha and so is inhibited. Here we use a combination of structural and biochemical methods to define the basis for TPX2 binding to importin-alpha. A 2.2 angstrom resolution crystal structure shows that the primary nuclear localization signal ((KRKH287)-K-284) of TPX2, which has been shown to be crucial for inhibition, binds to the minor NLS-binding site on importin-alpha. This atypical interaction pattern was confirmed using complementary binding studies that employed importin-alpha 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-alpha could account for much of the selectivity necessary during mitosis because this would reduce the competition for binding to importin-alpha from other NLS-containing proteins.
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