Phosphorylation of Maskin by Aurora-A Is Regulated by RanGTP and Importin β

Alison J Albee,Wei Tao,Christiane Wiese

doi:10.1074/jbc.m607203200

Alison J Albee, Wei Tao + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m607203200

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Abstract

Mitotic spindle assembly in Xenopus egg extracts is regulated at least in part by importin beta and its regulator, the small GTPase, Ran. RanGTP stabilizes microtubules near the chromosomes during spindle assembly by selectively releasing spindle assembly factors from inhibition by importin alpha/beta in the vicinity of the chromosomes. Several spindle assembly factors are regulated in this manner. We identified maskin, the Xenopus member of the transforming acidic coiled coil family of proteins, as a potential candidate in a two-step affinity chromatography approach designed to uncover additional downstream targets of importin alpha/beta in mitosis. Here, we show that although maskin lacks a canonical nuclear localization sequence, it binds importin beta in a RanGTP-regulated manner. We further show that importin beta inhibits the regulatory phosphorylation of maskin by Aurora-A. This suggests a novel mechanism by which importin beta regulates the activity of a spindle assembly factor.

Highlights

It has become increasingly clear over the past few years that spindle assembly is regulated by proteins that, during inter
We show that maskin interacts with importin ␤ in vitro, and this interaction is regulated by RanGTP
The Xenopus Spindle Assembly Factor Maskin Is a Potential Importin ␤ Target—Several proteins that function to regulate mitotic spindle assembly or MT bundling have been shown to interact with importin ␤ in mitosis [7, 49]

Summary

EXPERIMENTAL PROCEDURES

Expression and Cloning—Fusion proteins were expressed in Escherichia coli strain C41(DE3) [42] and purified by affinity chromatography using their respective tags: His6-importin ␣ (described in Ref. 8); His6-importin ␤ ( has an S-tag; Ref. 8); His6-RanL43E or RanT24N (described in Ref. 5); His6-Aurora-A [43]; GST-maskin [27] and GST-maskin truncation mutants (see below). Maskin (0 – 40 ␮M) and/or importin ␤ (as indicated in Fig. 4) were added to mitotic extract on ice; the reaction was moved to 22–25 °C and incubated for 15 min. Concentrated mitotic extracts (5 ml/purification) were diluted with 1 volume of buffer (50 mM sucrose, 10 mM K-HEPES (pH 7.7), 1 mM EGTA, 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride) This mixture was centrifuged at 192,000 ϫ g for 40 min at 4 °C. The beads and associated proteins were collected by a brief spin and washed 3 times with H100 (50 mM HEPES, 1 mM EGTA, 1 mM MgCl2, 100 mM NaCl, pH 7.6) and once with the same buffer containing 250 mM NaCl and 0.1% Triton X-100. Incorporation of label was detected by autoradiography using a PhosphorImager and was quantitated using Adobe Photoshop software

RESULTS

DISCUSSION

Methods