Aurora-A: an expedition to the pole of the spindle in Xenopus egg extracts

TPX2 has multiple functions during mitosis, including microtubule nucleation around the chromosomes and the targeting of Xklp2 and Aurora A to the spindle. We have performed a detailed domain functional analysis of TPX2 and found that a large N-terminal domain containing the Aurora A binding peptide interacts directly with and nucleates microtubules in pure tubulin solutions. However, it cannot substitute the endogenous TPX2 to support microtubule nucleation in response to Ran guanosine triphosphate (GTP) and spindle assembly in egg extracts. By contrast, a large C-terminal domain of TPX2 that does not bind directly to pure microtubules and does not bind Aurora A kinase rescues microtubule nucleation in response to RanGTP and spindle assembly in TPX2-depleted extract. These and previous results suggest that under physiological conditions, TPX2 is essential for microtubule nucleation around chromatin and functions in a network of other molecules, some of which also are regulated by RanGTP.

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Bora phosphorylation substitutes in trans for T-loop phosphorylation in Aurora A to promote mitotic entry

Nature Communications ◽

10.1038/s41467-021-21922-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

N. Tavernier ◽

Y. Thomas ◽

S. Vigneron ◽

P. Maisonneuve ◽

S. Orlicky ◽

...

Keyword(s):

Kinase Domain ◽

Activation Mechanism ◽

Aurora A Kinase ◽

Aurora A ◽

Mitotic Entry ◽

Amino Acid Region ◽

Egg Extracts ◽

Activation Segment ◽

Xenopus Egg ◽

Xenopus Egg Extracts

AbstractPolo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.

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Aurora A Phosphorylates MCAK to Control Ran-dependent Spindle Bipolarity

Molecular Biology of the Cell ◽

10.1091/mbc.e08-02-0198 ◽

2008 ◽

Vol 19 (7) ◽

pp. 2752-2765 ◽

Cited By ~ 82

Author(s):

Xin Zhang ◽

Stephanie C. Ems-McClung ◽

Claire E. Walczak

Keyword(s):

Phosphorylation Site ◽

Aurora B ◽

Aurora A ◽

Spindle Formation ◽

Chromatin Region ◽

Spindle Poles ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts ◽

Bipolar Spindles

During mitosis, mitotic centromere-associated kinesin (MCAK) localizes to chromatin/kinetochores, a cytoplasmic pool, and spindle poles. Its localization and activity in the chromatin region are regulated by Aurora B kinase; however, how the cytoplasmic- and pole-localized MCAK are regulated is currently not clear. In this study, we used Xenopus egg extracts to form spindles in the absence of chromatin and centrosomes and found that MCAK localization and activity are tightly regulated by Aurora A. This regulation is important to focus microtubules at aster centers and to facilitate the transition from asters to bipolar spindles. In particular, we found that MCAK colocalized with NuMA and XMAP215 at the center of Ran asters where its activity is regulated by Aurora A-dependent phosphorylation of S196, which contributes to proper pole focusing. In addition, we found that MCAK localization at spindle poles was regulated through another Aurora A phosphorylation site (S719), which positively enhances bipolar spindle formation. This is the first study that clearly defines a role for MCAK at the spindle poles as well as identifies another key Aurora A substrate that contributes to spindle bipolarity.

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Faculty Opinions recommendation of Greatwall kinase participates in the Cdc2 autoregulatory loop in Xenopus egg extracts.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1014405.370925 ◽

2006 ◽

Author(s):

Iain Hagan

Keyword(s):

Egg Extracts ◽

Xenopus Egg ◽

Greatwall Kinase ◽

Xenopus Egg Extracts

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Faculty Opinions recommendation of Cell cycle-regulated recognition of the destruction box of cyclin B by the APC/C in Xenopus egg extracts.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1017047.202474 ◽

2004 ◽

Author(s):

Kevin Hardwick

Keyword(s):

Cell Cycle ◽

Cyclin B ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts ◽

Destruction Box

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Faculty Opinions recommendation of Control of replication origin density and firing time in Xenopus egg extracts: role of a caffeine-sensitive, ATR-dependent checkpoint.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1020314.231528 ◽

2004 ◽

Author(s):

Stephen Kearsey

Keyword(s):

Replication Origin ◽

Firing Time ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

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Faculty Opinions recommendation of Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717980323.793475374 ◽

2013 ◽

Author(s):

Xueliang Zhu

Keyword(s):

Microtubule Nucleation ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

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The bulk of unpolymerized actin in Xenopus egg extracts is ATP-bound.

Molecular Biology of the Cell ◽

10.1091/mbc.6.2.227 ◽

1995 ◽

Vol 6 (2) ◽

pp. 227-236 ◽

Cited By ~ 32

Author(s):

J Rosenblatt ◽

P Peluso ◽

T J Mitchison

Keyword(s):

Actin Polymerization ◽

Critical Concentration ◽

Large Fraction ◽

Nucleotide Exchange ◽

Chromatography Analysis ◽

Thymosin Beta 4 ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

Non-muscle cells contain 15-500 microM actin, a large fraction of which is unpolymerized. Thus, the concentration of unpolymerized actin is well above the critical concentration for polymerization in vitro (0.2 microM). This fraction of actin could be prevented from polymerization by being ADP bound (therefore less favored to polymerize) or by being ATP bound and sequestered by a protein such as thymosin beta 4, or both. We isolated the unpolymerized actin from Xenopus egg extracts using immobilized DNase 1 and assayed the bound nucleotide. High-pressure liquid chromatography analysis showed that the bulk of soluble actin is ATP bound. Analysis of actin-bound nucleotide exchange rates suggested the existence of two pools of unpolymerized actin, one of which exchanges nucleotide relatively rapidly and another that apparently does not exchange. Native gel electrophoresis of Xenopus egg extracts demonstrated that most of the soluble actin exists in complexes with other proteins, one of which might be thymosin beta 4. These results are consistent with actin polymerization being controlled by the sequestration and release of ATP-bound actin, and argue against nucleotide exchange playing a major role in regulating actin polymerization.

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