Characterization of the TPX2 domains involved in microtubule nucleation and spindle assembly 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.
Brunet
Stéphane
S
Cell Biology and Biophysics Program, European Molecular Biology Laboratory, Heidelberg 69 117, Germany.
Sardon
Teresa
T
Zimmerman
Timo
T
Wittmann
Torsten
T
Pepperkok
Rainer
R
Karsenti
Eric
E
Vernos
Isabelle
I
D000818:Animals; D064127:Aurora Kinases; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002457:Cell Extracts / Q000737:chemistry*; D016764:Cell Polarity; D004789:Enzyme Activation; D016547:Kinesins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D008941:Spindle Apparatus / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D002457:Cell Extracts; C097723:KIF15 protein, Xenopus; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; C486551:TPX2 protein, Xenopus; D029867:Xenopus Proteins; D011494:Protein Kinases; C112134:AURKA protein, Xenopus; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D016547:Kinesins