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???displayArticle.abstract??? p53 is an important tumor suppressor regulating the cell cycle at multiple stages in higher vertebrates. The p53 gene is frequently deleted or mutated in human cancers, resulting in loss of p53 activity. This leads to centrosome amplification, aneuploidy, and tumorigenesis, three phenotypes also observed after overexpression of the oncogenic kinase Aurora A. Accordingly, recent studies have focused on the relationship between these two proteins. p53 and Aurora A have been reported to interact in mammalian cells, but the function of this interaction remains unclear. We recently reported that Xenopus p53 can inhibit Aurora A activity in vitro but only in the absence of TPX2. Here we investigate the interplay between Xenopus Aurora A, TPX2, and p53 and show that newly synthesized TPX2 is required for nearly all Aurora A activation and for full p53 synthesis and phosphorylation in vivo during oocyte maturation. In vitro, phosphorylation mediated by Aurora A targets serines 129 and 190 within the DNA binding domain of p53. Glutathione S-transferase pull-down studies indicate that the interaction occurs via the p53 transactivation domain and the Aurora A catalytic domain around the T-loop. Our studies suggest that targeting of TPX2 might be an effective strategy for specifically inhibiting the phosphorylation of Aurora A substrates, including p53.
FIGURE 1. p53 phosphorylation during Xenopus oocyte maturation is
regulated by Aurora A. A, Western blot of endogenous p53 during
oocyte maturation. Pro I, G2-arrested oocytes;
GVBD, progesterone-treated oocytes collected after germinal vesicle
breakdown (meiosis I); Meta II, progesterone-treated oocytes
collected at metaphase II (cytostatic factor arrest). The same Western blot
was probed for actin as a loading control. B, immature oocytes were
injected with mRNA encoding FLAG-tagged p53 and then induced to mature by the
addition of progesterone. Expression of FLAG-p53 was checked with either
anti-FLAG (top) or anti-p53 (bottom) antibodies, before (Pro
I) or after (Meta II) maturation. Progression to Meta II was monitored by
testing histone H1 kinase activity of the extract (autoradiograph;
bottom). C, metaphase II-arrested oocyte extract was treated
(+) or not (â) with λ-phosphatase, as described under
âExperimental Procedures,â and the electrophoretic mobility of
endogenous p53 was checked by Western blot. D, resting prophase
I-arrested oocytes were injected with the Aurora A inhibitor C1368
(right) or the control vehicle (DMSO) alone (left). After 30
min, half of the oocytes were treated with progesterone and incubated until
reaching Meta II, whereas the other half was maintained in Pro I for the same
period of time. Both sets of oocytes were homogenized and subjected to Western
blotting analysis for the indicated proteins. Hatch marks on the
left indicate phosphorylated and dephoshorylated forms of the
proteins. Actin was monitored as a loading control.
FIGURE 2. Activation of Aurora A by TPX2 is required for full accumulation and
phosphorylation of p53. Immature oocytes were injected with control
morpholinos (left) or morpholinos against TPX2 (right) and
then stimulated to mature by the addition of progesterone. Expression and
electrophoretic behavior of the indicated proteins were checked by Western
blot with the indicated antibodies before (Pro I) or after (Meta II)
maturation. Actin was monitored as a loading control. Hatch marks on
the left indicate phosphorylated and dephoshorylated forms of the
proteins. For the phospho-Thr-295 blot, the asterisk denotes a
nonspecific band detected by the Cell Signaling antibody
(35).
FIGURE 3. p53 and Aurora A interact in vivo in Xenopus
oocytes. mRNA encoding 6Ã Myc-Aurora A was injected into resting
Stage VI Xenopus oocytes. Oocytes were stimulated by progesterone and
extracts prepared when oocytes reached Meta II. Myc-Aurora A was then
immunoprecipitated from the extract using anti-Myc antibody (lanes 3
and 5), and anti-mouse IgG was used as a control (lane 4).
Immunoprecipitation of Myc-Aurora A was monitored by Western blot with
anti-Myc/horseradish peroxidase antibody (bottom panel), whereas the
co-immunoprecipitation of endogenous p53 was monitored by Western blot
(IB) using anti-p53 antibody (top). Lanes 1 and
2 display levels of endogenous p53 and Myc-Aurora A in 10% of extract
input as judged by Western blot (IB). Actin was monitored as a
loading control.
FIGURE 4. p53 interacts with the catalytic domain of Aurora A. A,
Aurora A is composed of an N-terminal regulatory domain containing two Aurora
boxes and a C-terminal kinase catalytic domain. DNA plasmids containing
full-length Aurora A (Fl) as well as the N-terminal domain
(Nt) or the catalytic domain (Ct) of Aurora A were
transcribed and translated in vitro in the presence of
[35S]methionine as described under âExperimental
Procedures.â B, GST-tagged full-length p53 (GST-p53 WT
Fl) was used as a bait in an in vitro pull-down assay to assess
its interaction with the 35S-labeled Aurora A constructs described
in A (lanes 3â5). Radiolabeled Aurora A proteins
pulled down by the GST-tagged proteins were resolved by SDS-PAGE and revealed
by autoradiography. GST-Nt TPX2 was used as a positive control for the
pull-down of Aurora A (lane 2), whereas GST alone served as a
negative control (lane 1). C, smaller pieces of the Aurora
catalytic domain expressed by in vitro transcription/translation in
the presence of [35S]methionine were assessed in a manner similar
to that in B, using GST-p53 as bait. A plus sign indicates
an interaction between GST-p53 WT Fl and the Aurora A piece equivalent to or
stronger than the one observed with full-length Aurora A (B, lane 3),
whereas a minus sign indicates no significant interaction (equivalent
to or weaker than with GST alone; B, lane 1).
FIGURE 5. Aurora A interacts with the transactivation domain of p53 in
vitro. A, GST-p53 constructs were expressed containing the
transactivation domain (TA), the DNA binding domain (DNABD),
the oligomerization domain (OD), or a combination of these domains
(TADNABD, DNABDOD), with (+) or without the adjacent linker region,
as indicated. B, these constructs were bacterially expressed,
purified on glutathione-agarose beads, and used as bait in in vitro
pull-down assays in the presence of 35S-labeled Aurora A.
35S-Labeled Aurora A pulled down by the GST-tagged proteins was
resolved by SDS-PAGE and revealed by autoradiography.
FIGURE 6. Aurora A phosphorylates p53 on serines 129 and 190 in
vitro. A, three residues matching the minimum consensus site
for Aurora A (RX(S/T)) were found in Xenopus p53: Ser-129,
Thr-134, and Ser-190. Ser-283 and -284 is equivalent to human Ser-313, -314,
and -315, where Ser-315 has been reported to be phosphorylated by human Aurora
A in vitro (28,
29). Serine to alanine mutants
were created for all of these putative Aurora A sites. B,
phosphorylation by Aurora A of the p53 proteins mutated on the sites described
in A was tested in an in vitro kinase assay, as described
under âExperimental Procedures.â The graph represents the
percentage phosphorylation of these mutants compared with phosphorylation of
wild-type p53 at the same concentration. TA, transactivation domain;
DNABD, DNA binding domain; OD, oligomerization domain.
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