Pascreau G et al. (2009), Phosphorylation of p53 is regulated by TPX2-Aur...

XB-ART-39010

J Biol Chem 2009 Feb 27;2849:5497-505. doi: 10.1074/jbc.M805959200.

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Phosphorylation of p53 is regulated by TPX2-Aurora A in xenopus oocytes.

Pascreau G, Eckerdt F, Lewellyn AL, Prigent C, Maller JL.

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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.

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Species referenced: Xenopus laevis
Genes referenced: actl6a aurka myc tp53 tpx2
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	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 (–) with λ-phosphatase, as described under “Experimental Procedures,” 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 6× 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 “Experimental Procedures.” 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 3–5). 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 “Experimental Procedures.” 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.

References [+] :

Amariglio, A functional analysis of p53 during early development of Xenopus laevis. 1997, Pubmed, Xenbase