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Gaetan Pascreau Frank Eckerdt Andrea L. Lewellyn Claude Prigent James L. Maller 《The Journal of biological chemistry》2009,284(9):5497-5505
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.Aurora A is an oncogenic protein kinase that is active in mitosis and plays
important roles in spindle assembly and centrosome function
(1). Overexpression of either
human or Xenopus Aurora A transforms mammalian cells, but only when
the p53 pathway is altered
(2–4).
Aurora A is localized on centrosomes during mitosis, and overexpression of the
protein leads to centrosome amplification and aneuploidy
(2,
3,
5,
6), two likely contributors to
genomic instability (7,
8). Because of its oncogenic
potential and amplification in human tumors, considerable attention has been
focused on the mechanism of Aurora A activation in mitosis. Evidence from
several laboratories indicates that activation occurs as a result of
phosphorylation of a threonine residue in the T-loop of the kinase
(4,
9,
10). Purification of Aurora
A-activating activity from M phase Xenopus egg extracts led to an
apparent activation mechanism in which autophosphorylation at the T-loop is
stimulated by binding of the targeting protein for Xklp2 (TPX2)
(11–14).
On the other hand, it has been shown that Aurora A activity can be inhibited
by interaction with several proteins, including PP1 (protein phosphatase 1),
AIP (Aurora A kinase-interacting protein), and, more recently, p53
(9,
15–17).p53 is a well known tumor suppressor able to drive cell cycle arrest,
apoptosis, or senescence when DNA is damaged or cell integrity is threatened
(18,
19). In human cancers, the p53
gene is frequently deleted or mutated, leading to inactivation of p53
functions (20). p53 protein is
almost undetectable in “normal cells,” mainly due to its
instability. Indeed, during a normal cell cycle, p53 associates with Mdm2 in
the nucleus and thereafter undergoes nuclear exclusion, allowing its
ubiquitination and subsequent degradation
(21). In cells under stress,
p53 is stabilized through the disruption of its interaction with Mdm2
(21), leading to p53
accumulation in the nucleus and triggering different responses, as described
above.Although p53 has mostly been characterized as a nuclear protein, it has
also been shown to localize on centrosomes
(22–24)
and regulate centrosome duplication
(23,
24). Centrosomes are believed
to act as scaffolds that concentrate many regulatory molecules involved in
signal transduction, including multiple protein kinases
(25). Thus, centrosomal
localization of p53 might be important for its own regulation by
phosphorylation/dephosphorylation, and one of its regulators could be the
mitotic kinase Aurora A. Indeed, phenotypes associated with the misexpression
of these two proteins are very similar. For example, overexpression of Aurora
A kinase leads to centrosome amplification, aneuploidy, and tumorigenesis, and
the same effects are often observed after down-regulation of p53
transactivation activity or deletion/mutation of its gene
(26,
27).Several recent studies performed in mammalian models show interplay between
p53 and Aurora A, with each protein having the ability to inhibit the other,
depending on the stage of the cell cycle and the stress level of the cell
(17,
28,
29). These studies reported
that p53 is a substrate of Aurora A, and serines 215 and 315 were demonstrated
to be the two major Aurora A phosphorylation sites in human p53 in
vitro and in vivo. Phosphorylation of Ser-215 within the DNA
binding domain of human p53 inhibited both p53 DNA binding and transactivation
activities (29). Recently, our
group showed that Xenopus p53 is able to inhibit Aurora A kinase
activity in vitro, but this inhibitory effect can be suppressed by
prior binding of Aurora A to TPX2
(9). Contrary to somatic cells,
where p53 is nuclear, unstable, and expressed at a very low level, p53 is
highly expressed in the cytoplasm of Xenopus oocytes and stable until
later stages of development
(30,
31). The high concentration of
both p53 and Aurora A in the oocyte provided a suitable basis for
investigating p53-Aurora A interaction and also evaluating Xenopus
p53 as a substrate of Aurora A. 相似文献
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Mario A. Pennella Yue Liu Jennifer L. Woo Chongwoo A. Kim Arnold J. Berk 《Journal of virology》2010,84(23):12210-12225
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Identification of rCop-1, a New Member of the CCN Protein Family,as a Negative Regulator for Cell Transformation
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Rong Zhang Lidia Averboukh Weimin Zhu Hong Zhang Hakryul Jo Peter J. Dempsey Robert J. Coffey Arthur B. Pardee Peng Liang 《Molecular and cellular biology》1998,18(10):6131-6141
By using a model system for cell transformation mediated by the cooperation of the activated H-ras oncogene and the inactivated p53 tumor suppressor gene, rCop-1 was identified by mRNA differential display as a gene whose expression became lost after cell transformation. Homology analysis indicates that rCop-1 belongs to an emerging cysteine-rich growth regulator family called CCN, which includes connective-tissue growth factor, CYR61, CEF10 (v-src inducible), and the product of the nov proto-oncogene. Unlike the other members of the CCN gene family, rCop-1 is not an immediate-early gene, it lacks the conserved C-terminal domain which was shown to confer both growth-stimulating and heparin-binding activities, and its expression is lost in cells transformed by a variety of mechanisms. Ectopic expression of rCop-1 by retroviral gene transfers led to cell death in a transformation-specific manner. These results suggest that rCop-1 represents a new class of CCN family proteins that have functions opposing those of the previously identified members.Oncogenic conversion of a normal cell into a tumor cell requires multiple genetic alterations (12). Of particular interest is the fact that mutations in both ras oncogenes (3) and the p53 tumor suppressor gene cooperate in transformation of mammalian cells (11). Mutations in both ras and the p53 gene were also found at high frequencies in a variety of human cancers, including those of the colon, lung, and pancreas (2, 18). It has been proposed that both p53 and Ras function, whether directly or through other signaling molecules, to control expression of genes that are important for cell growth and differentiation (13, 17, 37). To this end, several ras target genes (10) and p53 target genes, including those encoding p21/CIP1/WAF1, an inhibitor of G1 cyclin-dependent kinase (9); Mdm-2, a negative regulator of p53 (1); GADD45, a protein involved in DNA repair (36); and Bax, which promotes apoptosis (28), have been identified. Most of these genes, except p21/CIP1/WAF1, which was cloned by subtractive hybridization, were identified by the candidate gene hypothesis. Recently, more p53 target genes have been isolated by the differential display technique, including those coding for cyclin G (31); MAP4, a microtubule-associated protein negatively regulated by p53 (29); and PAG608, a novel nuclear zinc finger protein whose overexpression promotes apoptosis (14). Functional characterizations of these genes have shed light on the role of p53 in cell cycle control and apoptosis. However, genes that mediate tumor suppression activity by p53 remain elusive.The fact that neither the inactivation of p53 nor the activation of Ras alone is able to transform primary mammalian cells (34), whereas both mutations together can do so, suggests that genes regulated by p53 and Ras cooperate in upsetting normal cell growth control cells (11). Using differential display (22), we set out to identify genes whose expression is altered by both mutant ras and p53 by comparing the mRNA expression profiles of normal rat embryo fibroblasts (REFs) and their derivatives transformed by either a constitutively inactivated or a temperature-sensitive mutant p53 in cooperation with the activated H-ras oncogene (11, 27). In this report we describe the identification and give a functional characterization of rCop-1, a gene whose expression is abolished by cell transformation. By sequence homology, rCop-1 was found to belong to an emerging cysteine-rich growth regulator family called CCN (which stands for connective-tissue growth factor [CTGF], CEF10/Cyr61, and Nov) (4). Here we show that rCop-1 may represent a novel class of CCN family proteins based on its unique cell cycle expression pattern, its lack of the C-terminal (CT) domain conserved in all CCN proteins, its loss of expression in all transformed cells analyzed, and its ability to confer cytotoxicity to the transformed cells. 相似文献