| pmid | 11080164 | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| title | Regulation of p53 activity in nuclear bodies by a specific PML isoform. | ||||||||||
| authors |
|
||||||||||
| journal | EMBO J | ||||||||||
| year | 2000 | ||||||||||
| full_text_available | true | ||||||||||
| full_text_extraction_method | html | ||||||||||
| pmcid | PMC305840 | ||||||||||
| doi | 10.1093/emboj/19.22.6185 |
Authors: Fogal V, Gostissa M, Sandy P, Zacchi P, Sternsdorf T, Jensen K, Pandolfi PP, Will H, Schneider C, Del Sal G Journal: EMBO J (2000) DOI: 10.1093/emboj/19.22.6185 PMC: PMC305840
- EMBO J. 2000 Nov 15;19(22):6185-95. doi: 10.1093/emboj/19.22.6185.
Regulation of p53 activity in nuclear bodies by a specific PML isoform.
Fogal V(1), Gostissa M, Sandy P, Zacchi P, Sternsdorf T, Jensen K, Pandolfi PP, Will H, Schneider C, Del Sal G.
Author information: (1)Laboratorio Nazionale CIB, Area Science Park, Padriciano 99, 34012 Trieste, Italy.
Covalent modification of the promyelocytic leukaemia protein (PML) by SUMO-1 is a prerequisite for the assembly of nuclear bodies (NBs), subnuclear structures disrupted in various human diseases and linked to transcriptional and growth control. Here we demonstrate that p53 is recruited into NBs by a specific PML isoform (PML3) or by coexpression of SUMO-1 and hUbc9. NB targeting depends on the direct association of p53, through its core domain, with a C-terminal region of PML3. The relocalization of p53 into NBs enhances p53 transactivation in a promoter-specific manner and affects cell survival. Our results indicate the existence of a cross-talk between PML- and p53-dependent growth suppression pathways, implying an important role for NBs and their resident proteins as modulators of p53 functions.
DOI: 10.1093/emboj/19.22.6185 PMCID: PMC305840 PMID: 11080164 [Indexed for MEDLINE]
Abstract
Covalent modification of the promyelocytic leukaemia protein (PML) by SUMO-1 is a prerequisite for the assembly of nuclear bodies (NBs), subnuclear structures disrupted in various human diseases and linked to transcriptional and growth control. Here we demonstrate that p53 is recruited into NBs by a specific PML isoform (PML3) or by coexpression of SUMO-1 and hUbc9. NB targeting depends on the direct association of p53, through its core domain, with a C-terminal region of PML3. The relocalization of p53 into NBs enhances p53 transactivation in a promoter-specific manner and affects cell survival. Our results indicate the existence of a cross-talk between PML- and p53-dependent growth suppression pathways, implying an important role for NBs and their resident proteins as modulators of p53 functions.
Introduction
The tumour suppressor protein p53 is a key element in the control of human cell growth and differentiation and plays an important role in the maintenance of genome integrity ( Ko and Prives, 1996 ; Levine, 1997 ). Most of its functions are exerted by transcriptional activation of genes involved in cell cycle, apoptosis and DNA repair ( Ko and Prives, 1996 ). Under various stress conditions p53 becomes activated by post-translational modifications that affect its conformation and binding to several proteins, resulting in its stabilization and increased DNA-binding potential ( Giaccia and Kastan, 1998 ).
Another way to modulate p53 activity involves changes in its subcellular distribution. Certain tumours constitutively accumulate wild-type p53, which is functionally inactive because it is sequestered in the cytoplasm ( Moll et al ., 1996 ; Ostermayer et al ., 1996 ). In addition, treatment of human primary cells with Leptomycin B, a drug that specifically blocks nuclear export, induces the relocalization of p53 into punctate subnuclear structures, reminiscent of the so-called nuclear bodies (NBs) ( Lain et al ., 1999 ). A similar p53 distribution has also been observed upon coexpression with Mdm2 and ARF and this relocalization has been correlated with ARF-mediated inhibition of Mdm2–p53 nuclear export ( Zhang and Xiong, 1999 ).
NBs are cell cycle-regulated, matrix-associated subnuclear structures that appear as punctate foci in the interphase nuclei ( Seeler and Dejean, 1999 ). The structural integrity of these large multiprotein complexes appears to be important for normal cell growth and development, since in some human diseases, like acute promyelocytic leukaemia (APL) and spinocerebellar ataxia type I (SCA1), disruption of NBs leads to malignancy or neurodegenerative disorder, respectively ( Hodges et al ., 1998 ). Moreover, these structures are targeted and subsequently destroyed by numerous immediate early viral proteins ( Maul, 1998 ).
Promyelocytic leukaemia protein (PML), the most prominent component of the NBs (also referred to as PML oncogenic domains, PODs), was first identified in APL patients, where, as a result of a reciprocal translocation event, it is fused to the retinoic acid receptor α (RARα) ( de The et al ., 1991 ; Kakizuka et al ., 1991 ). The fundamental role of PML in directing the complex protein–protein interactions that mediate PODs formation is underlined by recent findings that the organization of several NB-associated components is impaired in PML –/– cells ( Zhong et al ., 2000a ). The possible role of PML and NBs in control of cell growth is suggested by studies on PML knockout mice, revealing tumour suppressor and pro-apoptotic functions for PML ( Quignon et al ., 1998 ; Wang et al ., 1998b ). In APL cells, the expression of the PML–RARα fusion compromises the integrity of PODs, while treatment with therapeutic agents, such as arsenic trioxide (As 2 O 3 ) or interferons, leads to the normalization of NB pattern and simultaneously induces differentiation or apoptosis of the malignant cells ( Lavau et al ., 1995 ; Muller et al ., 1998b ).
Other evidence implies NBs in the control of gene expression ( Zhong et al ., 2000b ). The reported interaction with pRb ( Alcalay et al ., 1998 ) and the direct binding to the histone acetyltransferase CBP ( LaMorte et al ., 1998 ) suggest a relevant role for PML in the regulation of transcription.
PML, Sp100 and probably other NB-resident proteins are post-translationally modified by SUMO-1, a small ubiquitin-related modifier ( Sternsdorf et al ., 1997 ), and recently we and others reported that p53 is also conjugated to SUMO-1 ( Gostissa et al ., 1999 ; Rodriguez et al ., 1999 ; Muller et al ., 2000 ). It has been demonstrated that sumolation of PML is absolutely required for NB formation and for recruitment of other factors to these structures ( Ishov et al ., 1999 ; Zhong et al ., 2000a ).
Here we provide evidence that PML recruits p53 into NBs. The relocalization of p53 depends on its direct association with a specific PML splice variant, PML3. Moreover, we demonstrate that binding to PML3 and NB targeting of p53 result in increased transcriptional activation of a p53-regulated pro-apoptotic gene and affect cell survival.
Discussion
A possible link between NBs and p53 has been suggested recently ( Gostissa et al ., 1999 ) by the finding that similarly to PML and Sp100, known components of NBs, p53 is covalently conjugated to the small ubiquitin-related modifier SUMO-1. In this study we demonstrated that PML3 mediates the recruitment of p53 into these structures (Figure 1 B). Overexpression of SUMO-1 and its conjugating enzyme, hUbc9, induced a similar relocalization of p53 that, however, was not dependent on the direct sumolation of the protein, since the conjugation-deficient mutant K386R was efficiently targeted to NBs as well (Figure 1 A). A recent report demonstrating that conjugation of SUMO-1 to PML is a prerequisite for its ability to form NBs and consequently to recruit other proteins into these structures ( Zhong et al ., 2000a ) let us hypothesize that enforced expression of SUMO-1 and hUbc9 results in the augmented assembly of NBs where p53 is also targeted. That this is likely to be the case is further supported by the finding that treatment of UV-irradiated cells with As 2 O 3 , a known inducer of PML sumolation and used for therapy of APL patients ( Chen et al ., 1997 ; Muller et al ., 1998a ), led to recruitment of endogenous p53 into PML3-containing NBs (Figure 1 C).
A detailed microinjection and coimmunoprecipitation analysis with a set of p53 deletions demonstrated that the observed relocalization of p53 into NBs is dependent on a direct association through its core domain with PML3 (Figure 2 ). Interestingly, amino acids from 355 to 363 of p53 play a negative role in NB targeting (Figure 2 B). The region comprising the last 40 amino acids of p53 is a well known target for various post-translational modifications and serves as a surface for intense protein–protein interactions that may modulate p53 functions ( Ko and Prives, 1996 ; Giaccia and Kastan, 1998 ). Therefore, it is tempting to speculate that the segment between residues 355 and 363 is a binding site for a factor that removes or, more probably, keeps p53 out from the NBs. Alternatively, the presence of a serine at position 362 raises the possibility that phosphorylation of this residue may have the same effect.
PML exists in numerous alternatively spliced variants that mostly differ in their C-terminal sequences ( de The et al ., 1991 ; Fagioli et al ., 1992 ). All of them described so far contain the RING finger, B-box and coiled-coil motifs (RBCC) and a nuclear localization signal, which together have been shown to be required and sufficient to target PML into NBs. Nevertheless, the available data do not exclude the possibility that the different PML proteins may interact with diverse cellular partners, thus affecting NB composition and functions. Supporting this interpretation, here we provided evidence that the interaction between p53 and PML is specific for the PML3 variant, since no significant binding was detected with the PML-L isoform and consequently p53 was not found in PML-L NBs (Figure 3 ). These findings are, to our knowledge, the first data on functional differences between the various PML proteins and raise the possibility that the complex splicing pattern of PML represents a cellular mechanism generating alternative binding interfaces for a variety of factors. In addition to splicing, enhanced SUMO-1 modification of PML may provide another level of complexity either by directly affecting these interactions or by enhancing the ability of PML to form NBs.
The function of the NBs is not yet fully understood; however, their possible involvement in growth suppression has been postulated since the discovery that leukaemia cells from APL patients have an aberrant nuclear dot organization ( Dyck et al ., 1994 ; Koken et al ., 1994 ; Weis et al ., 1994 ). Moreover, fibroblasts derived from PML –/– mice show an increased proportion of cells in S phase ( Wang et al ., 1998a ) and these knockout mice are less sensitive to lethal doses of gamma irradiation or Fas antibody treatment, proposing a pro-apoptotic role for PML ( Wang et al ., 1998b ). Several additional correlative results suggested that PML is a critical component for death induction, probably due to its activity in recruiting apoptotic proteins into NBs ( Quignon et al ., 1998 ). Since the role of p53 in induction of apoptosis and growth suppression is well established ( Sionov and Haupt, 1999 ), we hypothesized that recruitment of p53 into NBs may contribute to PML3-dependent growth inhibition. In line with this idea, we consistently observed a significant reduction of cell survival when PML3 was introduced into the p53-expressing U2OS cells but not in the p53-null MG63 cell line (Figure 4 A). Moreover, the effect observed with the p53 1–355 deletion mutant that shows complete relocalization into NBs suggests that recruitment of p53 into NBs is instrumental for PML3-mediated reduction of cell survival (Figure 4 B).
The apoptotic function of p53 has been shown to involve transcription-dependent activities as well as its capability to associate with other cellular factors ( Sionov and Haupt, 1999 ). Here we demonstrated that PML3 selectively increases the p53-dependent activation of the PIG3 promoter (Figure 5 ). PIG3 was originally isolated as one of several p53-regulated genes with the potential to induce or mimic oxidative stress ( Polyak et al ., 1997 ). The evidence that reactive oxygen species are involved in apoptosis and in cell aging ( Migliaccio et al ., 1999 ) suggests that PML3-dependent relocalization of transcriptionally active p53 into NBs could contribute to these physiological processes. Furthermore, the lack of p53 activation observed with PML3S – , which still binds to p53 but does not organize the NB structures, definitively proves the importance of p53 recruitment into NBs for modulation of its functions.
Which mechanisms control this promoter specificity? The interaction of p53 with PML3 and other NB-targeted factors involved in transcriptional control, like p300/CBP, which is also a p53 coactivator ( Avantaggiati et al ., 1997 ), can regulate the recognition of p53 target genes.
In addition, distinct post-translational modifications taking place in NBs could also contribute to activate p53 transcriptional functions in a promoter-specific manner. Interestingly, it was recently shown that expression of PML3 induces phosphorylation of p53 on serine 15 and enhances its acetylation ( Ferbeyre et al ., 2000 ; Pearson et al ., 2000 ). In this context the p53 1–355 protein, which exhibits an increased PML3-dependent ability to activate the PIG3 promoter, could mimic a special conformational change of the full-length protein that is a result of a post-translational modification upon relocalization to NBs. Our subcellular fractionation experiments as well as our immunofluorescence data strongly argue for the functional modification of p53 in the NBs and not in the nucleoplasm. Whether this is true for all types of cells remains to be investigated, since NB composition may vary among the various cell types and may depend on the differentiation state of the cells. The latter is consistent with very recent findings demonstrating that PML is induced by oncogenic ras and promotes premature senescence in fibroblasts in a p53-dependent manner ( Ferbeyre et al ., 2000 ; Pearson et al ., 2000 ), while in transformed cells PML may activate p53 apoptotic pathways.
Finally, since it has been suggested that NBs are also involved in chromatin remodelling ( Seeler et al ., 1998 ), access to a particular promoter region may depend on the association between transcription factors and non-histone chromosomal proteins.
Of note, hDaxx, a protein involved in Fas-mediated apoptosis, has recently been found to bind to PML and exert its apoptotic function in NBs ( Ishov et al ., 1999 ; Torii et al ., 1999 ). The physical interaction between hDaxx and p53 (M.Gostissa and G.Del Sal, unpublished results) may add another level of complexity in the role of cell death control of NBs.
Approximately 5–15% of p53 mutations occur in the C-terminal domain and result in truncated proteins that, although transcriptionally active, are defective in apoptosis induction ( Zhou et al ., 1999 ). It is tempting to speculate that in cells expressing such p53 mutants, apoptosis can still be induced by stimulating the relocalization of p53 into PML-containing NBs following treatment with agents that modulate the expression (or the sumolation) of PML3.
Control of cell death and differentiation may proceed through pathways involving either PML or p53 and, as demonstrated in this work, at least some of them are converging. Relocalization of various factors involved in transcriptional and growth control into NBs may allow the formation of specific protein–protein interactions and lead to the transactivation of particular promoters (Figure 8 ). The knowledge of integration and cross-talks between different apoptotic pathways could therefore allow the implementation of methods for blocking the transformation process and the design of novel therapeutic strategies.