In order to safeguard the genome against mutation or DNA loss, cells respond to stress by transiently delaying proliferation and mobilizing repair factors. We previously demonstrated that, during heat shock, nucleolin relocalizes to the nucleoplasm, where it sequesters RPA in nuclear foci away from sites of ongoing DNA synthesis (10
). Additional repair-related functions for nucleolin are suggested by biochemical data revealing that nucleolin stimulates DNA strand annealing (17
). In this study, we demonstrate that nucleolin mobilization occurs in a p53-dependent manner in response to γ-irradiation and CPT treatment. Although nucleolin mobilization is closely tied to p53 activation and nucleolin-p53 complex formation, it is independent of the transcriptional transactivation function of p53. Thus, unlike the G1
checkpoint, the role of p53 in nucleolin mobilization does not require the induction of p53-responsive genes, and therefore p53 exerts its effects through physical interaction with other factors. Interestingly, topoisomerase I has also been recently found to undergo p53-dependent relocalization from the nucleolus to the nucleoplasm following DNA damage (30
Our data indicate that nucleolin-p53 complex formation is required for nucleolin mobilization, but the mechanism of relocalization remains unclear. Our favored explanation is that, like other factors, nucleolar nucleolin exists in dynamic equilibrium with the nucleoplasm (37
). Under normal conditions, in which nuclear p53 levels are low, nucleolin exists only transiently in the nucleoplasm. Upon activation of p53 by stress, the increased level of this activated p53 promotes complex formation with nucleolin and thus shifts the majority of the nucleolin pool toward nucleoplasmic localization, allowing interaction with RPA. Alternatively, it has been proposed that a pool of p53 exists in the nucleolus (3
). Although this is less likely, it is possible that this p53 actively exports nucleolin to the nucleoplasm, where it becomes stably associated with a nucleoplasmic structure (see reference 10
), perhaps matrix attachment region (MAR) elements on chromosomal DNA (11
). In either case, the nucleolin-p53 interaction is likely transient, because a significantly larger fraction of the nucleolin pool is seen to relocalize relative to the fraction bound to p53.
The C-terminal 30 residues of p53 are required for nucleolin mobilization and for nucleolin-p53 complex formation in vivo and in vitro. This basic region has been termed the “regulatory” domain and is involved in a variety of protein-protein interactions, including interactions with the TFIIH subunits XPB and XPD factors involved in nucleotide excision repair, the Cockayne syndrome group B protein (CSB) also involved in DNA damage repair (31
), and the WRN gene product which is found mutated in the progeroid Werner syndrome (2
). The regulatory domain has been implicated in apoptotic functions of p53 and can also modulate its sequence-specific DNA-binding activity (31
). Because nucleolin appears to interact with the regulatory domain of p53, nucleolin thus has the potential to alter the ability of p53 to bind its DNA recognition elements and hence may modulate the induction of p53-responsive genes.
The nucleolin response is stress selective in that it is observed only under certain traumatic conditions but not under others. Interestingly, p53 activation occurs following UV irradiation (Fig. ) and HU treatment (data not shown), yet nucleolin does not mobilize in response to these stresses. Since it is accepted that different cellular stresses induce discrete signaling cascades, which in turn activate distinctive, sometimes overlapping responses (1
), it is likely that nucleolin is mobilized by only a subset of these stress-related pathways. More specifically, one can argue that nucleolin only associates with p53 in a particular posttranslational modification state. Clearly, the specific p53 phosphorylation profile is a regulatory determinant controlling its interaction with other factors, including the p53 antagonist Mdm2 (42
) and the transcription factors TFIID and CBP/p300 (27
). The nucleolin-p53 interaction does not appear to be directly regulated by the phosphorylation state of Ser15, as nucleolin is still mobilized in AT cells (in which the ATM kinase is defective) following γ-irradiation (data not shown). Because the C-terminal region contains various phosphorylation and acetylation sites, some of which are modulated by stress, it is perhaps more likely that the modification of this region will be found to alter nucleolin-p53 complex formation. Conversely, mammalian nucleolin is also a target of various kinases including casein kinase II, the cyclin B-cdc2 complex, and protein kinase C ζ (15
). Although little is known about the effect of stress on nucleolin phosphorylation, it would not be surprising to find that the phosphorylation state of nucleolin modulates both its cellular localization and its interaction with p53.
The unique compartmentalization of the nucleolus allows the cell to both sequester and rapidly mobilize critical factors involved in the regulation of DNA metabolism in response to stress (49
). Prominent examples include the sequestration of the Mdm2 oncoprotein by the nucleolar ARF protein, facilitating p53 stabilization (32
), and the relocalization of the human homologue of the Schizosaccharomyces pombe
Rad17 protein, a key damage response factor, from the nucleolus to the nucleoplasm following UV irradiation (6
). Combined with our findings, these data add to the growing body of evidence indicating that the nucleolus, along with its primary role in supporting ribosome biogenesis, is also intimately tied to the cellular stress response.