Energy-dependent nucleolar localization of p53 in vitro requires two discrete regions within the p53 carboxyl terminus.

The p53 tumor suppressor is a nucleocytoplasmic shuttling protein that is found predominantly in the nucleus of cells. In addition to mutation, abnormal p53 cellular localization is one of the mechanisms that inactivate p53 function. To further understand features of p53 that contribute to the regulation of its trafficking within the cell, we analysed the subnuclear localization of wild-type and mutant p53 in human cells that were either permeabilized with detergent or treated with the proteasome inhibitor MG132. We, here, show that either endogenously expressed or exogenously added p53 protein localizes to the nucleolus in detergent-permeabilized cells in a concentration- and ATP hydrolysis-dependent manner. Two discrete regions within the carboxyl terminus of p53 are essential for nucleolar localization in permeabilized cells. Similarly, localization of p53 to the nucleolus after proteasome inhibition in unpermeabilized cells requires sequences within the carboxyl terminus of p53. Interestingly, genotoxic stress markedly decreases the association of p53 with the nucleolus, and phosphorylation of p53 at S392, a site that is modified by such stress, partially impairs its nucleolar localization. The possible significance of these findings is discussed.

Correction: Energy-dependent nucleolar localization of p53 in vitro requires two discrete regions within the p53 carboxyl terminus.

Following the publication of this article the authors noted that two images were duplicated in Figure 2B. The corrected figure 2B is below. The authors wish to apologize for any inconvenience caused.

p53 down-regulates CHK1 through p21 and the retinoblastoma protein.

Both fission yeast and mammalian cells require the function of the checkpoint kinase CHK1 for G2 arrest after DNA damage. The tumor suppressor p53, a well-studied stress response factor, has also been shown to play a role in DNA damage G2 arrest, although in a manner that is probably independent of CHK1. p53, however, can be phosphorylated and regulated by both CHK1 as well as another checkpoint kinase, hCds1 (also called CHK2). It was therefore of interest to determine whether reciprocally, p53 affects either CHK1 or CHK2. We found that induction of p53 either by diverse stress signals or ectopically using a tetracycline-regulated promoter causes a marked reduction in CHK1 protein levels. CHK1 downregulation by p53 occurs as a result of reduced CHK1 RNA accumulation, indicating that repression occurs at the level of transcription. Repression of CHK1 by p53 requires p21, since p21 alone is sufficient for this to occur and cells lacking p21 cannot downregulate CHK1. Interestingly, pRB is also required for CHK1 downregulation, suggesting the possible involvement of E2F-dependent transcription in the regulation of CHK1. Our results identify a new repression target of p53 and suggest that p53 and CHK1 play interdependent and complementary roles in regulating both the arrest and resumption of G2 after DNA damage.