Stress-mediated nuclear stabilization of p53 is regulated by ubiquitination and importin-alpha3 binding

The activity of p53 as an inducible transcription factor depends on its rapid nuclear stabilization after stress. However, surprisingly, mechanism(s) that regulate nuclear p53 accumulation are not well understood. The current model of stress-induced nuclear accumulation holds that a decrease in p53 nuclear export leads to its nuclear stabilization. We show here that regulated nuclear import of p53 also has a critical function. p53 import is mediated by binding to the importin-alpha3 adapter and is negatively regulated by ubiquitination. p53 harbors several nuclear localization signals (NLS), with the major NLS I located at amino-acids 305-322. We find that direct binding of p53 to importin-alpha3 depends on the positive charge contributed by lysine residues 319-321 within NLS I. The same lysines are also targets of MDM2-mediated ubiquitination. p53 ubiquitination occurs primarily in unstressed cells, but decreases dramatically after stress. Importin-alpha3 preferentially interacts with non-ubiquitinated p53. Thus, under normal growth conditions, ubiquitination of Lys 319-321 negatively regulates p53-importin-alpha3 binding, thereby restraining p53 import. Conversely, stress-induced accumulation of non-ubiquitinated p53 in the cytoplasm promotes interaction with importin-alpha3 and rapid import. In later phases of the stress response, blocked nuclear export also takes effect. We propose that p53 nuclear import defines an important novel level of regulation in the p53-mediated stress response.