P19 ARF stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2

The INK4a-ARF locus encodes two distinct tumor suppressors, p16 INK4a and p19 ARF . Whereas p16 INK4a restrains cell growth through preventing phosphorylation of the retinoblastoma protein, p19 ARF acts by attenuating Mdm2-mediated degradation of p53, thereby stabilizing p53. Recent data indicate that Mdm2 shuttles between the nucleus and the cytoplasm and that nucleo-cytoplasmic shuttling of Mdm2 is essential for Mdm2’s ability to promote p53 degradation. Therefore, Mdm2 must export p53 from the nucleus to the cytoplasm where it targets p53 for degradation. We show here that coexpression of p19 ARF blocks the nucleo-cytoplasmic shuttling of Mdm2. Moreover, subnuclear localization of Mdm2 changes from the nucleoplasm to the nucleolus in a shuttling time-dependent manner, whereas p19 ARF is exclusively located in the nucleolus. In heterokaryons containing Mdm2 and p19 ARF , the longer the Mdm2 shuttling is allowed, the more Mdm2 protein colocalizes with p19 ARF in the nucleolus, implying that Mdm2 moves from the nucleoplasm to the nucleolus and then associates with p19 ARF there. Furthermore, whether or not Mdm2 colocalizes with p19 ARF in the nucleolus, p19 ARF prevents Mdm2 shuttling. This observation suggests that Mdm2 might be exported through the nucleolus and p19 ARF could inhibit the nuclear export of Mdm2 by tethering Mdm2 in the nucleolus. Taken together, p19 ARF could stabilize p53 by inhibiting the nuclear export of Mdm2.