Abstract
The biological mechanisms for maintaining the basal level of p53 in normal cells require nuclear exclusion and cytoplasmic degradation. Here, we showed that Jab1 facilitates p53 nuclear exclusion and its subsequent degradation in coordination with Hdm2. p53 was excluded from the nucleus in the presence of Jab1; this exclusion was prevented by leptomycin B treatment. Nuclear export of p53 was accompanied by a decrease in the levels of p53, as well as of its target proteins, which include p21 and Bax. Domain analyses of Jab1 showed that the N-terminal domain, 1-110, was capable of inducing cytoplasmic translocation of p53. Furthermore, 110-191 was required to facilitate the degradation of p53. Neither of these mutants incorporated into the CSN complex, indicating that Jab1 could affect the levels of p53 independent of intact CSN complex. Conversely, Jab1 was incapable of translocating and degrading two p53 mutants, W23S and 6KR, neither of which could be modified by Hdm2. Moreover, Jab1 did not affect the cellular localization or protein levels of p53 in p53 and Hdm2 double-null mouse embryo fibroblasts. We further observed that the ablation of endogenous Jab1 by small interfering RNA prevented Hdm2-mediated p53 nuclear exclusion. Under stressed conditions, which could sequester Hdm2 in its inactive state, Jab1 did not affect p53. Our studies implicate that Jab1 is required to remove post-translationally modified p53 and provide a novel target for p53-related cancer therapies.
Highlights
Under normal conditions, p53 is maintained at low steady-state levels by its rapid turnover, which is mediated by ubiquitination followed by proteasome-dependent proteolysis [2, 4, 5]
Jun activation domain-binding protein 1 (Jab1) Induces the Nuclear Exclusion and Degradation of p53—Because Jab1 is known to be a nuclear/cytoplasmic shuttle protein and interacts with p53, the effects of Jab1 on p53 cytoplasmic localization were first examined in H1299 and U2OS cells [17]
When leptomycin B (LMB), a CRM1 inhibitor, was added, it blocked Jab1-facilitated p53 nuclear export, which implied that this process requires the CRM1-dependent pathway (Fig. 1A, panels 9 –12) [11]
Summary
P53 is maintained at low steady-state levels by its rapid turnover, which is mediated by ubiquitination followed by proteasome-dependent proteolysis [2, 4, 5]. When leptomycin B (LMB), a CRM1 inhibitor, was added, it blocked Jab1-facilitated p53 nuclear export, which implied that this process requires the CRM1-dependent pathway (Fig. 1A, panels 9 –12) [11]. Immunofluorescent and biochemical analyses showed that Jab1 was incapable of translocating or degrading W23S and 6KR, which suggested that the post-translational modification of p53 by Hdm2 might be required in these processes (Fig. 4, B and C).
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