The current study identifies USP10 as a p53 deubiquitinase. Unlike HAUSP, a previously identified deubiquitinase for p53 and Mdm2, USP10 only interacts with p53, but not Mdm2. In addition, while the majority of HAUSP localizes in the nucleus, USP10 seems to be predominantly cytoplasmic in unstressed cells. Although ubiquitination of p53 has long been known to induce its nuclear export, whether or not the exported p53 could be recycled back to the nucleus was unknown. Here we demonstrate that USP10 is able to reverse Mdm2-mediated p53 nuclear export. This is the first time that a deubiquitinase has been demonstrated to deubiquitinate cytoplasmic p53. Therefore, Mdm2 induced p53 ubiquitination could be counteracted at two locations: either by HAUSP in the nucleus or USP10 in the cytoplasm.
We also show that following genotoxic stress, a fraction of USP10 translocates to the nucleus, and this translocation is required for the stabilization and activation of p53. Mechanistically, the phosphorylation of Thr42 and Ser337 of USP10 by ATM is required for USP10 translocation and stabilization. Currently, we do not know how the phosphorylation of USP10 affects its stability and localization. Neither do we know the relationship between USP10 translocation and stabilization. USP10 contains a potential nuclear localization sequence (NLS) and several Leu/Ile rich regions, which are potential nuclear export signals (NES). Treatment of cells with LMB induced USP10 accumulation in the nucleus, suggesting that USP10 is actively exported out of nucleus in unstressed cells. It is possible that USP10 phosphorylation by ATM hinders USP10 nuclear export and shields it from degradation in the cytoplasm. We will examine this further in the future.
There are several mechanisms have been shown to stabilize and activate p53 following DNA damage. The phosphorylation of p53 by ATM or Chk2 has been shown to inhibit Mdm2 binding (Banin et al., 1998
; Chehab et al., 2000
; Shieh et al., 2000
; Shieh et al., 1997
). Furthermore, ATM-dependent phosphorylation of Mdm2 and Mdmx could destabilize these proteins and compromise their activity (Khosravi et al., 1999
; Maya et al., 2001
; Meulmeester et al., 2005
; Pereg et al., 2005
; Stommel and Wahl, 2004
). Our results that USP10 is important for p53 stabilization and activation following DNA damage provide another regulatory route. Our results suggest that p53 is still ubiquitinated by residual Mdm2/Mdmx activity following DNA damage (Figure S3A
), and the nuclear translocation of USP10 could further boost p53 activation. Since p53 is such an important factor in the determination of cell fate, such multiple regulatory mechanisms ensure optimal stress response.
Through its action toward p53, USP10 could function as a tumor suppressor. We show downregulation of USP10 in a high percentage of renal cell carcinoma samples. Furthermore, we provide evidence that USP10 inhibits cancer cell proliferation in cells with WT p53. These results suggest that USP10 could suppress tumorigenesis. Since very low percentage of RCC cases are found to have p53 mutations (Soussi et al., 2000
) and IARC p53 Database: www.IARC.fr/p53/index.html
), decreased expression of USP10 could be another mechanism to inhibit p53 function in RCC.
Paradoxically, USP10 could promote cancer cell proliferation in mutant p53 background. We found USP10 is overexpressed in RCC cells with mutant p53. Recent papers also suggest that increased USP10 expression in some breast cancer and glioblastoma samples (Deng et al., 2007
; Grunda et al., 2006
), although it is not clear whether p53 is mutated in these samples. One mystery in the p53 field of research is that mutant p53 is usually overexpressed in tumor samples, however, the mechanism responsible for this phenotype is not clear. Although it was thought that mutant p53 is intrinsically stable, a recent paper suggest that downregulation of Mdm2 might be one mechanism (Terzian et al., 2008
). In contrast to mice with WT p53 background, loss of the Mdm2 in mutant p53 background increases p53 expression and promotes tumorigenesis. The explanation is that mutant p53 has a gain of function, and loss of Mdm2 increases mutant p53 levels and further promotes tumor development. Consistent with this study, we show that increased USP10 expression in mutant p53 background increases p53 levels and promotes cancer cell proliferation, while downregulation of USP10 inhibits cancer cell growth. Therefore, increased expression of USP10 could be another mechanism responsible for increased mutant p53 expression in human cancers. Future studies, for example generating USP10 knockout mice, are needed to establish the physiological role of USP10 in tumorigenesis.