4. Pirh2 (p53-Induced Protein with a RING-H2 Domain) and p53 Regulation
Pirh2,
also called ring finger and CHY (conserved cysteine and histidine
involved in the binding of one zinc atom) zinc finger domain-containing 1
(Rchy1), is an E3 ubiquitin ligase that has three distinct zinc
fingers: the CHY-type, the CTCHY-type (located at the C-terminus of the
CHY-type), and a RING finger domain [102].
Pirh2 physically binds to p53 (residues 82–292) [102], which is distinct from Mdm2’s binding site (residues 1–51 as well as the C-terminus of p53) [103,104,105]. By binding to p53, Pirh2 promotes ubiquitination and decreases the level of p53 protein in cells.
In contrast, silencing of endogenous Pirh2 expression leads to elevation of p53. The Pirh2-dependent ubiquitination and degradation of p53 suppress p53 tumor suppressor function, including transactivation and growth inhibition [102]. A set of studies using NMR spectroscopy revealed that p53 binds to both the N- and C-terminal domains of Pirh2. The C-terminal domain of Pirh2 binds to the tetramerization domain (TET) of p53, which can be enhanced by a weak interaction between the N-terminus domain of Pirh2 and the p53 DNA binding domain. By binding to the TET domain, Pirh2 preferably ubiquitinates the tetrameric form of p53 in vitro and in vivo, suggesting that Pirh2 can effectively downregulate the transcriptional active form of p53 in the cell [106].
Pirh2 physically binds to p53 (residues 82–292) [102], which is distinct from Mdm2’s binding site (residues 1–51 as well as the C-terminus of p53) [103,104,105]. By binding to p53, Pirh2 promotes ubiquitination and decreases the level of p53 protein in cells.
In contrast, silencing of endogenous Pirh2 expression leads to elevation of p53. The Pirh2-dependent ubiquitination and degradation of p53 suppress p53 tumor suppressor function, including transactivation and growth inhibition [102]. A set of studies using NMR spectroscopy revealed that p53 binds to both the N- and C-terminal domains of Pirh2. The C-terminal domain of Pirh2 binds to the tetramerization domain (TET) of p53, which can be enhanced by a weak interaction between the N-terminus domain of Pirh2 and the p53 DNA binding domain. By binding to the TET domain, Pirh2 preferably ubiquitinates the tetrameric form of p53 in vitro and in vivo, suggesting that Pirh2 can effectively downregulate the transcriptional active form of p53 in the cell [106].
While
Pirh2 targets and ubiquitinates p53 independently of Mdm2, current
evidence indicates that Pirh2 and Mdm2 could simultaneously bind to a
single p53 protein and efficiently enhance its ubiquitination [46,107].
Like the Mdm2–p53 feedback mechanism, Pirh2 gene expression is
regulated by p53, indicating the presence of a feedback mechanism that
regulates p53 protein levels and functions [102].
It has been shown that Tip60 (Tat-interactive protein of 60 kDa) binds
to Pirh2 and increases the half-life of Pirh2 protein in a COS-7 (CV-1
in Origin with SV40 genes) fibroblast-like cell line. Further studies
will determine whether Tip60 can stabilize Pirh2 in cancer cells and if
Tip60 alteration can change the development of tumors both in vitro and
in vivo [108].
A
set of in vivo experiments showed the level of p53 proteins is mildly
affected in Pirh2-deficient mice. However, Pirh2 deficiency leads to
higher p53 levels in response to DNA damage in several tissues.
Whole-body irradiation of Pirh2−/− mice or irradiation of
Pirh2 knockout cells leads to elevation of p53, p53’s downstream target
proteins, and apoptosis, in comparison to WT mice [109].
In addition to p53, Hakem et al. showed Pirh2 binds and mediates the
ubiquitination and proteasome degradation of c-Myc, an oncoprotein
frequently overexpressed in various human cancers, including lung,
breast, and ovarian cancer [110]. Development of solid tumors such as sarcoma in Pirh2+/− and Pirh2−/− mice as well as double knockout p53−/− and Pirh2−/− mice indicates that Pirh2 can function as a tumor suppressor protein (summarized in Figure 2B).
While
Pirh2 directly targets and inhibits p53, it also binds to the
Axin–HIPK2 complex, which is involved in p53 activation through
phosphorylation of p53 at Ser 46. The inhibitory effect of Pirh2 on the
Axin–HIPK2 complex depends on the level of DNA damage. In cells treated
with sublethal doses of doxorubicin or ultra-violet (UV) radiation,
Pirh2 blocks Axin-induced p53 activation by competing with HIPK2 for
binding to Axin. With a lethal dose of UV or doxorubicin, cells
overexpress another regulatory protein called Tip60. Tip60 binds to Axin
and suppresses formation of the Pirh2–Axin complex. A generated
Axin–Tip60–HIPK2–p53 complex in the presence of a lethal dose of
genotoxic factors allows full activation of p53, resulting in
p53-dependent apoptosis [111].
Interestingly, Sho et al. reported that overexpression of TRIM29
enhances degradation and alters subcellular localization of Tip60,
resulting in the abrogation of p53 acetylation mediated by Tip60. By
suppressing p53, TRIM29 promotes cell growth, suppresses apoptosis, and
triggers transforming activity. Upregulation of TRIM29 by UV irradiation
suggests that TRIM29 can function as an oncogene by reversing
Tip60-dependent activation of p53 [112].
A
recent study completed by Yang et al. reported that a microRNA named
miR-100 can enhance ubiquitination and proteasomal degradation of p53
protein in poorly differentiated gastric cancer cells while
non-cancerous gastric cells remain intact. Their results indicate that
knocking down miR-100 reduces the expression of Pirh2, a key E3
ubiquitin ligase for p53 ubiquitination in gastric cancer cells,
resulting in stabilization and upregulation of p53 in gastric cancer
cells and activation of p53’s downstream apoptosis pathway. Further
studies by Yang et al showed Pirh2 is not a direct target for miR-100.
In fact, miR-100 suppresses expression of RNF114B, which functions as an
E3 ubiquitin ligase for Pirh2. RNF144B binds and ubiquitinates Pirh2
for proteasomal degradation. Taken together, the above studies indicate
that miR-100 can indirectly trigger ubiquitination and proteasomal
degradation of the p53 tumor suppressor protein in poorly differentiated
gastric cancer via the miR-100–RNF144B–Pirh2–p53 pathway in both in
vitro and in vivo models [113].
The ability of Pirh2 to negatively regulate the protein levels of p53 and c-Myc plus upregulation [114,115] and downregulation [109] of Pirh2 and its regulators in different types of solid tumors (summarized in Figure 2B)
suggests Pirh2 may have a dual function during tumorigenesis as an
oncoprotein and tumor suppressor protein in tissue-, stress-, and
grade-dependent manners. The subcellular localization of Pirh2 could be
another factor for its dual function, as reported for other proteins
such as p27Kip1 [116].
