p53-dependent functions of Pirh2. Leng et al.(9) were the first to show that Pirh2 can directly bind to p53 in vitro and in vivo. Pirh2 catalyses the ubiquitylation of tetrameric p53 primarily through the interaction between the C-terminal domain residues 249–256 of Pirh2 and the tetramerisation domain of p53. The disruption of this interaction could represent a potential target for some cancer therapies. Additionally, a much weaker interaction was detected between the DNA binding domain of p53 and the N-terminal domain of Pirh2.(13) Intron 3 of Pirh2 has been shown to contain a p53 binding site. Pirh2 can repress p53-dependent transactivation, and p53 transactivates Pirh2, which can target p53 for ubiquitin-proteasome degradation; therefore, a feedback loop exists (Fig. S1).(9) The interaction inhibits apoptosis and the growth-inhibiting ability of p53, and it ultimately contributes to tumorigenicity, caused by the accumulation of genomic mutations that disrupt p53 homeostasis.(9,27) Tight regulation of p53 levels is crucial for maintaining normal cell growth and preventing tumorigenesis. These data might explain why Pirh2 is overexpressed in many types of cancer cells. Sheng et al.(13) observed Pirh2-mediated p53 ubiquitylation in vitro. However, Li et al.(26) showed that Pirh2 did not exhibit any E3 ubiquitin ligase activity towards p53 in vitro.
In response to sublethal DNA damage, Pirh2 also inhibits Axin-HIPK2-induced p53 phosphorylation of serine 46 by competing with HIPK2 for binding to Axin in a manner independent of the RING domain.(26) An in vitro reconstitution assay showed that high levels of Pirh2 correlated with higher dissociation rates of HIPK2 from Axin. The competition results in a decreased level of activated p53, thus inhibiting p53-induced apoptosis. The interaction was abolished by Tat-interactive protein of 60 kDa (Tip60) because an Axin-Tip60-HIPK2-p53 complex was formed when a lethal treatment was administered to reverse the Pirh2-Axin-induced p53 inactivation; this allowed for the maximal activation of p53, which triggered apoptosis (Fig. S1). Because Pirh2 and Tip60 can bind to the same sites of Axin, it was inferred that the two are in competition. ATM and ATR are also involved in this process by promoting the assembly of the Axin–Tip60 complex.(26) DNA polymerase η, a Y-family DNA polymerase that plays an important role in translesion DNA synthesis (TLS) through UV-induced cyclobutane pyrimidine dimers and photoproducts, can be targeted for proteasomal degradation by Pirh2.(45–47) DNA polymerase η might activate p53 in an ATM-dependent manner, therefore activating p53 via the phosphorylation of p53 serine 15.(48) DNA polymerase η is also involved in the regulation of ATM activity towards Chk2, which can phosphorylate p53 on serine 20 and promote its stability; this leads to cell cycle arrest and provides the cell with extra time to repair its DNA.(48) Cells deficient in DNA polymerase η are hypersensitive to UV-induced cell death. Pirh2-mediated proteasomal degradation of DNA polymerase η might play a role in UV-induced cancer formation.(45)
In addition to directly ubiquitylating p53 and targeting it for degradation, Pirh2 can also act synergistically with three additional major negative regulators of p53, MDM2, MDMX and COP1, to inhibit p53-mediated transcriptional activity (Fig. S1).(49–53) MDM2 and COP1 can both dramatically increase Pirh2 protein levels, and the overexpression of Pirh2 leads to a substantial increase in MDM2 and MDMX levels and a milder increase in COP1 levels.(50) Pirh2 can also increase MDM2 levels indirectly by targeting the SCY1-like 1 binding protein 1 (SCYL1-BP1) for proteasomal degradation, which can accelerate MDM2 self-ubiquitylation.(54)
Notably, Pirh2 can also degrade histone deacetylases 1 (HDAC1), which can inactivate p53 transcriptional activity. Recent biochemical studies revealed that Pirh2 can ubiquitylate HDAC1 and reduce HDAC1 levels, thus reducing the repressive activity of HDAC1 on transcription (Fig. S1).(18) Previous reports have shown that acetylation controls p53 stability by potentially interfering with MDM2-mediated ubiquitylation.(55,56) The recruitment of HDAC1 by MDM2 promotes p53 degradation by removing these acetyl groups.(57) HDAC1 also repressed the transcriptional activation of p53 and p21.(58,59) Moreover, HDAC1 is involved in the repression of the E2F1 transcription factor that determines the timely expression of many genes that are required for entry into and progression through S phase of the cell cycle.(60) E2F1 also indirectly regulates the levels and activity of p53.(61) Based on these data, Pirh2 has a dual role as a tumor suppressor and as an oncoprotein. Additionally, the ubiquitin modification has been shown to promote the transcriptional activity of some transcription factors,(62,63) so the ubiquitylation of HDAC1 might also promote its transcriptional repression of p53, p21 and E2F1. It is unclear whether HDAC1-mediated deacetylation of p53 is essential for Pirh2 to facilitate p53 degradation.
p53-independent functions of Pirh2. Under the stress of DNA damage, cells first initiate cell cycle arrest to make time for DNA repair.(64) p27Kip1 can inhibit the activation of cyclinE-CDK2 and cyclinA-CDK2, which can activate the transcription of genes that are required for the G1-S transition.(65,66) Pirh2 is a key regulator of p27Kip1 (Fig. S1) and degrades p27Kip1 late in the G1 phase in a ubiquitin-proteasome-dependent manner.(67) These data are in agreement with the fact that the Pirh2 expression level is low in G0 and early G1 phases and gradually increases toward the S phase in a cell cycle-dependent manner. Skp2 and the KPC are able to ubiquitylate p27Kip1 for degradation. However, neither protein can degrade p27Kip1 at the G1-S transition.(68,69) High expression levels of Pirh2 are associated with low expression levels of p27 and a poor prognosis in head and neck cancers(17) and lung cancer.(20) Pirh2 might also affect the function of p21, another important cell cycle-dependent kinase inhibitor. p21 is targeted by p53 and can be transactivated by p53. Transfection studies have shown that p21 accumulated in Pirh2 knockdown H1299 (p53−/−) cells.(45) p21 can be targeted by several E3 ubiquitin ligases for degradation,(70) but whether Pirh2 can directly regulate p21 warrants further investigation.
Cells are targeted for apoptosis if DNA damage is too severe for recovery. Pirh2 can inhibit the apoptosis pathway by binding to Kertain8/18 (K8/18); the phosphorylation of either target leads to apoptosis.(71) Duan et al. showed that the Pirh2-K8/18 association is significant for cells to maintain the K8/18 filament and that the mitochondria in these cells are normal (Fig. S1).(71) Mitochondria play a key role in controlling cell life and death by releasing cytochrome c into the cytoplasm; cytochrome c is a primary activator of the caspase cascade and its release activates the apoptotic process.(72) K8/18 is preferentially bound to unphosphorylated Pirh2 in the cytoplasm. The disruption of this association sensitises the cells to UV-induced apoptosis. This process is caused, in part, by enhancing the release of pro-apoptotic proteins, such as cytochrome c and Smac/DIABLO, from the mitochondria to the cytoplasm.(71) This partially explains why Pirh2 mainly exists in its unphosphorylated form in most tumor cell lines. Pancreatic tumor epithelial cells contained an increased level of phosphorylated K8/18,(73) and another group showed that K8/18 was hyperphosphorylated after an apoptotic challenge.(74) These data suggest that the phosphorylation of K8/18 by JNK or p38 leads to morphological changes in the mitochondria, caused by the dissociation of Pirh2-K8/18.(71,73,74) Studies also revealed that K8/18 can modulate the cellular response to specific pro-apoptotic signals. Moreover, it is involved in resisting TNF- and Fas-induced cytotoxicity or apoptosis.(75,76) Until now, it was unclear whether the Pirh2-K8/18 interaction was involved in these processes.
Although it was determined long ago that Pirh2 and p73 interacted(9) and Pirh2 could not directly degrade p73,(43) no new information about this interaction has emerged. p73 can induce cell apoptosis by functioning with many cofactors in both a p53-dependent and p53-independent manner.(77) Therefore, it is worthwhile to further investigate the relationship between them.
It has been shown that Pirh2 inhibits the androgen-dependent secretion of prostate-specific antigen (PSA); this implies that Pirh2 can negatively regulate protein secretion.(78) Previous studies have shown that some cellular proteins can utilise ubiquitin modification as their targeting signal.(79) Pirh2 ubiquitylates the signal recognition particle receptor β (SR β), which is a subunit of the signal recognition particle receptor (SR) heterodimer composed of SR α and SR β.(80) SR β contains a transmembrane segment, providing the membrane anchor for SR α. SR β also plays an important role in both the assembly and disassociation of SR subunits, which are essential for protein transportation.(81–83) The SR is located on the endoplasmic reticulum membrane and can associate with a signal recognition particle that recognises the N-terminal hydrophobic signal sequence of the secretory protein; this process targets the ribosome-nascent chain complex to the endoplasmic reticulum (ER). Once a protein has passed the ER quality control process it is transported to the Golgi. The continuous loss of material at the trans-face of the ER is antagonised by retrograde cargo retrieval to the ER, which is predominantly mediated by the coatomer complex. Pirh2 can promote the ubiquitylation of ε-COP, a subunit of COPI, and target it for degradation.(78) Because ε-COP plays an important role in the assembly and disassembly of COPI, the ubiquitylation and degradation of ε-COP can block the normal transportation of secretory proteins (Fig. S1).(78) Notably, the overexpression of Pirh2 causes a morphological change in the trans-Golgi network.(78) Previous studies have shown that K8/18, another Pirh2-interacting protein, helps to orchestrate the positioning and function of Golgi and protein secretion.(84) Further studies will help elucidate this process. Finally, it is worth noting that knockdown of Rchy1 specifically downregulated epidermal growth factor (EGF) internalisation.(85)
Pirh2, which was first identified as an ARNIP,(8) was able to enhance androgen receptor (AR) signalling by inhibiting HDAC1’s repressive activity towards AR.(18) Pirh2 can inhibit the AR N-C terminal interaction, which helps determine its transcriptional activity by regulating the androgen dissociation rate and the efficient recruitment of coactivators.(86–88) Although two groups showed that Pirh2 upregulates AR-mediated transcription of the PSA target gene,(18,78) Beitel et al.(8) reported that Pirh2 does not significantly affect AR transcriptional activation. Because AR signalling plays an important role in prostate cancer and Pirh2 overexpression was detected in prostate cancer cells, Pirh2 might contribute to prostate cancer formation via the AR signalling pathway.(89)