Pirh2

Abstract

Ubiquitylation is currently recognized as a major posttranslational modification that regulates diverse cellular processes. Pirh2 is a ubiquitin E3 ligase that regulates the turnover and functionality of several proteins involved in cell proliferation and differentiation, cell cycle checkpoints, and cell death. Here we review the role of Pirh2 as a regulator of the DNA damage response through the ubiquitylation of p53, Chk2, p73, and PolH. By ubiquitylating these proteins, Pirh2 regulates cell cycle checkpoints and cell death in response to DNA double-strand breaks or the formation of bulky DNA lesions. We also discuss how Pirh2 affects cell proliferation and differentiation in unstressed conditions through ubiquitylation and degradation of c-Myc, p63, and p27^kip1. Finally, we link these different functions of Pirh2 to its role as a tumor suppressor in mice and as a prognosis marker in various human cancer subtypes.

Introduction

Ubiquitylation has recently emerged as a key posttranslational modification in various cellular responses, regulating important cellular processes such as cell growth and proliferation, apoptosis, and the response to DNA damage. Pirh2 (p53-induced protein with a RING-H2 domain, also known as Rchy1) is an ubiquitin E3 ligase that was initially identified as a protein that interacts with the androgen receptor.¹ It was subsequently identified as a transcriptional target of p53 and a regulator of p53 turnover following DNA damage.² It has since been implicated in the ubiquitylation and degradation of several key proteins involved in the regulation of cell cycle, cell death, and proliferation such as p73, p63, p27^kip1, and c-Myc.^3-6 We have recently identified the tumor suppressor and cell cycle regulator Chk2 as an additional target of Pirh2.⁷ In what follows we show that through its E3 ligase activity Pirh2 acts as a key regulator of cell proliferation and cell cycle progression under both normal and stressed conditions.

Pirh2 and p53

The tumor suppressor p53 is a transcription factor that plays key roles in the regulation of cellular responses to stress such as DNA damage, oncogene activation and hypoxia.⁸ In response to DNA damage p53 activates numerous downstream transcriptional targets involved in cell cycle arrest and apoptosis such as p21, Puma, Bax, and Noxa.⁹ The levels and activity p53 in the cell are largely regulated through its posttranslational modifications such as ubiquitylation, phosphorylation, and acetylation. Monoubiquitylation of p53 can result in its export from the nucleus and its mitochondrial translocation, thus preventing it from acting as a transcription factor while allowing it to induce mitochondrial mediated apoptosis.¹⁰ Polyubiquitylation of p53 leads to its proteasomal degradation and several E3 ligases for p53 ubiquitylation have been identified including Mdm2, Cop1, ARF-BP1, synoviolin, p300/CBP, E4F1, and Pirh2.¹¹ Similar to Mdm2, Pirh2 is a transcriptional target of p53 and its transcription is upregulated in a cell type and DNA damage sp.ecific manner.² Pirh2 binds to p53 and mediates its polyubiquitylation in concert with the E2 ligase ubcH5b.² Overexpression of Pirh2 leads to a decrease in p53-mediated functions including apoptosis and cell cycle arrest.² Studies of the interaction between Pirh2 and p53 showed that Pirh2 preferentially interacts with and leads to the degradation of p53 in its active tetrameric form rather than its monomeric form.¹² Despite the data showing Pirh2 ubiquitylation of p53 and other key cellular proteins, the in vivo functions of this E3 ligase remained elusive. Recently we reported that in contrast to Mdm2 knockouts, Pirh2^−/− mice were viable and displayed normal development.⁶ Analysis of Pirh2^−/− mice supports a role for Pirh2 in mediating degradation of activated tetrameric p53, as under untreated conditions, these mutants displayed only a mild increase in the basal levels of the p53 protein and no significant transactivation of p53 targets was observed.⁶ In contrast, irradiated cells derived from various organs of Pirh2^−/− mice displayed a greater increase in the expression levels of p53 and its transcriptional targets involved in cell cycle arrest (e.g., p21) and apoptosis (e.g., Puma, Noxa, and Bax). Furthermore, cells derived from Pirh2^−/− mice were more radiosensitive, owing to their increased p53 activity.⁶ These results support an important physiological role for Pirh2 in the recovery from DNA damage.

Pirh2 and the p53 Homologs p63 and p73

The p63 and p73 proteins are homologs of p53.¹³ They share several of the functional domains of p53 including its DNA-binding domain, oligomerization domain, and transactivation domain.^14,15 Both p63 and p73 act as transcription factors to activate pro-apoptotic proteins and cell cycle regulators.¹⁶ They play a role in G₁/S cell cycle arrest by upregulating p21.¹⁵ In addition, p63 and p73 play important roles in development.¹⁷ The p63 protein is involved in development of limbs and all organs forming stratified epithelium such as the skin, mammary and salivary glands.^17,18 In addition, p63 has also been involved in tumor suppression.¹⁹ Mice that are p73-deficient display abnormal neuronal development.^18,20 They were also found to develop tumors.^19,21

Recently, it was found that p63 is a target of Pirh2 ubiquitylation. Pirh2 can ubiquitylate p63 and its dominant negative form ΔN-p63, leading to their proteasomal degradation.⁴ ΔN-p63 was found to promote epidermal cell proliferation whereas keratinocyte differentiation requires restraining of proliferation.²² Pirh2 overexpression in HaCaT cells promoted their in vitro differentiation into keratinocytes through the downregulation of ΔN-p63.⁴ This study suggests an important role for Pirh2 in controlling cell proliferation in the context of cell differentiation.

The p73 protein is responsive to DNA damage.¹⁵ Previous studies indicated that p73 is the target of HECT ubiquitin ligase Itch and F-box protein FBox45 that regulate its proteosomal degradation.^23,24 The activity of these E3 ligases is downregulated following DNA damage, leading to accumulation of p73 protein.^23,24 Because of the high homology between p73 and p53 it was hypothesized that p73 might also be a target of Pirh2 ubiquitylation. Indeed Pirh2 was found to polyubiquitylate p73 using K11, K29, K48, and K63 linkages in vivo and predominantly K63 linkages in vitro. This ubiquitylation of p73 leads to its proteasomal degradation and is downregulated following DNA damage.^25,26 Pirh2 was found to be important for p73-mediated G₁ cell cycle arrest and loss of Pirh2 protein was shown to increase p73-dependent and p53-independent cell death following exposure to the DNA damaging agent doxorubicin.^25,26 By regulating the levels of p53 homologs p63 and p73, Pirh2 is able to induce G₁/S cell cycle arrest or induce apoptosis in response to DNA damage in a p53-independent manner.

Pirh2 and p27^kip1

The tumor suppressor p27^kip1 is a cyclin-dependent kinase (CDK) inhibitor that regulates the transition from G₀/G₁ phases to the S phase of the cell cycle during normal cell proliferation.²⁷ Levels of p27^kip1 are tightly regulated throughout the cell cycle. They are usually high during G₀/G₁ phase and drop sharply just before cell entry into the S phase.²⁸ Levels of p27^kip1 are regulated trancriptionally, translationally and posttranslationally through phosphorylation and ubiquitylation.^27,28 The ubiquitin ligase Skp2 polyubiquitylates p27^kip1 and mediates its degradation. However, in its absence, p27^kip1 was still degraded, prompting investigators to look for other ubiquitin ligases that may regulate p27^kip1 turnover. Pirh2 was found to interact with p27^kip1 and to mediate its polyubiquitylation and proteasomal degradation.²⁹ Knockdown of Pirh2 prevented serum-starved cells to be released from G₀ cell cycle arrest following serum stimulation.²⁹ Therefore, Pirh2 can regulate normal cellular proliferation in response to mitogenic signals.

Pirh2 and c-Myc

Although Pirh2^−/− mice display increased levels of the tumor suppressor p53 in response to DNA damage, we found that about 25% of Pirh2^−/− mice display sp.ontaneous solid tumor development.⁶ This unexpected increased cancer predisposition was explained by the observation that the expression level of the oncogene c-Myc was elevated in various tissues derived from Pirh2^−/− mice. c-Myc was then found to be a target of Pirh2 polyubiquitylation and protesomal degradation. Interestingly, overexpression of c-Myc in Pirh2^−/− mice was associated with lymphoproliferation and sp.lenomegaly caused by accumulation of plasma cells.⁶ Increased proliferation in activated Pirh2^−/− B and T-cells was observed, although there was a concomitant increase in apoptosis in these cells, probably caused by c-Myc-induced accumulation and activation of p19/ARF and p53.⁶ Interestingly, double mutant Pirh2^−/−p53^−/− mice die significantly faster than their single mutant counterparts and 60% of these mice develop tumors. This could be caused by the unrestricted c-Myc-induced cell proliferation in the absence of p53.⁶ Studies of the expression of PIRH2 in human cancer indicated that decreased PIRH2 mRNA levels were associated with poor outcome in patients with breast, ovarian, and squamous cell lung carcinoma.⁶ Conversely, increased levels of PIRH2 expression were reported for lung cancer³⁰ and were also found to correlate with disease progression in prostate cancer.³¹ Thus, this E3 ligase might play different roles in cancer progression and tumorigenesis depending on cell and tissue type. Because its targets include both tumor suppressors (e.g., p53) and oncogenes (e.g., c-Myc), it is possible that PIRH2 itself acts as a tumor suppressor in some contexts and as an oncoprotein in others.

Pirh2 and DNA Polymerase Eta

DNA polymerase Eta (polH) is required for translesion DNA synthesis following exposure to UV irradiation. It was found that Pirh2 monoubiquitylates polH.³² This ubiquitylation of polH suppresses its interaction with PCDNA and prevents it from acting as a polymerase that bypasses residual DNA lesions during the S phase of the cell cycle. This results in increased cellular sensitivity to UV light.³² Thus, Pirh2 can regulate cellular responses to different types of DNA damage including DNA double strand breaks caused by ionizing radiation and bulky DNA lesions caused by UV irradiation.

Pirh2 and Chk2

Chk2 is a serine-threonine protein kinase involved in the regulation of cell cycle checkpoints. Chk2 is also a tumor suppressor and multi-organ cancer susceptibility gene. Indeed, germline mutations in the human CHK2 gene have been observed in different types of familial cancers including breast and prostate cancer.³³ Li-Fraumeni syndrome is an autosomal dominant hereditary disease which is typically associated with p53 mutations. Li-Fraumeni patients usually develop multiple tumors over their lifetime. CHK2 mutations that destabilize the CHK2 protein and impair its function have been identified in a subset of Li-Fraumeni patients.³⁴ In response to DNA double strand breaks, CHK2 is phosphorylated and activated by its upstream kinase ATM.³⁵ It then acts to phosphorylate multiple downstream substrates involved in cell cycle checkpoint, DNA repair, and apoptosis such as p53 and Brca1.³³ CHK2 is involved in the G₁-S, intra-S, and G₂-M cell cycle checkpoints in response to DNA double strand break formation.³⁶ Our most recent study of Pirh2^−/− mice revealed an increase in Chk2 levels both under unstressed conditions and following DNA damage.⁷ Despite the similar level of expression and activation of Atm in the absence or presence of Pirh2, γ-irradiation resulted in higher phosphorylation levels of p53 and E2F1, two Chk2 substrates in Pirh2^−/− cells compared with wildtype controls. This finding suggested the possibility that accumulated Chk2 in Pirh2^−/− cells might be due to defective ubiquitylation of this kinase. Chk2 was previously reported to be a target of ubiquitylation and its stability was reported to be regulated by its phosphorylation at Ser456 following DNA damage.³⁷ Mdm2 and PCAF were both found to be important for Chk2 polyubiquitylation.³⁸ However, the fact that Mdm2 and PCAF E3 ligase mutants are still able to induce Chk2 polyubiquitylation suggests that these E3 ligases do not directly ubiquitylate Chk2.³⁸ Ionizing radiation-induced Chk2 autophosphorylation at Ser379 was also found to be required for its ubiquitylation by a Cul1-containig complex.³⁹ Our data indicated that Chk2 and Pirh2 interact and that Pirh2 polyubiquitylates Chk2 both in vivo and in vitro in the presence of ubcH5b. Similar to what was previously observed, Chk2 phosphorylation at Ser460 in mouse cells prevented Chk2 ubiquitylation and mutation of this serine residue to alanine resulted in increased ubiquitylation of Chk2 by Pirh2. This suggests a strong role for Chk2 phosphorylation for the regulation of its stability, especially following DNA damage.⁷ USP28, a deubiquitylating enzyme that is a downstream target of ATM in response to DNA damage, is needed to stabilize Chk2 in response to DNA damage.⁴⁰ We found that USP28 counteracts Pirh2 ubiquitylation of Chk2.⁷ To examine the effect of Pirh2 deficiency on Chk2 physiological activity we examined cell cycle checkpoint responses of Pirh2^−/− and Pirh2^−/−Chk2^−/− mouse embryonic fibroblasts following ionizing radiation. We found that loss of Pirh2 elicited a stronger G₁-S and G₂-M cell cycle arrest compared with what was observed in WT cells. Interestingly, concomitant loss of Chk2 completely rescued this phenotype suggesting that increased Chk2 levels and activity were responsible for this hyperactivation of the cell cycle checkpoints.⁷

Conclusion

Recent studies have highlighted the role of the E3 ubiquitin ligase Pirh2 in the regulation of cell cycle checkpoints, cell proliferation, differentiation, and apoptosis. Pirh2 is especially important in the regulation of cellular homeostasis following the DNA damage response as it ubiquitylates and leads to the degradation of several transducers and effectors in this pathway including Chk2, p53, p73, p63, and polH. Pirh2 also has important roles in keeping cell proliferation in check through its regulation of c-Myc, p27^kip1 and ΔN-p63. Depending on the cellular context Pirh2 can act as a tumor suppressor that inhibits excessive cell proliferation and promotes cell differentiation or an oncogene that hinders DNA damage responses and drives tumorigenesis. We expect that identification of novel substrates of the E3 ligase substrates will help further understand the cellular and physiological functions of this protein. (Fig. 1)

Figure 1. Involvement of Pirh2 in the regulation of different cellular processes through ubiquitylation of several key proteins. The E3 ligase Pirh2 ubiquitylates several substrates including c-Myc, Chk2,S p53, PolH, p63, DN-p63, p73, and p27^kip1 (See text). Through these ubiquitylation events, Pirh2 contributes to the regulation of DNA damage repair, cell cycle, cell death, and differentiation.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.