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DNA polymerase eta (PolH), a Y family translesion polymerase, is required for repairing UV-induced DNA damage, and loss of PolH is responsible for early onset of malignant skin cancers in patients with xeroderma pigmentosum variant (XPV), an autosomal recessive disorder. Here, we show that PolH, a target of the p53 tumor suppressor, is a short-half-life protein. We found that PolH is degraded by proteasome, which is enhanced upon UV irradiation. We also found that PolH interacts with Pirh2 E3 ligase, another target of the p53 tumor suppressor, via the polymerase-associated domain in PolH and the RING finger domain in Pirh2. In addition, we show that overexpression of Pirh2 decreases PolH protein stability, whereas knockdown of Pirh2 increases it. Interestingly, we found that PolH is recruited by Pirh2 and degraded by 20S proteasome in a ubiquitin-independent manner. Finally, we observed that Pirh2 knockdown leads to accumulation of PolH and, subsequently, enhances the survival of UV-irradiated cells. We postulate that UV irradiation promotes cancer formation in part by destabilizing PolH via Pirh2-mediated 20S proteasomal degradation.
Polymerase eta (PolH) is a member of the Y family translesion DNA polymerases and capable of translesion synthesis over UV-induced cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (7). PolH is also involved in double-stranded break repair via homologous recombination (15, 23). Human PolH is the product of the xeroderma pigmentosum variant (XPV) gene (14, 22). XPV, an autosomal recessive disorder, exhibits clinical phenotypes of extreme sun sensibility, cutaneous and ocular deterioration, and early onset of malignant skin cancers. Thus, it is postulated that loss of PolH is responsible for accumulation of UV-induced lesions, which lead to early onset of multiple skin cancers in XPV patients.
The ubiquitin-dependent degradation pathway plays a key role in many cellular processes, including cell proliferation, differentiation, and DNA repair (6, 10, 11). The pathway involves multiple enzymatic reactions catalyzed by a single ubiquitin-activating enzyme (E1), several ubiquitin-conjugating enzymes (E2s), and a large number of ubiquitin ligases (E3s). Protein polyubiquitination serves as a signal for rapid degradation by 26S proteasome, whereas monoubiquitination modulates protein function (3, 30). 26S proteasome is a multisubunit protease consisting of a core 20S proteasome and two 19S regulatory particles (24). 20S proteasome on its own is a broad-spectrum ATP- and ubiquitin-independent protease. 19S regulatory particles recognize and thread polyubiquitinated proteins into 20S proteasome for degradation in an ATP-dependent manner.
The RING-H2 type E3 ligase (Pirh2) is regulated by p53 and targets p53 for degradation (19). Recently, studies showed that Pirh2 interacts with and potentially serves as an E3 ligase for TIP60 (21) and p27Kip1 (8). Here, we show that PolH protein stability is reduced by UV irradiation via Pirh2 in a ubiquitin-independent manner. We also showed that upon knockdown of Pirh2, PolH is accumulated and, consequently, desensitizes cells to UV-induced cell killing. Based on these observations, we postulate that UV irradiation promotes cancer formation in part by destabilizing PolH via Pirh2-mediated 20S proteasome degradation.
Antibodies used in this study were as follows: rabbit polyclonal and mouse monoclonal anti-PolH (Santa Cruz Biotechnology), mouse monoclonal anti-ubiquitin (Santa Cruz Biotechnology), anti-20S (PW8155; Affiniti), mouse monoclonal anti-19S (p45-110; Affiniti), rabbit polyclonal anti-Pirh2 antibody (Bethyl Laboratories), monoclonal anti-HA (HA11; Covance), anti-FLAG monoclonal antibody (Sigma), anti-p53 monoclonal antibodies (DO-1, PAb1801, PAb240, and PAb421), antiactin (Sigma), and anti-p21 (C-19) (Santa Cruz Biotechnology).
RKO cells were incubated with cycloheximide (CHX, 10 μg/ml; Sigma) to inhibit de novo protein synthesis for different time points before analysis along with MG132 (5 μM; Sigma) or lactacystin (5 μM; A.G. Scientific). Protein levels were quantified from three independent assays and plotted as log scale versus time (h), which was then used to calculate the half-life of PolH and p53.
All constructs were verified by DNA sequencing.
Pirh2 cDNA was amplified with total RNAs purified from RKO cells with forward primer Pirh2-FF (5′-GGAGAATTCCACCATGGCGGCGACGGCCCGG-3′) and reverse primer Pirh2-FR (5′-GTACTCGAGTCATTGCTGATCCAGTGT-3′) and then cloned into a pcDNA4 expression vector (Invitrogen). To generate 2× FLAG-tagged Pirh2, the cDNA fragment was amplified with Pirh2-FF1 (5′-GGATGGATCCATGGCGGCGACGGCCCGGGAAG-3′) and Pirh2-FR. Various Pirh2 mutants were generated by PCR with forward primer Pirh2-FF1 along with reverse primer Pirh2-137R (5′-ACACCTCGAGAATACACTTGTGTCTTCCTTGAAG-3′) for Pirh2(1-137), Pirh2-179R (5′-GTCTCTCGAGTTCTTTCAACATTTCTTCATAACACG-3′) for Pirh2(1-179), and Pirh2-186R (5′-CAGACTCGAGACATAATGGACATCTGTAGCCTTC-3′) for Pirh2(1-186). Pirh2 with mutations at amino acids (aa) 137 to 261 [Pirh2(137-261)] was amplified with forward primer 5′-GAGGGGATCCATTGAAAATGTGTCCCGACAGAATTG-3′ and reverse primer Pirh2-FR. Pirh2 which lacks the putative p53 binding domain (aa 120 to 137) [Pirh2(Δ120-137)] was produced by ligation of the fragment bearing aa 1 to 119 (amplified by forward primer Pirh2-FF1 and reverse primer Pirh2-119R [5′-TACAGATATCACAATGGAAAAAATCTTCCTTTGGACC-3′]) and the fragment bearing aa 138 to 261 (amplified by forward primer Pirh2-138F [5′-GGATATCGAAAATGTGTCCCGACAGAATTGTC-3′] and reverse primer Pirh2-FR). Pirh2(Δ145-186), which lacks the RING finger domain (aa 145 to 186), was produced by ligation of the fragment bearing aa 1 to 144 (amplified by forward primer Pirh2-FF1 and reverse primer Pirh2-144R [5′-CATATGATATCATTCTGTCGGGACACATTTTCAATACAC-3′]) and the fragment bearing aa 187 to 261 (amplified by forward primer Pirh2-187F [5′-CCAGATATCATGCACTCTGCTTTAGATATGACCAGG-3′] and reverse primer Pirh2-FR). Pirh2(Δ171-179) was produced by ligation of the fragment bearing aa 1 to 170 (amplified by forward primer Pirh2-FF1 and reverse primer Pirh2-170R [5′-TCTATGTAAAAGATGTCCACATGGCAAG-3′]) and the fragment encoding aa 180 to 261 (amplified by forward primer Pirh2-180F [5′-GGCTACAGATGTCCATTATGTATGCAC-3′] and reverse primer Pirh2-FR). These products were cloned into a pcDNA3-2× FLAG vector via BamHI and XhoI sites.
To generate hemagglutinin (HA)-tagged PolH, cDNA fragment was amplified from pcDNA3-PolH (20) with forward primer PolH-FF (5′-GAATTCATGTACCCATACGATGTTCCAGATTACGCTGCTACTGGACAGGATCGAGTG GTTG-3′) and reverse primer PolH-FR (5′-CTCGAGGGATCCCTAATGTGTTAATGGCTTAAAAAATG-3′). Various PolH mutants were amplified with forward primer PolH-FF1 (5′-CCGCGGATCCGCTACTGGACAGGATCGAGTGGTTGC-3′) along with reverse primer PolH-365R (5′-TCACCTCGAGCTGGGTGGCTACCCTGTCATTATTATC-3′) for PolH(1-365); with reverse primer PolH-505R (5′-GGGCTCGAGAGTGGGAGCAGTAAGAGATGATTG-3′) for PolH(1-505); and with PolH-635R (5′-CTTCTCGAGGGGCACTTGGTCCTCAGCAGCTAG-3′) for PolH(1-635). PolH(351-713) was amplified with forward primer PolH-351F (5′-CCCAGGATCCAGACTGACTAAAGACCGAAATGATAATG-3′) and reverse primer PolH-FR. PolH(506-713) was amplified with forward primer PolH-506F (5′-GCTGGATCCCAGGCTCCCATGAGCAATTCACCATC-3′) and reverse primer PolH-FR. PolH(594-713) was amplified with forward primer PolH-594F (5′-CAACTGGATCCGAGATGGATTTGGCCCACAACAGCCAAAG-3′) and reverse primer PolH-FR. PolH(Δ394-505), which lacks the polymerase-associated domain (aa 394 to 505), was produced by ligation of the fragment bearing aa 1 to 393 (amplified with forward primer PolH-FF1 and reverse primer PolH-393R [5′-GGCTGATATCGTGAGCATCATAGCGGGTAAGGGCAC-3′]) and the fragment bearing aa 506 to 713 (amplified by forward primer PolH-506F [5′-CTGCTGATATCCAGGCTCCCATGAGCAATTCACC-3′] and reverse primer PolH-FR). These fragments were then cloned into a pcDNA3-2× FLAG vector.
Pirh2(C145S/C148S) and PoH(D652A) were generated by site-directed mutagenesis (QuikChange; Stratagene). Conserved cysteines in the Pirh2 RING domain were altered to serines to generate Pirh2(C145S/C148S) with sense primer 5′-CGACAGAATTCTCCAATATCTTTGGAGGACATTC-3′ and antisense primer 5′-GAATGTCCTCCAAAGATATTGGAGAATTCTGTCG-3′. PolH(D652A), which has an amino acid change at codon 652 from aspartate to alanine, was generated with sense primer 5′-GATATGCCAGAACACATGGCCTATCATTTTGCATTG-3′ and antisense primer 5′-CAATGCAAAATGATAGGCCATGTGTTCTGGCATATC-3′.
To generate 2× FLAG-tagged ubiquitin, cDNA fragment was amplified with forward primer 5′-CCGGGATCCATGCAGATTTTCGTGAAAACCCTTACG-3′ and reverse primer 5′-GAAAGTCGACACCACCACGAAGTCTCAACACAAG-3′, which was then cloned into a pcDNA3-2× FLAG vector.
To generate a construct that expresses short hairpin RNA (shRNA) against Pirh2 under the control of the tetracycline-regulated H1 promoter, one pair of oligonucleotides was synthesized, annealed, and then cloned into pBabe-H1 at HindIII and BglII sites (20). The resulting construct was designated pBabe-H1-siPirh2. The sense oligonucleotide is 5′-GATCCCCCATGCCCAACAGACTTGTGTTCAAGAGACACAAGTCTGTTGGGCATGTTTTTA-3′ and the antisense oligonucleotide is 5′-AGCTTAAAAACATGCCCAACAGACTTGTGTCTCTTGAACACAAGTCTGTTGGGCATGGGG-3′ (the siRNA targeting regions are underlined).
Scramble and Pirh2 small interfering RNAs (siRNAs) were purchased from Dharmacon. Pirh2 siRNA sequences are 5′-CAUGCCCAACAGACUUGUG-dTdT-3′ (sense) and 5′-CACAAGUCUGUUGGGCAUG-dTdT-3′ (antisense).
H1299 cell lines, which inducibly express Pirh2 or in which Pirh2 can be inducibly knocked down, were generated as reported previously (20).
In vitro 35S-labeled PolH or PolH(D652A) was incubated in a buffer (20 mM Tris-Cl [pH 7.2], 50 mM NaCl, 10 mM MgCl2, and 1 mM dithiothreitol [DTT]) supplemented with 1 μg 20S proteasome (Affiniti) along with 80 μM MG132 at 37°C for the indicated times.
H1299 cells were seeded at 1 × 104 per well in triplicate in a six-well plate and then counted over the next few days.
H1299 cells seeded at 500 per well in a six-well plate were cultured for 11 days and then stained with crystal violet.
Both floating cells in the medium and live cells on the plates were collected 16 h following 15 J/m2 UV irradiation from normal and XPV cells, which were transfected with scramble or Pirh2 siRNA for 3 days. DNA histogram analysis was performed as previously described (20).
The significances were calculated by Student's t test.
Previously, we showed that PolH is transcriptionally regulated by DNA damage in a p53-dependent manner (20). Since PolH is required for DNA repair, it is likely that PolH is tightly controlled via multiple pathways. To test this, we measured the half-life of PolH protein and found that PolH protein was unstable, with a calculated half-life of ~28 min (Fig. (Fig.1A,1A, lanes 1 to 6, and Fig. Fig.1B).1B). As a control, p53 had a calculated half-life of ~25 min, consistent with previous reports (25). We also found that PolH degradation was blocked by the MG132 proteasome inhibitor (Fig. (Fig.1A,1A, lanes 7 to 12). To further test this, we measured the steady-state level of PolH and found that the level of PolH protein was increased by MG132 and proteasome inhibitor lactacystin in a time-dependent manner (Fig. (Fig.1C,1C, compare lanes 1 to 5 with lanes 6 to 10 and 11 to 15, respectively). As a control, the levels of p53 were increased by these proteasome inhibitors. Since PolH is necessary for repairing UV-induced DNA damage (7), we measured the effect of UV irradiation on PolH stability and showed that the level of PolH protein was decreased by UV in a time-dependent manner, which was abrogated by proteasome inhibitors (Fig. (Fig.1D,1D, compare lanes 1 to 5 with lanes 6 to 10 and 11 to 15, respectively). This is consistent with a recent study that showed that Caenorhabditis elegans PolH stability is decreased by UV (16).
Since proteasomal degradation of PolH is enhanced by UV, we examined the Pirh2 E3 ligase, which is known to be induced by DNA damage in a p53-dependent manner (19). We showed that endogenous Pirh2 was coimmunoprecipitated with endogenous PolH, and exogenous PolH was coimmunoprecipitated with endogenous Pirh2 in RKO and MCF7 cells (Fig. 2A and B). Similarly, exogenous Pirh2 was coimmunoprecipitated with exogenous PolH, and exogenous PolH was also coimmunoprecipitated with exogenous Pirh2 in RKO cells (Fig. 2C and D). Next, we examined the region in PolH responsible for interaction with Pirh2 (Fig. (Fig.2E).2E). We showed that the polymerase-associated domain (aa 394 to 505) in PolH was required, since Pirh2 interacted with PolH(1-505), PolH(1-635), and PolH(351-713), but not PolH(1-365), PolH(506-713), PolH(594-713), and PolH(Δ394-505) (Fig. (Fig.2F).2F). Similarly, we examined the region in Pirh2 responsible for interaction with PolH (Fig. (Fig.2G).2G). We showed that the RING finger domain (aa 145 to 186) in Pirh2 was required, since PolH interacted with Pirh2(1-179), Pirh2(1-186), and Pirh2(137-261), but not Pirh2(1-137), Pirh2(Δ145-186), and Pirh2(Δ171-179) (Fig. (Fig.2H).2H). Interestingly, we showed that a small quantity of Pirh2(Δ120-137) was found to interact with PolH (Fig. (Fig.2H,2H, lane 9). This may reflect the effect of the small deletion on the folding of Pirh2, as suggested by Sheng et al. (28).
To investigate whether PolH stability is regulated by Pirh2, we generated H1299 cell lines in which endogenous Pirh2 can be inducibly knocked down. We showed that the level of PolH was increased in a manner inversely correlated with the level of Pirh2 (Fig. (Fig.3A).3A). In addition, knockdown of Pirh2 abrogated UV-induced destabilization of PolH protein (Fig. (Fig.3B,3B, compare lanes 1 to 5 with lanes 6 to 10, respectively). Conversely, we showed that overexpression of Pirh2 had an opposite effect on PolH stability in RKO cells transiently expressing PolH and Pirh2, as the levels of exogenous and total PolH were decreased in a manner directly correlated with the expression levels of Pirh2 (Fig. (Fig.3C).3C). Moreover, overexpression of Pirh2 led to p53 destabilization (Fig. (Fig.3C),3C), consistent with previous reports (19, 28). We would like to mention that p21 was accumulated by Pirh2-KD in p53 null H1299 cells (Fig. (Fig.3A3A).
To determine whether ubiquitination is required for PolH degradation, Pirh2 and PolH were coexpressed in temperature-sensitive murine ts20 cells in which E1 ubiquitin-activating enzyme is not active at the restrictive temperature of 39°C (4). We showed that the level of endogenous wild-type p53 was increased at the restrictive temperature (39°C) (Fig. (Fig.3D,3D, compare lane 1 with lane 3), consistent with the report that p53 is accumulated in ts20 cells (4). We also showed that Pirh2 was capable of inhibiting p53 expression at the permissive temperature (35°C), but much less efficient at 39°C (Fig. (Fig.3D,3D, compare lanes 1 and 3 with lanes 2 and 4, respectively). However, PolH expression was inhibited by Pirh2 at both 35°C and 39°C (Fig. (Fig.3D,3D, compare lanes 1 and 3 with lanes 2 and 4, respectively). In addition, the stability of PolH and p53 was restored upon treatment with MG132 (Fig. (Fig.3D,3D, lanes 5 to 8). To further verify the effect of Pirh2 on the degradation of PolH, cells were treated as shown in Fig. Fig.3D3D and subjected to immunoprecipitation with anti-FLAG or anti-p53 antibodies. We found that p53 but not PolH was polyubiquitinated at 35°C (Fig. 3E and F). These results indicated that Pirh2 promotes PolH proteasomal turnover independently of ubiquitination.
Based on the results above, PolH is degraded by proteasome in a ubiquitin-independent manner, suggesting that 20S proteasome plays a role. Indeed, we found that HA-tagged PolH physically associated with 20S proteasome but not 19S regulatory particle (Fig. (Fig.4A).4A). We also found that the extent of PolH interaction with 20S proteasome was progressively increased as the level of Pirh2 was gradually increased (Fig. (Fig.4B).4B). Nevertheless, a minute amount of 19S regulatory particle was found to associate with PolH when Pirh2 was highly expressed (Fig. (Fig.4B,4B, lanes 5 to 6). Next, an in vitro protein degradation assay was performed and showed that PolH was degraded in a time-dependent manner by 20S proteasome, which was inhibited by MG132, an inhibitor also known to repress 20S proteasome (Fig. (Fig.4C).4C). Since PolH can be monoubiquitinated, it is possible that monoubiquitination facilitates PolH degradation. Thus, mutant PolH(D652A) was generated, which cannot be monoubiquitinated (2) or polyubiquitinated (data not shown). We found that like wild-type PolH, PolH(D652A) was degraded by 20S proteasome, which was inhibited by MG132 (Fig. (Fig.4C).4C). We also showed that PolH(D652A) was decreased in a manner correlated with the expression levels of Pirh2 (Fig. (Fig.4D).4D). Moreover, like wild-type PolH (Fig. (Fig.4E),4E), PolH(D652A) was able to interact with 20S proteasome but not 19S particle (Fig. (Fig.4F).4F). To further demonstrate that ubiquitination is not required, Pirh2-DN, an E3 ligase-defective mutant (C145S/C148S) (28), was made and found to be capable of promoting PolH degradation in a dose-dependent manner when coexpressed with PolH (Fig. (Fig.4G4G).
Cells deficient in PolH are hypersensitive to UV-induced cell death, due to accumulation of UV-induced DNA damage (7). Thus, a colony formation assay was performed to examine whether Pirh2 has an effect on cell survival via PolH in H1299 cells. We showed that tetracycline alone had no effect on cell survival regardless of UV irradiation (Fig. (Fig.5A).5A). However, in the absence of UV irradiation, knockdown of Pirh2 decreased the colony-forming ability of the H1299 cells (Fig. (Fig.5B),5B), which is probably due to increased levels of p21 (Fig. (Fig.3A).3A). Interestingly, upon exposure to UV, cell survival was markedly increased when Pirh2 was knocked down (Fig. (Fig.5B).5B). Next, a short-term cell proliferation assay was performed. We showed that tetracycline alone had no effect (Fig. (Fig.5C,5C, left panel). However, knockdown of Pirh2 desensitized cells to UV irradiation, whereas in the absence of UV irradiation, knockdown of Pirh2 decreased cell proliferation (Fig. (Fig.5C,5C, right panel). Therefore, Pirh2-KD had similar effects on cell proliferation (Fig. (Fig.5C)5C) and survival (Fig. 5A and B). To demonstrate the role of PolH, Pirh2 was transiently knocked down in normal and XPV fibroblasts (Fig. (Fig.5D).5D). Consistent with the observation in H1299 cells (Fig. (Fig.3A),3A), Pirh2 knockdown led to a marked increase in PolH in normal, but not in XPV, fibroblasts (Fig. (Fig.5D,5D, compare lanes 1 and 3 with lanes 2 and 4, respectively). We also showed that upon knockdown of Pirh2, p53 is accumulated in both normal and XPV cells (Fig. (Fig.5D).5D). Next, DNA histogram analysis was performed and showed that upon knockdown of Pirh2, UV-induced apoptosis was significantly decreased in normal fibroblasts (13.27% versus 8.18%) (Fig. (Fig.5E).5E). This is likely due to the possibility that an increased level of PolH would quickly eliminate UV-induced lesions, which would then decrease UV-induced cell killing by reducing the duration and extent of accumulation of proapoptotic proteins, such as p53. However, in XPV cells, UV-induced apoptosis was not decreased, but instead increased, by knockdown of Pirh2 (16.45% versus 22.23%) (Fig. (Fig.5F).5F). This is likely due to the possibility that XPV cells (without PolH) are deficient in repairing UV-induced lesions, which would prolong accumulation of proapoptotic proteins negatively regulated by Pirh2, such as p53, and subsequently increase UV-induced cell killing.
Here, we present evidence that upon UV irradiation, the stability of PolH protein is compromised via Pirh2 in a ubiquitin-independent manner. We also showed that upon knockdown of Pirh2, PolH is accumulated and, consequently, desensitizes cells to UV-induced cell killing. Based on these observations, we postulate that UV irradiation promotes cancer formation in part by destabilizing PolH via Pirh2-mediated 20S proteasome degradation.
Pirh2 targets p53 for ubiquitin-dependent degradation (19). Here, we showed that Pirh2 recruits 20S proteasome to degrade PolH. Thus, Pirh2 has properties similar to those of Mdm2. It is well defined that Mdm2 targets p53 for degradation via a ubiquitin-dependent pathway (9, 12, 17, 31). In addition, Mdm2 interacts with, and degrades, p21 and RB through 20S proteasome in a ubiquitin-independent manner (13, 27, 32). Furthermore, Mdmx, which does not have ubiquitin E3 ligase activity, acts as an adaptor of Mdm2 to degrade p21 in a ubiquitin-independent manner (13). Therefore, future studies are warranted to examine the possibility that PolH may be regulated by Mdm2 and/or Mdmx.
Although the level of PolH was decreased upon UV irradiation, the level of Pirh2 was not substantially altered in H1299 cells (Fig. (Fig.3B).3B). Thus, the question is whether and how Pirh2 is regulated upon UV irradiation. In cells with endogenous wild-type p53, it is likely that the activation of p53 upon UV irradiation would lead to increased Pirh2 expression, since Pirh2 is a well-defined p53 target gene (19). However, in cells with no functional p53, such as the H1299 cells used in Fig. Fig.3B,3B, other mechanisms are likely to regulate Pirh2 activity. Previous studies showed that Pirh2 can be phosphorylated in a calmodulin-dependent manner and that phosphorylated Pirh2 is deficient in polyubiquitinating and degrading p53 (5). Since UV irradiation leads to activation of various DNA damage-induced kinases, it is possible that the activity of Pirh2 to target PolH may be increased by phosphorylation. Additionally, Pirh2 exists as a homodimer or a heterodimer with PLAGL2 (pleomorphic adenoma gene-like 2) (33). Thus, it is possible that UV irradiation may alter the partner of Pirh2, which then enhances Pirh2 to target PolH.
Although Pirh2 promotes PolH degradation independent of ubiquitination, it remains possible that Pirh2 may still ubiquitinate PolH, albeit not polyubiquitinate it. This is significant, especially considering that PolH is known to be monoubiquitinated and that monoubiquitination modulates PolH functions in repairing UV-induced DNA damage and homologous recombination repair (2, 26). For example, PolH(D652A), which has a mutation in the UBZ (ubiquitin binding zinc finger) domain, cannot be monoubiquitinated and is incapable of interacting with monoubiquitin (2). Thus, future studies are necessary to determine whether Pirh2 and other DNA damage-inducible E3 ligases catalyze PolH monoubiquitination.
We showed that upon knockdown of Pirh2, p21 expression was increased in H1299 cells (Fig. (Fig.3),3), which might be responsible for decreased cell proliferation and survival (Fig. 5A to C). Like p53 and PolH, p21 is a short-half-life protein and subject to proteasome-dependent degradation. Several ubiquitin E3 ligases are known to target p21 for degradation, including CRL4Cdt2 and CRL4Ddb2, which polyubiquitinate p21 for 26S proteasomal degradation (1, 29), and SCFSkp2 and Mdm2, which target p21 for 20S proteasomal degradation (13, 18, 32). Thus, whether p21 is directly regulated by Pirh2 is worth further investigation.
This work is supported in part by NIH grants (CA076069 and CA081237).
We thank Jim Xiao and H. Ozer for providing ts20 cells.
Published ahead of print on 14 December 2009.