Control of human PIRH2 protein stability: involvement of TIP60 and the proteosome.

Murine PIRH2 (mPIRH2) was recently identified as a RING finger-containing ubiquitin-protein isopeptide ligase that interacts with both p53 and the human androgen receptor. mpirh2 is a p53-responsive gene that is up-regulated by UV, and mPIRH2 protein has the capacity to polyubiquitylate p53, perhaps leading to p53 destruction. mpirh2 therefore has properties similar to those of the oncogene mdm2. Here, we have identified human PIRH2 (hPIRH2) as a TIP60-interacting protein. To investigate its regulation, we characterized hPIRH2 in parallel with hPIRH2 variants possessing mutations of conserved RING finger residues. We observed that wild-type hPIRH2 is an unstable protein with a short half-life and is a target for RING domain-dependent proteasomal degradation. Accordingly, we found that hPIRH2 was ubiquitylated in cells. The TIP60-hPIRH2 association appeared to regulate hPIRH2 stability; coexpression of TIP60 enhanced hPIRH2 protein stability and altered hPIRH2 subcellular localization. These results suggest that hPIRH2 activities can be controlled, at the post-translational level, in multiple ways.

The tumor suppressor p53 is a transcription factor that, through modulation of gene expression, can induce apoptosis or cell cycle arrest in response to cellular insults, including DNA damage (1). Several regulatory mechanisms control p53 activities, including post-translational modifications (2,3) such as ubiquitylation (4). Ubiquitylation can be an acute mechanism controlling the activities of proteins in numerous cellular processes, including transcription (5,6), and can occur once (monoubiquitylation) or in chains (polyubiquitylation) on target proteins (7). Attachment of polyubiquitin chains by ubiquitinprotein isopeptide ligase (E3) 1 enzymes results in discriminate irreversible destruction of the p53 protein by the 26 S proteasome (8). Other key proteins are controlled the same way; polyubiquitylation leads to their destruction, especially upon modification by Lys 48 -linked polyubiquitin chains (7).
MDM2 has become the focus of research (8) establishing regulatory pathways controlling its enzymatic activity, subcellular localization, and abundance, all of which influence MDM2. MDM2 enzymatic activity is inhibited by association with the murine p19 ARF (p14 in humans) tumor suppressor (21)(22)(23)(24)(25) in response to oncogene activation (26). MDM2 phosphorylation by ATM in response to DNA damage also inhibits MDM2-p53 interaction (27,28), and sumoylation can also control MDM2 activity (29). Other mechanisms indirectly control MDM2 activities; HAUSP is a ubiquitin protease that can remove ubiquitin groups from p53 upon DNA damage (30), reversing MDM2 effects. Nucleolar compartmentalization of MDM2 by alternative reading frame (ARF) or the ribosomal protein L11 can separate MDM2 from p53, producing a p53dependent response to stress (31)(32)(33). Evidence also exists that ARF can regulate MDM2 without nucleolar sequestration (34). MDM2 is an unstable protein capable of autoubiquitylation, which leads to its own destruction (13,35). Association of MDM2 with other proteins can stabilize MDM2. MDM2-TSG101 interaction appears to inhibit MDM2 destruction (36), resulting in decreased p53 levels (37). Although nucleolar accumulation inhibits MDM2, it also stabilizes MDM2 (33). Although this is unlikely to increase MDM2 activity, it demonstrates MDM2 levels can undergo dynamic alterations. Other E3 enzymes such as E6-AP, p300, and PIRH2 (p53-induced RING-H2) can also modify p53 (38 -40). The murine PIRH2 (mPIRH2) protein was initially cloned from mouse as ARNIP (androgen receptor N-terminal interacting protein) (41), but was then demonstrated to interact directly with and ubiquitylate p53 (40). mpirh2 is a p53-regulated gene; its transcript and protein levels increase in response to UV irradiation and cisplatin treatment (40). These findings demonstrate that mpirh2 has a similar relationship to p53 as mdm2.
Here, we demonstrate that human PIRH2 (hPIRH2) is an unstable protein that is ubiquitylated and targeted for RING domain-dependent proteasomal destruction. We also demonstrate that hPIRH2 can interact with TIP60, which bears similarities to the MDM2-ARF/L11 associations; we show that TIP60 can stabilize hPIRH2 protein levels and produce hPIRH2 subcellular relocation and, in some cells, nucleolar compartmentalization. We propose that hPIRH2 stability is likely to be regulated by multiple post-translational mechanisms, some of which are described herein.
DNA Manipulation-Sequencing results compared with human expressed sequence tags using BLAST identified one clone containing a partial cDNA encoding hPIRH2. Full-length hPIRH2 cDNA from the IMAGE consortium (clone 4651778) was subsequently characterized in this study. Primers 5Ј-ggatccatggcggcgacggcccgg-3Ј and 5Ј-gaatccttgctgatccagtgaaat-3Ј were used to amplify full-length hPIRH2, and cDNA was cloned into pcDNA4.1 (Invitrogen) using BamHI and EcoRI to produce Myc-tagged hPIRH2 (hPIRH2-Myc).
Immunoprecipitation and Antibodies-The antibodies used were as follows: anti-Myc antibody 9B11 (Cell Signaling), anti-ubiquitin antibody PD41 (Santa Cruz Biotechnology), anti-TIP60 antibody N-17 (Santa Cruz Biotechnology), anti-pentahistidine antibody (QIAGEN Inc.), anti-FLAG antibody M2 (Sigma), anti-GFP antibody (Santa Cruz Biotechnology), anti-nucleolin antibody 4E2 (Research Diagnostics Inc.), and Alexa 350-conjugated goat anti-mouse antibody (Molecular Probes, Inc.). For immunoprecipitation, 5 ϫ 10 5 293 cells seeded on 90-mm dishes (Corning Inc.) were transfected with the indicated plasmids. 48 h later, cells were lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride with protease inhibitors, and lysates were clarified by ultracentrifugation at 15,000 rpm for 20 min and then precleared in 50% protein G-Sepharose (Amersham Biosciences) at 4°C for 4 h. Samples were mixed with 2 g of antibody and fresh protein G-Sepharose at 4°C for 12 h. Immune complexes recovered by centrifugation were washed three times with 100 mM Tris (pH 7.4) and 150 mM NaCl and then resuspended in 40 l of Laemmli buffer. Samples were resolved by SDS-PAGE and subjected to Western blotting as shown.

RESULTS
TIP60 Interacts with the RING Finger Protein hPIRH2-Yeast two-hybrid screening with a human brain cDNA library fused to the Gal4 activation domain identified a clone encoding amino acids 31-261 of the RING domain-containing hPIRH2 protein that interacted robustly with TIP60 fused to the Gal4 DNA-binding domain (Fig. 1A). The TIP60-hPIRH2 interaction is specific, as hPIRH2 did not interact with pLAM5Ј (Fig. 1A), and TIP60 did not interact with another RING domain-containing protein (BRCA1) in yeast (Fig. 1C).
To confirm TIP60-hPIRH2 association, co-immunoprecipitation of proteins produced in vitro and from transiently transfected 293 cells was performed. FLAG-TIP60 and full-length wild-type hPIRH2-Myc proteins were specifically co-immunoprecipitated from 293 cells with anti-TIP60 antibody N-17 as shown in Fig. 1B. Western blotting showed that immunoprecipitation did not recover large amounts of TIP60 protein, in agreement with reports of low TIP60 solubility (60). Western blotting for the Myc epitope showed specific hPIRH2-Myc coimmunoprecipitation with FLAG-TIP60, demonstrating that the two proteins complex in 293 cells (Fig. 1B).
hPIRH2 contains a RING finger motif, identical to that of mPIRH2, between residues 145 and 186. To investigate whether the hPIRH2 RING structure mediates the TIP60 interaction, two conserved key cysteine residues were mutated (Fig. 1D). The mutant hPIRH2 protein carrying mutations of Cys 145 and Cys 148 (C145S/C148S hPIRH2-Myc) also co-immunoprecipitated with TIP60 ( Fig. 1B), suggesting that the hPIRH2 RING domain structure is dispensable for the TIP60-hPIRH2 interaction. 35 S-Radiolabeled TIP60 and hPIRH2 proteins, produced in vitro using a coupled transcription/translation system, were also specifically co-immunoprecipitated with anti-His antibody directed against TIP60 (data not shown).
TIP60-hPIRH2 interaction was analyzed further in yeast. First, full-length wild-type hPIRH2 was assayed for TIP60 interaction and demonstrated a similar interaction strength as the clone encoding amino acids 31-261 of hPIRH2 (ϳ60-fold over background levels) (Fig. 1C). Next, to delineate regions of hPIRH2 that interact with TIP60, hPIRH2 deletion constructs were assayed with TIP60 in yeast. A construct encoding amino acids 40 -261 of hPIRH2 was capable of TIP60 interaction, although to a much lesser degree than full-length hPIRH2 (Fig.  1C). Additionally, hPIRH2 residues 80 -261 interacted with TIP60 to the same degree as residues 40 -261, whereas residues 94 -261 failed to interact with TIP60 (Fig. 1C). This suggests that hPIRH2 amino acids 31-40 likely mediate the TIP60 interaction, but that amino acids 80 -94 may also participate in TIP60 interaction. Overall, it is clear the hPIRH2 N terminus mediates TIP60 interaction(s).
TIP60 interacted with full-length C145S/C148S hPIRH2 to the same degree as full-length wild-type hPIRH2 in yeast, but not with the N-terminal deletion construct encoding residues 120 -261 containing the hPIRH2 RING domain (Fig. 1C). Together, the deletion construct and C145S/C148S mutation data show that the hPIRH2 RING domain is dispensable for TIP60-hPIRH2 interactions. The mPIRH2 RING domain is also dispensable for p53 interaction (40), which is mediated by mPIRH2 residues 120 -137 (40). TIP60 therefore does not bind to the same residues as p53. Pilot data indicated that the TIP60 atypical zinc finger contributed to the TIP60-hPIRH2 interaction and that histone acetylase-deficient TIP60 could also interact with hPIRH2. 3 hPIRH2 Has a Short Half-life and Is a Target for Proteasomal Destruction-The presence of a RING domain in several proteins has identified their role as E3 enzymes that covalently attach ubiquitin peptides to substrates (61). mPIRH2 is a bona fide RING domain-dependent E3 enzyme that can mediates p53 ubiquitylation (40). Several E3 enzymes can also autoubiquitylate in cells, targeting themselves for proteasomal destruction (13,61). One such RING domain-containing E3 enzyme (MDM2) not only modifies itself, but also p53 and other proteins such as TIP60 by attachment of polyubiquitin chains (13,35,60). Proteins ubiquitylated (either in cis or in trans) may be expected to exhibit a relatively short half-life due to rapid turnover by the proteasome (7) and accumulation in high molecular mass polyubiquitylated species upon proteasomal inhibition. Experiments to examine the possibility that hPIRH2 may regulate its own expression were undertaken. First, the half-life of hPIRH2 was examined. hPIRH2 expression was examined in COS-7 cells transiently transfected with either wild-type or C145S/C148S hPIRH2-Myc and then treated with cycloheximide, a potent inhibitor of protein translation in eukaryotes (62). Western analysis showed wild-type hPIRH2 exhibited a relatively short half-life, as hPIRH2 protein levels decreased within 3 h and more dramatically within 5 h ( Fig.  2A, left panels). Densitometry analysis indicated that the hPIRH2 half-life (normalized to ␣-tubulin levels) was ϳ3.5 h. The C145S/C148S hPIRH2 RING domain double mutant showed no reduction in protein levels with cycloheximide treatment ( Fig. 2A, right panels), illustrating that it is a stable protein and suggesting that destruction of hPIRH2 is dependent upon an intact hPIRH2 RING domain. Interestingly, high molecular mass C145S/C148S hPIRH2 RING domain mutant species were also detected that were not present in wild-type hPIRH2-transfected cells. Similar results were obtained in other cell lines tested. 3 To assess whether hPIRH2 protein instability might be a result of proteasome-mediated degradation, further experiments examined hPIRH2 levels in the presence of proteasomal inhibition. Upon transfection of wild-type hPIRH2-Myc into COS-7 cells and subsequent treatment with the proteasomal inhibitor MG132 (5 M) (63) for 24 h, hPIRH2 protein levels were dramatically increased, as shown by Myc Western blotting, whereas ␣-tubulin levels remained unaltered (Fig. 2B, left panels). Multiple higher and lower molecular mass wild-type hPIRH2 bands also appeared in these MG132-treated cells, but not in untreated cells (Fig. 2B, left panels). MG132 time course treatment revealed that the increase in hPIRH2 protein levels and appearance of other molecular mass hPIRH2 species occurred within 3 h of proteasomal blockade (Fig. 2B, right panels). These data show hPIRH2 is a target for proteasomal destruction. High molecular mass hPIRH2 appeared exclusively upon MG132 treatment, showing that these species in particular are targets for efficient proteasomal destruction. These are reminiscent of polyubiquitylated proteins.
To examine whether the presence of unstable high molecular mass hPIRH2 (likely to be ubiquitin-conjugated hPIRH2) is dependent upon an intact hPIRH2 RING domain, the behavior of hPIRH2 RING domain mutant proteins was examined in parallel with wild-type hPIRH2. COS-7 cells were transfected with wild-type hPIRH2-Myc, the C145S/C148S hPIRH2-Myc RING domain double mutant, or hPIRH2-Myc with a C164S  1D). As seen previously, Western blotting showed that MG132 time course treatment resulted in the appearance of high molecular mass wild-type hPIRH2, particularly the ϳ98-kDa species (Fig. 2C, lanes 1, 4, 7, 10, and 13). These high molecular mass wild-type hPIRH2 species appeared only upon proteasomal blockade (Fig. 2C, compare lanes 1 and 13), suggesting that they are targets for proteasomal degradation. Interestingly, the C145S/C148S and C164S hPIRH2 RING domain mutant proteins were present in the same high molecular mass species in the absence of proteasomal inhibition (Fig. 2C, lanes 2 and 3). This suggests that high molecular mass unstable hPIRH2 species are destroyed by the proteasome in a RING domain-dependent manner. Destruction of these high molecular forms of hPIRH2, but not their occurrence, is strictly dependent upon an intact hPIRH2 RING domain. These data corroborate the fact that hPIRH2 RING domain mutations enhanced hPIRH2 stability during cycloheximide treatment ( Fig. 2A, right panels). Overall, it seems that hPIRH2 is an unstable protein that is targeted for proteasomal destruction and that the RING domain participates in and is required for efficient proteasomemediated destruction of hPIRH2. Proteins targeted for destruction may contain a consensus PEST sequence that is a target for cleavage by proteases. hPIRH2 sequence analysis using PESTFind 4 showed a weak putative PEST sequence in hPIRH2, outside the RING domain (Fig. 2D).
The characteristics described above imply that hPIRH2 is a candidate for ubiquitylation. To determine whether hPIRH2 is ubiquitylated within cells, a vector encoding cytomegalovirusdriven ubiquitin (CMV-Ub) was cotransfected into COS-7 cells with wild-type hPIRH2-Myc in a dose-range experiment (Fig.  3A). Myc Western blotting showed that two high molecular mass hPIRH2 species (likely to be ubiquitylated hPIRH2) increased in correlation with increasing levels of cotransfected CMV-Ub (Fig. 3A, compare lanes 1-4). These bands were also apparent upon MG132 treatment (Fig. 3A, lane 5), further FIG. 2. hPIRH2 is unstable and targeted for proteasomal degradation. A, COS-7 cells transfected with wild-type (wt) hPIRH2-Myc (left panels) or C145S/C148S (C145/8S) hPIRH2-Myc (right panels). After 48 h, cells were treated for the indicated times with 40 g/ml cycloheximide. Cell lysates were Western-blotted as shown. The arrow indicates unmodified hPIRH2. B, transfection of wild-type hPIRH2-Myc as described for A. Cells were treated with 5 M MG132 for the indicated times. Western blotting was performed as described for A. C, transfection of wild-type, C145S/C148S, or C164S hPIRH2-Myc as described for B. Cells were treated for the indicated times with 5 M MG132. Western blotting was performed as described for A, with samples loaded in groups of three per time point (from left to right in each group: wild-type hPIRH2, C145S/C148S hPIRH2, and C164S hPIRH2). Lane 16 was untransfected. D, a weak PEST sequence located using PESTFind between the indicated hPIRH2 residues.
suggesting that they are ubiquitylated hPIRH2 species. No high molecular mass hPIRH2 was detected in the absence of CMV-Ub (Fig. 3A, lane 1). Next, hPIRH2-Myc was transiently cotransfected into 293 cells with or without CMV-Ub, and immunoprecipitation was performed using anti-ubiquitin antibody. Immunoprecipitated material was then Western-blotted with anti-Myc antibody (Fig. 3B). Myc Western blotting detected several hPIRH2 species of different molecular masses that were specific to hPIRH2-transfected cells (Fig. 3B, lanes  2-5). These species represent oligoubiquitylated hPIRH2 proteins. Importantly, two of the most prominent of these ubiquitylated hPIRH2 bands corresponded to the 98-kDa unstable wild-type hPIRH2 proteins shown in Fig. 2C, suggesting that the high molecular mass bands are polyubiquitylated hPIRH2. Also, a 45-kDa hPIRH2 band immunoprecipitated from hPIRH2-transfected cells migrated at the correct position corresponding to monoubiquitylated hPIRH2 (Fig. 3B). MG132 treatment further increased recovery of ubiquitylated hPIRH2 (Fig. 3B, lane 4), showing that ubiquitylated hPIRH2 is targeted for proteasome-mediated degradation. hPIRH2 appears to be both mono-and polyubiquitylated in cells. The same results were obtained for the C145S/C148S hPIRH2 RING domain double mutant, 3 suggesting that hPIRH2 does not mediate autoubiquitylation in cells.
The findings that hPIRH2 is short-lived, that proteasomal blockade results in increased hPIRH2 expression and high molecular mass hPIRH2, and that hPIRH2 is ubiquitylated demonstrate that expression of hPIRH2 is controlled, at least in part, by the proteasome. It would seem that high molecular mass ubiquitylated hPIRH2 is targeted for proteasomal degradation, but that ubiquitylation of hPIRH2 can also be uncoupled from hPIRH2 degradation by disruption of the hPIRH2 RING domain; mutation of the hPIRH2 RING domain stabilizes expression of ubiquitylated hPIRH2. This implicates both the RING domain and ubiquitylation in proteasomal degradation of hPIRH2.
Stabilization of the hPIRH2 Protein upon TIP60 Coexpression-Having established a TIP60-hPIRH2 interaction and that hPIRH2 is an unstable protein that is a target for proteasomal degradation, TIP60 was investigated for its effects upon hPIRH2 stability. Having shown hPIRH2 degradation in COS-7 cells, these cells were transiently transfected with wildtype hPIRH2-Myc and either empty vector or increasing amounts of full-length FLAG-TIP60 (Fig. 4A). Myc Western blotting revealed that TIP60 coexpression increased hPIRH2 protein levels in a dose-dependent manner (Fig. 4A, lanes 1-5). TIP60 is a histone acetylase and is known to participate in transcriptional regulation (42,44,45). To exclude the possibility that expression of TIP60 could increase hPIRH2 abundance by increased transcription from the hPIRH2-encoding vector, further analysis was performed in the presence of cycloheximide to inhibit de novo protein synthesis. Transfection of COS-7 cells with wild-type hPIRH2-Myc and FLAG-TIP60 in the absence of cycloheximide resulted in dramatically increased levels of hPIRH2 protein compared with cells transfected with wild-type hPIRH2-Myc and vector, as shown by Myc Western blotting (Fig. 4B, compare lanes 1 and 2). Cells transfected with hPIRH2 and vector and treated with cycloheximide showed reduced expression of the hPIRH2 protein over vehicle control-treated cells (Fig. 4B, compare lanes 1 and 3), in agreement with the short hPIRH2 half-life. Cells cotransfected with hPIRH2-Myc and FLAG-TIP60 did not exhibit reduced hPIRH2 expression upon cycloheximide application (Fig. 4B,  compare lanes 2 and 4). These data show that TIP60 can stabilize the hPIRH2 protein, resulting in an extended hPIRH2 protein half-life. The acetylase activity of TIP60 was not required for increased hPIRH2 levels, as cotransfection of hPIRH2-Myc with the acetylase-dead FLAG-TIP60 mutant FLAG-TIP60D (46,57) had the same effect upon hPIRH2 as wild-type FLAG-TIP60 expression (Fig. 4B, lane 5). Cotransfection of an alternative acetylase, CMV-driven p300, did result in increased hPIRH2 levels, but did not increase the half-life of the hPIRH2 protein, as shown by cycloheximide application (Fig. 4B, lanes 6 and 7). The p300 effect is therefore probably transcriptional. Conditional expression of FLAG-TIP60 in a doxycycline-induced PC3M-derived cell line also produced increased levels of transfected enhanced GFP-hPIRH2 in response to doxycycline induction of TIP60 for 24 -48 h (Fig. 4C,  compare lanes 2-4). Uninduced cells transfected with GFP-hPIRH2 exhibited almost undetectable levels of transfected hPIRH2. Overall, hPIRH2 is an unstable protein that can be stabilized by TIP60 expression independent of cell type and hPIRH2 fusion protein type.
Subcellular Movement of the hPIRH2 Protein with TIP60 Coexpression-For some proteins targeted for ubiquitindependent destruction, cellular localization is a mechanism that can regulate protein stability. For example, nuclear export of p53 is thought to be required for proteasome-mediated destruction (64), whereas containment of MDM2 in the nucleolus stabilizes MDM2 (33). It has been previously reported that mPIRH2, 86% identical to hPIRH2, co-localizes with exogenously expressed androgen receptor in COS-1 cells (41).
In an attempt to explain the observed stabilization of the hPIRH2 protein upon TIP60 expression, the cellular localization of hPIRH2 was examined in the presence or absence of TIP60. Full-length GFP-hPIRH2 was used to study hPIRH2 cellular localization in the FLAG-TIP60-expressing doxycycline-induced PC3M cell line, previously shown to stabilize transfected hPIRH2 upon FLAG-TIP60 expression (Fig. 4C). The localization of GFP-hPIRH2 in a TIP60D-expressing doxycycline-induced PC3M cell line that conditionally expresses histone acetylase-deficient TIP60 was also examined. GFP-hPIRH2 was transiently transfected, and cells were either immediately induced to express TIP60 or left uninduced. GFP-hPIRH2 exhibited a diffuse nuclear and cytoplasmic distribution in untreated cells (Fig. 5A, left  column). Induction of FLAG-TIP60 expression with doxycycline produced a striking redistribution of GFP-hPIRH2 into nuclear speckles, with little cytoplasmic fluorescence (Fig. 5A, middle column) in ϳ50% of the transfected cells. The same phenomenon occurred in TIP60D-expressing induced cells upon TIP60D induction with doxycycline (Fig. 5A, right column), showing TIP60 acetylase activity to be dispensable for this effect. These nuclear speckles, into which GFP-hPIRH2 accumulated, did not overlap with 4Ј,6-diamidino-2-phenylindole staining and may be nucleoli. Western blotting with anti-FLAG antibody of cells seeded and induced in parallel confirmed induction of FLAG-TIP60 expression. 3 Overexpression of TIP60 therefore causes a redistribution of hPIRH2 into nuclear speckles. Although hPIRH2 can clearly residue in the nucleus, we have not been able to identify a consensus nuclear localization signal within hPIRH2.
GFP-hPIRH2 was next used to study the cellular localization of the hPIRH2 protein in human LNCaP prostate cancer cells expressing the endogenous androgen receptor. The C145S/ C148S GFP-hPIRH2 RING domain double mutant was also examined. LNCaP cells exhibited the same nuclear and cytoplasmic diffuse fluorescence for both wild-type and C145S/ C148S GFP-hPIRH2, with some perinuclear protein accumulation (Fig. 5B, upper row). 3 However, GFP also accumulated to some degree in perinuclear regions in LNCaP cells (data not shown). It is possible that the observed relocation of GFP-hPIRH2 upon TIP60 expression may be due to direct interaction between the two proteins. To assess whether the hPIRH2 nuclear speckles witnessed upon TIP60 coexpression actually contained TIP60, GFP-hPIRH2 was cotransfected into LNCaP cells with constructs encoding TIP60␣-RFP or TIP60␤-RFP. Empty vectors were used as controls. LNCaP cells transfected with TIP60␣-RFP alone exhibited almost exclusively nuclear red fluorescence contained within large speckles, which are likely to be nucleoli (Fig. 5B, middle row), as shown previously for TIP60 by immunofluorescence and fluorescent protein tagging in live cells (44,48). Cotransfection of both TIP60␣-RFP and GFP-hPIRH2 expression vectors produced a striking redistribution of GFP-hPIRH2. Most of the nuclear hPIRH2 protein was redistributed into distinct nuclear bodies (as seen in FLAG-TIP60-expressing doxycycline-induced PC3M-derived cells), this time resulting in co-localization of GFP-hPIRH2 and TIP60␣-RFP proteins (Fig. 5B, lower row) in Ͼ90% of the cotransfected cells. The redistribution of GFP-hPIRH2 was apparently not accompanied by altered distribution of TIP60␣-RFP because the speckles containing the two proteins did not coincide with 4Ј,6-diamidino-2-phenylindole staining. The same phenomenon was observed upon expression of TIP60␤-RFP in PC3M, 293, COS-7, MCF-7, U-2OS cells and in other cell lines (Fig. 5C). 3 It would thus appear that, upon TIP60 coexpression, the hPIRH2 protein is sequestered into subnuclear regions containing TIP60. These results also suggest that the normal diffuse cellular localization of hPIRH2 and the punctuate hPIRH2 foci upon TIP60 expression are not dependent upon association with the human androgen receptor or p53 because PC3M cells lack expression of both these factors.
We previously proposed that TIP60 has the capacity to reside within the nucleolus (48) and later found that TIP60 can participate in ribosomal gene transcription and is contained to some degree in the nucleolus. 3 Depletion of steroids from serum-containing medium results in more prominent expression of TIP60 in distinct nuclear foci (48) that we have shown to be nucleoli. 3 We tested whether TIP60 might have the capacity to retain hPIRH2 in the nucleolus under these conditions. PC3M or LNCaP cells were cotransfected with GFP-hPIRH2-and TIP60␣-RFP-encoding vectors. After 24 h, cells were cultured in steroid-depleted medium containing 10% dextran-coated charcoal-treated fetal calf serum. Dextran-coated charcoal treatment depletes fetal calf serum of steroids (45). Most cells cultured in steroid-depleted medium exhibited some TIP60␣-RFP nucleolar accumulation, as shown by indirect immunofluorescence counterstaining with the nucleolar marker nucleolin. Steroid-depleted medium treatment also resulted in some degree of speckled nucleolar localization of GFP-hPIRH2 in ϳ60% of the cotransfected cells (Fig. 6), resulting in TIP60␣-RFP and GFP-hPIRH2 nucleolar co-localization. The remaining cotransfected cells still exhibited speckled co-localization of TIP60-RFP and GFP-hPIRH2, but fewer of these speckles coincided with nucleolin staining. These results show that TIP60 can modulate hPIRH2 subcellular localization by sequestering hPIRH2 into the nucleolus. Cells cultured in complete medium containing steroids also exhibited some nucleolar co-localization of TIP60␣-RFP and GFP-hPIRH2, but to a lesser extent than cells cultured in steroid-depleted medium (data not shown). The effects were independent of PC3M or LNCaP cell type. This may represent a mechanism by which TIP60 can stabilize hPIRH2. DISCUSSION Here, we have further characterized the human version of the recently discovered p53 ubiquitin ligase-and androgen receptor-interacting protein mPIRH2 (40,41). First, we identified an interaction between human TIP60 and hPIRH2 in yeast that was confirmed in mammalian cells. The presence of an hPIRH2 RING domain prompted investigation into hPIRH2 regulation. hPIRH2 proved to be an unstable protein with a relatively short half-life. Furthermore, proteasomal blockade produced increased hPIRH2 levels and accumulation of high molecular mass polyubiquitylated hPIRH2. These data show that hPIRH2 is a short-lived protein that is targeted for proteasomal degradation. Appropriately, we demonstrated that hPIRH2 is polyubiquitylated, a modification that is involved in targeting proteins for proteasomal destruction (7). Polyubiquitylation seems to target hPIRH2 for destruction, as these forms of hPIRH2 are unstable. An intact RING domain also appears to regulate destruction of hPIRH2, as mutation of consensus RING residues stabilized hPIRH2. hPIRH2 carrying RING domain mutations was still capable of being ubiquitylated and readily accumulated in high molecular mass polyubiquitylated species, which were targeted for proteasomal degradation in the case of wild-type hPIRH2. This argues that stabilization of hPIRH2 through RING domain mutation is likely to be a consequence of disruption of RING domain-dependent destruction of hPIRH2 by the proteasome. Because an intact hPIRH2 RING domain is required for hPIRH2 degradation, this domain represents a degron within hPIRH2. It would be of interest to isolate hPIRH2 RING domain-interacting factors that may control destruction of hPIRH2. These data suggest that hPIRH2 is not autoubiquitylated in cells and that hPIRH2 ubiquitylation is not sufficient for its degradation; the RING domain is also required. To our knowledge, this is the first demonstration that the RING domain is required for efficient proteasomal destruction in a protein that is not autoubiquitylated in cells.
The hPIRH2 RING domain and ubiquitylation represent factors that can negatively impact upon hPIRH2. One factor that can positively impact upon hPIRH2 expression is TIP60, most likely through direct protein-protein interactions. TIP60 coexpression increased the hPIRH2 half-life and caused hPIRH2 redistribution into TIP60-containing nuclear speckles in cell lines that express the androgen receptor and p53 or neither transcription factor. TIP60 also recruited hPIRH2 into nucleoli, which was enhanced by steroid depletion. We have previously found that TIP60 can reside in the nucleolus (48) and participate in ribosomal gene transcription. However, TIP60 and hPIRH2 readily FIG. 6. Partial nucleolar co-localization of hPIRH2 and TIP60. PC3M cells were cotransfected with GFP-hPIRH2 and TIP60␣-RFP. After 48 h, cells were subjected to indirect immunofluorescence for nucleolin (blue). Panels from left to right: green/blue, red/blue, red/green, and total merged images. Nucleoli are indicated by arrows. co-localize in non-nucleolar speckles within the nucleus. We have not definitively demonstrated that nucleolar accumulation is the hPIRH2-stabilizing mechanism, and no nucleolar targeting signal has been identified yet in TIP60. However, it is unlikely that the histone acetylase activity of TIP60 is required for any of these effects, as expression of TIP60D, which is deficient in histone acetylase activity, still resulted in accumulation of hPIRH2 proteins and localization to nuclear speckles. We have also observed that TIP60 purified from insect cells, although active, is not capable of acetylating bacterially expressed hPIRH2 in vitro (data not shown). Whatever the case, it is likely that hPIRH2 redistribution by TIP60 reflects direct protein-protein interactions that produce co-localization of the two proteins and simultaneous hPIRH2 stabilization.
Overall, these data identify two opposing factors (TIP60 and the proteasome) that can associate directly or indirectly with different regions of hPIRH2 to regulate hPIRH2 expression. The diametrically opposite effects of TIP60 and the proteasome on hPIRH2 stability would be expected, at least in part, to control the fate of hPIRH2 depending upon their relative activities. These data bear similarities to the regulation of MDM2, which is controlled, in part, by ARF and L11. Both ARF and L11 can recruit MDM2 into nucleoli as well as stabilize expression of, but simultaneously inactivate, the enzymatic activity of MDM2 (31,33). Other reports have confirmed that ARF can control MDM2 ubiquitin ligase activities, but without requirement for nucleolar recruitment (34). We are currently examining whether regulation of hPIRH2 by TIP60 modulates the ubiquitin ligase function of hPIRH2 and subsequent p53 stability, in a fashion similar to regulation of p53 stability by the MDM2-ARF/L11 associations.
Mechanisms controlling the activities of ubiquitin ligase coregulators are becoming more numerous. For example, MDM2 can be phosphorylated (27,28), sumoylated (29), and ubiquitylated (13,35), all of which likely impact upon enzymatic activity. Subcellular localization also seems to be key in regulating E3 activities. The data presented here provoke ideas that hPIRH2 activities are controlled in a similar way to MDM2 activities. These regulatory mechanisms could emerge to be key requirements in the control of p53 activities and therefore successful targets for therapeutic intervention.
Ubiquitylation has an emerging role in transcriptional regulation (5,6). Aside from histones, several prominent transcription factors are ubiquitylated, including p53, Myc, and the androgen receptor (12,62,65). Ubiquitylation appears to have a conserved role in the network of transcriptional regulators discussed here; hPIRH2, TIP60, p53, and MDM2 can all be ubiquitylated, likely resulting in their discriminate proteasomal degradation. It has been proposed that transcription factors may be unstable, and possibly ubiquitylated, when active (5,6). We are now attempting to delineate cellular signals that control hPIRH2-mediated ubiquitylation of p53 and other transcription factors, as well as the spatial and temporal ubiquitylation/destruction of hPIRH2 and TIP60 co-regulators, in an attempt to correlate the timing of destruction of these factors with the occurrence of transcriptional regulation of target genes. This may provide an insight into why the cell possesses several different ubiquitin ligase enzymes that regulate p53 activities.
In summary, we have discovered roles for the proteasome and TIP60 in hPIRH2 stability. Regulation of hPIRH2 could be multifaceted and is likely to involve control of hPIRH2 degradation by the proteasome and sequestration of hPIRH2 into TIP60-containing nuclear bodies. The consequence of the TIP60-hPIRH2 association with respect to transcription factor activities should now be assessed.