PML-RARa and Its Phosphorylation Regulate PML ... · that PML-RARa disrupts PML nuclear bodies by inhibiting PML oligomerization. The N-terminal or the coiled-coil domain of PML-RARa

Molecular and Cellular Pathobiology

PML-RARa and Its Phosphorylation Regulate PMLOligomerization and HIPK2 Stability

Yutaka Shima, Yuki Honma, and Issay Kitabayashi

AbstractThe PML gene is frequently fused to the retinoic acid receptor a (RARa) gene in acute promyelocytic leukemia

(APL), generating a characteristic PML-RARa oncogenic chimera. PML-RARa disrupts the discrete nuclearspeckles termed nuclear bodies, which are formed in PML, suggesting that nuclear body disruption is involved inleukemogenesis. Nuclear body formation that relies upon PML oligomerization and its stabilization of thehypoxia-inducible protein kinase (HIPK)-2 is disrupted by expression of the PML-RARa chimera. Here, we reportthat disruption of nuclear bodies is also mediated by PML-RARa inhibition of PML oligomerization. PKA-mediated phosphorylation of PML-RARa blocked its ability to inhibit PML oligomerization and destabilizeHIPK2. Our results establish that both PML oligomerization and HIPK2 stabilization at nuclear bodies areimportant for APL cell differentiation, offering insights into the basis for the most common prodifferentiationtherapies of APL used clinically. Cancer Res; 73(14); 4278–88. �2013 AACR.

IntroductionIn human leukemia, specific chromosomal translocations

result in the expression of fusion proteins promoting malig-nancy (1, 2). The PML gene is the target of the t(15;17)chromosome translocation that is observed in more than90% of acute promyelocytic leukemia (APL) cases, in whichfusion of the PML and retinoic acid receptor a (RARa) genesleads to the expression of the aberrant PML-RARa fusionprotein (3–6). The PML protein normally forms discretenuclear speckles in the nucleus called PML nuclear bodies;in these, PML recruits other proteins, including transcrip-tion factors such as p53 and acute myeloid leukemia 1 (7–9),transcription coactivators such as hypoxia-inducible proteinkinase (HIPK)-2 and p300 (10, 11), SUMO (12), and DAXX (13,14). Nuclear bodies have been implicated in the regulation ofapoptosis, cellular senescence, and antiviral responses. It hasbeen reported that PML stabilizes transcription coactiva-tors, such as HIPK2 and p300, to assemble transcriptionfactor/coactivator complexes within nuclear bodies (15). Incontrast, nuclear bodies are disrupted in t(15;17) APL(12, 16–19); in the presence of the PML-RARa fusion protein,nuclear bodies appear as dispersed microspeckles in whichHIPK2 is destabilized (15). All-trans-retinoic acid (ATRA)and As2O3, which are used clinically in APL, restore the

normal appearance of nuclear bodies (12, 17, 19–21). In t(15;17) APL, it remains unclear that the disruption of nuclearbodies is related to leukemogenesis and that their restora-tion would lead to therapy. The molecular mechanism bywhich PML-RARa disrupts nuclear bodies has remainedelusive, hampering progress in the understanding ofleukemogenesis.

The present study reveals that PML-RARa blocks PMLoligomerization and disrupts nuclear bodies, and that theeffect is reversed upon cAMP/PKA-dependent phosphoryla-tion of PML-RARa. In addition, pharmacologic activation ofadenylyl cyclase by forskolin restores PML nuclear bodies andpromotes ATRA-induced APL cell differentiation. Further-more, nuclear body restoration induced HIPK2 stabilization.These results suggest that nuclear body formation is regulatedby PML-RARa phosphorylation, and that the restoration ofnuclear bodies is important for APL cell differentiation.

Materials and MethodsCell culture, infection, and antibodies

293FT cells, which were purchased from Invitrogen, andU2OS cells were cultured in Dulbecco's Modified EagleMedium (DMEM) supplemented with 10% fetal calf serum(FCS). Plat-E cells were obtained from Dr. T. Kitamura(University of Tokyo, Tokyo, Japan) and were cultured inDMEM supplemented with 10% FCS, 10 mg/mL blasticidin,and 1 mg/mL puromycin. NB4 cells and K562 cells werecultured in RPMI-1640 medium supplemented with 10%FCS. Anti-HIPK2 antibody was described previously (15).Other antibodies were purchased commercially: anti-HA(3F10, Roche and Y11, Santa Cruz), anti-FLAG (M2, Sigma),anti-Myc (9E10, Upstate), anti-tubulin (H235, Santa Cruz),anti-PML (16.1–104, Upstate; H238, Santa Cruz; 001, MBL),anti-SUMO (Zymed), anti-DAXX (Exbio), and anti-RARa(C20, Santa Cruz).

Authors' Affiliation: Division of Hematological Malignancy, NationalCancer Center Research Institute, Chuo-ku, Tokyo, Japan

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Issay Kitabayashi, Division of HematologicalMalignancy, National Cancer Center Research Institute, 5-1-1 Tsukiji,Chuo-ku, Tokyo 104-0045, Japan. Phone: 81-3-3547-5274; Fax: 81-3-3542-0688; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-3814

�2013 American Association for Cancer Research.

CancerResearch

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PlasmidsThe PML, PML-RARa, and HIPK2 expression vectors were

generated as described previously (15). PML and PML-RARadeletion mutants were generated by PCR using pLNCX-HA-PML IV or pLNCX-HA-PML-RARa as the template.

Immunoprecipitation and Western blotting293FT cells were transfected with the desired vectors and

lysed as described previously (15). Cell lysates and immu-noprecipitates were fractionated on SDS-polyacrylamidegels and transferred onto nitrocellulose membranes (Amer-sham). The membranes were incubated with primary anti-bodies and horseradish peroxidase-conjugated secondaryantibodies. The immune complexes were visualized by theenhanced chemiluminescence (ECL) or ECL-Plus technique(Amersham), and the images were analyzed by ImageGauge(FUJIFILM).

ImmunofluorescenceU2OS cells were cultured in 4-well chamber slides and

transfected as described previously (15). The cells wereexposed to 50 mmol/L forskolin for 18 hours. After forskolinexposure, the cells were fixed and incubated with antibodies asdescribed previously. PML-RARa–expressing mouse c-kitþ

cells were exposed to 50 mmol/L forskolin for 24 hours. NB4cells were exposed to 50 mmol/L forskolin or 1 mmol/L ATRAfor 24 or 72 hours. The cells were analyzed by the sametechniques as U2OS cells. Images were captured on an Olym-pus microscope and analyzed by deconvolution.

Serial replating assaypMSCV, pMSCV-HA-PML-RARa, pMSCV-HA-PML-RARa

DE, pMSCV-HA-PML-RARa S704A, pMSCV-HA-PML-RARaS704D, and pMSCV-HA-HIPK2 were transfected into Plat-Ecells using GeneJuice (Novagen), and supernatants containingretrovirus were collected 48 hours after transfection. C-kitþ

cells were selected from the femurs of C57BL/6 mice usingCD117-specific MicroBeads (Miltenyi Biotech), transducedwith retroviruses using RetroNectin (Takara), and plated inmethylcellulose medium (M3434, StemCell Technologies). Thecells were cultured and replated every 4 to 6 days in methyl-cellulosemedium under G418 selection. The cells from the firstround (empty vector and PML-RARa DE) or the third round(PML-RARa point mutants) of colonies were harvested andanalyzed by using immunofluorescence as described above.With respect to HIPK2 stabilization, c-kitþ cells were infectedwith retroviruses containing HA-HIPK2. The next day, the cellswere infected with retroviruses containing wild-type ormutants of HA-PML-RARa. The cells were cultured in meth-ylcellulose medium under G418 and puromycin selection. Thecells of the third-round colonies were harvested and subjectedto Western blot analysis.

RT-PCRReverse transcriptase PCR was conducted as described

previously (15). HIPK2 mRNA expression was analyzed usingthe following primers: forward (50-CCCCTCAAATACATT-CGCCC-30) and reverse (50-TGGTGTCTTCAGTCTCCACA-

AAGG-30). The glyceraldehyde-3-phosphate dehydrogenaseprimer set was described previously (15).

Flow cytometric analysisBefore incubationwith anti-Mac1-FITC (M1/70, eBioscience),

NB4 cells were preincubated with immunoglobulin G from ratserum (Sigma) to prevent nonspecific binding of the antibody.The stained cells were analyzed by JSAN (Bay Bioscience), andthe results were analyzed using FLOWJO software.

ResultsPML oligomerization is required for nuclear bodyformation

The coiled-coil domain of PML mediates homo-oligomer-ization and hetero-oligomerization (18, 22–24). We have pre-viously shown that PML IV interacts with 2 transcriptionfactors important for granulocytic differentiation, PU.1 andCAAT/enhancer-binding protein (C/EBPe; refs. 25, 26).Therefore, we used PML IV and a PML mutant lacking thecoiled-coil domain (PML IV DCC; Fig. 1A). As expected, PMLIV DCC could not form homo-oligomers (SupplementaryFig. S1A). Immunofluorescence was conducted to assess thelocalization of the PML IV DCC mutant. As shown previously(21, 27), wild-type PML IV localized to the nucleus andformed nuclear bodies of normal appearance; however, thePML IV DCC mutant was expressed uniformly throughoutthe nucleus and did not form nuclear bodies (SupplementaryFig. S1B). These results suggest that PML oligomerization isassociated with nuclear body formation. HIPK2 is stabilizedby PML in nuclear bodies and destabilized by PML-RARa(15). To test whether the PML mutant destabilized HIPK2,FLAG-tagged HIPK2 was cotransfected with HA-tagged PMLIV or PML IV DCC. Results showed that PML IV stabilizedHIPK2, which is consistent with previous observations (15);however, PML IV DCC destabilized HIPK2 (Fig. 1B). The dest-abilization was rescued by a proteasome inhibitor, MG132(Fig. 1B). These results suggest that PML oligomerization is re-quired for PML-mediated HIPK2 stabilization.

PML-RARa disrupts nuclear bodies; however, the molecularmechanism underlying this effect remains unclear. The defectin nuclear body formation by the oligomerization-deficientPML IVDCCmutant led to the hypothesis that PML-RARamayprevent PML oligomerization. To test this hypothesis, theeffect of PML-RARa on nuclear body disruption was evaluatedby immunofluorescence. Wild-type PML and PML-RARa areexpressed in APL cells. The localization of PML is diffused incells expressing PML-RARa (18). We transfected U2OS cellswith expression vectors encoding FLAG-tagged PML IV andhemagglutinin (HA)-tagged PML IV or HA-tagged PML-RARa. PML nuclear bodies were detected using anti-FLAGantibody. As shown in Supplementary Fig. S2B, PML IVformed large, discrete, and distinct nuclear foci in theabsence of PML-RARa (top) but small and disperse foci inthe presence of PML-RARa (bottom). The oligomerizationcapacity of PML was subsequently assessed in the presenceand absence of PML-RARa. As shown in Supplementary Fig.S2C, PML-RARa inhibited PML–PML interaction, suggesting

Mechanism of PML Nuclear Body Disruption by PML-RARa

www.aacrjournals.org Cancer Res; 73(14) July 15, 2013 4279




that PML-RARa disrupts PML nuclear bodies by inhibitingPML oligomerization. The N-terminal or the coiled-coildomain of PML-RARa has been reported to be importantfor nuclear body disruption (28). PML 1–394, which is thePML moiety of PML-RARa and lacks the PML C-terminal,did not disrupt PML nuclear bodies (Supplementary Fig.S2D), suggesting that the RARa moiety of the fusion proteinis also important for nuclear body disruption.

The ligand-binding domain of the RARa moiety of PML-RARa is essential for nuclear body disruption

Because the RARa moiety of PML-RARa is important forthe disruption of PML nuclear bodies, PML-RARa deletionmutants were generated (Fig. 2A) to identify the region ofRARa required for the effect. HA-tagged deletion mutantswere cotransfected with FLAG-tagged PML IV. Immunoflu-orescence analysis indicated that PML-RARa 1–748 led todispersed microspeckles, as did wild-type PML-RARa, butthat deletion mutants lacking the ligand-binding domain,such as PML-RARa 1–567, 1–492, and 1–420, did not (Fig.2B). These results suggest that the ligand-binding domain ofRARa plays an important role in PML-RARa–mediatednuclear body disruption. The DE PML-RARa mutant, whichlacks the ligand-binding domain (Fig. 2A), failed to disruptnuclear bodies (Fig. 2B). The DC PML-RARa mutant, whichlacks the DNA-binding domain, disrupted nuclear bodies(Fig. 2B). All of the PML-RARa deletion mutants wereexpressed at similar levels (Fig. 2C). We also tested theeffect of the wild-type PML-RARa and the DE PML-RARa

mutant on endogenous nuclear body formation in normalmyeloid stem/progenitor cells. As the cells transduced withPML-RARa DE were not immortalized (Fig. 2D), we assayedfor PML localization using the cells of first-round colonieswith an anti-PML antibody specific for mouse Pml. Nuclearbodies were not disrupted in PML-RARa DE–infected cells(Fig. 2E). The localization of DE was different from that ofwild-type PML-RARa. The ligand-binding domain of PML-RARa might be important for its dispersion. These resultssuggest that the ligand-binding domain of PML-RARa isindeed important for nuclear body disruption.

The effect of these PML-RARamutations on HIPK2 stabilitywas assessed. As shown in Fig. 2F, wild-type PML-RARa andPML-RARa 1–748, which disrupted nuclear bodies, destabi-lized HIPK2, whereas the other PML-RARamutants, which didnot disrupt nuclear bodies, did not destabilize HIPK2. Toassess the effect of the same PML-RARa mutations on PMLoligomerization, 293FT cells were cotransfected with Myc-tagged and FLAG-tagged PML IV together with HA-taggedPML-RARa mutants. As shown in Fig. 2G, immunoprecipita-tion andWestern blotting indicated that wild-type PML-RARaand 1–748 inhibited the interaction between Myc-tagged andFLAG-tagged PML, whereas the other PML-RARamutants didnot. These results support the essential role of the ligand-binding domain of PML-RARa in the disruption of PMLnuclear bodies.

The PKA phosphorylation site of PML-RARa is requiredfor nuclear body disruption

Experiments were carried out to identify the sites respon-sible for PMLnuclear body inhibitionwithin the ligand-bindingdomain. As shown in Supplementary Fig. S3A, HA-tagged PML-RARa 1–708 or 1–661 was cotransfected with FLAG-taggedPML IV. PML-RARa 1–708 led to the formation of micro-speckle PML nuclear bodies, whereas PML-RARa 1–661 didnot (Supplementary Fig. S3B). These results indicate that thePML-RARa region spanning amino acids 662 to 708 is requiredfor PML nuclear body inhibition. RARa and PML-RARa arephosphorylated by PKA (29, 30). Because serine 704, the PKA-dependent phosphorylation site of PML-RARa, is locatedwithin the region required for nuclear body disruption (Fig.3A), mutants were generated in which serine 704 was substi-tuted by alanine and aspartate to simulate dephosphorylatedand phosphorylated serine, respectively. The effect of thesemutants on PML nuclear body formation was assessed by thetransfection of U2OS cells. Representative immunofluores-cence data are shown in Fig. 3B (left), and the expressionlevels of PML and PML-RARa are shown in Fig. 3C. Quanti-fication was done by counting the number of cells that formedlarge, discrete, and distinct PML nuclear foci in all transfectedcells (Fig. 3D). Interestingly, wild-type PML-RARa and thealanine mutant (S704A) disrupted nuclear bodies, as shownby the presence of microspeckles, whereas the aspartatemutant (S704D) did not. Both mutants interacted with PMLIV as strongly as did wild-type PML-RARa and the DE mutant(Supplementary Fig. S4), suggesting that the inability to disruptnuclear bodies is not due to any deficiency in binding to PML.Forskolin was then used to activate adenylyl cyclase to

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Figure 1. The coiled-coil domain of PML is required for HIPK2stabilization. A, diagram of the PML deletion mutant. The proline-richregion (Pro), the ring-finger domain (RING), the coiled-coil domain (CC),the nuclear-import signal (NLS), and the serine-proline–rich region (SP)are indicated. B, PML IV DCC destabilizes HIPK2. 293FT cells weretransfected with pLNCX-FLAG-HIPK2 and empty vector, pLPCX-HA-PML IV, or pLPCX-HA-DCC. The cells were treated with or without 10mmol/L MG132 for 16 hours. The expression of HIPK2 (top), PML IV, orPML IV DCC (middle) and tubulin (bottom) was detected byimmunoblotting using anti-FLAG, anti-HA and anti-tubulin antibodies,respectively.

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Cancer Res; 73(14) July 15, 2013 Cancer Research4280




determine whether cAMP/PKA might restore nuclear bodiesby phosphorylating the serine residue of PML-RARa. In thepresence of forskolin, wild-type PML-RARa did not affectnuclear bodies (Fig. 3B and D); however, the S704A mutant,which lacks the serine residue phosphorylated by PKA, inhib-ited PML nuclear body formation even in the presence offorskolin.We also tested the effect of wild-type and mutant PML-

RARa in normal myeloid stem/progenitor cells. C-kitþ

mouse myeloid stem/progenitor cells were infected with aretrovirus encoding HA-tagged PML-RARa, HA-taggedS704A, or HA-tagged S704D and cultured in methylcellulosemedium. The location of Pml in the immortalized cells wasassessed using an anti-PML antibody specific for mouse Pml.Representative immunofluorescence data are shown in Fig.4A, and quantification is shown in Fig. 4B. Nuclear bodyformation was maintained in S704D-expressing cells but waslargely disrupted in wild-type PML-RARa- and S704A-expressing cells. The cells transduced with S704D wereimmortalized as well as those transduced with PML-RARaand S704A (Fig. 4C). Wild-type PML-RARa, S704A, andS704D were expressed at similar levels in the immortalizedcells (Fig. 4D). Forskolin restored PML nuclear bodies in thewild-type PML-RARa–expressing cells but could hardlyrestore nuclear bodies in the S704A-expressing cells (Fig.4E). These results suggest that the ability of PML-RARa todisrupt nuclear bodies is inhibited by cAMP/PKA-mediatedphosphorylation of PML-RARa at serine 704.

Disruption of PML nuclear bodies by PML-RARa isstrongly correlatedwith thedestabilizationofHIPK2andthe inhibition of PML oligomerization by PML-RARaAs shown in Fig. 1B and Supplementary Fig. S1B, PML IV

DCC did not form nuclear bodies and destabilized HIPK2.Similarly, the PML-RARa deletion mutants that inhibitedPML nuclear body formation also destabilized HIPK2 (Fig.2F). These results suggest a correlation between nuclearbody formation and HIPK2 stability. Therefore, the PML-RARa point mutants described above were assessed for theireffect on HIPK2 stability. As shown in Fig. 5A, the level ofHIPK2 decreased when HIPK2 was cotransfected with wild-type PML-RARa or S704A but not when HIPK2 was cotrans-fected with S704D. The effect of the point mutants on HIPK2stability was also assessed in a stable system as shown in Fig.4. Endogenous mouse HIPK2 could not be detected (data notshown). The expression of HA-tagged human HIPK2, intro-duced in mouse c-kitþ cells, was detected only in S704D-expressing cells (Fig. 5B) of the third-round colonies.However, HIPK2 mRNA levels in cells expressing wild-typePML-RARa, S704A, or S704D were not appreciably different(Supplementary Fig. S5). These data support the hypothesisthat HIPK2 destabilization is associated with PML nuclearbody disruption. The point mutants were then assessed fortheir effect on PML oligomerization. S704A inhibited PMLhomo-oligomerization, as did wild-type PML-RARa, whereasS704D did not (Fig. 5C), implying that the protection of PMLoligomerization from PML-RARa promotes PML nuclearbody formation.

PKA-dependent phosphorylation of PML-RARa restoresnuclear bodies and promotes ATRA-induced APL celldifferentiation

As shown in Figs. 3–5, PKA-dependent phosphorylation ofPML-RARa may be the switch that restores nuclear bodies.Cyclic AMP (cAMP) alone has no effect on nuclear bodyrestoration, but cAMP and ATRA cooperatively restore nuclearbodies in NB4-R1 cells, which are ATRA-maturation–resistantcell lines (31). These data suggest that the cAMP/PKA pathwayplays an important role in nuclear body formation. To deter-mine whether the cAMP/PKA pathway actually regulatesnuclear body formation in APL cells, APL-derived NB4 cells,which express endogenous PML-RARa, were exposed to for-skolin alone. Nuclear bodies were detected using an anti-PMLantibody; nuclear bodies were disrupted in NB4 cells, andbecame clear in the presence of forskolin (Supplementary Fig.S6A). Forskolin did not induce NB4 cell differentiation (Sup-plementary Fig. S6A). To characterize the forskolin-inducedclear particles, the localization of SUMO and DAXX, which arerecruited to nuclear bodies, was assessed using anti-SUMOandanti-DAXX antibodies. In the absence of forskolin or ATRA, thecolocalization of SUMO and PML, or DAXX and PML, was verylimited (Supplementary Fig. S6B). In contrast, ATRA restorednuclear bodies and recruited SUMO and DAXX to nuclearbodies. Forskolin changed the appearance of PML nuclearbodies from microspeckles to clear particles. In the clearparticles, PML colocalized with SUMO and DAXX (Supple-mentary Fig. S6B). Thus, forskolin alone, like ATRA, restorednuclear bodies. ATRA induced PML-RARadegradation, where-as forskolin did not (Supplementary Fig. S6C). These datamight reflect the smaller size of nuclear bodies in the presenceof forskolin compared with that of ATRA. These data alsosuggest that PML-RARa phosphorylated by cAMP/PKA inhi-bits nuclear body disruption.

Because forskolin restored nuclear bodies in NB4 cells, thestability of endogenous HIPK2 was assessed in NB4 cellsexposed to forskolin. As shown in Fig. 6A, forskolin increasedHIPK2 protein levels but not HIPK2 mRNA levels. In contrast,forskolin did not increase HIPK2 protein levels in the non-APLK562 cells (Fig. 6B). HIPK2 protein levels increased in NB4 cellsexposed to ATRA for 24 hours (Fig. 6C). Time-course analysisindicated that the increase in HIPK2 expression was correlatedwith nuclear body restoration (Fig. 6C and D). These resultssuggest that HIPK2 is stabilized in nuclear bodies restoredupon cAMP/PKA-mediated phosphorylation of PML-RARa.

Finally, NB4 cells were exposed to forskolin or/and ATRA todetermine whether nuclear body restoration may promoteNB4 cell differentiation. As shown previously, forskolin wasnot sufficient to induce NB4 cell differentiation (Fig. 6E and F);however, forskolin enhanced ATRA-induced differentiation(Fig. 6E and F). The combination of ATRA and forskolinresulted in the differentiation of NB4 cells into segmentedgranulocytes (Fig. 6F) and increased the expression of thedifferentiation marker Mac-1 (Fig. 6G) more efficiently thaneither drug alone. Other studies have also shown the efficacy ofcAMP against APL (32–35). These results and reports suggestthat cAMP/PKA promotes ATRA-induced APL cell differenti-ation by restoring nuclear bodies.






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DiscussionIn more than 90% of APL cases, the PML-RARa fusion

protein is generated by the t(15;17) chromosomal transloca-tion. PML-RARa disrupts PML nuclear bodies by amechanismthat was not understood in detail. The present study reveals

that PML-RARa blocks PML oligomerization, resulting in thedisruption of nuclear bodies, and that cAMP/PKA phosphor-ylation of PML-RARa restores nuclear bodies. Our resultssuggest that nuclear body restoration enhances APL celldifferentiation.

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Figure 3. The PKA-dependentphosphorylation site of PML-RARa regulates PML nuclear bodyformation. A, diagram of thelocation of the PKA-dependentphosphorylation site of PML-RARa. B, phosphorylation ofserine 704 is important for therestoration of PML nuclear bodies.FLAG-tagged PML IV and HA-tagged PML-RARa point mutantswere coexpressed in U2OS cells.Cells were exposed to 50 mmol/Lforskolin. PML nuclear bodieswere analyzed using an anti-FLAGantibody. The white bar represents10 mm. C, expression of PML-RARa wild-type and pointmutants. The expression of PML IV(top), PML, PML-RARa wild-typeor point mutants (middle), andtubulin (bottom) in U2OS cells wasdetected by immunoblot analysiswith anti-FLAG, anti-HA, and anti-tubulin antibodies, respectively. D,quantification of nuclear bodyformation. The number of cellswithPML nuclear bodies was counted.Values represent the mean � SEMof 4 independent experiments.�, P < 0.01 compared with thePML-RARa value withoutforskolin. DAPI, 40, 6-diamidino-2-phenylindole; NB, nuclear body.

Figure 2. The ligand-binding domain of PML-RARa is essential for nuclear body disruption. A, diagram of PML-RARa deletion mutants. B, the ligand-bindingdomain of PML-RARa is required for the disruption of PML nuclear bodies. U2OS cells were transfected with pLNCX-FLAG-PML IV and pLNCX-HA-PML-RARa deletion constructs or only with pLNCX-HA-PML-RARa deletion constructs as described. PML nuclear bodies were analyzed using anti-FLAGantibody. The white bar represents 10 mm. C, expression of PML-RARa deletion mutants. The expression of PML IV (top), PML-RARa deletion mutants(middle), and tubulin (bottom) in U2OS cells was detected by immunoblotting using anti-FLAG, anti-HA, and anti-tubulin antibodies, respectively. D, the cellsexpressing PML-RARa DE are not immortalized. C-kitþ mouse bone marrow cells were infected with empty vector (mock), pMSCV-HA-PML-RARawild-type, or DE and cultured in methylcellulose medium. The colony number from the third to the fifth round of colonies is indicated (top). Values representmean�SEM from 3 independent experiments. E, PML-RARaDE does not inhibit nuclear body formation. C-kitþmouse bonemarrow cells were infectedwithempty vector, pMSCV-HA-PML-RARa wild-type, or DE, and plated in methylcellulose medium. Endogenous murine PML nuclear bodies of the cellsat first round of colonies were analyzed using mouse Pml-specific antibody (16.1–104). The white bar represents 10 mm. E, effect of PML-RARa deletionmutants on HIPK2 stability. 293FT cells were transfected with pLNCX-FLAG-HIPK2 and either empty vector or pLNCX-HA-PML-RARa deletion constructs.The expression of HIPK2 (top), PML-RARa deletion mutants (middle), and tubulin (bottom) was detected by immunoblotting using anti-FLAG, anti-HA,and anti-tubulin antibodies, respectively. G, effect of PML-RARa deletion mutants on PML oligomerization. 293FT cells were transfected with pLNCX-Myc-PML IV and either pLNCX-HA-PML-RARa deletion constructs or pLNCX-FLAG-PML IV (empty vectors were used as negative controls). The expression ofMyc-tagged PML IV andHA-tagged PML-RARa deletionmutants in the lysates of transfectantswas detected by immunoblotting using anti-Myc and anti-HAantibodies, respectively (input). The lysates of transfectants were incubated with anti-FLAG antibody. The immunoprecipitates were analyzed byimmunoblotting using anti-Myc, anti-HA, and anti-FLAG antibodies (IP). The values of IP/input intensity of Myc-PML IVwere quantifiedwith ImageGauge andnormalized to the value of Myc-PML IV/empty vector/FLAG-PML IV. DAPI, 40, 6-diamidino-2-phenylindole; IP, immunoprecipitation.






PML nuclear body disruption and restorationNuclear bodies are disrupted in APL cells harboring the t

(15;17) chromosomal translocation (16, 17, 19). Results

showed that wild-type PML-RARa blocked PML oligomer-ization (Supplementary Fig. S2C). Deletion analysis showedthat PML-RARa mutants that block PML oligomerization

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Figure 4. Nuclear body formation ismaintained in cells stably expressing PML-RARaS704D. A, PML-RARaS704Ddoes not inhibit nuclear body formation. C-kitþmouse bonemarrowcellswere infectedwith pMSCV-HA-PML-RARawild-type, S704A, or S704D. EndogenousmurinePMLnuclear bodies of cells at thethird round of colonies were analyzed using mouse Pml-specific antibody (16.1–104). The white bar represents 10 mm. The thick arrow representsPMLnuclear bodies, arrowhead represents intermediatenuclear bodies, and thin arrow representsmicrospeckles.B, quantification of nuclear body formation.The number of cells with PML nuclear bodies, intermediate nuclear bodies, and microspeckles was counted. Values represent the average of 4 independentexperiments. C, the cells expressing PML-RARa S704D are immortalized. C-kitþ mouse bone marrow cells were infected with empty vector (mock),pMSCV-HA-PML-RARawild-type, S704A, or S704Dandcultured inmethylcellulosemedium. The colony number from the third to the fifth roundof colonies isindicated (top). Values represent mean� SEM from 3 independent experiments. The cells at the third round of colonies were stained with May–Giemsa stain(bottom). D, expression of PML-RARa wild-type, S704A, and S704D. The expression of wild-type PML-RARa and mutants and of tubulin in cells atthe third round of colonies was analyzed by immunoblotting using anti-PML and anti-tubulin antibodies, respectively. E, quantification of nuclear bodyrestoration. The cells expressing wild-type PML-RARa and S704A at the third round of colonies were collected and exposed to 50 mmol/L forskolinfor 24 hours. Endogenous murine PML nuclear bodies were analyzed as described in A. The number of cells with PML nuclear bodies, intermediate nuclearbodies, ormicrospeckleswas counted. Values represent the average of 4 independent experiments. DAPI, 40, 6-diamidino-2-phenylindole; NB, nuclear body;WT, wild-type.

Shima et al.





also disrupt nuclear bodies (Figs. 2B and G, 3B, and 5C). Theblocking of PML oligomerization by PML-RARa occursindependent of their interaction (Figs 2G and 5C). Theseresults suggest that the inability of inhibition of PML olig-omerization by PML-RARa mutants is not due to the inabil-ity of interaction with PML IV, and imply that PML-RARa–induced nuclear body disruption is due to impaired PMLoligomerization.RARa and PML-RARa are phosphorylated by the cAMP/

PKA pathway at a site located within the ligand-bindingdomain (29, 30). The PML-RARa S704D mutant, which isexpected to simulate phosphorylated PML-RARa, did notblock PML oligomerization and did not disrupt nuclearbodies. Moreover forskolin restored nuclear bodies dis-rupted in U2OS cells expressing wild-type PML-RARa (Fig.3B and D), in mouse myeloid stem/progenitor cells expres-sing wild-type PML-RARa (Fig. 4E), or in APL-derived NB4cells (Fig. 6D). Our results indicate that the ligand-bindingdomain of PML-RARa is key to the inhibition of PMLoligomerization and to the disruption of PML nuclear bod-

ies, and that PKA-dependent phosphorylation of the serineresidue in that region reverses those effects and restoresnuclear body formation. Phosphorylated PML-RARa isknown to be easily degraded by ATRA (30); however, in theabsence of ATRA, phosphorylated PML-RARa was stable(Figs. 4D, 5A and B, and Supplementary Fig. S6C). Themechanism by which phosphorylation of PML-RARa pre-vents its inhibition of PML oligomerization remains unclear.However, there are precedents for this; the phosphorylationof RARa by PKA enhances the interaction between RARaand cyclin H/cdk2 (29), suggesting that phosphorylation ofRARa increases its interaction with cyclin H/cdk2. Thus,PML might find it easier to access a complex of phosphor-ylated PML-PML-RARa than a complex of dephosphorylatedPML-PML-RARa.

Nuclear body restoration is important for differentiationof APL cells

The PKA phosphorylation site of PML-RARa regulatesnuclear body formation. Forskolin restored nuclear bodies in

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Figure 5. HIPK2 destabilization and inhibition of PML oligomerization are correlated with PML nuclear body disruption by PML-RARa. A, effect of the PML-RARa point mutants on HIPK2 stability. FLAG-tagged HIPK2 was expressed with either empty vector or HA-tagged PML-RARa point mutants. Theexpression of HIPK2 (top), PML-RARa point mutants (middle), and tubulin (bottom) was detected by immunoblotting using anti-FLAG, anti-HA, andanti-tubulin antibodies, respectively. B, effect of the PML-RARa point mutants on HIPK2 stability in a stable expression system. C-kitþ mouse bone marrowcells were infected with pMSCV-HA-HIPK2 and pMSCV-HA-PML-RARa encoding either wild-type, S704A, or S704D. The expression of HIPK2, PML-RARa(wild-type and mutants), and tubulin at the third round of colonies was analyzed by immunoblotting using anti-HIPK2, anti-PML, and anti-tubulin antibodies,respectively. C, effect of thePML-RARapointmutants onPMLoligomerization. 293FTcellswere transfectedwith pLNCX-Myc-PML IV andeither pLNCX-HA-PML-RARa substitution constructs or pLNCX-FLAG-PML IV (empty vectors were used as negative constructs). The expression of Myc-tagged PML IV andHA-tagged PML-RARa substitution mutants in the lysates of transfectants was detected by immunoblotting using anti-Myc and anti-HA antibodies,respectively (Input). The lysates of transfectants were incubated with anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblotting usinganti-Myc, anti-HA, andanti-FLAGantibodies (IP). The valuesof IP/Input intensityofMyc-PML IVwerequantifiedwith ImageGauge, andnormalized to the valueof Myc-PML IV/empty vector/FLAG-PML IV. IP, immunoprecipitation.






APL-derived NB4 cells (Fig. 6D). Although forskolin alone wasnot sufficient to induce NB4 cell differentiation, it promotedATRA-induced differentiation (Fig. 6G). Published studiesshowed that cAMP enhances retinoic acid-induced APL dif-ferentiation and PML-RARa transactivation (32–35). Thesestudies also showed that PKA dissociates RARa from SMRTand activates transcription. Moreover, a recent report indicat-ed that cAMP-dependent phosphorylation of PML-RARa wascrucial for the eradication of APL-initiating cells (30). Takentogether, these reports and the present data suggest that

nuclear body restoration is one of the reasons why cAMP/PKAcould be useful as an APL therapy.

We previously showed that HIPK2 is stabled in PMLnuclear bodies and degraded outside of nuclear bodies bySCFFbx3, suggesting that HIPK2 is destabilized by disrup-tion of nuclear bodies (15). In this paper, we showed thatPML IV DCC, wild-type PML-RARa, PML-RARa 1–748, andS704A, which disrupted PML nuclear bodies, also destabi-lized HIPK2. In contrast, PML, PML-RARa 1–567, 1–492,1–420, DE, and S704D, which did not disrupt nuclear bodies,

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Figure 6. Nuclear body restorationpromotes ATRA-induced APLcell differentiation. A, forskolinincreases HIPK2 proteinexpression in NB4 cells. NB4 cellswere treated with forskolin for 72hours. The expression of HIPK2and tubulin was analyzed byimmunoblotting (right) using anti-HIPK2 and anti-tubulin antibodies,respectively. B, forskolin does notincrease HIPK2 protein expressionin K562 cells. The expression ofHIPK2 and tubulin in K562 cellswas analyzed as described in A.C, effect of forskolin or ATRA onHIPK2 expression. NB4 cells weretreated with forskolin or ATRA for24, 48, and 72 hours and wereharvested. The lysates wereanalyzed as described in A. D,effect of forskolin or ATRA onnuclear body restoration.NB4cellswere exposed to forskolin or ATRAfor 24 or 72 hours and stained withanti-PML antibody. The white barrepresents 10 mm. E, growth curveof NB4 cells exposed to ATRAand/or forskolin. Cells werecounted every other day for 6 days.Values were normalized to thevalue obtained at the zero timepoint. Values represent themean � SEM of 4 independentexperiments. F, forskolin increasesATRA-induced NB4 celldifferentiation. NB4 cells werestained with May–Giemsa stain5 days after adding ATRA and/orforskolin. G, the combination ofATRA and forskolin increases theexpression of Mac-1 in NB4 cells.NB4 cells were treated with ATRAand/or forskolin for 5 days, andincubated with anti-Mac-1-FITC.The cells were analyzed by flowcytometry.

Shima et al.





did not destabilize HIPK2. Forskolin and ATRA, whichrestore nuclear bodies in NB4 cells, increased HIPK2 expres-sion (Fig. 6A and C), and the increase in HIPK2 expressionwas correlated with nuclear body restoration (Fig. 6D).These data indicate that HIPK2 destabilization is stronglycorrelated with nuclear body disruption and that nuclearbody formation is important for HIPK2 stabilization. HIPK2is important for PML-dependent transcriptional activation(15). We have also found that PML stabilizes the PU.1/p300complex to regulate PU.1-dependent transcription and mye-loid differentiation (26). Mutations of HIPK2 are foundin AML and myelodysplastic syndrome (36). PML-RARa alsodisrupts PU.1/p300 complexes and inhibits myeloid differ-entiation (26). Therefore, nuclear body formation by PMLoligomerization may lead to the recruitment of transcriptionfactors/coactivators and to their stabilization for transcrip-tional activation and regulation of granulopoiesis. As sug-gested by the results of the present study, this might bebecause cAMP/PKA-dependent nuclear body restorationenhances APL cell differentiation.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Shima, I. KitabayashiDevelopment of methodology: Y. ShimaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Shima, Y. HonmaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Shima, I. KitabayashiWriting, review, and/or revision of themanuscript: Y. Shima, I. Kitabayashi

AcknowledgmentsThe authors thank Dr. de Th�e for helpful discussions.

Grant SupportThisworkwas supported in part byGrants-in-Aid from theMinistry ofHealth,

Labor, and Welfare, the Ministry of Education, Culture, Sports, Science, andTechnology, and National Cancer Center Research and Development Fund.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 2, 2012; revised April 7, 2013; accepted April 22, 2013;published OnlineFirst May 30, 2013.

References1. Gilliland DG. Molecular genetics of human leukemia. Leukemia

1998;12 Suppl 1:S7–12.2. Look AT. Oncogenic transcription factors in the human acute leuke-

mias. Science 1997;278:1059–64.3. de Th�e H, Chomienne C, Lanotte M, Degos L, Dejean A. The t(15;17)

translocation of acute promyelocytic leukaemia fuses the retinoic acidreceptor alpha gene to a novel transcribed locus. Nature 1990;347:558–61.

4. de Th�e H, Lavau C, Marchio A, Chomienne C, Degos L, Dejean A. ThePML-RAR alpha fusion mRNA generated by the t(15;17) translocationin acute promyelocytic leukemia encodes a functionally altered RAR.Cell 1991;66:675–84.

5. Goddard AD, Borrow J, Freemont PS, Solomon E. Characterization ofa zinc finger gene disrupted by the t(15;17) in acute promyelocyticleukemia. Science 1991;254:1371–4.

6. Kakizuka A,MillerWH Jr, Umesono K,Warrell RP Jr, Frankel SR,MurtyVV, et al. Chromosomal translocation t(15;17) in human acute pro-myelocytic leukemia fuses RAR alpha with a novel putative transcrip-tion factor, PML. Cell 1991;66:663–74.

7. GuoA, Salomoni P, Luo J, Shih A, Zhong S,GuW, et al. The function ofPML in p53-dependent apoptosis. Nat Cell Biol 2000;2:730–6.

8. Nguyen LA, Pandolfi PP, Aikawa Y, Tagata Y, Ohki M, Kitabayashi I.Physical and functional link of the leukemia-associated factors AML1and PML. Blood 2005;105:292–300.

9. PearsonM, CarboneR, Sebastiani C, CioceM, Fagioli M, Saito S, et al.PML regulates p53 acetylation and premature senescence induced byoncogenic Ras. Nature 2000;406:207–10.

10. M€oller A, Sirma H, Hofmann TG, Rueffer S, Klimczak E, DrogeW, et al.PML is required for homeodomain-interacting protein kinase 2(HIPK2)-mediated p53 phosphorylation and cell cycle arrest but isdispensable for the formation of HIPK domains. Cancer Res 2003;63:4310–4.

11. von Mikecz A, Zhang S, Montminy M, Tan EM, Hemmerich P. CREB-binding protein (CBP)/p300 and RNA polymerase II colocalize intranscriptionally active domains in the nucleus. J Cell Biol 2000;150:265–73.

12. Weis K, Rambaud S, Lavau C, Jansen J, Carvalho T, Carmo-FonsecaM, et al. Retinoic acid regulates aberrant nuclear localization of PML-RARalpha in acutepromyelocytic leukemia cells.Cell 1994;76:345–56.

13. Ishov AM, Sotnikov AG, Negorev D, Vladimirova OV, Neff N, KamitaniT, et al. PML is critical for ND10 formation and recruits the PML-

interacting protein daxx to this nuclear structure when modified bySUMO-1. J Cell Biol 1999;147:221–34.

14. Torii S, Egan DA, Evans RA, Reed JC. Human Daxx regulates Fas-induced apoptosis from nuclear PML oncogenic domains (PODs).EMBO J 1999;18:6037–49.

15. Shima Y, Shima T, Chiba T, Irimura T, Pandolfi PP, Kitabayashi I. PMLactivates transcription by protecting HIPK2 and p300 from SCFFbx3-mediated degradation. Mol Cell Biol 2008;28:7126–38.

16. Daniel MT, Koken M, Romagne O, Barbey S, Bazarbachi A, Stadler M,et al. PML protein expression in hematopoietic and acute promyelo-cytic leukemia cells. Blood 1993;82:1858–67.

17. Dyck JA, Maul GG, Miller WH Jr, Chen JD, Kakizuka A, Evans RM. Anovel macromolecular structure is a target of the promyelocyte-reti-noic acid receptor oncoprotein. Cell 1994;76:333–43.

18. Kastner P, Perez A, Lutz Y, Rochette-EglyC,GaubMP,DurandB, et al.Structure, localization and transcriptional properties of two classes ofretinoic acid receptor alpha fusion proteins in acute promyelocyticleukemia (APL): structural similarities with a new family of oncopro-teins. EMBO J 1992;11:629–42.

19. Koken MH, Puvion-Dutilleul F, Guillemin MC, Viron A, Linares-CruzG, Stuurman N, et al. The t(15;17) translocation alters a nuclearbody in a retinoic acid-reversible fashion. EMBO J 1994;13:1073–83.

20. ChenGQ, Shi XG, TangW, Xiong SM, Zhu J, Cai X, et al. Use of arsenictrioxide (As2O3) in the treatment of acute promyelocytic leukemia(APL): I. As2O3 exerts dose-dependent dual effects on APL cells.Blood 1997;89:3345–53.

21. Zhu J, KokenMH,Quignon F, Chelbi-AlixMK, Degos L,WangZY, et al.Arsenic-induced PML targeting onto nuclear bodies: implications forthe treatment of acute promyelocytic leukemia. Proc Natl Acad SciU S A 1997;94:3978–83.

22. Lin RJ, EvansRM. Acquisition of oncogenic potential byRARchimerasin acute promyelocytic leukemia through formation of homodimers.Mol Cell 2000;5:821–30.

23. Minucci S, Maccarana M, Cioce M, De Luca P, Gelmetti V, SegallaS, et al. Oligomerization of RAR and AML1 transcription factors asa novel mechanism of oncogenic activation. Mol Cell 2000;5:811–20.

24. Perez A, Kastner P, Sethi S, Lutz Y, Reibel C, Chambon P. PMLRARhomodimers: distinct DNA binding properties and heteromeric inter-actions with RXR. EMBO J 1993;12:3171–82.






25. Tagata Y, Yoshida H, Nguyen LA, Kato H, Ichikawa H, Tashiro F, et al.Phosphorylation of PML is essential for activation of C/EBP epsilonandPU.1 to accelerate granulocytic differentiation. Leukemia 2008;22:273–80.

26. Yoshida H, Ichikawa H, Tagata Y, Katsumoto T, Ohnishi K, Akao Y,et al. PML-retinoic acid receptor alpha inhibits PML IV enhancement ofPU.1-inducedC/EBPepsilon expression inmyeloid differentiation.MolCell Biol 2007;27:5819–34.

27. Jeanne M, Lallemand-Breitenbach V, Ferhi O, Koken M, Le Bras M,Duffort S, et al. PML/RARA oxidation and arsenic binding initiate theantileukemia response of As2O3. Cancer Cell 2010;18:88–98.

28. Grignani F, Testa U, Rogaia D, Ferrucci PF, Samoggia P, Pinto A,et al. Effects on differentiation by the promyelocytic leukemia PML/RARalpha protein depend on the fusion of the PML protein dimer-ization and RARalpha DNA binding domains. EMBO J 1996;15:4949–58.

29. Gaillard E, Bruck N, Brelivet Y, Bour G, Lalevee S, Bauer A, et al.Phosphorylation by PKA potentiates retinoic acid receptoralpha activity by means of increasing interaction with and phos-phorylation by cyclin H/cdk7. Proc Natl Acad Sci U S A 2006;103:9548–53.

30. Nasr R, Guillemin MC, Ferhi O, Soilihi H, Peres L, Berthier C, et al.Eradication of acute promyelocytic leukemia-initiating cells throughPML-RARA degradation. Nat Med 2008;14:1333–42.

31. Duprez E, Lillehaug JR, Naoe T, LanotteM. cAMP signalling is decisivefor recovery of nuclear bodies (PODs) during maturation of RA-resis-tant t(15;17) promyelocytic leukemia NB4 cells expressing PML-RARalpha. Oncogene 1996;12:2451–9.

32. Altucci L, Rossin A, Hirsch O, Nebbioso A, Vitoux D, Wilhelm E, et al.Rexinoid-triggered differentiation and tumor-selective apoptosis ofacutemyeloid leukemia byprotein kinaseA-mediateddesubordinationof retinoid X receptor. Cancer Res 2005;65:8754–65.

33. Duprez E, Lillehaug JR, Gaub MP, Lanotte M. Differential changes ofretinoid-X-receptor (RXRalpha) and itsRARalpha andPML-RARalphapartners induced by retinoic acid and cAMP distinguish maturationsensitive and resistant t(15;17) promyelocytic leukemia NB4 cells.Oncogene 1996;12:2443–50.

34. Guillemin MC, Raffoux E, Vitoux D, Kogan S, Soilihi H, Lallemand-Breitenbach V, et al. In vivo activation of cAMP signaling inducesgrowth arrest and differentiation in acute promyelocytic leukemia.J Exp Med 2002;196:1373–80.

35. Kamashev D, Vitoux D, De Th�e H. PML-RARA-RXR oligomers mediateretinoid and rexinoid/cAMP cross-talk in acute promyelocytic leuke-mia cell differentiation. J Exp Med 2004;199:1163–74.

36. Li XL,Arai Y,HaradaH,ShimaY,YoshidaH,Rokudai S, et al.Mutationsof the HIPK2 gene in acute myeloid leukemia and myelodysplasticsyndrome impair AML1- and p53-mediated transcription. Oncogene2007;26:7231–9.

Shima et al.





2013;73:4278-4288. Published OnlineFirst May 30, 2013.Cancer Res Yutaka Shima, Yuki Honma and Issay Kitabayashi and HIPK2 Stability

and Its Phosphorylation Regulate PML OligomerizationαPML-RAR

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PML-RARa and Its Phosphorylation Regulate PML ... · that PML-RARa disrupts PML nuclear bodies by inhibiting PML oligomerization. The N-terminal or the coiled-coil domain of PML-RARa