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Previous Article | Next Article 
Molecular and Cellular Biology, February 2003, p. 950-960, Vol. 23, No. 3
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.3.950-960.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
and Philip Leder*
Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115
Received 16 April 2002/ Returned for modification 11 June 2002/ Accepted 24 October 2002
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ABSTRACT |
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INTRODUCTION |
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The HIPKs are related to a group of kinases that includes Yak1 (Saccharomyces cerevisiae), YakA (Dictyostelium discoideum), and Minibrain (Drosophila melanogaster, rodents, and humans) (15, 23). This group represents a family of protein kinases that regulate the transition from growth to differentiation in eukaryotic cells. The assignment of HIPKs to this group suggests a common function in growth regulation. In fact, growth-regulatory and tumor suppressor functions have been attributed to the HIPKs (5, 10, 26, 28). For example, HIPK2 transfection into cells results in a dramatic decrease in colony formation (5, 10). This outcome occurs through the cooperative activities of HIPK2 and the tumor suppressor protein p53 (5, 10). HIPK2 phosphorylates p53 on Ser 46, resulting in activation of p53-dependent transcription, cell growth regulation, and apoptosis initiation (5, 10, 31).
A potential apoptotic function was also assigned to HIPK3, as it interacts with the cell surface death receptor Fas and phosphorylates the Fas-associated death domain (FADD), a transducer of Fas-mediated apoptotic signaling (28). The association of HIPK3, a nuclear protein, with a cell surface receptor was reconciled by demonstrating diffuse cytoplasmic HIPK3 localization.
Daxx is also a nuclear protein that was shown to associate with cytoplasmic and cell surface molecules, including transforming growth factor beta and Fas (25, 32). Overexpressed Daxx was shown to bind the Fas death domain and to mediate apoptotic signaling by activating ASK-1 and subsequently the Jun-N-terminal kinase (JNK) pathway, independent of the FADD/procaspase-8 pathway (3, 32).
Within the nucleus, Daxx interacts with the promyelocytic leukemia protein (PML) and localizes to PML oncogenic domains (PODs) (17, 30, 33). Localization of Daxx to PODs correlates with Daxx's proapoptotic activity. For example, Daxx mutants that fail to localize to PODs do not facilitate Fas-induced cell death (30). Furthermore, in the absence of PML, Daxx is dispersed throughout the nucleus, and activated cell death is diminished (33). These findings indicate that Daxx and PML may cooperate in PODs in mediating apoptotic signals. This may be important in understanding the biological nature of acute promyelocytic leukemia, which is characterized by reciprocal chromosomal translocation between the pml and retinoic acid receptor 
genes, resulting in the oncogenic fusion product PML-retinoic acid receptor . In acute promyelocytic leukemia cells, Daxx does not localize to PODs. However, upon treatment with retinoic acid, which induces disease remission, Daxx relocalizes to PODs (33).
Despite evidence supporting a proapoptotic function for Daxx, other studies have demonstrated that Daxx is essential to cell survival or is antiapoptotic. Targeted disruption of Daxx in mice results in embryonic lethality accompanied by extensive apoptosis (21). Elevated apoptosis was also observed in Daxx-null embryonic stem cells (21) as well as in fibroblasts in which endogenous Daxx was depleted by RNA interference treatment (J. S. Michaelson and P. Leder, unpublished data). In myeloid precursor cells, Daxx overexpression inhibited activated cell death, indicating an antiapoptotic role for Daxx (2). Furthermore, an antiapoptotic role for Daxx in acute promyelocytic leukemia cells was proposed, as Daxx expression decreased after apoptotic induction with HDAC1 inhibitors (1). It is possible that Daxx provides bipartite functions. Under certain circumstances, Daxx may be essential for cell survival, and under other circumstances, Daxx may propagate apoptotic signals.
Similar to the HIPKs, Daxx functions as a transcriptional regulator. For example, Daxx repressed the transcriptional activities of the Pax3 and ETS1 transcription factors (11, 18). Interestingly, Daxx was not able to repress the transcriptional activities of the oncogenic fusion protein Pax3-FKHR present in an alveolar rhabdomyosarcoma. This suggests that Pax3-FKHR circumvents the transcriptional controls normally applied to Pax3 (11). Daxx also either repressed or activated Pax5-mediated transcription (6). The specific effect of Daxx on Pax5 activity varied in different B-cell lines. Activation of Pax5-mediated transcription by Daxx depended on recruitment of the histone acetyltransferase CREB binding protein.
To date, the regulation of Daxx transcriptional activity is poorly understood. It was suggested that in the absence of PML, Daxx localized to chromatin, where it recruited HDAC1 and repressed transcription (12, 17). Overexpression of PML but not the oncogenic fusion PML-retinoic acid receptor recruits Daxx to PODs, thereby inhibiting Daxx repression (16, 17). The recruitment of Daxx from chromatin to PODs requires the secondary modification of PML by the ubiquitin-like molecule SUMO-1 (12, 16, 17). These results demonstrate that Daxx transcriptional regulatory activity is controlled in part by PML, which sequesters Daxx from condensed chromatin to PODs.
In this study, we characterized the expression and localization of HIPK1. In addition, we propose two novel mechanisms for regulating Daxx behavior, both mediated by HIPK1. HIPK1 relocates Daxx from PODs, presumably to chromatin, where Daxx participates in transcriptional regulation. In addition, HIPK1 phosphorylates Daxx on Ser 669, a site important in modulating Daxx activity.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A HIPK1 probe (open reading frame bp 2456 to 3267) was generated by PCR from mouse brain cDNA. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was a SacI-BamHI restriction product from plasmid pGEM-GAPDH encompassing exons 5 to 8 of mouse GAPDH. For HIPK expression analysis in humans, a multiple-tissue Northern blot and an RNA master blot (Clontech Laboratories) were hybridized with the HIPK1 probe described above with the manufacturer's recommended methods. The multiple-tissue Northern blot was also hybridized with a probe specific for ß-actin, which was provided by the manufacturer.
Cloning of full-length HIPK1. Full-length HIPK1 was generated from adult FVB mouse RNA (isolated as described above) by reverse transcription-PCR. Single-stranded cDNA was constructed from total RNA (5 µg) with avian myeloblastosis virus reverse transcriptase (Seikagaku America) and oligo(dT) according to the manufacturer's recommended methods. cDNA (5 µl) was used for PCR amplification of full-length HIPK1. The HIPK1 primers forward (5'-TAAGAATTCGCCACCATGGCCTCACAGCTGCAGGTG-3') and reverse (5'-CAAGTAAGAATACTGACTGATCTTGG-3') amplified the region from open reading frame bp 1 to 3627. HIPK1 cDNA was amplified with the Advantage-GC PCR kit (Clontech Laboratories) according to the manufacturer's recommended methods. Overhangs (3'-A) were added by incubating amplification products with 1 µl of Taq DNA polymerase (Promega) at 72°C for 15 min. The amplification product was ligated into pCR2.1 with the Original TA cloning kit (Invitrogen).
Generation of a kinase-inactive HIPK1. A kinase-inactive version of HIPK1 (K219A) was made by changing Lys 219 to an Ala with the QuikChange site-directed mutagenesis kit (Stratagene). The following primers were used for the mutagenesis; forward, 5'-CCAAGGAAATTGTGGCCATTGCAATCTTGAAGAACCACCCCTCC-3'; and reverse, 5'-GGAGGGGTGGTTCTTCAAGATTGCAATGGCCACAATTTCCTTGG-3'.
Generation of HIPK1 antibodies. A glutathione S-transferase (GST)-HIPK1 fusion protein was made by cloning a HIPK1 PCR amplification product (open reading frame bp 2842 to 3144) into the EcoRI and NotI sites of pGEX-6P-1 (Amersham Pharmacia). GST-HIPK1 fusions were expressed in bacteria and purified as described elsewhere (13). Covance Research Products prepared the anti-HIPK1 rabbit serum with the purified GST-HIPK1 fusion protein. HIPK1 antibodies were affinity purified from serum against the GST-HIPK1 fusion with the AminoLink Plus immobilization kit (Pierce).
Expression vectors. Full-length HIPK1 or HIPK1 K219A containing either a Flag or Myc C-terminal epitope tag was cloned into the EcoRI site of the pß expression vector (27). Full-length Daxx containing a Myc C-terminal epitope was cut with EcoRV and cloned into the blunted XhoI site of the pCAGGS expression vector (24). Plasmid pSG5-PML (8) was obtained from P. P. Pandolfi. The E2F1 and SP1-luciferase reporter vectors (29) were obtained from P. Farnham. The Met-luciferase reporter vector (7) was obtained from R. Maas. The CRE-luciferase reporter vector was supplied by Stratagene. The pCMVß vector (19) was obtained from I. Skerjanc.
Immunoprecipitation and Western blot analysis. For analysis of transfected proteins, human embryonic kidney 293 cells (2 x 104 to 2.5 x 104/cm2) were grown for 12 h in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum (HyClone), 4 mM L-glutamine, and antibiotics (50 U of penicillin and 50 µg of streptomycin per ml). The cells were transfected with pß, Flag epitope-tagged pß/HIPK1, or pß/K219A, with or without Myc epitope-tagged pCAGGS/Daxx or vectors encoding the Daxx phosphorylation mutants described below. Transfections were performed with FuGene 6 (Roche) according to the manufacturer's recommended methods. The cells were lysed 24 h after transfection in immunoprecipitation buffer (1% NP-40, 0.15 M NaCl, 0.01 M NaH2PO4, 2 mM EDTA, 50 mM NaF, 0.2 mM Na3VO4, complete protease inhibitor cocktail tablet [Roche]) and centrifuged to remove insoluble debris. Cell lysates were incubated with antibody (1 µg/300 to 500 µg of total protein) for 2 h at 4°C.
Antibodies used for immunoprecipitations included anti-HIPK1 antibody (above), anti-Flag M2 antibody (Sigma), anti-Daxx antibody (21), and anti-Myc antibody (Invitrogen). Protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) or UltraLink immobilized protein A/G (Pierce) (for rabbit and mouse antibodies, respectively) (20 µl of a 1:1 solution per µg of antibody) was added and incubated for an additional 2 h at 4°C. The immobilized proteins were collected by centrifugation, washed three times with immunoprecipitation buffer, and solubilized by boiling for 5 min in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (0.01 M Tris-HCl [pH 6.8], 1 mM EDTA, 10% glycerol, 0.004% bromophenol blue, 4% SDS, 0.02 M dithiothreitol).
For analysis of endogenous HIPK1, nuclear lysates were prepared from confluent 293 cells with the NE-PER nuclear and cytoplasmic extraction reagents (Pierce). Immunoprecipitations were performed with anti-HIPK1 antibody or anti-Fyn antibody (Santa Cruz) with protein A-Sepharose CL-4B as described above. The proteins were resolved by SDS-PAGE and Western blotted with electrochemiluminescence (Amersham Pharmacia Biotech). Primary antibodies used for Western blotting included anti-HIPK1 antibody (1:1,000), anti-Daxx antibody (1:1,000), anti-Myc antibody (1:1,000), anti-PML (H-238) antibody (1:200; Santa Cruz), and anti-HDAC1 antibody (Upstate Biotechnology; 1:500). Secondary antibodies used for Western blotting included goat anti-rabbit immunoglobulin G (1:10,000; Southern Biotechnology) and goat anti-mouse immunoglobulin G (heavy and light chains) conjugated to horseradish peroxidase (1:10,000; Southern Biotechnology).
In vitro pulldown assay. Daxx (open reading frame bp 1 to 2220) was cloned into the BamHI and NotI sites of pGex-6P-1. GST and Daxx-GST fusion proteins were expressed in bacteria and purified as described above. Full-length HIPK1 and MEK1 were cloned into pcDNA3.1(-)/Myc-HisB (Invitrogen), and proteins were radiolabeled from these constructs with Easy Tag Express-[35S] protein labeling mix (1,175.0 Ci/mmol) (Perkin-Elmer) with the TNT coupled reticulocyte lysate system (Promega) according to the manufacturer's recommended methods. The translation products (10 µl) were incubated with equal amounts of GST or Daxx-GST fusion proteins in GST buffer (0.5% NP-40, 20 mM Tris-Cl [pH 8], 100 mM NaCl, 1 mM EDTA, complete protease inhibitor cocktail tablet) for 3 h at 4°C. The samples were collected by centrifugation, washed three times with GST buffer, and solubilized by boiling for 5 min in SDS-PAGE sample buffer. The samples were resolved by SDS-PAGE, and the gels were dried on a Speed Gel gel drying system (Savant) and visualized by autoradiography.
In vitro kinase assay.
293 cells were grown for 12 h as described above and transfected with pß, Flag epitope-tagged pß/HIPK1, Flag epitope-tagged pß/K219A, or Myc epitope-tagged pCAGGS/Daxx. The cells were lysed 24 h after transfection in immunoprecipitation buffer. Proteins were immunoprecipitated from cell lysates as described above with anti-Flag antibody (HIPK1 and K219A) or anti-Myc antibody (Daxx). The immobilized proteins were washed three times in immunoprecipitation buffer and once in kinase buffer (20 mM Tris-Cl [pH 7.5], 10 mM MgCl2, 2 mM MnCl2, 1 mM dithiothreitol). Immobilized HIPK1 or K219A was incubated with 50 µM ATP, 5 µCi of [
-32P]ATP, and substrate, including 1.25 µg of myelin basic protein (Sigma) or immobilized Daxx. Phosphorylation reactions were performed for 30 min at 30°C and stopped by adding 4x SDS-PAGE sample buffer containing 40 mM EDTA. Proteins were resolved by SDS-PAGE, and the gels were dried on a Speed Gel gel-drying system. Labeled proteins were visualized by autoradiography.
Immunofluorescence. NIH 3T3 mouse fibroblasts or U2OS human fibroblasts (6.25 x 103/cm2) were grown for 12 h on two- to four-well plastic slides (Nalge Nunc) in the cell culture medium described above. The cells were transfected with Flag or Myc epitope-tagged pß/HIPK1 or pß/K219A, with or without pSG5-PML, Myc epitope-tagged pCAGGS/Daxx, or a vector encoding the Daxx phosphorylation mutant described below. The cells were fixed 15 to 24 h after transfection with 3.7% formaldehyde-phosphate-buffered saline (PBS), washed three times with PBS, and permeabilized with PBS containing 0.1% Triton X-100. After being washed once with PBS, the cells were stained with the indicated antibodies in PBS with 1% bovine serum albumin for 1 h at room temperature. Antibodies used included anti-HIPK1 antibody (1:200), anti-Myc antibody (1:200), anti-Flag antibody (10 µg/ml), anti-Daxx antibody (1:200), and anti-PML antibody (1:50). The cells were washed three times with PBS and stained with fluorescein isothiocyanate- or tetramethyl rhodamine isothiocyanate-conjugated goat anti-rabbit immunoglobulin antibody (1:200; Jackson Immunoresearch), fluorescein isothiocyanate- or tetramethyl rhodamine isothiocyanate-conjugated goat anti-mouse immunoglobulin antibody (1:200; Jackson Immunoresearch), and Hoechst 33258 at 10 mg/ml (1:1,000; Molecular Probes) in PBS with 1% bovine serum albumin for 30 min at room temperature. The slides were washed three times with PBS and once with distilled H2O and mounted with glass coverslips in PBS containing 15% Gelvatol (20). Fluorescent cells were examined with a Zeiss Axiophot upright fluorescence microscope, and images were captured with a SPOT-RT cooled digital camera (Diagnostic Instruments).
Identification of phosphorylated Daxx residues. 293 cells were grown for 12 h as described above on ten 10-cm cell culture dishes and transfected with Myc epitope-tagged pCAGGS/Daxx and Flag epitope-tagged pß/HIPK1 or pß/K219A. The Daxx protein was immunoprecipitated 24 h after transfection with an anti-Myc antibody as described above. The Daxx protein was resolved by SDS-PAGE and visualized by staining the gel with colloidal blue (Invitrogen). The various Daxx bands were excised from the gels and submitted to the Taplin Biological Mass Spectrometry Facility (Department of Cell Biology, Harvard Medical School) for microcapillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Phosphorylated Daxx residues were identified as described previously (9).
Generation of Daxx phosphorylation mutants. Mutations in phosphorylated DAXX residues were generated with the QuikChange multisite-directed mutagenesis kit (Stratagene) with Myc epitope-tagged pCAGGS/Daxx as a template. The following primers were used for the mutagenesis; Ser 502 to Ala, 5'-GATAATGAAGGAAATGAGGCACCCACATCGCCTTCAG-3', and Ser 669 to Ala, 5'-GTGTCCAGCCTATGCCAGCACCCCCCTTGGCCTCTG-3'. pCAGGS/Daxx-S502/669A had both Ser 502 and Ser 669 altered to alanine, while pCAGGS/Daxx-S502A and pCAGGS/Daxx-S669A had only Ser 502 or Ser 669, respectively, altered to alanine.
Transcriptional reporter assays. HeLa human fibroblasts (2 x 104/cm2) were grown for 24 h in 35-mm cell culture dishes in the culture medium described above. The cells were transfected with pß, pCAGGS/Daxx, or the vector encoding the Daxx phosphorylation mutant described above with the indicated luciferase reporter and pCMVß. The cells were lysed in 1x passive lysis buffer (Promega), and the luciferase activity was measured with an Automat LB953 luminometer (Berthold) with automatic injection of the luciferase reagent (Fisher). The absolute values of luciferase activity were normalized to transfection efficiency by measuring ß-galactosidase activity with the Galactostar ß-galactosidase reporter kit (Tropix).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HIPK1 interacts with and phosphorylates Daxx. A previous study identified an interaction between HIPK3 and Daxx, although the physiological relevance of that interaction was not pursued (28). We found that HIPK1 and Daxx also interact in cells. Endogenous Daxx coimmunoprecipitated with endogenous HIPK1 (Fig. 2A). In addition, overexpressed HIPK1 coimmunoprecipitated with endogenous Daxx (HIPK1 lane) and to a much greater degree with overexpressed Daxx (Daxx/HIPK1 lane) (Fig. 2B). In this experiment, no HIPK1 was detected when an empty vector (pß lane) or Daxx alone (Daxx lane) was expressed in cells due to the relatively low levels of endogenous HIPK1 protein.
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To determine whether Daxx serves as a substrate for HIPK1, Daxx was immunoprecipitated from cells cotransfected with pß, HIPK1, or a kinase-inactive HIPK1 (K219A) (Fig. 3A). K219A was generated by mutating lysine 219 of HIPK1 to alanine, thereby disrupting the phosphotransfer reaction carried out by the kinase domain. By Western blot derived from large SDS-PAGE separations, Daxx presented as multiple bands when expressed with pß, HIPK1, or K219A. However, when expressed with pß or K219A, the lowest-migrating or basal phosphorylated band (Fig. 3A, Daxx) was the predominant form of Daxx. When expressed with HIPK1, the highest-migrating or hyperphosphorylated band (Fig. 3A, Daxx*) was the predominant form of Daxx. This indicated that HIPK1 shifted Daxx from a basal phosphorylated to a hyperphosphorylated state in vivo. Lambda phosphatase treatment of immunoprecipitated Daxx caused it to migrate as a single band in all samples, demonstrating that the HIPK1 modification of Daxx resulted from phosphorylation and not another form of secondary modification.
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HIPK1 relocates Daxx from PODs, providing evidence for altered Daxx activity. Endogenous Daxx normally localizes in part to PODs, which appear as small spots within the nucleus. Immunofluorescence revealed that increased HIPK1 expression relocalized endogenous Daxx from PODs to a diffuse localization throughout the nucleus (Fig. 4A). Increased K219A expression did not relocate endogenous Daxx from PODs (Fig. 4A). HIPK1 but not K219A relocalization of Daxx was observed in multiple human and mouse fibroblast lines (data not shown). Within PODs, Daxx is known to interact with PML (12, 16, 17). Expression of HIPK1 but not pß or K219A disrupted this interaction, as determined by Daxx-PML coimmunoprecipitation (Fig. 4C, PML). Direct Western blot analysis of the lysates demonstrated equal transfection efficiencies for HIPK1 and K219A (Fig. 4B). In addition, equal amounts of Daxx were immunoprecipitated in each sample (Fig. 4C, Daxx). The apparent absence of phosphorylated bands in the Daxx Western blot results from a small SDS-PAGE separation. Together, the relocalization of Daxx from PODs and the disrupted Daxx-PML interaction suggest that HIPK1 sequesters Daxx from PODs to a diffuse localization throughout the nucleus, likely to chromatin.
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When not present in PODs, Daxx localizes to condensed chromatin, where it functions as a transcriptional regulator by interacting with proteins such as HDAC1 (12, 17). An association between Daxx and HDAC1 is likely required for Daxx to act as a transcriptional repressor. Therefore, to determine whether relocalization of Daxx by HIPK1 affected Daxx activity, we examined the association of Daxx with HDAC1 in the presence of pß, HIPK1, and K219A by coimmunoprecipitation (Fig. 4C, HDAC1). Western blots containing Daxx immunoprecipitates and probed for HDAC1 showed that a Daxx-HDAC1 association was present in all samples. However, this association was increased in the presence of HIPK1 relative to the association in the presence of pß or K219A. These findings indicate that HIPK1 may influence Daxx transcriptional repressive activity by enhancing its association with HDAC1.
Identification of phosphorylated Daxx residues. Microcapillary liquid chromatography-tandem mass spectrometry was used to identify phosphorylated Daxx residues from samples immunoprecipitated from cells expressing increased HIPK1 or K219A (Table 1). Seven phosphorylated residues were identified by this method and were either serines or threonines. Five residues were found to be phosphorylated in the basal and hyperphosphorylated forms of Daxx and therefore are likely constitutively phosphorylated. Two residues were found phosphorylated only in the hyperphosphorylated form of Daxx, Ser 502 and Ser 669. Therefore, phosphorylation of Daxx at these sites appears to be regulated, likely by HIPK1.
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Phosphorylation of Daxx Ser 669 is important in modulating Daxx activity. To determine the functional significance of Daxx Ser 669 phosphorylation with respect to transcriptional repression, reporter assays were performed with pß, wild-type Daxx, and Daxx-S669A (Fig. 7). Increased expression of wild-type Daxx significantly repressed the transcriptional activities of the CRE-luciferase (Fig. 7A), E2F1-luciferase (Fig. 7B), Met-luciferase (Fig. 7C), and Sp1-luciferase (Fig. 7D) reporters relative to the levels with pß. This is consistent with previous findings characterizing Daxx as a transcriptional repressor (6, 11, 18). Interestingly, Daxx-S669A repressed the CRE-luciferase, E2F1-luciferase, and Sp1-luciferase reporters to a greater degree than wild-type Daxx. For the Met-luciferase reporter, however, there was no significant difference in repression between wild-type Daxx and Daxx-S669A. These experiments demonstrate that phosphorylation of Daxx Ser 669 is important in modulating Daxx activity, possibly by downregulating Daxx-mediated repression.
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DISCUSSION |
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One mechanism for controlling Daxx transcriptional regulatory activity was described previously. SUMO-1-conjugated PML sequesters Daxx to PODs, thereby inhibiting transcriptional regulation by Daxx (12, 16, 17). In the absence of SUMO-1-conjugated PML or PML altogether, Daxx localizes to chromatin. When localized to chromatin, Daxx likely recruits HDAC1 and represses transcription (17). Here, we propose two novel mechanisms, mediated by HIPK1, that regulate Daxx activity. HIPK1 relocalizes Daxx from PODs, presumably to chromatin, where Daxx interacts with HDAC1. In addition, HIPK1 phosphorylates Daxx on Ser 669, a site important in modulating Daxx's transcription-repressive activity.
Transcription-repressive activities were assigned to both HIPK1 and Daxx (4, 15). HIPK2 was shown to provide a regulatory role in a large transcription-repressive complex containing the repressive elements Groucho and HDAC1 (4). Daxx localized to chromatin, where it interacted with HDAC1 and repressed transcription (12, 17). Our data indicate that HIPK1 recruits Daxx from PODs to chromatin, where these proteins likely associate with a large transcription-repressive complex containing HDAC1. Within this complex, HIPK1 provides a modulatory role, at least in part by phosphorylating Daxx.
The relocalization of Daxx from PODs by HIPK1 is dependent on an active kinase domain, as the kinase-inactive HIPK1 K219A was unable to relocate Daxx (Fig. 4A). Although K219A did not relocate Daxx from PODs, relocalization was independent of Daxx phosphorylation on Ser 669, as HIPK1 relocated a Daxx mutant with an alteration in this residue (Fig. 6A). The preferential interaction of Daxx with HIPK1 compared to K219A (Fig. 6C) indicates that Daxx relocalization is dependent on a direct HIPK1-Daxx interaction. It is possible that HIPK1 autophosphorylation is important in this interaction, justifying the necessity for an active kinase domain. Autophosphorylation may alter the HIPK1 conformation, thereby influencing its interactions.
Although increased HIPK1 expression relocalizes Daxx from PODs, POD integrity is not compromised (Fig. 4D), indicating that the HIPK1 effect is specific to Daxx. Daxx has a dynamic localization between PODs and chromatin and may oscillate between these sites in response to various cellular signals. Increased HIPK1 expression dramatically shifts Daxx localization towards chromatin. It is possible that PODs function as a Daxx warehouse, storing Daxx until its transcriptional functions are required. Alternatively, it is possible that Daxx functions both inside and outside of PODs. This is conceivable, as Daxx localization to PODs has been suggested to be necessary for it to propagate apoptotic signals (30, 33).
An opportunity exists for an overlap in HIPK1 and Daxx localization within PODs, as HIPK1 partially localized to PODs. This seems unlikely for several reasons. First, the majority of HIPK1 remains in nuclear speckles different from PODs or is diffused throughout the nucleus. Second, the relocalization of Daxx from PODs suggests that the HIPK1-Daxx interaction occurs elsewhere in the nucleus. This is supported by HIPK1 and Daxx colocalization outside of PODs (Fig. 6A).
Previous work identified an interaction between HIPK3 and Daxx (28). It was inferred that this interaction occurs at the cell periphery, as it was shown to be important in the early steps of Fas-mediated apoptosis. We found that the HIPK1-Daxx interaction occurs within the nucleus. It is possible that the HIPK3-Daxx interaction previously identified also occurred within the nucleus. It will be important to determine the extent to which the functions of the HIPKs overlap.
HIPK1 phosphorylates Daxx on Ser 669. A Daxx mutant containing an alanine in place of the serine at position 669 demonstrated the significance of phosphorylation at this site. This mutant repressed three of the four luciferase reporters examined to a greater degree than wild-type Daxx (Fig. 7). Therefore, phosphorylation at Ser 669 by HIPK1 diminishes the ability of Daxx to repress transcription. However, because the mutated Daxx influenced only three of the four luciferase reporters tested, phosphorylation of Ser 669 may provide specificity in modulating Daxx activity at specific promoters.
A role for HIPK1 in decreasing Daxx transcriptional repression by phosphorylation of Ser 669 appears contradictory to a role for HIPK1 in increasing Daxx transcriptional repression by sequestering Daxx from PODs to chromatin. However, we view these HIPK1 roles as exclusive of one another. Sequestration of Daxx to chromatin may be important in initiating or enhancing Daxx-mediated transcriptional repression. HIPK1 phosphorylation of Daxx may provide precise modifications to Daxx activity, as the Daxx phosphorylation mutant had only a modest effect on transcriptional repression relative to wild-type Daxx and only in three of the four reporters tested.
Ser 669 is phosphorylated even when HIPK1 levels are not increased, demonstrated by the decrease in the residual levels of hyperphosphorylated Daxx in the presence of K219A when this site was mutated (Fig. 5, compare wild-type Daxx with K219A to Daxx-S669 with K219A). Therefore, Daxx Ser 669 is likely phosphorylated by endogenous HIPK1. Ideally, we would like to have examined the affect of HIPK1 on Daxx more directly by including HIPK1 and K219A in the transcriptional reporter assay. Unfortunately, it was difficult to distinguish direct HIPK1 repressive effects from the indirect HIPK1 repressive effects through Daxx.
In addition to Ser 669, six other Daxx residues were found to be phosphorylated by microcapillary liquid chromatography-tandem mass spectrometry. Ser 502 was the only other regulated site of Daxx phosphorylation identified. However, our data demonstrate that Ser 502 is not phosphorylated by HIPK1. An unidentified regulated site of Daxx phosphorylation may also exist, as Daxx-Ser 669 presented as two bands when expressed with HIPK1. This is not surprising, as microcapillary liquid chromatography-tandem mass spectrometry does not normally yield complete protein sequence. The significance of phosphorylation at this site and S502A is unclear. Five Daxx residues were defined as being constitutively phosphorylated. However, lambda phosphatase treatment of Daxx did not cause a significant downshift in basal phosphorylated Daxx by Western blot (Fig. 3A). It is possible that these sites are insensitive to the phosphatase treatment. Alternatively, we have not corroborated phosphorylation at these sites by secondary methods and cannot exclude the possibility that these represent artifactual phosphorylation sites.
Our findings reveal that HIPK1 and Daxx collaborate in regulating transcription. This collaboration is likely important in cell growth and viability control. The HIPKs are related to a group of protein kinases that regulate the transition from growth to differentiation in eukaryotic cells (15, 23). More recent evidence showed that the HIPKs themselves participate in important growth-regulatory pathways (5, 10, 26, 28). In addition, Daxx is an essential gene and may under certain circumstances mediate apoptotic signals (3, 21, 25, 32). Consequently, the regulation of Daxx by HIPK1 may ultimately prove to be important in further elucidating their individual and cooperative roles in cell growth and viability.
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ACKNOWLEDGMENTS |
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J.A.E. is a Howard Hughes Medical Institute Research Fellow. J.S.M. was supported by a Breast Cancer Research Fellowship from the Department of Defense.
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FOOTNOTES |
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Present address: Department of Exploratory Science, Biogen Inc., Cambridge, MA 02142.
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REFERENCES |
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2. Cermak, L., A. Imova, A. Pintzas, V. Horeji, and L. Andera. 2001. Molecular mechanisms involved in CD43-mediated apoptosis of TF-1 cells: roles of transcription, Daxx expression and adhesion molecules. J. Biol. Chem. 31:31.
3. Chang, H. Y., H. Nishitoh, X. Yang, H. Ichijo, and D. Baltimore. 1998. Activation of apoptosis signal-regulating kinase 1 (ASK1) by the adapter protein Daxx. Science 281:1860-1863.
4. Choi, C. Y., Y. H. Kim, H. J. Kwon, and Y. Kim. 1999. The homeodomain protein NK-3 recruits Groucho and a histone deacetylase complex to repress transcription. J. Biol. Chem. 274:33194-33197.
5. D'Orazi, G., B. Cecchinelli, T. Bruno, I. Manni, Y. Higashimoto, S. Saito, M. Gostissa, S. Coen, A. Marchetti, G. Del Sal, G. Piaggio, M. Fanciulli, E. Appella, and S. Soddu. 2002. Homeodomain-interacting protein kinase-2 phosphorylates p53 at Ser 46 and mediates apoptosis. Nat. Cell Biol. 4:11-19.[CrossRef][Medline]
6. Emelyanov, A. V., C. R. Kovac, M. A. Sepulveda, and B. K. Birshtein. 2002. The interaction of Pax5 (BSAP) with Daxx can result in transcriptional activation in B cells. J. Biol. Chem. 17:17.
7. Epstein, J. A., D. N. Shapiro, J. Cheng, P. Y. Lam, and R. L. Maas. 1996. Pax3 modulates expression of the c-Met receptor during limb muscle development. Proc. Natl. Acad. Sci. USA 93:4213-4218.
8. Fagioli, M., M. Alcalay, P. P. Pandolfi, L. Venturini, A. Mencarelli, A. Simeone, D. Acampora, F. Grignani, and P. G. Pelicci. 1992. Alternative splicing of PML transcripts predicts coexpression of several carboxy-terminally different protein isoforms. Oncogene 7:1083-1091.[Medline]
9. Gygi, S. P., D. K. Han, A. C. Gingras, N. Sonenberg, and R. Aebersold. 1999. Protein analysis by mass spectrometry and sequence database searching: tools for cancer research in the post-genomic era. Electrophoresis 20:310-319.[CrossRef][Medline]
10. Hofmann, T. G., A. Moller, H. Sirma, H. Zentgraf, Y. Taya, W. Droge, H. Will, and M. L. Schmitz. 2001. Regulation of p53 activity by its interaction with homeodomain-interacting protein kinase-2. Nat. Cell Biol. 4:1-10.
11. Hollenbach, A. D., J. E. Sublett, C. J. McPherson, and G. Grosveld. 1999. The Pax3-FKHR oncoprotein is unresponsive to the Pax3-associated repressor hDaxx. EMBO J. 18:3702-3711.[CrossRef][Medline]
12. Ishov, A. M., A. G. Sotnikov, D. Negorev, O. V. Vladimirova, N. Neff, T. Kamitani, E. T. Yeh, J. F. Strauss 3rd, and G. G. Maul. 1999. PML is critical for ND10 formation and recruits the PML-interacting protein Daxx to this nuclear structure when modified by SUMO-1. J. Cell Biol. 147:221-234.
13. Kaelin, W. G., Jr., W. Krek, W. R. Sellers, J. A. DeCaprio, F. Ajchenbaum, C. S. Fuchs, T. Chittenden, Y. Li, P. J. Farnham, M. A. Blanar, et al. 1992. Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell 70:351-364.[CrossRef][Medline]
14. Kim, Y. H., C. Y. Choi, and Y. Kim. 1999. Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1. Proc. Natl. Acad. Sci. USA 96:12350-12355.
15. Kim, Y. H., C. Y. Choi, S. J. Lee, M. A. Conti, and Y. Kim. 1998. Homeodomain-interacting protein kinases, a novel family of co-repressors for homeodomain transcription factors. J. Biol. Chem. 273:25875-25879.
16. Lehembre, F., S. Muller, P. P. Pandolfi, and A. Dejean. 2001. Regulation of Pax3 transcriptional activity by SUMO-1-modified PML. Oncogene 20:1-9.[CrossRef][Medline]
17. Li, H., C. Leo, J. Zhu, X. Wu, J. O'Neil, E. J. Park, and J. D. Chen. 2000. Sequestration and inhibition of Daxx-mediated transcriptional repression by promyelocytic leukemia protein. Mol. Cell. Biol. 20:1784-1796.
18. Li, R., H. Pei, D. K. Watson, and T. S. Papas. 2000. EAP1/Daxx interacts with ETS1 and represses transcriptional activation of ETS1 target genes. Oncogene 19:745-753.[CrossRef][Medline]
19. MacGregor, G. R., and C. T. Caskey. 1989. Construction of plasmids that express E. coli beta-galactosidase in mammalian cells. Nucleic Acids Res. 17:2365.
20. Martin, S. S., and P. Leder. 2001. Human MCF10A mammary epithelial cells undergo apoptosis following actin depolymerization that is independent of attachment and rescued by Bcl-2. Mol. Cell. Biol. 21:6529-6536.
21. Michaelson, J. S., D. Bader, F. Kuo, C. Kozak, and P. Leder. 1999. Loss of Daxx, a promiscuously interacting protein, results in extensive apoptosis in early mouse development. Genes Dev. 13:1918-1923.
22. Missero, C., M. T. Pirro, S. Simeone, M. Pischetola, and R. Di Lauro. 2001. The DNA glycosylase T:G mismatch-specific thymine DNA glycosylase represses thyroid transcription factor-1-activated transcription. J. Biol. Chem. 276:33569-33575.
23. Moilanen, A. M., U. Karvonen, H. Poukka, O. A. Janne, and J. J. Palvimo. 1998. Activation of androgen receptor function by a novel nuclear protein kinase. Mol. Biol. Cell 9:2527-2543.
24. Niwa, H., K. Yamamura, and J. Miyazaki. 1991. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193-199.[CrossRef][Medline]
25. Perlman, R., W. P. Schiemann, M. W. Brooks, H. F. Lodish, and R. A. Weinberg. 2001. transforming growth factor beta-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation. Nat. Cell Biol. 3:708-714.[CrossRef][Medline]
26. Pierantoni, G. M., M. Fedele, F. Pentimalli, G. Benvenuto, R. Pero, G. Viglietto, M. Santoro, L. Chiariotti, and A. Fusco. 2001. High mobility group I (Y) proteins bind HIPK2, a serine-threonine kinase protein which inhibits cell growth. Oncogene 20:6132-6141.[CrossRef][Medline]
27. Pinkas, J., and P. Leder. 2002. MEK1 signaling mediates transformation and metastasis of EpH4 mammary epithelial cells independent of epithelial to mesenchymal transition. Cancer Res. 65:4781-4790.
28. Rochat-Steiner, V., K. Becker, O. Micheau, P. Schneider, K. Burns, and J. Tschopp. 2000. FIST/HIPK3: a Fas/FADD-interacting serine/threonine kinase that induces FADD phosphorylation and inhibits Fas-mediated Jun NH2-terminal kinase activation. J. Exp. Med. 192:1165-1174.
29. Slansky, J. E., Y. Li, W. G. Kaelin, and P. J. Farnham. 1993. A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter. Mol. Cell. Biol. 13:1610-1618.
30. Torii, S., D. A. Egan, R. A. Evans, and J. C. Reed. 1999. Human Daxx regulates Fas-induced apoptosis from nuclear PML oncogenic domains (PODs). EMBO J. 18:6037-6049.[CrossRef][Medline]
31. Wang, Y., K. M. Debatin, and H. Hug. 2001. HIPK2 overexpression leads to stabilization of p53 protein and increased p53 transcriptional activity by decreasing Mdm2 protein levels. BMC Mol. Biol. 2:8.
32. Yang, X., R. Khosravi-Far, H. Y. Chang, and D. Baltimore. 1997. Daxx, a novel Fas-binding protein that activates JNK and apoptosis. Cell 89:1067-1076.[CrossRef][Medline]
33. Zhong, S., P. Salomoni, S. Ronchetti, A. Guo, D. Ruggero, and P. P. Pandolfi. 2000. Promyelocytic leukemia protein (PML) and Daxx participate in a novel nuclear pathway for apoptosis. J. Exp. Med. 191:631-640.
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