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Originally published In Press as doi:10.1074/jbc.M507227200 on January 9, 2006

J. Biol. Chem., Vol. 281, Issue 11, 7489-7497, March 17, 2006
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Phosphorylation and Transactivation of Pax6 by Homeodomain-interacting Protein Kinase 2*

Eun A. Kim{ddagger}1, Yoon Tae Noh{ddagger}1, Myung-Jeom Ryu§, Hyun-Taek Kim2, Sung-Eun Lee||, Cheol-Hee Kim2, Cheolju Lee§3, Young Ho Kim**4, and Cheol Yong Choi{ddagger}5

From the {ddagger}Department of Biological Science, Sungkyunkwan University, 300 Chunchundong, Jangangu, Suwon 440-746, South Korea, §Life Sciences Division, Korea Institute of Science and Technology, Seongbuk-Gu, Seoul 136-791, South Korea, the Department of Biology, Chungnam National University, Taejeon 305-764, South Korea, the ||Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, Hawaii 96822, and **Digital Biotech, 1227 Shingildong, Ansan 425-839, South Korea

Received for publication, July 5, 2005 , and in revised form, January 9, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pax6 is a transcriptional activator that contains two DNA binding domains and a potent transcription activation domain in the C terminus, which regulates organogenesis of the eye, nose, pancreas, and central nervous system. Homeodomain-interacting protein kinase 2 (HIPK2) interacts with transcription factors, including homeoproteins, and regulates activities of transcription factors. Here we show that HIPK2 phosphorylates the activation domain of Pax6, which augments Pax6 transactivation by enhancing its interaction with p300. Mass spectrometric analysis identified three Pax6 phosphorylation sites as threonines 281, 304, and 373. The substitutions of these threonines with alanines decreased Pax6 transactivation, whereas substitutions to glutamic acids increased transactivation in mimicry of phosphorylation. Furthermore, the knock-down of either endogenous or exogenous HIPK2 expression with HIPK2 shRNA markedly inhibited Pax6 phosphorylation and its transactivating function on proglucagon promoter in cultured cells. These results strongly indicate that HIPK2 is an upstream protein kinase for Pax6 and suggest that it modulates Pax6-mediated transcriptional regulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pax proteins are key regulators of vertebrate organogenesis and are involved in embryonic pattern formation (1). Pax6 is a member of the Pax gene family and is expressed in eye, nose, pancreas, and central nervous system from the early stages of embryonic development (2). Moreover, Pax6 plays an important role in eye morphogenesis in animals. The human Pax6 gene was initially identified by the positional cloning of the 11p13 aniridia locus (3). Evolutionary conservation of Pax6 function is evidenced by the fact that mutations in the Pax6 homologue cause ocular phenotypes in Drosophila, mice, and humans (4, 5), and the ectopic expressions of Drosophila or mammalian Pax6 genes in the developing Drosophila eye leads to the induction of ectopic eyes (6, 7). In addition to its role in eye morphogenesis, Pax6 has important functions in the development of the brain, spinal cord, and pancreas. Much of the information about the roles of Pax6 in development comes from the analysis of mutant mice. Homozygous Pax6Sey and Pax6Sey-Neu mutants lack eyes and a nose and die at birth with severe abnormalities of the central nervous system, and the numbers of all four types of endocrine cells in the pancreas are decreased, and islet morphology is abnormal (8, 9). In addition, mice with heterozygous mutations in Pax6 have lower levels of pancreatic hormones (10), and the conditional inactivation of Pax6 in the pancreas causes early onset diabetes (11). Consistently, consensus Pax6-binding sites have been described in the promoter regions of proglucagon, somatostatin, and insulin genes, and Pax6 has been shown to activate their gene expression (10, 12-14).

The Pax6 protein is composed of several distinct domains, an amino-terminal paired domain, a glycine-rich hinge region, a homeodomain, and a carboxyl-terminal proline/serine/threonine (PST)-rich transactivation domain (1). Both the paired domain and homeodomain have independent DNA binding activities, but they have also been shown to act cooperatively to mediate transcription (1, 15-19). It has been repeatedly reported that various nonsense or missense mutations of Pax6 are associated with aniridia, keratitis, and familial foveal dysplasia. In many cases, Pax6 mutations are found in either the paired domain or the homeodomain, pointing to the importance of their cooperative action of two DNA binding domains (20, 21). However, several truncation mutations have been found to occur in the C-terminal half of Pax6 in patients with aniridia (22). Such truncations result in mutant proteins that retain the DNA binding domains but have lost all or part of the transactivation domain. In addition, the position of the premature stop codon found in the Pax6Sey-Neu mutant mouse strain is located within the activation domain (23, 24). Therefore, both the DNA binding activity and the transactivation activity of Pax6 appear to be critical for the proper functioning of Pax6 during development.

Several signaling pathways are known to be involved in the Pax6-mediated developmental program. In the spinal cord and hind brain, Pax6 establishes distinct ventral progenitor cell populations and controls the identity of motor neurons and ventral interneurons by mediating graded Shh signaling (25, 26). The expressions of two Drosophila orthologs of Pax6, eyeless and twin of eyeless, are induced by Notch signaling. In Xenopus embryos, the activation of Notch signaling causes eye duplication and proximal eye defects, but the molecular mechanism of its control remains largely unknown (6). In addition, Pax6 is essential for the insulin responsiveness of proglucagon promoter (27). Insulin inhibits proglucagon gene transcription through the conserved regulatory elements onto which Pax6 binds, and the inhibition of glucagon synthesis and the secretion of insulin are important for the coordinated synthesis and secretion of biologically antagonistic islet hormones. However, the regulations of Pax6 at the molecular level for each signaling pathway remain to be understood.

Homeodomain-interacting protein kinase 2 (HIPK2)6 interacts with transcription factors, including homeoproteins, and has been demonstrated to regulate the activities of transcription factor (28, 29). HIPK2 also interacts with Groucho corepressor and p300/CREB-binding protein coactivator and regulates the transcription of various genes in a context-dependent manner (30, 31). Previously, we expressed constitutive active DHIPK2(KD) and dominant negative kinase-dead DHIPK2(KR) in the developing eye of Drosophila and observed occasional ectopic eyes and small eyes, respectively. Also, DHIPK2 interacted with and phosphorylated Eyeless in vitro and in vivo. Moreover, the transcriptional activities of Eyeless increased upon co-expression of DHIPK2 in transient transcription assays (30).

In an attempt to understand the mechanisms underlying the phosphorylation-dependent transactivation of Pax6, we examined the trans-activating properties of Pax6 induced by HIPK2-mediated phosphorylation. We report here that Pax6 transactivation can be augmented by HIPK2-mediated phosphorylation of Pax6. Using both LC-MALDI-MS/MS and LC-electrospray ionization-MS/MS analysis, we identified three phosphorylation sites of Pax6 as Thr-281, Thr-304, and Thr-373. Mutation analysis of these phosphorylation sites further demonstrated that multiple phosphorylations cooperatively contribute to the recruitment of p300 and consequently enhance Pax6 transactivation. However, the knock-down of either endogenous or exogenous HIPK2 expression via HIPK2 shRNA reduced Pax6 phosphorylations and transactivation. This finding suggests that HIPK2 is an upstream kinase for Pax6.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfection—U2OS cells (human osteosarcoma cell line) and STC-1 cells (mouse intestinal endocrine cell line) were grown in Dulbecco's modified Eagle's medium and RPMI 1640, respectively, and the both media were supplemented with 10% fetal bovine serum. For immunoblot analysis, U2OS cells were seeded onto 6-well plates, and DNA transfection was carried out using Fugene6 reagent (Roche Applied Science). For reverse transcription-PCR analysis, STC-1 cells were transfected with Pax6 and HIPK2 expression plasmids using magnetofection kits (OZ Biosciences).

Plasmid Construction and Site-directed Mutagenesis—Various HIPK2 constructs, p300 expression plasmid, and its deletion mutants have been described previously (29, 30, 32). pEntr-Pax6 plasmid was constructed by the insertion of PCR-amplified cDNA from a mouse embryonic cDNA library (Clontech) into the EcoRI and XhoI sites of pEntr3C (Invitrogen). GAL4-Pax6, GST-Pax6, and Myc-Pax6 expression plasmids were generated using Gateway Technology (Invitrogen). Point mutants of Pax6 phosphorylation sites were generated using a QuikChange mutagenesis kit (Stratagene) according to the manufacturer's recommendations. Mutations were verified by DNA sequencing. Mutagenesis was conducted on pEntr-derived Pax6 plasmid, and Myc-tagged and GST fusion Pax6 expression plasmids were generated using Gateway Technology. HIPK2 shRNA plasmid was constructed by inserting double-stranded oligonucleotides, which contain the HIPK2 sequence (5'-GAAAGTACATTTTCAACTG-3'), into the BglII and HindIII sites of pSUPER (OligoEngine) as per the manufacturer's recommendations. The reporter plasmid containing the proglucagon promoter upstream of the luciferase gene was constructed by inserting DNA fragments for proglucagon promoter (from -350 to +63) into the NheI and XhoI sites of pGL3-basic (Promega). The proglucagon promoter was PCR-amplified from rat genomic DNA with specific primers as follows: Glu-350 (forward), 5'-GATGCTAGCAATACCAAATCAAGGGATAAG-3'; Glu-63 (reverse), 5'-GATCTCGAGATCTAGACAGAGGGAGTCCCC-3'. pEntr-p300 and pEntr-p300{Delta}HAT (deletion from aa 1472 to 1522, corresponding to the HAT domain) mutant were constructed by insertion of PCR-amplified full-length and combined DNA fragments (SalI-HindIII and HindIII-NotI fragments) into the SalI and NotI sites of pEntr4, respectively. Myc-p300 and Myc-p300{Delta}HAT expression plasmids were generated using Gateway Technology. The primers (restriction enzyme sites are underlined) for the PCR amplifications were as follows: p300-ATG (forward), 5'-GATGTCGACCATGGCCGAGAATGTGGTGGAA-3'; p300-term (backward), 5'-GATGCGGCCGCCTAGTGTATGTCTAGTGTACT-3'; p300-aa1471 (backward), 5'-GATAAGCTTTTTTTTGTACCATTCCTGCAG-3'; p300-aa1523 (forward), 5'-GATAAGCTTGAGGAAGAAGAGAGAAAACGA-3'.

Luciferase Reporter Assay—For luciferase reporter assays, U2OS cells seeded onto 6-well plates were transfected with G5-TK-Luc reporter plasmid, which harbored the luciferase gene under the control of a thymidine kinase minimal promoter and five copies of GAL4 binding sites, and with combinations of GAL4-Pax6 and HIPK2 or HIPK2 K221R mutant expression plasmids, together with pCMV-beta-gal, which was used to normalize transfection efficiencies. For the proglucagon promoter-luciferase reporter assay, cells were transfected with Pax6, HIPK2, and p300 expression plasmids as indicated in Figs. 2 and 5. Total plasmid amounts were adjusted using empty vectors. Thirty-six hours after transfection, luciferase activity was measured from duplicate plates using the Luciferase Reporter Assay System (Promega) and a Genios luminometer (TECAN). Each experiment was repeated at least three times. Statistical analyses were performed by Student's t test in which calculations were performed by using the INSTAT program (GraphPad, San Diego, CA).

In Vitro Pull-down Assays—p300 deletion constructs were subjected to in vitro translation using a TNT-coupled reticulocyte lysate system (Promega). Pull-down assays were performed by incubating equal amounts of GST or GST-Pax6 fusion proteins, immobilized onto glutathione-Sepharose beads, with the in vitro translated 35S-labeled truncated form of p300, as described previously (33). This mixture was placed onto a rocking platform for 2 h and washed five times with buffers containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% Nonidet P-40. Bound proteins were then eluted, separated by 8% SDS-polyacrylamide gel electrophoresis, and autoradiographed.

Chromatin Immunoprecipitation (ChIP) Assays—ChIP assays were performed using a ChIP assay kit (Upstate%20Biotechnology">Upstate Biotechnology) as previously described (29). Cells were transfected with the plasmids (3 µg of reporter plasmids and 6 µg of expression plasmids) and fixed with 1% formaldehyde for 10 min before harvesting. Cross-linked chromatin was immunoprecipitated with antibodies to p300 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or anti-Myc antibodies. Total DNA (50 µl) recovered from the immunoprecipitates was subjected to quantitative PCR (22 cycles of 30 s at 94 °C, 30 s at 65 °C, 45 s at 72 °C) using specific primers (5'-GATGCTAGCAATACCAAATCAAGGGATAAG-3' and 5'-GATCTCGAGATCTAGACAGAGGGAGTCCCC-3') for proglucagon promoter.

In Vitro Phosphorylation and Electrophoretic Mobility Shift Assay—Equal amounts (0.5 µg) of GST-Pax6 fusion protein or mutant proteins were mixed with purified GST-HIPK2(KD)-(1-629) and 0.4 µCi of [{gamma}-32P]ATP in 30 µl of kinase buffer (50 mM HEPES, pH 7.0, 0.1 mM EDTA, 0.01% Brij, 0.1 mg/ml bovine serum albumin, 0.1% beta-mercaptoethanol, 0.15 M NaCl) and incubated for 30 min at 30 °C. The phosphorylated Pax6 proteins were resolved in 8% SDS-PAGE and autoradiographed. In order to detect phosphorylated Pax6 with phospho-specific antibody (Cell Signaling), kinase reactions were performed using 0.1 mM cold ATP. Electrophoretic mobility shift assays were performed as previously described (28) with a 32P-labeled double-stranded DNA probe (5'-CCCATTATTTACAGATGAGAAATTTATATGTCAGCGTAAA-3') that contains the Pax6 target sequences. Nuclear extracts prepared from cells transfected with wild-type Pax6 or point mutants were incubated with 32P-labeled DNA probe in binding buffer containing 25 mM HEPES (pH 7.5), 3 mM MgCl2, 1 mM EDTA, 0.5% Nonidet P-40, 10% glycerol, and 1 µg of poly(dI-dC). Reactions were incubated at room temperature for 15 min and analyzed on 5% polyacrylamide gels in 0.5x Tris borate buffer.


Figure 1
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  		FIGURE 1.
Phosphorylation of Pax6 by HIPK2. A, wild-type (WT) GFP-HIPK2 or deletion mutants were transfected into U2OS cells along with Myc-Pax6 expression plasmids. Cell lysates were immunoprecipitated (IP) with anti-Myc antibody, and the resulting precipitates were visualized by Western blotting (WB) using anti-GFP antibody. Lanes 1-4 and 5-8 indicate the proteins before and after co-immunoprecipitation, respectively. B, in vitro translated 35S-labeled HIPK2 was subjected to GST pull-down analysis with equal amounts (1 µg) of affinity-purified GST or GST-Pax6. Input, 10% of protein used. C, wild-type GFP-HIPK2 or deletion mutants were transfected into U2OS cells along with Myc-Pax6 expression plasmids. The phosphorylated Myc-Pax6 was detected by Western blotting using anti-Myc antibody. P-Pax6, indicates phosphorylated Pax6 with delayed migration. The schematics of the HIPK2 expression plasmid used are shown at the top. Both KR and KDKR denote kinase-dead mutants in which lysine 221 residues were substituted with arginines. D, wild-type GST-Pax6 or deletion mutants were expressed in E. coli BL21 and affinity-purified and then subjected to in vitro phosphorylation using purified GST-HIPK2(KD)-(1-629), as described previously (30). The schematics for Pax6 proteins are shown at the top. CBB, protein staining with Coomassie Brilliant Blue.

 
Reverse Transcription-PCR—First strand cDNA synthesis was performed with 2 µg of total RNA using oligo(dT) primers and avian myeloblastosis virus reverse transcriptase (Roche Applied Science). One-twentieth of the reaction product was used for PCR amplification (DNA denaturing at 94 °C for 30 s, primer annealing at 62 °C for 30 s, primer extension at 72 °C for 40 s). The following proglucagon-specific primers were used for PCR amplification: Glu nt65, 5'-CCCTTCAAGACACAGAGGAGAA-3'; Glu nt392, 5'-TCTCGCCTTCCTCGGCCTTTCA-3'.

Immunocytochemistry—U2OS cells were grown on coverslips and transfected with 0.2 µg of EGFP-C2 plasmids and 3 µg of the HIPK2 shRNA expression plasmid. Thirty-six hours after transfection, cells were fixed with 100% methanol for 5 min at -20 °C and incubated with a solution containing 1x phosphate-buffered saline and 0.5% Triton X-100. Cells were rinsed with 1x phosphate-buffered saline containing 1% bovine serum albumin and incubated with anti-HIPK2 rabbit polyclonal antibody for 1 h. After washing five times with 1x phosphate-buffered saline, cells were incubated with anti-rabbit secondary antibody conjugated with rhodamine red (Molecular Probes, Inc., Eugene, OR). Fluorescence microscopy was performed with a Zeiss Axiopgoto 2 microscope, using excitation wavelengths of 543 nm (rhodamine red) and 488 nm (GFP). The acquired images were processed with Adobe Photoshop.

Determination of Phosphorylation Sites by Tandem MS (MS/MS)—Electrophoretically separated phosphorylated GST-PAX6 protein was excised and stain-stripped in 50% acetonitrile, 25 mM ammonium bicarbonate. Proteolytic peptides were recovered from the gel by in-gel digestion using 12 ng/µl sequencing grade chymotrypsin (Roche Applied Science) in 25 mM ammonium bicarbonate. Protein digests were separated using an Agilent 1100 Series Capillary LC system (Agilent Technologies) running at a flow rate of 1.2 µl/min on a microcapillary analytical column (150-µm inner diameter x 150 mm). The column was prepared by packing fused silica capillary with 200-Å Magic C18AQ resin (Michrom BioResources Inc.). Peptides were eluted using a linear gradient from solution A (0.1% trifluoroacetic acid, 5% acetonitrile, 95% water) to solution B (0.1% trifluoroacetic acid, 40% acetonitrile, and 60% water) over 60 min. Eluent was delivered to an on-line AccuSpot micro-fractionation system (Shimadzu Corp.), mixed coaxially with a solution of MALDI matrix (7 mg/ml {alpha}-cyano-4-hydroxycinnamic acid), and deposited as discrete spots on 576-well MALDI target plates. Each spot represented a 10-s fraction of a 1-h reversed phase gradient. MALDI-MS/MS analyses were performed using a 4700 Proteomics Analyzer (Applied Biosystems), a tandem time-of-flight mass spectrometer. The mass spectrometer was set to acquire positive ion MS survey scans over the mass range of 700-3500 Da. Once the MS survey scans had been completed, data were processed to generate a list of precursor ions for interrogation by tandem MS. MS/MS was performed with air as the collision gas at a pressure of 10-6 torr. The resultant data were first quality-filtered such that spectra lacking neutral loss of 98 Da were removed prior to manual inspection.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phosphorylation of Pax6 by HIPK2—We have previously demonstrated that DHIPK2 interacted with and phosphorylated Eyeless, a Drosophila homologue of mammalian Pax6, and augmented the transactivation of Eyeless, according to the results of transient transcription assays (30). In order to gain further insight into HIPK2-mediated Pax6 regulation in the mammalian system, we first examined the interaction between HIPK2 and Pax6 in vivo and in vitro. U2OS cells were transfected with plasmids encoding wild-type GFP-HIPK2 or deletion mutants (Fig. 1C) along with plasmid encoding Myc-Pax6. Immunoprecipitation of the lysates of transfected cells revealed that both the N- and C-terminal HIPK2 as well as full-length HIPK2 were associated with Pax6 in cultured cells (Fig. 1A). Pax6 was shown to strongly interact with the kinase-dead N-terminal domain (KDKR), but not with catalytically active N-terminal domain (KD), suggesting that Pax6 is a phosphorylation target of HIPK2. Direct physical interactions between HIPK2 and Pax6 were confirmed by GST pull-down analysis with GST-Pax6 in vitro (Fig. 1B). The phosphorylation of Pax6 by HIPK2 was also assessed in cultured cells, in which Pax6 and various HIPK2 deletion mutants were co-expressed (Fig. 1C). The migrations of Pax6 in Western blot were delayed by the co-expression of wild-type or the kinase domain of HIPK2 (lanes 2 and 4) but not by the co-expression of C-terminal or kinase-dead HIPK2 (lanes 3, 5, and 6). These results suggest that Pax6 is a phosphorylation target of HIPK2 in cultured cells. In order to delineate the site(s) at which Pax6 is phosphorylated by HIPK2, the affinity-purified GST-Pax6 deletion mutants were assessed with regard to whether it could be phosphorylated by HIPK2 in vitro. As shown in Fig. 1D, only the activation domain, not the paired domain or homeodomain, of Pax6 was strongly phosphorylated by affinity-purified GST-HIPK2. These results indicated that HIPK2 interacts with and phosphorylates mammalian Pax6 both in vitro and in vivo in the same manner as Drosophila DHIPK2 interacts with and phosphorylates Eyeless.


Figure 2
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  		FIGURE 2.
Transactivation of Pax6 by HIPK2 and p300. A, expression plasmids encoding wild-type Pax6 or deletion mutants fused to DNA binding domain of GAL4 were transfected into U2OS cells along with G5-TK-luciferase reporter plasmids in combination with HIPK2 or kinase-dead HIPK2 K221R expression plasmids. Thirty-six hours after transfection, luciferase activity was measured using the luciferase reporter assay system (Promega). Transfection efficiency was normalized by the expression of beta-galactosidase. The S.E. value is indicated. Note the statistical significance (*, p < 0.05). B, the expression plasmids encoding HIPK2, HIPK2 KR, Pax6, and p300 were transfected into U2OS cells in combination as indicated in the figure. The plasmid containing the luciferase gene under the control of proglucagon promoter (-350 to +63) was used as reporter. Transcription assays were performed described above. Each experiment was repeated at least three times. The S.E. value is indicated. C, reverse transcription-PCR (RT-PCR) analysis was performed with specific primers for the proglucagon gene using mRNA isolated from transfected STC-1 cells. Lane 1, no transfection; lane 2, Pax6 expression; lane 3, both Pax6 and HIPK2 expression. Actin gene was used as a negative control.

 


Figure 3
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  		FIGURE 3.
Recruitment of p300 by phosphorylated Pax6. A, the expression plasmids encoding Pax6, HIPK2, HIPK2 KR, and p300, in combination as indicated in the figure, were transfected into U2OS cells together with reporter plasmid containing the proglucagon promoter. Cells were fixed 36 h after transfection. Cell lysates of these transfected cells were immunoprecipitated (IP) with anti-p300 antibody (Santa Cruz Biotechnology), and co-precipitated proglucagon promoter was PCR-amplified using specific primers after reverse reaction for cross-linking. Input (lanes 1-4), DNA in cell lysates before immunoprecipitation. B, p300 deletion mutants were in vitro translated in the presence of [35S]methionine and then subjected to GST pull-down analyses using equal amounts of affinity-purified GST-Pax6. The p300 plasmids used in this assay are depicted at the bottom, and the results of p300 binding to Pax6 are summarized to the right of the schematics. I, G, and P, input, GST, and GST-Pax6, respectively. C, full-length GST-Pax6 or phosphorylated GST-Pax6 was incubated with increasing amounts of 35S-labeled p300 C/H3 (aa 1620-1891), and the bound proteins were separated on 8% SDS-PAGE and autoradiographed. Affinity-purified GST-Pax6 and phosphorylated GST-Pax6 used in this assay are shown at the bottom (lanes 8 and 9).

 
Both p300 and HIPK2 Augment Transactivation of Pax6—We previously observed that Eyeless transactivation is increased by DHIPK2 in transcription assays (30). To assess the relationship between Pax6 phosphorylation and its enhanced transactivation by HIPK2, U2OS cells were transfected with plasmids encoding wild-type GAL4-Pax6 or deletion mutants along with reporter plasmid, in combination with HIPK2 expression plasmids, as shown in Fig. 2A. The transcriptional activities of GAL4-Pax6 were increased by the expression of wild-type HIPK2 but not by kinase-dead HIPK2 (Fig. 2A, lanes 3 and 4). The transactivation of Pax6 by HIPK2 in a heterologous GAL4 system suggests that its transactivation might be induced by an increase in the transactivating properties of Pax6 rather than by an alteration in its DNA binding activity. Consistently, dissections of the Pax6 domain showed that the activation domain, not the paired domain or homeodomain, mediated the phosphorylation-dependent transactivation of Pax6 by HIPK2 (Fig. 2A, lanes 5-8). It was previously reported that the transactivation of Pax6 is mediated through its interaction with the transcriptional co-activator p300 (34). This observation compelled us to examine whether p300 and HIPK2 exert any synergistic effects on the transcriptional activities of Pax6. U2OS cells were transfected in combination with expression plasmids encoding Pax6, HIPK2, kinase-dead HIPK2 KR, and p300 along with reporter plasmids containing the promoter region (-350 to +63) of the proglucagon gene, a well known target of Pax6. Consistent with the results obtained in conjunction with GAL4-Pax6, HIPK2 was found to augment the transcriptional activities of Pax6 in a phosphorylation-dependent manner (Fig. 2B, lanes 2-5). Consistent with this result, reverse transcription-PCR analysis using mRNA isolated from transfected STC-1 cells indicated that the expression of endogenous mRNA for proglucagon was enhanced by Pax6 expression (Fig. 2C, lane 2) and was further increased by coexpression of Pax6 and HIPK2 (Fig. 2C, lane 3). Furthermore, p300 and HIPK2 additively enhanced Pax6 transcriptional activities, whereas kinase-dead HIPK2 did not (Fig. 2B, lanes 6-9). These results suggest that recruitment of p300 to the proglucagon promoter might be increased by HIPK2-mediated Pax6 phosphorylation.

Recruitment of p300 by Pax6 Target Gene in a Phosphorylation-dependent Manner—As described above, the additive activations of Pax6 by HIPK2 and p300 prompted us to examine the recruitment of p300 to proglucagon promoter in the presence or absence of HIPK2 using chromatin immunoprecipitation assays. Pax6 expression plasmids and reporter plasmid containing the proglucagon promoter were transfected into U2OS cells in combination with expression plasmids encoding p300, HIPK2, or HIPK2 KR, as indicated in Fig. 3A. Lysates prepared from transfected cells were immunoprecipitated with anti-p300 antibodies, and the amounts of co-precipitated proglucagon promoter were analyzed by PCR. As shown in Fig. 3A, the amount of p300 recruited by the proglucagon promoter was enhanced by wild-type HIPK2 but not by kinase-dead HIPK2. The same experiments using plasmids encoding Pax6 devoid of an activation domain exhibited no recruitment of p300 to the proglucagon promoter, thus indicating specific p300 recruitment by Pax6 activation domain (data not shown). These results strongly suggest that p300 is efficiently recruited to the proglucagon promoter after Pax6 phosphorylation by HIPK2. We then employed a GST pull-down analysis in order to identify the p300 domain responsible for its interaction with Pax6. GST-Pax6 was found to bind the p300 C terminus containing the C/H3 domain, which is known to interact with many transcription factors (35). Further analysis revealed that the C/H3 domain was primarily responsible for the interaction of p300 with Pax6 (Fig. 3B). Therefore, we examined the effects of Pax6 phosphorylation on the interaction with p300 by GST pull-down analysis. Increasing amounts of 35S-labeled p300 C/H3 domain (aa 1629-1891) were subjected to GST pull-down analysis with affinity-purified GST-Pax6 or GST-Pax6 phosphorylated by HIPK2 in vitro (Fig. 3C, top). The p300 C/H3 domain was found to bind phosphorylated GST-Pax6 more efficiently than unphosphorylated GST-Pax6 (Fig. 3C, bottom). Taken together, the phosphorylation of Pax6 by HIPK2 accelerated the recruitment of p300 to proglucagon promoter, both in vitro and in cultured cells.


Figure 4
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  		FIGURE 4.
Identification of phosphorylation sites by LC-MALDI-MS/MS. A, mass spectra were recorded using a tandem time-of-flight mass analyzer for RQASNpTPSHIPISSSF at m/z 1808.97, QPIPQPTpTPVSSF at m/z 1478.78, and pTP-PHMQTHM at m/z 1028.48. Each peptide contains a phosphorylation site of Thr-281, Thr-304, and Thr-373, respectively. Amino acids in the three-letter codes reading vertically denote immonium ions. Fragments of b ions (for RQASNpTPSHIPISSSF and QPIPQPTpTPVSSF) or b-H3PO4 ions (for pTP-PHMQTHM) are marked by dotted lines to annotate the spectra with sequence information. Peaks marked with asterisks are unassigned peaks. B, GST-Pax6 was phosphorylated in vitro using GST-HIPK2(KD), followed by Western blotting using anti-phospho-specific antibodies as indicated in the figure. CBB, protein staining with Coomassie Brilliant Blue.

 
HIPK2-mediated Pax6 Phosphorylations at Multiple Threonine Residues—To identify the phosphorylation sites in the activation domain of Pax6, GST-Pax6-(270-422) was expressed and affinity-purified and phosphorylated in vitro with HIPK2. After running the sample on SDS-gel, the phosphorylated Pax6 protein band was excised, in situ digested with chymotrypsin, and analyzed by LC-MALDI-MS/MS (Fig. 4A). Three different phosphopeptides were detected, with 65% sequence coverage. Accordingly, three phosphorylation sites were identified as Thr-281, Thr-304, and Thr-373. Phosphothreonine-containing peptides are known to lose the phosphoric acid moiety in MS/MS (36). In Fig. 4A, the MS/MS spectra of the peptides RQASNpTPSHIPISSSF and pTPPHMQTHM (where pT represents phosphothreonine) indicated that the fragment ion from the neutral loss was notably abundant and clearly showed a decrease of 98.0 Da (-H3PO4). A loss of 80.0 Da (-HPO3) was also detected, which is consistent with other observations gleaned by tandem time-of-flight mass analysis (37). The neutral loss of phosphoric acid from the peptide QPIPQPTpTPVSSF was less prominent, and its y10 fragment resulting from cleavage between Ile and Pro was stronger. The intensity of the y8 fragment in the mass spectrum of QPIPQPTpTPVSSF was also high. It has been noted that cleavage to C terminus of Pro tends to be fairly low, and there is a strong preference to cleave at its N-terminal side (38). Preferential cleavage also has been known to occur at the C terminus of beta-branched aliphatic residues, such as Ile. These results suggest that during the fragmentation process of QPIPQPTpTPVSSF, peptide cleavage is more favorable than the neutral loss of phosphoric acid because of the relative abundance of Pro residues and the labile Ile-Pro peptide bond. The other two peptides also showed preferential cleavage at the N terminus of the internal Pro residues. We also identified these three phosphorylation sites by LC-electrospray ionization-MS/MS (data not shown).


Figure 5
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  		FIGURE 5.
Mutation analysis of Pax6 phosphorylation sites. A, an equal amount of wild-type (WT) GST-Pax6 and point mutant GST-Pax6 were phosphorylated in vitro using GST-HIPK2(KD) in the presence of [{gamma}-32P]ATP (top). The same experiments were performed using cold ATP, and phosphorylated proteins were detected by Western blotting (WB) using anti-phosphothreonine antibody (middle). CBB, Coomassie Brilliant Blue staining (bottom). B, expression plasmids encoding wild-type Pax6 or triple mutant (T3A; T281A/T304A/T373A) were transfected into U2OS cells, either with or without HIPK2 expression plasmids. Pax6 and GFP-HIPK2 protein were detected by Western blotting using anti-Myc and anti-GFP antibody, respectively (lanes 1-4). Lysates from cells transfected with plasmids encoding GFP-HIPK2 and Myc-pax6 or triple mutant were immunoprecipitated with anti-Myc antibody. Precipitates were analyzed by Western blotting using anti-Myc or anti-phosphothreonine antibody (lanes 5 and 6). C, the plasmids encoding wild-type Pax6 or point mutants (Thr to Ala substitutes) were transfected into U2OS cells, either with (lanes 6-9) or without (lanes 2-5) HIPK2 expression plasmids. Plasmids encoding Pax6 point mutants (Thr to Glu mutant (lanes 11-13) and Thr to Ala mutant (lanes 14-17)) were also transfected into cells together with (lanes 15-17) or without (lanes 11-13) p300 expression plasmid. T1E, T2E, and T3E, Pax6(T304E), Pax6(T304E/T373E), and Pax6(T304E/T373E/T281E) mutant, respectively. A plasmid containing the luciferase gene under the control of the proglucagon promoter (-350 to +63) was used as a reporter. Transcription assays were performed as described above. Each experiment was repeated at least three times. Electrophoretic mobility shift assays were performed using the 32P-labeled G1 element of the proglucagon promoter and nuclear extracts from cells transfected with expression plasmids encoding wild-type Pax6 or point mutant as indicated in the figure (inset). The S.E. is indicated. D, ChIP analysis was performed as described in the legend to Fig. 3A. The expression plasmids encoding Pax6 wild type or Pax6 T3A mutant, HIPK2, HIPK2 KR, and Myc-p300 in combination as indicated in the figure were transfected into U2OS cells, together with reporter plasmids containing the proglucagon promoter. Lane 1, negative control without reporter plasmid. Cell lysates of these transfected cells were immunoprecipitated with anti-Myc antibodies, and co-precipitated proglucagon promoter was PCR-amplified using specific primers. Input (top), DNA in cell lysates before immunoprecipitation. E, GST pull-down analysis was performed as described in the legend to Fig. 3C. Full-length GST-Pax6 or GST-Pax6 T3A mutant was phosphorylated with HIPK2 and incubated with increasing amounts of 35S-labeled p300 C/H3 (aa 1620-1891), and the bound proteins were separated on 8% SDS-PAGE and autoradiographed. Affinity-purified GST-Pax6 and GST-Pax6 T3A mutant before and after phosphorylation with HIPK2 are shown at the bottom (lanes 8-11). F, the expression plasmids encoding HIPK2, Pax6, p300 (2 µg), and increasing amounts of p300{Delta}HAT (0.5, 1, and 2 µg) were transfected into U2OS cells in combination as indicated in the figure. The plasmid containing the luciferase gene under the control of proglucagon promoter was used as reporter. Transcription assays were performed described above. The S.E. is indicated.

 
To confirm the MS/MS data, in vitro phosphorylated Pax6 was subjected to Western blotting with the respective phospho-specific antibodies (Fig. 4B). Phosphorylated Pax6 was strongly detected using anti-phosphothreonine and anti-phosphoserine antibodies (Fig. 4B, lanes 4 and 6), whereas HIPK2 was detected by all phospho-specific antibodies. Western blotting signals corresponding to Pax6 phosphorylations at threonine residues were stronger than those due to phosphorylation at serine residues. Also, Pax6 phosphorylation at the serine residues was observed under longer reaction time conditions. These results indicate that the threonine residues of Pax6 are primarily phosphorylated by HIPK2. In order to validate the identified phosphorylation sites, we replaced the Thr-304, Thr-373, and Thr-281 of Pax6 with alanines. Affinity-purified double or triple mutants displayed markedly lower levels of phosphorylation by HIPK2 using [{gamma}-32P]ATP (Fig. 5A, top) and lower detection levels using anti-phosphothreonine antibodies (Fig. 5A, middle). The co-expression of Pax6 and HIPK2 in cultured cells resulted in a shift in the migration of Pax6 (Figs. 1C and 5B, lane 2). However, the migration of the Pax6 triple mutant (T281A/T304A/T373A) was not changed with overexpression of HIPK2 (Fig. 5B, lane 4). Furthermore, phosphorylation of wild-type Pax6, following the immunoprecipitation from transfected cell lysates, was detected by Western blotting using anti-phosphothreonine antibody, whereas the phosphorylation of T3A triple mutant was not (Fig. 5B, lanes 5 and 6). These results indicate that the Thr-304, Thr-373, and Thr-281 are indeed the Pax6 sites phosphorylated by HIPK2 in vitro and in cultured cells. The transcriptional activities of Pax6 mutants were examined with transcription assays using reporter plasmid containing the proglucagon promoter. The transactivating function of Pax6 triple mutant was substantially impaired, and the Pax6 activation by HIPK2 was also diminished (Fig. 5C, lanes 2-9). To rule out the possibility that this decreased transcription activity was due to reduced DNA binding activity, we conducted an electrophoretic mobility shift assay with the lysates of cells transfected with plasmids encoding wild-type or mutant Pax6. As shown in the inset of Fig. 5C, the DNA binding activities of Pax6 mutants were unaltered. In order to determine whether phosphorylation of the identified threonine residues is directly linked to the transactivation function of Pax6, transcription assays were performed using mutant Pax6 in which threonine residues were substituted by glutamic acid to confer a negative charge in mimicry of phosphorylation. These threonine-glutamic acid substitutes showed enhanced transcription activities in proportion to the number of substitutions (Fig. 5C, lanes 10-13). Furthermore, the threonine-alanine substitution mutant (T3A) failed to display transactivation upon coexpression of p300 co-activator (compare Fig. 2B, lane 9, with Fig. 5C, lane 17). Taken together, the amino acid residues identified as phosphorylation sites by tandem mass analysis could be phosphorylated by HIPK2 both in vitro and in vivo, and these sites appear to enhance Pax6 transactivation by accelerating p300 recruitment. To analyze further the correlation between Pax6 phosphorylation and p300 recruitment to the proglucagon promoter, ChIP assays were performed with wild-type and Pax6 T3A mutant as shown in Fig. 5D. Lysates prepared from transfected cells were immunoprecipitated with anti-Myc antibodies, and the amounts of co-precipitated proglucagon promoter were analyzed by PCR. Recruitment of p300 to the proglucagon promoter was enhanced by the combinatorial expression of Pax6 and HIPK2 in a phosphorylation-dependent manner (lanes 4 and 5) but not by expression of Pax6 T3A phosphorylation mutant and HIPK2 (lane 7). GST pull-down analysis also indicated that binding affinity of phosphorylated GST-Pax6 T3A mutant to the C/H3 domain of p300 was lower than that of wild-type Pax6 (Fig. 5E, lanes 2-7). These results indicate that phosphorylation of Pax6 by HIPK2 is required for efficient recruitment of p300 to the proglucagon promoter. In addition, expression of increasing amounts of p300{Delta}HAT mutant displayed dominant negative effects on the HIPK2-mediated phosphorylation-dependent Pax6 transactivation (Fig. 5F, lanes 4-6). These results suggest that phosphorylation of Pax6 by HIPK2 and concomitant recruitment of p300 result in the activation of the proglucagon promoter.


Figure 6
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  		FIGURE 6.
Inhibition of Pax6 transactivation by HIPK2 knock-down. A, HIPK2 shRNA expression plasmid and EGFP-C2 plasmid (Clontech) were transfected into U2OS cells. Thirty-six hours after transfection, cells were fixed, and endogenous HIPK2 was visualized by immunostaining using anti-HIPK2 antibody. The red signal (HIPK2) was obtained with anti-rabbit rhodamine red-conjugated secondary antibody, whereas the green signal (EGFP/HIPK2 shRNA) was obtained with GFP fluorescence. The superimposition of the two colors indicated the knock-down of endogenous HIPK2 expression in the presence of HIPK2 shRNA. B, plasmids encoding Myc-HIPK2 and Myc-Groucho were transfected into U2OS cells, either with or without plasmids for HIPK2 shRNA (lane 3) or control shRNA (lane 2). The levels of Myc-HIPK2 and Myc-Groucho were analyzed by Western blotting using anti-Myc antibody. C, plasmids encoding Myc-Pax6 were transfected into U2OS cells, either with or without plasmids expressing HIPK2 or HIPK2 shRNA. Myc-Pax6 was immunoprecipitated (IP) with anti-Myc antibody, and the precipitates were subjected to Western blotting using anti-Myc or anti-phosphothreonine antibody. D, plasmids encoding Pax6 and HIPK2 were transfected into U2OS cells, either with or without plasmid expressing HIPK2 shRNA as indicated in the figure. The plasmid containing the luciferase gene under the control of the proglucagon promoter (-350 to +63) was used as a reporter. Transcription assays were performed as described above. Experiments were repeated at least three times. The S.E. is indicated. Note the statistical significance (**, p < 0.01). DAPI, 4',6-diamidino-2-phenylindole.

 
Knock-down of either Endogenous or Exogenous HIPK2 Expression by shRNA Reduces Pax6 Transactivation—We found that HIPK2 enhances transcriptional potential of Pax6 by phosphorylation at multiple sites within the activation domain. To examine the trans-activating function of Pax6 in the absence of HIPK2, we tried to knock down either endogenous or exogenous HIPK2 expression by HIPK2 shRNA. HIPK2 shRNA plasmids were transfected into U2OS cells along with GFP expression plasmid, and the expression levels of endogenous HIPK2 were observed by immunostaining using anti-HIPK2 antibodies. The cells expressing HIPK2 shRNA could be indirectly monitored by co-transfection of GFP expression plasmid at a high ratio, of 3 µg (HIPK2 shRNA) to 0.2 µg (EGFP-C2). As shown in Fig. 6A, the GFP-positive cells expressed endogenous HIPK2 at a low level, whereas the cells expressing no GFP displayed relatively high levels of HIPK2. The effects of HIPK2 shRNA on HIPK2 levels were also examined by Western blotting. Both Myc-HIPK2 and Myc-Groucho expression plasmids were transfected into cells in the absence or presence of HIPK2 shRNA plasmid. Western blotting with anti-Myc antibody showed that HIPK2 expression was specifically knocked down (Fig. 6B, lane 3), whereas Groucho levels were unchanged (Fig. 6B, lanes 1-3), indicating that HIPK2 shRNA knocked down the expression of HIPK2. To study the phosphorylation status of Pax6 in the absence of endogenous HIPK2, we transfected Myc-Pax6 expression plasmids into cells, either with or without HIPK2 shRNA plasmid. Cell lysates were immunoprecipitated with anti-Myc antibody, and the precipitated Myc-Pax6 proteins were analyzed by Western blotting using anti-Myc antibody or anti-phosphothreonine antibody. The levels of phosphorylated Pax6 were found to be reduced with co-expression of HIPK2 shRNA, whereas Pax6 levels remained constant (Fig. 6C, lane 2). In addition, the forced expression of HIPK2 increased Pax6 phosphorylation (lane 3), which was also reduced by HIPK2 shRNA (lane 4). We next examined the transcription activities of Pax6 under HIPK2 knock-down conditions. The transactivations of Pax6 in the absence or presence of expressed HIPK2 were shown to decrease with the co-expression of HIPK2 shRNA (Fig. 6D, lanes 4 and 7). These results strongly suggest that the phosphorylation of Pax6 by HIPK2 is required for the transactivation of Pax6 and that HIPK2 is an upstream protein kinase for Pax6.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we have shown that Pax6 is a phosphorylation target of HIPK2 and that this phosphorylation is required for Pax6 transactivation. We identified three threonine residues in the activation domain of Pax6 as phosphorylation sites by HIPK2. Pax6 is composed of three functional domains, a paired domain, a homeodomain, and an activation domain. The paired/homeodomains are involved in target sequence recognition and the protein-protein interactions with its binding partners (39). The activation domain of Pax6 is also referred to as the PST domain, because this domain is rich in proline, serine, and threonine. A total of 45 serine and threonine residues constitute 29% of the activation domain. As can be predicted by viewing the amino acid composition of the serine/threonine-rich activation domain, multiple sites in the activation domain of Pax6 were phosphorylated by HIPK2. These multiple phosphorylations might contribute to the transactivation of Pax6 for the following reasons. First, serial deletion of the Pax6 activation domain gradually reduced transactivation activity. Moreover, the N- and C-terminal activation domains equally contributed to the Pax6 transactivation (40-42). Second, the transactivation function of the Pax6 activation domain is dispersed into the four constituent exons (exons 10-13). The four exon fragments act synergistically to stimulate transcription, but none of them can function individually as an independent transactivation domain (41). Third, the activation domain of mammalian Pax6 is functionally equivalent to that of Drosophila Eyeless, despite no significant homology to Eyeless except for a high proportion of proline, serine, and threonine and a large difference in the length of the activation domain (6, 7). In agreement with previous reports, we observed that the threonine-glutamic acid substitution mutants had higher transactivation activities than wild-type Pax6, and this effect was proportional to the number of Thr to Glu substitutions (Fig. 5C, lanes 11-13). Conversely, the threonine-alanine substitution mutants showed decreased transactivation in proportion to the number of substitutions (Fig. 5C, lanes 6-9). These results support the idea that multiple sites of the activation domain cooperatively contribute to the Pax6 transactivation after phosphorylation.

All of the identified phosphorylation sites of Pax6 by HIPK2 had a following proline (T281P, T304P, and T373P). The same was observed multiple phosphorylation sites in Groucho (S196 for P and PS297) (30) and well known target p53 (S46P) (43, 44). Moreover, it was recently reported that codon 47 proline is important for the phosphorylation of serine 46 and p53-mediated apoptosis (45). These findings raise the possibility that HIPK2 may be a proline-directed protein kinase and that it functionally cooperates with other proline-directed protein kinases. Serine 46 of p53 is known to be phosphorylated by HIPK2 and p38 MAP kinase (46). Also, Pax6 was known to be phosphorylated and regulated by p38 MAP kinase. The major phosphorylation site of zebrafish Pax6.1 by p38 was known to be S413P (40). In the present study, we failed to find any Pax6 serine residue phosphorylated by HIPK2 via tandem mass analysis. In addition, we did not observe any comparable change in the phosphorylation level or transcription activity of S398A Pax6 mutant, which correspond to the zebrafish Pax6.1 S413A, by HIPK2. However, we cannot rule out the possibility that the S398P is one of the multiple phosphorylation sites of Pax6 by HIPK2, because Pax6 could be phosphorylated at serine residue(s) in vitro (Fig. 4B, lane 6), and a simple single phosphorylation site mutation might lead to subtle alterations in Pax6 phosphorylation and transactivation. Given that Pax6 is a master regulator during eye development in Drosophila, mice, and humans and has been shown to have central function in neural tube formation and pancreas development, multiple signaling pathways might be implicated in the Pax6 phosphorylation and Pax6-mediated regulation of the developmental process. Therefore, investigations on functional cross-talk between various kinases upstream of Pax6 regulation might provide insights into the various organogenesis of the eye, neural tube, and pancreas.

The physiological relevance of Pax6 phosphorylation by HIPK2 could be discussed with the data obtained from targeted DHIPK2 expression during Drosophila eye development. We have expressed various forms of DHIPK2, a Drosophila homologue of mouse or human HIPK2, in the developing fly eye (30). The expression of constitutive active DHIPK2(KD) led to occasional ectopic eye formation, a typical marker of Eyeless activation. In contrast, the expression of the dominant negative kinase-dead mutant DHIPK2(KR) resulted in the small eye or eyeless phenotypes. Consistent with these eye phenotypes, DHIPK2 directly phosphorylated Eyeless and consequently enhanced the transactivation of Eyeless in a phosphorylation-dependent manner (30). Collectively, the regulation of Pax6/Eyeless function by HIPK2/DHIPK2 appears to be conserved between Drosophila and mammals.

Pax6 has been shown to be a critical regulator of brain, eye, pancreas, and central nervous system development. In particular, the transcriptional regulations of crystallin and proglucagon promoter during eye and pancreatic development, respectively, have been extensively investigated in efforts to elucidate the role of Pax6 as a transcription factor (39, 47). Here we investigated the transcriptional control of proglucagon promoter with regard to Pax6 phosphorylation by HIPK2. Upon the expression of HIPK2, transcription from the proglucagon promoter was found to be remarkably increased by Pax6 phosphorylation (Fig. 2B) but reduced by the knock-down of either endogenous or exogenous HIPK2 expression (Fig. 6D). Although it is evident from the results of this study that transcription from the proglucagon promoter is increased by HIPK2-mediated Pax6 phosphorylation, the physiological relevance of Pax6 phosphorylation by HIPK2 in glucose homeostasis remains to be elucidated. Many homeoproteins, such as Nkx6.1, Nkx2.2, Pdx1, Cdx2, and Isl1 are involved in the transcription regulation of insulin and proglucagon promoters (10, 48-51). Among these proteins, the NK family proteins are well known targets of HIPK2 (28) and other homeoproteins might also constitute targets of HIPK2. Thus, the effects of HIPK2 activation on glucose homeostasis might be complicated, and extensive biochemical and genetic analysis are required to provide an insight into the HIPK2-mediated regulation of the transcription factors involved in glucose homeostasis.


   FOOTNOTES
 
* This work was supported by Health Technology Planning and Evaluation Board Grant 02-PJ1-PG3-21001-0011 (to C. Y. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These authors contributed equally to this work. Back

2 Supported by the Neurobiology Research Program from the Korea Ministry of Science and Technology. Back

3 Supported by the Functional Proteomics Research Center of the 21st Century Frontier Research Program. Back

4 To whom correspondence may be addressed. E-mail: youngho{at}digitalbiotech.com.

5 To whom correspondence may be addressed. E-mail: choicy{at}skku.ac.kr.

6 The abbreviations used are: HIPK2, homeodomain-interacting protein kinase 2; DHIPK2, Drosophila homeodomain-interacting protein kinase 2; LC, liquid chromatography; MALDI, matrix-assisted laser desorption ionization; MS/MS, tandem mass spectrometry; Myc, Myc epitope; GST, glutathione S-transferase; GFP, green fluorescence protein; ChIP, chromatin immunoprecipitation; shRNA, small hairpin RNA; CREB, cAMP-response element-binding protein. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Yongsok Kim for helpful suggestions and for continuous support during the course this study. We also thank M. Lienhard Schmitz (University of Bern) for providing the anti-HIPK2 antibody.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Chi, N., and Epstein, J. A. (2002) Trends Genet. 18, 41-47[CrossRef][Medline] [Order article via Infotrieve]
  2. Walther, C., and Gruss, P. (1991) Development 113, 1435-1449[Abstract]
  3. Ton, C. C., Hirvonen, H., Miwa, H., Weil, M. M., Monaghan, P., Jordan, T., van Heyningen, V., Hastie, N. D., Meijers-Heijboer, H., Drechsler, M., Royer-Pokora, B., Collins, F., Swaroop, A., Strong, L. C., and Saunders, G. F. (1991) Cell 67, 1059-1074[CrossRef][Medline] [Order article via Infotrieve]
  4. Wawersik, S., and Maas, R. L. (2000) Hum. Mol. Genet. 9, 917-925[Abstract/Free Full Text]
  5. Sisodiya, S. M., Free, S. L., Williamson, K. A., Mitchell, T. N., Willis, C., Stevens, J. M., Kendall, B. E., Shorvon, S. D., Hanson, I. M., Moore, A. T., van Heyningen, V. (2001) Nat. Genet. 28, 214-216[CrossRef][Medline] [Order article via Infotrieve]
  6. Onuma, Y., Takahashi, S., Asashima, M., Kurata, S., and Gehring, W. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 2020-2025[Abstract/Free Full Text]
  7. Chow, R. L., Altmann, C. R., Lang, R. A., and Hemmati-Brivanlou, A. (1999) Development 126, 4213-4222[Abstract]
  8. Hogan, B. L., Horsburgh, G., Cohen, J., Hetherington, C. M., Fisher, G., and Lyon, M. F. (1986) J. Embryol. Exp. Morphol. 97, 95-110[Medline] [Order article via Infotrieve]
  9. Schmahl, W., Knoedlseder, M., Favor, J., and Davidson, D. (1993) Acta Neuropathol. 86, 126-135[CrossRef][Medline] [Order article via Infotrieve]
  10. Sander, M., Sussel, L., Conners, J., Scheel, D., Kalamaras, J., Dela Cruz, F., Schwitzgebel, V., Hayes-Jordan, A., and German, M. (2000) Development 127, 5533-5540[Abstract]
  11. Ashery-Padan, R., Zhou, X., Marquardt, T., Herrera, P., Toube, L., Berry, A., and Gruss, P. (2004) Dev. Biol. 269, 479-488[CrossRef][Medline] [Order article via Infotrieve]
  12. StOnge, L., SosaPineda, B., Chowdhury, K., Mansouri, A., and Gruss, P. (1997) Nature 387, 406-409[CrossRef][Medline] [Order article via Infotrieve]
  13. Andersen, F. G., Jensen, J., Heller, R. S., Petersen, H. V., Larsson, L. I., Madsen, O. D., and Serup, P. (1999) FEBS Lett. 445, 315-320[CrossRef][Medline] [Order article via Infotrieve]
  14. Ritz-Laser, B., Estreicher, A., Klages, N., Saule, S., and Philippe, J. (1999) J. Biol. Chem. 274, 4124-4132[Abstract/Free Full Text]
  15. Duncan, M. K., Haynes, J. I. 2nd, Cvekl, A., and Piatigorsky, J. (1998) Mol. Cell. Biol. 18, 5579-5586[Abstract/Free Full Text]
  16. Jun, S., and Desplan, C. (1996) Development 122, 2639-2650[Abstract]
  17. Mikkola, I., Bruun, J. A., Holm, T., and Johansen, T. (2001) J. Biol. Chem. 276, 4109-4118[Abstract/Free Full Text]
  18. Mishra, R., Gorlov, I. P., Chao, L. Y., Singh, S., and Saunders, G. F. (2002) J. Biol. Chem. 277, 49488-49494[Abstract/Free Full Text]
  19. Singh, S., Stellrecht, C. M., Tang, H. K., and Saunders, G. F. (2000) J. Biol. Chem. 275, 17306-17313[Abstract/Free Full Text]
  20. Glaser, T., Jepeal, L., Edeards, J. G., Young, S. R., Favor, J., and Maas, R. L. (1994) Nat. Genet. 7, 463-471[CrossRef][Medline] [Order article via Infotrieve]
  21. Hanson, I., Churchill, A., Love, J., Axton, R., Moore, T., Clarke, M., Meire, F., and van Heyningen, V. (1999) Hum. Mol. Genet. 8, 165-172[Abstract/Free Full Text]
  22. Prosser, J., and van Heyningen, V. (1998) Hum. Mutat. 11, 93-108[CrossRef][Medline] [Order article via Infotrieve]
  23. Favor, J., Peters, H., Hermann, T., Schmahl, W., Chatterjee, B., Neuhauser-Klaus, A., and Sandulache, R. (2001) Genetics 159, 1689-1700[Abstract/Free Full Text]
  24. Sander, M., Neubuser, A., Kalamaras, J., Ee, H. C., Martin, G. R., and German, M. S. (1997) Genes Dev. 11, 1662-1673[Abstract/Free Full Text]
  25. Ericson, J., Rashbass, P., Schedl, A., Brenner-Morton, S., Kawakami, A., van Heyningen, V., Jessell, T. M., and Briscoe, J. (1997) Cell 90, 169-180[CrossRef][Medline] [Order article via Infotrieve]
  26. Muhr, J., Andersson, E., Persson, M., Jessell, T. M., and Ericson, J. (2001) Cell 104, 861-873[CrossRef][Medline] [Order article via Infotrieve]
  27. Grzeskowiak, R., Amin, J., Oetjen, E., and Knepel, W. (2000) J. Biol. Chem. 275, 30037-30045[Abstract/Free Full Text]
  28. Kim, Y. H., Choi, C. Y., Lee, S-J., Conti, M. A., and Kim, Y. (1998) J. Biol. Chem. 273, 25875-25879[Abstract/Free Full Text]
  29. Choi, C. Y., Kim, Y. H., Kwon, H. J., and Kim, Y. (1999) J. Biol. Chem. 274, 33194-33197[Abstract/Free Full Text]
  30. Choi, C. Y., Kim, Y. H., Kim, Y. O., Park, S. J., Kim, E. A., Riemenschneider, W., Gajewski, K., Schulz, R. A., and Kim, Y. (2005) J. Biol. Chem. 280, 21427-21436[Abstract/Free Full Text]
  31. Kim, E. J., Park, J. S., and Um, S. J. (2002) J. Biol. Chem. 277, 32020-32028[Abstract/Free Full Text]
  32. Kim, Y. H., Choi, C. Y., and Kim, Y. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12350-12355[Abstract/Free Full Text]
  33. Choi, C. Y., Lee, Y. M., Kim, Y. H., Park, T., Jeon, B. H., Schulz, R. A., and Kim, Y. (1999) J. Biol. Chem. 274, 31543-31552[Abstract/Free Full Text]
  34. Hussain, M. A., and Habener, J. K. (1999) J. Biol. Chem. 274, 28950-28957[Abstract/Free Full Text]
  35. Chan, H. M., and La Thangue, N. B. (2001) J. Cell Sci. 114, 2363-2373[Abstract/Free Full Text]
  36. Tholey, A., Reed, J., and Lehmann, W. D. (1999) J. Mass Spectrom. 34, 117-123[Medline] [Order article via Infotrieve]
  37. Raska, C. S., Parker, C. E., Dominski, Z., Marzluff, W. F., Glish, G. L., Pope, R. M., and Borchers, C. H. (2002) Anal. Chem. 74, 3429-3433[Medline] [Order article via Infotrieve]
  38. Tabb, D. L., Smith, L. L., Breci, L. A., Wysocki, V. H., Lin, D., and Yates, J. R., III (2003) Anal. Chem. 75, 1155-1163[Medline] [Order article via Infotrieve]
  39. Simpson, T. L., and Price, D. J. (2002) BioEssays 24, 1041-1051[CrossRef][Medline] [Order article via Infotrieve]
  40. Mikkola, I., Bruun, J. A., Bjorkoy, G., Holm, T., and Johansen, T. (1999) J. Biol. Chem. 274, 15115-15126[Abstract/Free Full Text]
  41. Tang, H. K., Singh, S., and Saunders, G. F. (1998) J. Biol. Chem. 273, 7210-7221[Abstract/Free Full Text]
  42. Singh, S., Tang, H. K., Lee, J. Y., and Saunders, G. F. (1998) J. Biol. Chem. 273, 21531-21541[Abstract/Free Full Text]
  43. Hofmann, T. G., Moller, A., Sirma, H., Zentgraf, H., Taya, Y., Droge, W., Will, H., and Schmitz, M. L. (2002) Nat. Cell Biol. 4, 1-10[CrossRef][Medline] [Order article via Infotrieve]
  44. D'Orazi, G., Cecchinelli, B., Bruno, T., Manni, I., Higashimoto, Y., Saito, S., Gostissa, M., Coen, S., Marchetti, A., Del Sal, G., Piaggio, G., Fanciulli, M., Appella, E., and Soddu, S. (2002) Nat. Cell Biol. 4, 11-19[CrossRef][Medline] [Order article via Infotrieve]
  45. Li, X., Dumont, P., Pietra, A. D., Shetler, C., and Murphy, M. E. (2005) J. Biol. Chem. 280, 24245-24251[Abstract/Free Full Text]
  46. Bulavin, D. V., Saito, S., Hollander, M. C., Sakaguchi, K., Anderson, C. W., Appella, E., Fornace, A. J., Jr. (1999) EMBO J. 18, 6845-6854[CrossRef][Medline] [Order article via Infotrieve]
  47. Yang, Y., Chauhan, B. K., Cveklova, K., Cvekl, A. (2004) J. Mol. Biol. 344, 351-368[CrossRef][Medline] [Order article via Infotrieve]
  48. Schisler, J. C., Jensen, P. B., Taylor, D. G., Becker, T. C., Knop, F. K., Takekawa, S., German, M., Weir, G. C., Lu, D., Mirmira, R. G., and Newgard, C. B. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7297-7302[Abstract/Free Full Text]
  49. Li, Y., Cao, X., Li, L. X., Brubaker, P. L., Edlund, H., and Drucker, D. J. (2005) Diabetes 54, 482-491[Abstract/Free Full Text]
  50. Wang, M., and Drucker, D. J. (1995) J. Biol. Chem. 270, 12646-12652[Abstract/Free Full Text]
  51. Habener, J. F., Kemp, D. M., and Thomas, M. K. (2005) Endocrinology 146, 1025-1034[Abstract/Free Full Text]

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