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• W2146724496 abstract "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. 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. Pax proteins are key regulators of vertebrate organogenesis and are involved in embryonic pattern formation (1Chi N. Epstein J.A. Trends Genet. 2002; 18: 41-47Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). 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 (2Walther C. Gruss P. Development. 1991; 113: 1435-1449Crossref PubMed Google Scholar). 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 (3Ton 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. Saunders G.F. Cell. 1991; 67: 1059-1074Abstract Full Text PDF PubMed Scopus (746) Google Scholar). Evolutionary conservation of Pax6 function is evidenced by the fact that mutations in the Pax6 homologue cause ocular phenotypes in Drosophila, mice, and humans (4Wawersik S. Maas R.L. Hum. Mol. Genet. 2000; 9: 917-925Crossref PubMed Scopus (162) Google Scholar, 5Sisodiya 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. Nat. Genet. 2001; 28: 214-216Crossref PubMed Scopus (193) Google Scholar), and the ectopic expressions of Drosophila or mammalian Pax6 genes in the developing Drosophila eye leads to the induction of ectopic eyes (6Onuma Y. Takahashi S. Asashima M. Kurata S. Gehring W.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2020-2025Crossref PubMed Scopus (113) Google Scholar, 7Chow R.L. Altmann C.R. Lang R.A. Hemmati-Brivanlou A. Development. 1999; 126: 4213-4222Crossref PubMed Google Scholar). 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 (8Hogan B.L. Horsburgh G. Cohen J. Hetherington C.M. Fisher G. Lyon M.F. J. Embryol. Exp. Morphol. 1986; 97: 95-110PubMed Google Scholar, 9Schmahl W. Knoedlseder M. Favor J. Davidson D. Acta Neuropathol. 1993; 86: 126-135Crossref PubMed Scopus (180) Google Scholar). In addition, mice with heterozygous mutations in Pax6 have lower levels of pancreatic hormones (10Sander M. Sussel L. Conners J. Scheel D. Kalamaras J. Dela Cruz F. Schwitzgebel V. Hayes-Jordan A. German M. Development. 2000; 127: 5533-5540Crossref PubMed Google Scholar), and the conditional inactivation of Pax6 in the pancreas causes early onset diabetes (11Ashery-Padan R. Zhou X. Marquardt T. Herrera P. Toube L. Berry A. Gruss P. Dev. Biol. 2004; 269: 479-488Crossref PubMed Scopus (122) Google Scholar). 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 (10Sander M. Sussel L. Conners J. Scheel D. Kalamaras J. Dela Cruz F. Schwitzgebel V. Hayes-Jordan A. German M. Development. 2000; 127: 5533-5540Crossref PubMed Google Scholar, 12StOnge L. SosaPineda B. Chowdhury K. Mansouri A. Gruss P. Nature. 1997; 387: 406-409Crossref PubMed Scopus (664) Google Scholar, 13Andersen F.G. Jensen J. Heller R.S. Petersen H.V. Larsson L.I. Madsen O.D. Serup P. FEBS Lett. 1999; 445: 315-320Crossref PubMed Scopus (47) Google Scholar, 14Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). 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 (1Chi N. Epstein J.A. Trends Genet. 2002; 18: 41-47Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). Both the paired domain and homeodomain have independent DNA binding activities, but they have also been shown to act cooperatively to mediate transcription (1Chi N. Epstein J.A. Trends Genet. 2002; 18: 41-47Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 15Duncan M.K. Haynes 2nd, J.I. Cvekl A. Piatigorsky J. Mol. Cell. Biol. 1998; 18: 5579-5586Crossref PubMed Google Scholar, 16Jun S. Desplan C. Development. 1996; 122: 2639-2650Crossref PubMed Google Scholar, 17Mikkola I. Bruun J.A. Holm T. Johansen T. J. Biol. Chem. 2001; 276: 4109-4118Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 18Mishra R. Gorlov I.P. Chao L.Y. Singh S. Saunders G.F. J. Biol. Chem. 2002; 277: 49488-49494Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 19Singh S. Stellrecht C.M. Tang H.K. Saunders G.F. J. Biol. Chem. 2000; 275: 17306-17313Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). 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 (20Glaser T. Jepeal L. Edeards J.G. Young S.R. Favor J. Maas R.L. Nat. Genet. 1994; 7: 463-471Crossref PubMed Scopus (598) Google Scholar, 21Hanson I. Churchill A. Love J. Axton R. Moore T. Clarke M. Meire F. van Heyningen V. Hum. Mol. Genet. 1999; 8: 165-172Crossref PubMed Scopus (150) Google Scholar). However, several truncation mutations have been found to occur in the C-terminal half of Pax6 in patients with aniridia (22Prosser J. van Heyningen V. Hum. Mutat. 1998; 11: 93-108Crossref PubMed Scopus (229) Google Scholar). 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 (23Favor J. Peters H. Hermann T. Schmahl W. Chatterjee B. Neuhauser-Klaus A. Sandulache R. Genetics. 2001; 159: 1689-1700Crossref PubMed Google Scholar, 24Sander M. Neubuser A. Kalamaras J. Ee H.C. Martin G.R. German M.S. Genes Dev. 1997; 11: 1662-1673Crossref PubMed Scopus (461) Google Scholar). 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 (25Ericson J. Rashbass P. Schedl A. Brenner-Morton S. Kawakami A. van Heyningen V. Jessell T.M. Briscoe J. Cell. 1997; 90: 169-180Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar, 26Muhr J. Andersson E. Persson M. Jessell T.M. Ericson J. Cell. 2001; 104: 861-873Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). 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 (6Onuma Y. Takahashi S. Asashima M. Kurata S. Gehring W.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2020-2025Crossref PubMed Scopus (113) Google Scholar). In addition, Pax6 is essential for the insulin responsiveness of proglucagon promoter (27Grzeskowiak R. Amin J. Oetjen E. Knepel W. J. Biol. Chem. 2000; 275: 30037-30045Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). 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) 6The 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. interacts with transcription factors, including homeoproteins, and has been demonstrated to regulate the activities of transcription factor (28Kim Y.H. Choi C.Y. Lee S-J. Conti M.A. Kim Y. J. Biol. Chem. 1998; 273: 25875-25879Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 29Choi C.Y. Kim Y.H. Kwon H.J. Kim Y. J. Biol. Chem. 1999; 274: 33194-33197Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). HIPK2 also interacts with Groucho corepressor and p300/CREB-binding protein coactivator and regulates the transcription of various genes in a context-dependent manner (30Choi C.Y. Kim Y.H. Kim Y.O. Park S.J. Kim E.A. Riemenschneider W. Gajewski K. Schulz R.A. Kim Y. J. Biol. Chem. 2005; 280: 21427-21436Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 31Kim E.J. Park J.S. Um S.J. J. Biol. Chem. 2002; 277: 32020-32028Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). 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 (30Choi C.Y. Kim Y.H. Kim Y.O. Park S.J. Kim E.A. Riemenschneider W. Gajewski K. Schulz R.A. Kim Y. J. Biol. Chem. 2005; 280: 21427-21436Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). 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. 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 (29Choi C.Y. Kim Y.H. Kwon H.J. Kim Y. J. Biol. Chem. 1999; 274: 33194-33197Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 30Choi C.Y. Kim Y.H. Kim Y.O. Park S.J. Kim E.A. Riemenschneider W. Gajewski K. Schulz R.A. Kim Y. J. Biol. Chem. 2005; 280: 21427-21436Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 32Kim Y.H. Choi C.Y. Kim Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12350-12355Crossref PubMed Scopus (143) Google Scholar). 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-p300ΔHAT (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-p300ΔHAT 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-β-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).FIGURE 5Mutation 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 [γ-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 p300ΔHAT (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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 (33Choi C.Y. Lee Y.M. Kim Y.H. Park T. Jeon B.H. Schulz R.A. Kim Y. J. Biol. Chem. 1999; 274: 31543-31552Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). 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 Biotechnology) as previously described (29Choi C.Y. Kim Y.H. Kwon H.J. Kim Y. J. Biol. Chem. 1999; 274: 33194-33197Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). 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 [γ-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% β-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 (28Kim Y.H. Choi C.Y. Lee S-J. Conti M.A. Kim Y. J. Biol. Chem. 1998; 273: 25875-25879Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar) 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 0.5× Tris borate buffer. 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 1× phosphate-buffered saline and 0.5% Triton X-100. Cells were rinsed with 1× phosphate-buffered saline containing 1% bovine serum albumin and incubated with anti-HIPK2 rabbit polyclonal antibody for 1 h. After washing five times with 1× 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 × 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 α-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. 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 (30Choi C.Y. Kim Y.H. Kim Y.O. Park S.J. Kim E.A. Riemenschneider W. Gajewski K. Schulz R.A. Kim Y. J. Biol. Chem. 2005; 280: 21427-21436Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). 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" @default.
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• W2146724496 title "Phosphorylation and Transactivation of Pax6 by Homeodomain-interacting Protein Kinase 2" @default.
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