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• W1978548933 abstract "Human SIX1 (HSIX1) is a member of the Six class of homeodomain proteins implicated in muscle, eye, head, and brain development. To further understand the role of HSIX1 in the cell cycle and cancer, we developed an HSIX1-specific antibody to study protein expression at various stages of the cell cycle. Our previous work demonstrated that HSIX1 mRNA expression increases as cells exit S phase and that overexpression of HSIX1 can attenuate a DNA damage-induced G2 cell cycle checkpoint. Overexpression of HSIX1 mRNA was observed in 44% of primary breast cancers and 90% of metastatic lesions. Now we demonstrate that HSIX1 is a nuclear phosphoprotein that becomes hyperphosphorylated at mitosis in both MCF7 cells and in Xenopus extracts. The pattern of phosphorylation observed in mitosis is similar to that seen by treating recombinant HSIX1 with casein kinase II (CK2) in vitro. Apigenin, a selective CK2 inhibitor, diminishes interphase and mitotic phosphorylation of HSIX1. Treatment of MCF7 cells with apigenin leads to a dose-dependent arrest at the G2/M boundary, implicating CK2, like HSIX1, in the G2/M transition. HSIX1 hyperphosphorylated in vitro by CK2 loses its ability to bind the MEF3 sites of the aldolase A promoter (pM), and decreased binding to pM is observed during mitosis. Because CK2 and HSIX1 have both been implicated in cancer and in cell cycle control, we propose that HSIX1, whose activity is regulated by CK2, is a relevant target of CK2 in G2/M checkpoint control and that both molecules participate in the same pathway whose dysregulation leads to cancer. Human SIX1 (HSIX1) is a member of the Six class of homeodomain proteins implicated in muscle, eye, head, and brain development. To further understand the role of HSIX1 in the cell cycle and cancer, we developed an HSIX1-specific antibody to study protein expression at various stages of the cell cycle. Our previous work demonstrated that HSIX1 mRNA expression increases as cells exit S phase and that overexpression of HSIX1 can attenuate a DNA damage-induced G2 cell cycle checkpoint. Overexpression of HSIX1 mRNA was observed in 44% of primary breast cancers and 90% of metastatic lesions. Now we demonstrate that HSIX1 is a nuclear phosphoprotein that becomes hyperphosphorylated at mitosis in both MCF7 cells and in Xenopus extracts. The pattern of phosphorylation observed in mitosis is similar to that seen by treating recombinant HSIX1 with casein kinase II (CK2) in vitro. Apigenin, a selective CK2 inhibitor, diminishes interphase and mitotic phosphorylation of HSIX1. Treatment of MCF7 cells with apigenin leads to a dose-dependent arrest at the G2/M boundary, implicating CK2, like HSIX1, in the G2/M transition. HSIX1 hyperphosphorylated in vitro by CK2 loses its ability to bind the MEF3 sites of the aldolase A promoter (pM), and decreased binding to pM is observed during mitosis. Because CK2 and HSIX1 have both been implicated in cancer and in cell cycle control, we propose that HSIX1, whose activity is regulated by CK2, is a relevant target of CK2 in G2/M checkpoint control and that both molecules participate in the same pathway whose dysregulation leads to cancer. human SIX1 casein kinase II protein kinase C glutathioneS-transferase polymerase chain reaction phosphate-buffered saline in vitro translated electrophoretic mobility shift assays mitogen-activated protein kinase calf intestinal alkaline phosphatase The products of homeobox genes are characterized by a 60 amino acid DNA-binding region, the homeodomain, which enables them to activate the transcription of genes that are important for the regulation of cell growth, fate, differentiation, and body patterning. HSIX11 belongs to the Six class of homeodomain containing proteins, which share a lysine in position 50 of the recognition helix of the homeodomain (1.Oliver G. Wehr R. Jenkins N.A. Copeland B.G. Cheyette B.N.R. Hartenstein V. Zipursky S.L. Gruss P. Development. 1995; 121: 693-705Crossref PubMed Google Scholar). These proteins can be further subdivided into three distinct families that presumably originated from three different ancestral Six genes (2.Seo H.-C. Curtiss J. Mlodzik M. Fjose A. Mech. Dev. 1999; 83: 127-139Crossref PubMed Scopus (119) Google Scholar). In mammals two gene members have been identified for each family, thus accounting for the six known members of this class. To date, 12 Six gene homologues have been identified in lower vertebrates (2.Seo H.-C. Curtiss J. Mlodzik M. Fjose A. Mech. Dev. 1999; 83: 127-139Crossref PubMed Scopus (119) Google Scholar). Of the Six proteins discovered to date, several function in the development of the forebrain, eye, and muscle (2.Seo H.-C. Curtiss J. Mlodzik M. Fjose A. Mech. Dev. 1999; 83: 127-139Crossref PubMed Scopus (119) Google Scholar, 3.Heanue T.A. Reshef R. Davis R.J. Mardon G. Oliver G. Tomarev S. Lassar A.B. Tabin C.J. Genes Dev. 1999; 13: 3231-3243Crossref PubMed Scopus (301) Google Scholar). We previously cloned HSIX1 from late S phase 21PT mammary carcinoma cells, and demonstrated that its overexpression in MCF7 cells attenuated a DNA damage-induced G2 cell cycle checkpoint. HSIX1 overexpression was observed in 44% of primary breast cancers, and 90% of metastatic lesions examined. This suggested that HSIX1 has a role in tumor progression, possibly through its cell cycle checkpoint function (4.Ford H.L. Kabingu E.K. Bump E.A. Mutter G.L. Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12608-12613Crossref PubMed Scopus (148) Google Scholar). Recently, it was speculated that the c-metgene is a potential target of Six1 (5.Relaix F. Buckingham M. Genes Dev. 1999; 13: 3171-3178Crossref PubMed Scopus (87) Google Scholar). Additional targets that may explain the role of Six1 in the cell cycle and/or tumor progression are not known. However, myogenin was identified as a target of HSIX1 in muscle development (6.Spitz F. Demignon J. Porteu A. Kahn A. Concordet J.-P. Daegelen D. Maire P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14220-14225Crossref PubMed Scopus (182) Google Scholar). In general, very little is known about the targets of homeodomain proteins. Although most homeodomain containing proteins bind to similar short consensus DNA sequences in vitro, they have highly specific functions in vivo. Therefore, target specificityin vivo is achieved by other elements such as interaction with cofactors, translational regulation, subcellular localization, or protein phosphorylation (7.Kasahara H. Izumo S. Mol. Cell. Biol. 1999; 19: 526-536Crossref PubMed Scopus (82) Google Scholar). Protein phosphorylation regulates a number of homeodomain-containing transcription factors including Csx/Nkx2.5, Cut, Pit-1, Oct-1, andDrosophila Engrailed and Antennapedia by affecting protein-protein interactions, DNA binding, or nuclear localization (7.Kasahara H. Izumo S. Mol. Cell. Biol. 1999; 19: 526-536Crossref PubMed Scopus (82) Google Scholar). In some instances, phosphorylation is cell cycle-dependent (8.Stukenberg P.T. Lustig K.D. McGarry T.J. King R.W. Kuang J. Kirschner M.W. Curr. Biol. 1997; 7: 338-348Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 9.Caelles C. Hennemann H. Karin M. Mol. Cell. Biol. 1995; 15: 6694-6701Crossref PubMed Scopus (59) Google Scholar, 10.Segil N. Roberts S.B. Heintz N. Science. 1991; 254: 1814-1816Crossref PubMed Scopus (205) Google Scholar). Mitotic phosphorylation of both the POU transcription factor GHF-1 and the Oct-1 homeodomain containing protein inhibits their DNA binding activity (9.Caelles C. Hennemann H. Karin M. Mol. Cell. Biol. 1995; 15: 6694-6701Crossref PubMed Scopus (59) Google Scholar, 10.Segil N. Roberts S.B. Heintz N. Science. 1991; 254: 1814-1816Crossref PubMed Scopus (205) Google Scholar) and may represent a general mechanism for decreasing transcription during mitosis. Several kinases are known to phosphorylate homeodomain-containing proteins, including protein kinase casein kinase II (CK2), protein kinase C (PKC), and protein kinase A. In particular, protein kinase CK2, a serine/threonine kinase that is ubiquitously expressed, has been shown to phosphorylate transcription factors including those encoded by Csx/Nkx2.5 (7.Kasahara H. Izumo S. Mol. Cell. Biol. 1999; 19: 526-536Crossref PubMed Scopus (82) Google Scholar), Cut (11.Coqueret O. Martin N. Berube G. Rabbat M. Litchfield D. Nepvue A. J. Biol. Chem. 1997; 273: 2561-2566Abstract Full Text Full Text PDF Scopus (50) Google Scholar), Hoxb-6 (12.Fienberg A.A. Nordstedt C. Belting H.G. Czernik A.J. Nairn A.C. andy S. Greengard P. Ruddle F.H. J. Exp. Zoology. 1999; 285: 76-84Crossref PubMed Scopus (13) Google Scholar), even-skipped (13.Li C. Manley J.L. Mol. Cell. 1999; 3: 77-86Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), and Engrailed (14.Bourbon H.M. Martin-Blanco E. Rosen D. Kornberg T.B. J. Biol. Chem. 1995; 270: 11130-11139Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) homeobox genes. The phosphorylation of the DrosophilaAntennapedia protein by CK2 was shown to be important for its role in thoracic and abdominal development (15.Jaffe L. Ryoo H.-D. Mann R.S. Genes Dev. 1997; 11: 1327-1340Crossref PubMed Scopus (103) Google Scholar). To understand the regulation of the HSIX1 protein, we developed an HSIX1-specific antibody and examined protein levels and phosphorylation at various stages of the cell cycle. We find that HSIX1 is a phosphoprotein in both interphase and mitotic cells and that protein kinase CK2 is at least partly responsible for the phosphorylation of HSIX1 in both interphase and mitosis. In mitosis, the HSIX1 protein becomes hyperphosphorylated, and a concomitant loss in DNA binding activity is seen. The phosphorylation of HSIX1 by CK2 has implications for both cell cycle control and tumorigenesis. The GST C-terminal HSIX1 construct utilized for antibody production was generated by PCR amplification of the C terminus of HSIX1 (beginning from nucleotide 822, just after the homeodomain and terminating at the STOP codon) from the full-length SKMFL plasmid (wild type HSIX1 cloned into the BamHI/XbaI site of the Invitrogen pcDNA3.1/His plasmid) utilizing standard PCR conditions and a 5′ primer containing a XhoI restriction site (ACT CTC GAG GAG GCC AAG GAA AGG GAG AAC) and 3′ primer containing anXbaI restriction site (TGC TCT AGA CAC TTA GGA CCC CAA GTC CAC-pSixXba I). The C terminus was then subcloned into an Invitrogen TA cloning vector pCR2.1 according to the manufacturers recommendations, resulting in the pCR2.1Cterm plasmid. Partial digests (16.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 9.24-9.25Google Scholar) were performed on the pCR2.1Cterm plasmid with EcoRI to release the full-length C-terminal fragment of HSIX1. The C terminus of HSIX1 was then subcloned into the EcoRI sites of pGEX2TK (Amersham Pharmacia Biotech), and the resulting construct was sequenced to ensure the proper orientation and to ensure that no mutations were introduced. Deletion constructs were generated as follows. The N terminus of HSIX1 (from the start codon to nucleotide 689, which is in the first helix of the encoded homeodomain) was amplified as the C terminus (above), using a 5′ primer containing aBamHI site (CTG GGA TCC ATG TCG ATG CTG CCG TCG TTT- pSixBHI) and a 3′ primer containing a XhoI site (ATC CTC GAG GAC ACC CCT CGA CTT CTC CTT). The resulting N-terminal fragment was then subcloned into the TA cloning vector pCR2.1 as above (pCR2.1Nterm). The N-terminal and C-terminal portions of HSIX1 were then removed from pCR2.1 by digesting withBamHI/XhoI and XhoI/XbaI, respectively, and were subsequently ligated into the BamHI and XbaI sites of pcDNA3.1(+) to generate the ΔHD plasmid. Sequencing was performed to ensure that the two portions of HSIX1 were fused in frame and that the homeodomain was lacking. The homeobox and C-terminal portions (for ΔNterm) of HSIX1 were amplified using standard PCR conditions from the SIXFL plasmid (4.Ford H.L. Kabingu E.K. Bump E.A. Mutter G.L. Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12608-12613Crossref PubMed Scopus (148) Google Scholar) with a 5′ primer containing aBamHI site (CTG GGA TCC ATG AAA TTT CCA CTG CCG CGC ACC) and the pSixXbaI 3′ primer. The N-terminal and homeobox regions (for ΔCterm) were amplified as above using the pSixBHI 5′ primer and a 3′ primer containing a STOP codon as well as an XbaI site (TGC TCT AGA CTA GTT CTC CCT TTC CTT GGC CTC). The PCR products were then digested with BamHI and XbaI and subcloned into the pcDNA3.1(+) plasmid. Sequencing of both constructs was performed to ensure that no mutations were introduced. The GST C-terminal HSIX1 fusion protein was induced and purified on glutathione beads as described previously (17.Ford H.L. Zain S. Oncogene. 1995; 10: 1597-1605PubMed Google Scholar). The fusion protein was released into the supernatant by adding 50 mm Tris, pH 8, containing 10 mm reduced glutathione and incubating at 4 °C for 10 min. Bradford assays were performed to determine the protein concentration after which the protein was electrophoresed on a 12% SDS-polyacrylamide gel. The gel was lightly stained with Coomassie Brilliant Blue, and GST C-terminal HSIX1 was excised from the gel according to Harlow and Lane (18.Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 63Google Scholar). Approximately 500 μg of protein was sent to Spring Valley (Woodbine, MD) for antibody production in rabbits. Successive bleeds of GST C-terminal HSIX1 antibody were tested on lysates from MCF7 cells transfected with the SIXFLexpression construct (MCF7/SIXFL). When HSIX1-specific antibody was observed in plasma, the antibody was affinity purified, first over a GST column (to remove all antibodies recognizing the GST portion of the fusion protein) and then over a GST C-terminal HSIX1 column. The columns were made using the AminoLink Plus Immobilization Kit from Pierce. The affinity purified antibody was then tested and titered on MCF/SIXFL lysates. MCF7 mammary carcinoma cells were maintained in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum and antibiotics at 37 °C in 6.5% CO2. 21PT cells were maintained as described (4.Ford H.L. Kabingu E.K. Bump E.A. Mutter G.L. Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12608-12613Crossref PubMed Scopus (148) Google Scholar). For transfections, subconfluent 100-mm plates of MCF7 cells were split 1:4 into 100-mm plates. The following day, the 100-mm plate of cells was transfected with 10 μg of SIXFL or pcDNA3.1(+) (mock transfected control) using Superfect (Qiagen) according to the manufacturer's protocols. Transfected cells were lysed 24–48 h post-transfection in RIPA buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, 0.5% nadeoxycholate, 0.1% SDS, 5 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, 10 mm NaF, 200 μmNa3VO4) for 20 min. at 4 °C. Lysates were passed through a 25-gauge needle 5–6 times to shear the DNA and then microcentrifuged at 4 °C, 14,000 rpm for 15 min. Supernatants were treated with calf intestinal alkaline phosphatase according to Kasahara and Izumo (7.Kasahara H. Izumo S. Mol. Cell. Biol. 1999; 19: 526-536Crossref PubMed Scopus (82) Google Scholar), and Western blots were performed as described (18.Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 63Google Scholar) using a 1:1000 dilution of anti-HSIX1 as the primary antibody and a 1:10000 dilution of anti-rabbit IgG horseradish peroxidase (Sigma) as a secondary antibody. Chemiluminescence with ECL (Amersham Pharmacia Biotech) was utilized to detect the HSIX1 signal. MCF7 cells were plated in 6-well dishes on coverslips at 2.5 × 105 cells/well. 24 h later, cells were transfected with SIXFL using Fugene (Roche Molecular Biochemicals) according to the manufacturer's protocol. 24–48 h post-transfection, cells were fixed in 0.7% formaldehyde in PBS for 10 min. followed by 5–10 min in 0.5% Triton X-100. After several washes in PBS, cells were incubated in a 1:1000 dilution of anti-HSIX1 for 1 h at room temperature followed by several washes in PBS. The cells were then incubated in a 1:100 dilution of anti-rabbit IgG-fluorescein (Calbiochem, La Jolla, CA) for 45 min at room temperature. After five washes in PBS, the cells were mounted in Vectashield (Vector Labs, Burlingame, CA) containing 0.1 μg/ml 4,6-diamidino-2-phenylindole to counterstain the nuclei. Xenopus interphase and mitotic extracts were prepared as described by Stukenberg et al. (8.Stukenberg P.T. Lustig K.D. McGarry T.J. King R.W. Kuang J. Kirschner M.W. Curr. Biol. 1997; 7: 338-348Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). [35S]Methionine-labeled HSIX1 and the deleted proteins were in vitro translated (IVT) from the SIXFL, ΔHD, ΔNterm, and ΔCterm constructs using the TNT coupled reticulocyte lysate system from Promega (Madison, WI) according to the manufacturer's protocol. Proteins were then incubated for 1 h at room temperature in interphase or mitotic extracts (1 μl of IVT reaction plus 5 μl extract) and examined for phosphatase sensitive alterations in mobility according to Stukenberg et al. (8.Stukenberg P.T. Lustig K.D. McGarry T.J. King R.W. Kuang J. Kirschner M.W. Curr. Biol. 1997; 7: 338-348Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). 1 μl of this reaction was resolved on a 10% or 12% SDS-polyacrylamide gel and visualized by autoradiography. [35S]Methionine-labeled HSIX1 was incubated with various kinases as follows: CK2 for 30 min at 30 °C with 50 units of human recombinant casein kinase II (Calbiochem) in 20 mmTris, pH 7.5, 50 mm KCl, 10 mmMgCl2, 0.25 mm ATP; cyclin B/cdc2 for 20 min at 30 °C with 2 μl of purified Xenopus cyclin B-cdc2 (19.Wilhelm H. Andersen S.L. Karsenti E. Methods Enzymol. 1997; 283: 12-29Crossref PubMed Scopus (11) Google Scholar) in 50 mm Tris, pH 7.4, 10 mm MgCl2, 1 mm dithiothreitol; PKC for 15 min at 30 °C with 20 ng of the catalytic subunit of rat brain protein kinase C (Calbiochem) in 25 mm Tris, pH 7.4, 5 mm EGTA, 140 mm KCl, 6 mm MgCl2, 1 mm CaCl2, 1 mm ATP. Before use in EMSAs, salt concentrations were adjusted to give appropriate final molarities. Cells were transfected with SIXFL as described above. MCF7/SIXFL cells were incubated with various inhibitors at indicated concentrations in medium for 3–5 h at 37 °C, after which lysates were isolated as above. Densitometric scanning of Western blots developed with the HSIX1 antibody allowed determination of the percentage of HSIX1 phosphorylated in interphase in the presence of the various inhibitors. For assessment of kinases important for mitotic phosphorylation, mitotic Xenopus assays containing [35S]methionine-labeled HSIX1 were carried out as above by adding the indicated inhibitors at the time of HSIX1 addition. Densitometric scanning was utilized to determine the percentage of HSIX1 that was hyperphosphorylated in the presence of various inhibitors. The inhibitors utilized were: apigenin (Sigma), a selective CK2 inhibitor; roscovitine (Calbiochem), a cdc2 kinase inhibitor; bisindolylmaleimide I (Calbiochem), a PKC inhibitor; and PD98059 (New England Biolabs, Beverly, MA), a mitogen-activated protein kinase kinase 1 (MEKI) and MAPK cascade inhibitor. For kinase assays, 5 μg of protein extracted from MCF7 cells was incubated with or without 1 mm of the specific protein kinase CK2 peptide RRREEETEEE (Sigma-Genosys, The Woodlands, TX) in buffer (100 mm Tris, pH 8.0, 20 mmMgCl2, 100 mm NaCl, 50 mm KCl, 0.1 μg/μl bovine serum albumin, and 100 μmNa3VO4) and 5 μCi of [γ-32P]GTP at 30 °C for 10 min (20.Seldin D.C. Leder P. Science. 1995; 267: 894-897Crossref PubMed Scopus (360) Google Scholar). The kinase reaction was terminated by addition of 25 μl of 100 mmATP in 0.4 n HCl. Samples were spotted onto a P81 Whatman filter and washed four times for 5 min each with 150 mmH3PO4 to elute unincorporated counts. Incorporated counts were quantified in an automatic scintillation counter. Samples were assayed in triplicate. Kinase activity was calculated as the subtraction of the mean of samples without peptide from the mean of samples with peptide. MCF7 cells were cultured as described above. When cells reached 50–70% confluence, 20–80 μm apigenin (Sigma) or Me2SO alone was administered for 18 h. Cells were then resuspended in Nicoletti buffer (0.1% Triton X-100 and 0.1% sodium citrate) containing 0.5 mg/ml propidium iodide (Sigma), and the DNA content was analyzed on a flow cytometer (Becton Dickinson, Mountain View, CA) using the Cellquest software program. These assays were performed as described in Spitz et al. (6.Spitz F. Demignon J. Porteu A. Kahn A. Concordet J.-P. Daegelen D. Maire P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14220-14225Crossref PubMed Scopus (182) Google Scholar) using the aldolase A MEF3 site sequence (tgaatgtcaggggct t caggtttcccta). The buffer utilized for protein-DNA binding contained 25 mm Hepes, pH 7.6, 5 mm MgCl2, 10% glycerol, 34 mm KCl, 1 mm dithiothreitol. Unlabeled wild type and mutant oligonucleotides (bold nucleotide changed fromt to g above) were used as competitors at 50 times the radiolabeled oligonucleotide concentration. To study HSIX1 function, we generated an HSIX1-specific antibody by injecting a GST C-terminal HSIX1 fusion protein into rabbits. After affinity purifying the antibody, it was tested on lysates from MCF7 cells transiently transfected with HSIX1 (MCF7/SIXFL) and on lysates from control transfected MCF7 cells that had previously been shown to contain almost no endogenous HSIX1 mRNA. Western blot analysis identified three bands of molecular masses between 37 and 42 kDa in HSIX1 transfected MCF7 cells but not in controls, demonstrating the specificity of our antibody (Fig.1 A) and suggesting that the protein is post-translationally modified or processed. Immunocytochemistry with the HSIX1 antibody demonstrated that transfected HSIX1 is a nuclear protein (Fig. 1 B). A data-base search of the HSIX1 amino acid sequence revealed 11 potential phosphorylation sites in the protein (Fig.2), particularly in the C terminus. To address whether HSIX1 is a phosphoprotein, lysates obtained from asynchronous MCF7/SIXFL cells were treated with calf intestinal alkaline phosphatase (CIAP), which demonstrated the existence of a phosphatase sensitive form of HSIX1 (Fig.3 A). Dephosphorylation was blocked in the presence of excess phosphate. To determine whether endogenous HSIX1 also exists as a phosphoprotein, we prepared nuclear extracts from asynchronous 21PT breast cancer cells, previously shown to contain HSIX1 mRNA, and performed the CIAP reaction as described for MCF7/SIXFL cells. Fig. 3 B demonstrates that endogenous HSIX1 also exists as a phosphoprotein in an asynchronous population of 21PT cells.Figure 3HSIX1 exists as a phosphoprotein in asynchronous cells. A, Western blot analysis with HSIX1 antibody on lysates from MCF7/SIXFL cells (left lane), MCF7/SIXFL cells treated with CIAP (middle lane), and MCF7/SIXFL cells treated with both CIAP and Na2HPO4 to compete the phosphatase reaction (right lane). B, experiment carried out as inA with nuclear extracts from 21PT cells, which express HSIX1 endogenously.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The cdc2 kinase, which has catalytic specificity for a proline C-terminal to the site it phosphorylates, is only active in mitosis when it is partnered with its regulatory subunit, cyclin B, and is activated by various phosphorylation and dephosphorylation events (21.Jackman M.R. Pines J.N. Cancer Surveys. 1997; 29: 47-73PubMed Google Scholar). The HSIX1 sequence has several putative cdc2 phosphorylation sites (Fig. 2). This, in addition to previous findings that several homeodomain containing proteins are hyperphosphorylated in mitosis (8.Stukenberg P.T. Lustig K.D. McGarry T.J. King R.W. Kuang J. Kirschner M.W. Curr. Biol. 1997; 7: 338-348Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 9.Caelles C. Hennemann H. Karin M. Mol. Cell. Biol. 1995; 15: 6694-6701Crossref PubMed Scopus (59) Google Scholar, 10.Segil N. Roberts S.B. Heintz N. Science. 1991; 254: 1814-1816Crossref PubMed Scopus (205) Google Scholar), prompted us to examine the phosphorylation state of HSIX1 in interphaseversus mitosis. Western blot analysis on lysates from MCF7/SIXFL cells synchronized in mitosis by addition of nocodazole, as well as on lysates from mitotis-enriched MCF7/SIXFL cells that were sorted by flow cytometry, demonstrated the existence of a hyperphosphorylated form of HSIX1 (data not shown). Because biochemical analysis of this form of the protein was difficult in mammalian culture cells, where drug treatments must be used to obtain large numbers of mitotic cells, we chose the synchronousXenopus laevis system to carry out these studies. In vitro translated [35S]methionine-labeled HSIX1 was incubated with interphase and mitotic extracts from X. laevis and examined for phosphatase-sensitive alterations in mobility. In interphase, an HSIX1 triplet was observed, as seen in asynchronous MCF7 cells, where greater than 75–90% are generally in interphase (data not shown). HSIX1 incubated in mitotic extracts exhibited a higher molecular mass form of HSIX1, which could be eliminated by treatment with CIAP, indicating hyperphosphorylation of HSIX1 in mitosis (Fig.4 A). To determine the region of HSIX1 that is hyperphosphorylated in mitosis, deletion constructs were generated (Fig. 4 B). Proteins with deleted regions were translated in the presence of [35S]methionine and incubated in interphase and mitotic extracts. Those lacking the homeodomain (ΔHD) or the N terminus (ΔNterm) exhibited a shift to a slower mobility when incubated in mitotic extracts. However, the C-terminal deleted protein (ΔCterm) was not shifted (Fig. 4 C). This suggests that the majority of the mitotic-specific phosphorylation occurs in the C terminus, in accordance with the multiple phosphorylation sites observed in this region of the protein. Data base searching revealed that HSIX1 contains potential consensus phosphorylation sites for protein kinase CK2, PKC, and cyclin B/cdc2. We set out to determine which of these kinases are responsible for HSIX1 phosphorylation. [35S]Methionine-labeledin vitro translated HSIX1 was incubated with each of these three putative HSIX1 kinases (Fig.5 A). PKC, cyclin B/cdc2, and CK2 all can phosphorylate HSIX1 in vitro, and a greater extent of hyperphosphorylation is observed when the protein is incubated with cyclin B/cdc2 or CK2 than with PKC. Phosphorylation of HSIX1 by CK2 in vitro most closely resembled the hyperphosphorylation of the protein observed in mitotic extracts (Fig.5 A), although none of the kinases gave in vitrophosphorylation patterns of HSIX1 that were identical to those seen in interphase or mitotic extracts. To determine which kinases were responsible for phosphorylating HSIX1in vivo, MCF7/SIXFL cells were treated with inhibitors to each of these kinases. Apigenin, a selective CK2 inhibitor, diminished the phosphorylation of HSIX1 (Fig. 5 B). This inhibition of HSIX1 phosphorylation was paralleled by partial inhibition of CK2 activity (Fig. 5 C). Neither roscovitine, a cyclin B/cdc2 inhibitor, nor bisindolylmaleimide, a PKC inhibitor, significantly inhibited the phosphorylation of HSIX1 in asynchronous, primarily interphase cells (Fig. 5 B). [35S]Methionine-labeled HSIX1 was incubated in Xenopus mitotic extracts in the absence or presence of various kinase inhibitors. 100 μm apigenin reduced CK2 activity in the extract by approximately 40% (data not shown) and decreased the ratio of the higher molecular mass (hyperphosphorylated) form of HSIX1 to the total amount of protein by an average of 27%, a statistically significant difference as assessed by a Student'st test. However, treatment with either 100 μmroscovitine, a concentration known to inhibit MPF (cyclin B/cdc2) activity in Xenopus extracts, or 1 μmbisindolylmaleimide, which specifically inhibits PKC activity, did not significantly alter the extent of HSIX1 hyperphosphorylation (Fig.5 D). This suggests that CK2 is, at least in part, also responsible for the mitotic-specific hyperphosphorylation of HSIX1. Our previous work as well as that of others has implicated both HSIX1 and CK2 in the DNA damage-induced G2cell cycle checkpoint and in tumorigenesis. Discovery of HSIX1 as a target of CK2 in both mammalian and Xenopus systems implies that the two proteins may cooperate in cell cycle control and tumorigenicity. Because we have already demonstrated that overexpression of HSIX1 in MCF7 cells affects the transition of cells through G2, we set out to determine the effect of CK2 on the cell cycle. MCF 7 cells treated with apigenin were arrested at the G2/M boundary in a dose-dependent manner (Fig.6), suggesting that CK2 activity is important in the G2/M transition of mammary carcinoma cells. To determine whether hyperphosphorylation of HSIX1 by CK2 affects its DNA binding activity, we performed EMSA (Fig.7 A) using the MEF3 sites of the aldolase A promoter (pM), which were previously demonstrated to bind the mouse Six1 protein (6.Spitz F. Demignon J. Porteu A. Kahn A. Concordet J.-P. Daegelen D. Maire P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14220-14225Crossref PubMed Scopus (182) Google Scholar). IVT HSIX1 formed three complexes when incubated with the pM oligonucleotide, which were all competed by cold wild type pM. Only the fastest migrating complex was not competed with cold mutant pM, su" @default.
• W1978548933 created "2016-06-24" @default.
• W1978548933 creator A5002769265 @default.
• W1978548933 creator A5023441926 @default.
• W1978548933 creator A5026202946 @default.
• W1978548933 creator A5026644077 @default.
• W1978548933 creator A5072633316 @default.
• W1978548933 creator A5076515558 @default.
• W1978548933 date "2000-07-01" @default.
• W1978548933 modified "2023-10-12" @default.
• W1978548933 title "Cell Cycle-regulated Phosphorylation of the Human SIX1 Homeodomain Protein" @default.
• W1978548933 cites W1566906383 @default.
• W1978548933 cites W1576665385 @default.
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