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• W2013906846 abstract "The engrailed gene encodes a homeodomain-containing phosphoprotein that binds DNA. Here, we show that engrailed protein is posttranslationally modified in embryos and in embryo-derived cultured cells but is essentially unmodified when expressed in Escherichia coli. Engrailed protein produced by bacteria can be phosphorylated in nuclear extracts prepared from Drosophila embryos, and phosphotryptic peptides from this modified protein partly reproduce two-dimensional maps of phosphotryptic fragments obtained from metabolically labeled engrailed protein. The primary embryonic protein kinase modifying engrailed protein is casein kinase II (CK-II). Analysis of mutant proteins revealed that the in vitro phosphoacceptors are mainly clustered in a region outside the engrailed homeodomain and identified serines 394, 397, 401, and 402 as the targets for CK-II phosphorylation. CK-II-dependent phosphorylation of an N-truncated derivative of engrailed protein purified from bacteria increased its DNA binding 2–4-fold. The engrailed gene encodes a homeodomain-containing phosphoprotein that binds DNA. Here, we show that engrailed protein is posttranslationally modified in embryos and in embryo-derived cultured cells but is essentially unmodified when expressed in Escherichia coli. Engrailed protein produced by bacteria can be phosphorylated in nuclear extracts prepared from Drosophila embryos, and phosphotryptic peptides from this modified protein partly reproduce two-dimensional maps of phosphotryptic fragments obtained from metabolically labeled engrailed protein. The primary embryonic protein kinase modifying engrailed protein is casein kinase II (CK-II). Analysis of mutant proteins revealed that the in vitro phosphoacceptors are mainly clustered in a region outside the engrailed homeodomain and identified serines 394, 397, 401, and 402 as the targets for CK-II phosphorylation. CK-II-dependent phosphorylation of an N-truncated derivative of engrailed protein purified from bacteria increased its DNA binding 2–4-fold. Homeodomain proteins represent a large and evolutionarily conserved family of proteins that play critical regulatory roles in the development of most organisms. In Drosophila, homeodomain proteins have been shown to bind specific DNA sequences with high affinity (1Affolter M. Percival-Smith A. Muller M. Leupin W. Gehring W.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4093-4097Crossref PubMed Scopus (152) Google Scholar, 2Kissinger C.R. Liu B. Martin-Blanco E. Kornberg T.B. Pabo C.O. Cell. 1990; 63: 579-590Abstract Full Text PDF PubMed Scopus (803) Google Scholar) and to regulate RNA synthesis in vitro (3Biggin M. Tjian R. Cell. 1989; 58: 433-440Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 4Ohkuma Y. Horikoshi M. Roeder R. Desplan C. Cell. 1990; 61: 475-484Abstract Full Text PDF PubMed Scopus (44) Google Scholar, 5Johnson F. Krasnow M. Genes & Dev. 1990; 4: 1044-1052Crossref PubMed Scopus (19) Google Scholar) and in vivo (6Driever W. Nüsslein-Volhard C. Nature. 1989; 337: 138-143Crossref PubMed Scopus (468) Google Scholar, 7Schier A.F. Gehring W.J. Nature. 1992; 356: 804-807Crossref PubMed Scopus (127) Google Scholar). Despite these and many other related studies, our understanding of how the activity of homeodomain proteins is regulated is rudimentary. One important unresolved problem is the manner by which homeodomain proteins select their targets, since most bind DNA in vitro with similar affinities and specificities (2Kissinger C.R. Liu B. Martin-Blanco E. Kornberg T.B. Pabo C.O. Cell. 1990; 63: 579-590Abstract Full Text PDF PubMed Scopus (803) Google Scholar, 8Desplan C. Theis J. O'Farrell P. Cell. 1988; 54: 1081-1090Abstract Full Text PDF PubMed Scopus (337) Google Scholar, 9Hoey T. Levine M. Nature. 1988; 332: 858-861Crossref PubMed Scopus (309) Google Scholar, 10Müller M. Affolter M. Leupin W. Otting G. Wuthrich K. Gehring W.J. EMBO J. 1988; 7: 4299-4304Crossref PubMed Scopus (150) Google Scholar, 11Ekker S.C. Young K.E. von K.D. Beachy P.A. EMBO J. 1991; 10: 1179-1186Crossref PubMed Scopus (130) Google Scholar). It is likely that their specificity for promoter selection is achieved through interactions with other proteins, and examples of interactions with homeodomain proteins that are relevant include the interactions between the α-2 repressor and the MCM-1 and a1 proteins of Saccharomyces cerevisiae (12Smith D.L. Johnson A.D. Cell. 1992; 68: 133-142Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 13Goutte C. Johnson A.D. J. Mol. Biol. 1993; 233: 359-371Crossref PubMed Scopus (67) Google Scholar) and the interactions of the Drosophila extradenticle protein with the engrailed, Ultrabithorax, Antennapedia, and Abdominal-A homeodomain proteins (14Chan S.K. Jaffe L. Capovilla M. Botas J. Mann R.S. Cell. 1994; 78: 603-615Abstract Full Text PDF PubMed Scopus (333) Google Scholar, 15van Dijk M.A. Murre C. Cell. 1994; 78: 617-624Abstract Full Text PDF PubMed Scopus (235) Google Scholar). These interactions appear to involve contacts both within and outside of the homeodomain (16Kornberg T.B. J. Biol. Chem. 1993; 268: 26813-26816Abstract Full Text PDF PubMed Google Scholar, 17Stark M.R. Johnson A.D. Nature. 1994; 371: 429-432Crossref PubMed Scopus (54) Google Scholar). An alternative mechanism that might help to regulate the activity of homeodomain proteins is posttranslational modification. Protein phosphorylation has been demonstrated to be an effective means of regulating an increasing number of cellular processes, and a number of homeodomain proteins have been shown to be phosphorylated. These include engrailed (18Gay N.J. Poole S.J. Kornberg T.B. Nucleic Acids Res. 1988; 16: 6637-6647Crossref PubMed Scopus (29) Google Scholar), fushi tarazu (19Krause H.M. Gehring W.J. EMBO J. 1989; 8: 1197-1204Crossref PubMed Scopus (17) Google Scholar), bicoid (6Driever W. Nüsslein-Volhard C. Nature. 1989; 337: 138-143Crossref PubMed Scopus (468) Google Scholar), Hox-1.3 (20Odenwald W. Garben J. Arnheiter H. Tournier-Lasserve E. Lazzarini R. Genes & Dev. 1989; 3: 158-172Crossref PubMed Scopus (88) Google Scholar), Oct1 (21Tanaka M. Herr W. Cell. 1990; 60: 375-386Abstract Full Text PDF PubMed Scopus (517) Google Scholar), and Ultrabithorax proteins (22Gavis E.R. Hogness D.S. Development. 1991; 112: 1077-1093Crossref PubMed Google Scholar). Although the function of these modifications has not been established, phosphorylation of homeodomain proteins could in principle affect their interactions with DNA or other proteins. In the present study, we have analyzed the phosphorylation states of Drosophila engrailed protein (En). This homeodomain protein is required for the development of the posterior compartments during embryogenesis and subsequent larval and pupal development, and it is essential to the processes that maintain compartment and segment borders (23Lawrence P. Morata G. Dev. Biol. 1976; 50: 321-337Crossref PubMed Scopus (206) Google Scholar, 24Kornberg T. Dev. Biol. 1981; 86: 363-381Crossref PubMed Scopus (79) Google Scholar). En has 552 amino acids, with a homeodomain near its C terminus and with regions rich in glutamine, alanine, serine, or acidic residues distributed elsewhere in its sequence (25Poole S.J. Kauvar L. Drees B. Kornberg T. Cell. 1985; 40: 37-43Abstract Full Text PDF PubMed Scopus (427) Google Scholar). Here, we show that En is posttranslationally modified by casein kinase II (CK-II), 1The abbreviations used are: CK-IIcasein kinase IIIPTGisopropyl-1-thio-β-D-galactopyranosidePBSphosphate-buffered salineHPLChigh pressure liquid chromatography. a widely conserved growth-related protein kinase (reviewed in Ref. 26Issinger O.G. Pharmacol. & Ther. 1993; 59: 1-30Crossref PubMed Scopus (250) Google Scholar). Furthermore, we identify the major in vitro phosphoacceptors and show that phosphorylation by purified CK-II stimulates DNA binding. casein kinase II isopropyl-1-thio-β-D-galactopyranoside phosphate-buffered saline high pressure liquid chromatography. Plasmid Constructions and Polymerase Chain Reaction Mutagenesis—For routine subcloning and plasmid amplification, XL1-Blue (Stratagene) strain was used; for bacterial expression of En and its truncated derivatives, BL21(DE3) (27Studier W.F. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4839) Google Scholar) strain was used. To express En derivatives in Escherichia coli, the various portions of the engrailed coding sequence were inserted into appropriate pAR vectors (54Rosenberg A. Lade B. Chini D. Lin S. Dunn J. Studier F. Gene (Amst.). 1987; 56: 125-135Crossref PubMed Scopus (1044) Google Scholar). To express full-length En, the sequence surrounding the ATG of engrailed was converted to an NdeI site by site-directed mutagenesis, and a 1.76-kilobase NdeI fragment (nucleotides 178–1938 of the c-2.4 en cDNA (25Poole S.J. Kauvar L. Drees B. Kornberg T. Cell. 1985; 40: 37-43Abstract Full Text PDF PubMed Scopus (427) Google Scholar) containing the entire coding region was inserted into the unique NdeI of pAR3038. Most of the En derivatives were constructed from a BamHI partial cleavage of this expression plasmid, pT7En. Expression of these constructs yield fusion proteins with the first 11 amino acids of the gene 10 protein. The expression vector pT7En-1/298 was obtained by subcloning a 888-base pair NdeI-BamHI fragment into pAR3040. Note that En-1/298 contains a C-terminal dipeptide (GO derived from the parental vector. The expression vector pT7En-228/552 was obtained by inserting a blunted 1.23-kilobase NaeI-EcoRV fragment (obtained from pT7En) into the blunted/dephosphorylated BamHI site of pAR3039. pHB40P was constructed by digesting pAR3040 with XbaI, filling in and redigesting with NdeI and BamHI prior to inserting a polylinker using two complementary oligonucleotides: DEL-2 (5′-TATGGTACCTCTAGACTCGAGGG-3′) and DEL-1 (5′-GATCCCCTCGAGTCTAGAGGTACCA-3′). pHB90 was constructed by inserting a 908-base pair BamHI fragment (obtained from a partial BamHI digestion of pT7En) into pHB40P. Nested deletion mutants were generated by exonuclease III followed by S1 nuclease digestions of pHB90 cleaved at the KpnI and XbaI sites (see Fig. 6a) as described (55Henikoff S. Methods Enzymol. 1987; 155: 156-165Crossref PubMed Scopus (676) Google Scholar). BL2KDE3) strains freshly transformed with T7 expression vectors expressing N-deleted En versions were selected by immunodetection using mAbs 4D9 (56Patel N.H. Martin-Blanco E. Coleman K. Poole S. Ellis M.C. Kornberg T.B. Goodman C.S. Cell. 1989; 58: 955-968Abstract Full Text PDF PubMed Scopus (801) Google Scholar) as follows. Transformants were directly plated onto nitrocellulose filters on LB/amp plates. Colonies were grown at 37 °C until 1 mm in diameter. From the master filters, two replica nitrocellulose filters (pre-wetted with 10 mM IPTG) were obtained and were placed on LB/amp plate colonies facing upward. Colonies were grown until 1 mm in diameter, filters were washed in 1 × PBS (10 mM sodium phosphate, pH 7.4, 100 mM NaCl) and processed for immunodetection, as described below for Western blots. A number of selected constructs were analyzed by restriction mapping and sequenced using an oligonucleotide corresponding to a portion of the T7 promoter as a primer (oligonucleotide HB-T7, 5′-AATACGACTCACTATAG-3′). The pHB67 expression vector was constructed by inserting a 677-base pair SamHI fragment (from pT7En) into pHB40P. Expression of this construct yields a fusion protein (En-374/552) with the first 7 amino acids (MVPLDSR) being encoded by the synthetic polylinker of pHB40P. The two mutated forms of En-374/552 were obtained by two successive rounds of polymerase chain reaction amplifications, using appropriated oligonucleotides as primers. First, to construct the pHB67/2A expression vector, two oligonucleotides were used: MUT-1 (5′-GAACGCGTGTCATCCCGCGGCCTCCATTCC-3′) and MUT-2 (5′ATGGTACCTCTAGACGCGAGGGG-3′). Amplified DNA was filled-in, double digested with KpnI and MluI, gel purified, and inserted into gel-purified pHB67, which was linearized with KpnI and MluI. Mutated constructs were screened for the presence of a new SacII site (underlined in the MUT-2 sequence) and by DNA sequencing using the HB-T7 oligonucleotide as a primer. Second, to construct the pHB67/4A expression vector, a third oligonucleotide (along with MUT-2) was used, MUT-3 (5′-ATCCGCGGCCTCCATTCCGGCATCTCGGGCAACGGCGGA-3′). Amplified DNA was filled-in, double digested with SacII and KpnI, gel purified, and inserted into gel-purified pHB67/2A, which was linearized with KpnI and SacII. Expression and Purification of En—BL2KDE3) cells freshly transformed with pT7En or its derivatives were grown at 37 °C to an A600 of 0.8 in the presence of 100 μg/liter ampicillin. IPTG was added to 0.5 mM, and incubation was continued for an additional 3 h. Bacteria were harvested, frozen in dry ice for 30 min, stored at –80 °C, partially thawed on ice (about 15 min), and quickly resuspended in 1/100 of buffer HEDN (25 mM HEPES (pH 7.6), 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.1% (v/v) Nonidet P-40, 10 μg/ml leupeptin, 0.1 mM benzamidine, 10 μg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1 µg/ml 1,10-phenanthroline) + 100 mM KCl. 0.8-ml aliquots were subjected to sonication at 4 °C. The bacterial debris and insoluble materials were removed by ultracentrifugation (68,000 rpm, 45 min, 4 °C, in a Beckman TLA100.1 rotor), and supernatants were frozen in dry ice and stored at –80 °C. All subsequent steps were carried out at 4°C. To purify cloned full-length En, KCl was added to 225 mM, and nucleic acids were removed over a DE52 column (Whatman), which was equilibrated with HEDN + 225 mM KCl. bEn was recovered in the flow-through with 2 bed volumes of the same buffer, diluted with buffer HEDN + 150 mM KCl, loaded onto a heparin-Sepharose CL-6B column (Pharmacia Biotech Inc.), and finally eluted with a linear KCl gradient (0.15–1 M KCl in HEDN). Fractions were monitored by SDS-PAGE. bEn eluted with 0.4–0.6 M KCl and was purified to 90% homogeneity as determined by silver staining. 16 mg of partially purified En was obtained from 10 liters of cell culture. The total protein concentration was 0.8 mg/ml, as determined by a dye binding assay (Bio-Rad). To purify the two N-truncated versions of the En (En-297/552 and En-374/552), supernatants obtained from the high speed centrifugation step were loaded directly onto phosphocellulose columns (Whatman, P11), which were equilibrated with buffer HEDN + 100 mM KCl (flow rate of about 0.4 ml/min). Columns were washed with 4 bed volumes of the same buffer, and the En versions were eluted with a linear KCl gradient (0.1–1 M KCl in HEDN). Fractions were monitored by SDS-PAGE, and fractions containing the En fragment were pooled, diluted 10–20 times with buffer HE (25 mM HEPES, pH 7.6, 0.1 mM EDTA) to bring the KCl concentration to 50 mM, and loaded onto Mono-S columns (HR 5/5, Pharmacia). Fractionations were performed with an HPLC Gold System (Beckman) and were monitored by UV absorption at 280 nm. Columns were extensively washed with buffer HE + 50 mM KCl (flow rate, 2 ml/min), and En fragments were eluted with linear KCl gradients (0.05–1 M KCl in HE (flow rate, 1 ml/min)). 1-ml fractions were collected and monitored by SDS-PAGE. The two En fragments were purified to more than 95%, as determined by Coomassie Blue staining. Protein concentrations were 0.3 and 1.4 mg/ml for En-297/552 and En-374/552, respectively, as determined by UV absorption at 280 nm. For both proteins, 2 mg of highly purified preparation were obtained from 1-liter cell cultures. Aliquots were stored at –80 °C. For phosphorylation assays, bacterial protein extracts were prepared from 5-ml minicultures as follows. From freshly transformed BL21(DE3) cells, 10 colonies were grown at 37 °C until mid-exponential growth phase (about 2 h) in LB/amp medium. Expression was achieved with 1 mM IPTG, and cells were grown at 37 °C for an additional 3 h. Induced cells were harvested by low speed centrifugation, frozen in dry ice for 30 min, stored overnight at –80 °C, thawed on ice for 5 min, quickly resuspended in 200 μl of buffer HEDN by up/down pipetteing, and finally transferred to 1.5-ml microtubes. Keeping tubes on ice, cells were sonicated 10 times, 1 s each at setting 4 of a Branson Sonifier with a microtip, and bacteria debris was removed by centrifugation for 5 min at 13,000 × g; finally, the resulting supernatants (typically containing about 5 μg/μl total protein) were monitored by SDS-PAGE (typically 10 μl of extracts were analyzed) and directly used in our various phosphorylation assays. Expression, Metabolic Labeling, and Immunopurification of En from Drosophila Tissue Culture Cells—Nuclear extracts were prepared from Drosophila embryos or HSEN cells as previously described (Refs. 57Soeller W.C. Poole S.J. Kornberg T.B. Genes & Dev. 1988; 2: 68-81Crossref PubMed Scopus (190) Google Scholar, 58Gay N.J. Poole S.J. Kornberg T.B. EMBO J. 1988; 7: 4291-4298Crossref PubMed Scopus (11) Google Scholar, respectively) and stored in aliquots at –80 °C. HSEN cells were grown and metabolically labeled with 1 mCi/ml carrier-free [32P]phosphate (ICN Pharmaceuticals) essentially as previously described (18Gay N.J. Poole S.J. Kornberg T.B. Nucleic Acids Res. 1988; 16: 6637-6647Crossref PubMed Scopus (29) Google Scholar), except that cells were washed twice with 1 × PBS and lysed by three freeze-thaw cycles in 100 μl of 0.25 M Tris-HCl (pH 7.8). The cell lysate was clarified by centrifugation (14,000 × g, 5 min, 4 °C) and processed for immunopurification as follows. mAbs 4F11 (18Gay N.J. Poole S.J. Kornberg T.B. Nucleic Acids Res. 1988; 16: 6637-6647Crossref PubMed Scopus (29) Google Scholar) were linked to antimouse IgG-coated magnetic beads (MagniSort M, DuPont NEN), 1 volume of a 1:1 mixture of mAb 4F11-attached beads, and buffer IM (25 mM HEPES, pH 8, 100 mM KCl, 0.1 mM EDTA, 0.1% (v/v) Nonidet P-40, 1.25 mM MgCl2, 10% glycerol, 10 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 10 mM ammonium molybdate) was added and then incubated at 25 °C for 30 min. The beads were washed three times, 10 min each at 25 °C with 2 volumes of buffer IM. Bound En was removed from the beads by boiling for 5 min in 50 μl of SDS-PAGE sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 4 M urea, 0.0025% bromphenol blue) and finally analyzed by SDS-PAGE and autoradiography. Two-dimensional Gel Electrophoresis and Immunoblot Analysis of En Modification Patterns—Two-dimensional gels were run following procedures described by 0′Farrell et al. (59O'Farrell P.Z. Goodman H.M. O'Farrell P.H. Cell. 1977; 12: 1133-1142Abstract Full Text PDF PubMed Scopus (2583) Google Scholar). 10-h-old HS3 embryos were aged for 2 h, washed with 0.04% Triton X-100, 0.7% NaCl onto nitex netting, dechorionated by immersion in 50% commercial bleach for 2 min, and finally extensively washed with Triton X-100, NaCl solution. Embryos were heat shocked at 37 °C for 1 h, suspended in 4 μl/mg of embryos in isoelectric focusing buffer IF (9.5 M urea, 2% Nonidet P-40, 5% 2-mercaptoethanol, and 2% ampholytes (4 parts of pH 5–8 for 1 part of pH 3.5–10)), supplemented with a protease inhibitor mixture (the same that the mixture included in buffer HEDN), and were homogenized by strokes with a Teflon pestle fitting 1.5-ml microtubes. Samples obtained from soluble extracts were precipitated with 3 volumes of cold acetone and kept on dry ice for 4 h; proteins were recovered by centrifugation (14,000 × g, 15 min, 4 °C), ether washed, and solubilized in the buffer IF supplemented with protease inhibitors as above. Samples were warmed 5 min at 42 °C immediately before loading onto isofocusing gels. Isoelectric variants of En were detected by Western blotting as follows. Proteins were transferred electrophoretically to nitrocellulose membranes (HAHY, Millipore), essentially as described by Towbin et al. (60Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44933) Google Scholar), except that 0.1% SDS was added to the transfer buffer. All subsequent incubations were at 25 °C. Nonspecific binding of antibodies was blocked with 3% bovine serum albumin (fraction V from Sigma) in PBS for 30 min, and filters were washed in PTBS (PBS with 0.1% Triton X-100) for 15 min. Primary antibody was added (ascites supernatant from the 4D9 cell line diluted 2-fold in PBS + 3% bovine serum albumin) for 1 h. The filters were washed in PTBS (4 × 5 min) and then incubated for 30 min in PTBS that contained a dilution of 1:2000 of an alkaline phosphatase-conjugated goat anti-mouse IgG (H + L). The filters were washed four times in PTBS as above, washed 1 time in PBS for 5 min, and finally developed with 5-bromo-4-chloro-3-indolyl phosphate + nitro blue tetrazolium following supplier’s instructions (Kirkegaard and Perry). Protein Kinase Assays—Purified En or crude bacterial protein extracts were incubated with crude nuclear protein kinases or purified CK-II (a generous gift from C.V. Glover III). Reactions were carried out for 20 min at 30 °C in buffer P (50 mM Tris-HCl (pH 8), 100 mM NaCl, 5 mM MgCl2, 4 mM dithiothreitol, and 100 μμ [γ-32P]ATP (ICN Pharmaceuticals, 1 μCi/µl). Typically, 5 μg of purified En or 10 μl of crude bacterial protein extracts were incubated in a final volume of 200 μl in presence of 5 μg of nuclear proteins prepared from embryos as a source of protein kinases. For CK-II-dependent phosphorylation, the purified enzyme was added in the assay at a final concentration of 10 nM. Reactions were stopped with 25 mM EDTA, and phosphoprotein species were analyzed by SDS-PAGE followed by autoradiography of the dried gels. Exposures were at –80 °C with Kodak XAR-5 films and intensifying screens. For quantitation, the phosphorylated products were excised from gels, and Cerenkov radiation was measured. For gel mobility shift experiments, purified En-374/552 (10 μμ) was incubated in 50 μl of buffer P with or without purified CK-II (10 nM) for 30 min at 30 °C and directly diluted (a series of 2-fold dilutions) with binding buffer (buffer B) as described below. Phosphopeptide Mapping—For phosphotryptic peptide analysis, bands corresponding to En were cut from the gels with a razor blade, washed with 25% isopropanol for 1 h to remove SDS, and dried in a vacuum centrifuge (Savant). The gel slices were then incubated with a 50 μg/ml solution of tosylphenylalanyl chloromethyl ketone-treated trypsin (Sigma) in 50 mM ammonium bicarbonate (pH 8) overnight at 37 °C. Trypsin (25 μg) was again added, and samples were incubated for an additional 4 h. Samples were centrifuged to remove particulate material, and the resulting peptides were recovered by lyophilization (four times in H2O), dissolved in 5% acetic acid, and then were resolved in two dimensions on 100-μm cellulose thin layer plates (Eastman Kodak Co.; chromatogram 13255, 20 × 20 cm) as described by Ward and Kirschner (61Ward G. Kirschner M. Cell. 1990; 81: 561-577Abstract Full Text PDF Scopus (276) Google Scholar). Electrophoresis (pH 1.9) was for 20 min at 1 kV (15 °C). A solution containing 1% acid fuschin (Sigma) was spotted on the plates to monitor the course of electrophoresis and chromatography steps. Exposures were at –80 °C with Kodak X-Omat AR films and DuPont Cronex Lighting Plus intensifying screens. DNA Binding Assays—Reactions were assembled at 4 °C for 30 min in a final volume of 10 μl of binding buffer β (25 mM HEPES (pH 7.6), 100 mM KCl, 1% (w/v) polyvinyl alcohol, 0.1% Nonidet P-40, 1 mg/ml bovine serum albumin (Life Technologies, Inc.)) supplemented with 1 × 10–10 M 32P-labeled DBEN probe. Complexes were loaded onto 7.5% (29:1 (w/v) acrylamide, N,N′-methylenebisacrylamide; Bio-Rad) nondenaturing 0.5 × TBE (90 mM Tris, 90 mM borate, 2 mM EDTA) Polyacrylamide gels containing 0.1% Nonidet P-40. Gels (15 cm) were prerun at 4 °C during 2 h at 200 V and run at 4 °C during 2/3 h at the same voltage. Gels were dried onto DE51 paper (Whatman) and exposed to Kodak XAR films at –80 °C. Patterns of Posttranslational Modification of En in Embryos and Tissue Culture Cells—In a previous study, Drosophila embryos and cultured cells were labeled with 32PO4–, and phosphoamino acid analysis of immunoprecipitates revealed that En had been modified by a serine-specific protein kinase (18Gay N.J. Poole S.J. Kornberg T.B. Nucleic Acids Res. 1988; 16: 6637-6647Crossref PubMed Scopus (29) Google Scholar). We have extended these studies, first by determining the fraction of En molecules that are covalently modified and by quantifying the number of modified residues. To obtain unmodified protein, an engrailed cDNA clone was expressed in E. coli using a vector with an inducible T7 promoter (27Studier W.F. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4839) Google Scholar). En accumulated up to 20% of total soluble protein after appropriate induction (see below, Fig. 3b) and was partially purified on a heparin-Sepharose column. Two-dimensional Polyacrylamide gel electrophoresis of this enriched fraction revealed a major spot of silver staining material with an apparent pI of 7.75 (Fig. 1a). This value closely approximates the theoretical value of 7.8 calculated for the engrailed amino acid sequence. Not apparent in this figure are the several degradation products that were revealed by Western immunoblotting (not shown).FIG. 1Two-dimensional analysis of En modifications in Drosophila embryos and tissue culture cells. Proteins were resolved in the first dimension by isoelectric focusing (a, b, d; separation right to left) or by non-equilibrium pH gradient electrophoresis (c, separation left to right) and in the second dimension (top to bottom) by SDS-PAGE (9% gels). Proteins were electrotransferred to nitrocellulose membranes and Western blotted to detect En. a, silver staining analysis of a protein extract (1 μg) highly enriched in full-length En expressed in and purified from E. coli (as described under “Materials and Methods”). The apparent isoelectric point (7.75) was determined by measuring the pH value of solutions containing pieces of sectioned isoelectric focusing gels. b and d, Western analysis of En obtained from a nuclear extract prepared from 2–12-h Oregon-R embryos (40 μg of soluble nuclear proteins) or from heat-shocked HS3 embryos of 2–12 h of development (total embryonic proteins from about 300 embryos heat shocked at 37 °C for 1 h), respectively. c, Western analysis of En in heat-shocked cultured HSEN cells (50 μg). The relative position of each spot is marked and numbered. Lane M, molecular mass markers. Note that En migrates with an apparent molecular mass of approximately 70 kDa.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In contrast, En extracted from Drosophila embryos (2–16 h after egg laying) resolved into an array of four isoforms corresponding to the net addition of zero, one, two, or three negative charges (Fig. 1b). More than 75% of the En appeared to be modified. A similar pattern of modifications was observed for protein extracted from embryos of a transgenic line of flies (28Poole S.J. Kornberg T.B. French V. Ingham P. Cooke J. Smith J. Mechanisms of Segmentation. The Company of Biologists, Cambridge1988: 85-93Google Scholar) that expresses En uniformly and at high levels under heat shock control (Fig. 1d). These patterns of isoelectric variants represent the average of all isoforms produced between 2 and 16 h of embryogenesis. These isoforms are acidic with respect to the unmodified protein, and therefore their existence is consistent with the possibility that En is subjected to successive covalent additions of phosphate on at least three serine residues. A third source of modified En was a Schneider cell line (HSEN) that carries a transfected engrailed cDNA under heat shock control (18Gay N.J. Poole S.J. Kornberg T.B. Nucleic Acids Res. 1988; 16: 6637-6647Crossref PubMed Scopus (29) Google Scholar). After heat shock, En accumulated to high levels in the nuclei of these cells, apparently modified in a manner similar to protein in embryos (Fig. 1c). Treating a nuclear extract of these cells with calf intestine alkaline phosphatase eliminated all of the acidic isoforms (not shown), indicating the likely nature of the modifications. Analysis of in Vivo and in Vitro Phosphotryptic Peptides—To identify the metabolically modified residues, an in vitro system capable of phosphorylating En was developed. Sources of unmodified protein and protein kinases were, respectively, En produced in E. coli and a nuclear extract prepared from Drosophila embryos (EmNE). Highly purified En was readily modified by EmNE, as indicated by [γ-32P]αTP labeling (Fig. 2a). Two-dimensional PAGE resolved labeled En into three phosphorylated forms (data not shown). Phosphotryptic peptides derived from protein phosphorylated in vitro and in vivo were compared. In vivo phosphotryptic peptides were obtained from heat-shocked HSEN cells that had been metabolically labeled with [32P]orthophosphate. En was immunopurified, separated by SDS-PAGE (Fig. 2a, lane 3), digested with trypsin, and then fractionated, first by high voltage electrophoresis on thin layer cellulose plates and then by chromatography. 11 phosphotryptic peptides were detected by autoradiography, mostly distributed among a set of peptides that were not resolved by electrophoresis (Fig. 2c). In vitro phosphotryptic peptides were obtained and analyzed in the same manner, and a similar pattern of phosphopeptides was observed (Fig. 2b). Mixing experiments (not shown) indicated that the homologous" @default.
• W2013906846 created "2016-06-24" @default.
• W2013906846 creator A5009492471 @default.
• W2013906846 creator A5011046135 @default.
• W2013906846 creator A5054928899 @default.
• W2013906846 creator A5073751819 @default.
• W2013906846 date "1995-05-01" @default.
• W2013906846 modified "2023-09-28" @default.
• W2013906846 title "Phosphorylation of the Drosophila Engrailed Protein at a Site Outside Its Homeodomain Enhances DNA Binding" @default.
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