Identification of a Region within the Ubiquitin-activating Enzyme Required for Nuclear Targeting and Phosphorylation
1997; Elsevier BV; Volume: 272; Issue: 16 Linguagem: Inglês
10.1074/jbc.272.16.10895
ISSN1083-351X
AutoresAndrew Stephen, Julie S. Trausch‐Azar, P M Handley-Gearhart, Aaron Ciechanover, Alan L. Schwartz,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoThe ubiquitin-activating enzyme exists as two isoforms: E1a, localized predominantly in the nucleus, and E1b, localized in the cytoplasm. Previously we generated hemagglutinin (HA) epitope-tagged cDNA constructs, HA1-E1 (epitope tag placed after the first methionine) and HA2-E1 (epitope tag placed after the second methionine) (Handley-Gearhart, P. M., Stephen, A. G., Trausch-Azar, J. S., Ciechanover, A., and Schwartz, A. L. (1994) J. Biol. Chem.269, 33171–33178), which represent the native isoforms. HA1-E1 is exclusively nuclear, whereas HA2-E1 is found predominantly in the cytoplasm. Using high resolution isoelectric focusing and SDS-polyacrylamide gel electrophoresis, we confirm that these epitope-tagged constructs HA1-E1 and HA2-E1 represent the two isoforms E1a and E1b. HA1-E1/E1a exists as one non-phosphorylated and four phosphorylated forms, and HA2-E1/E1b exists as one predominant non-phosphorylated form and two minor phosphorylated forms. We demonstrate that the first 11 amino acids are essential for phosphorylation and exclusive nuclear localization of HA1-E1. Within this region are four serine residues and a putative nuclear localization sequence (NLS; 5PLSKKRR). Removal of these four serine residues reduced phosphorylation levels by 60% but had no effect on nuclear localization of HA1-E1. Each serine residue was independently mutated to an alanine and analyzed by two-dimensional electrophoresis; only serine 4 was phosphorylated. Disruption of the basic amino acids within the NLS resulted in loss of exclusive nuclear localization and a 90–95% decrease in the phosphorylation of HA1-E1. This putative NLS was able to confer nuclear import on a non-nuclear protein in digitonin-permeabilized cells in a temperature- and ATP-dependent manner. Thus the predominant requirement for efficient phosphorylation of HA1-E1/E1a is a functional NLS, suggesting that E1a may be phosphorylated within the nucleus. The ubiquitin-activating enzyme exists as two isoforms: E1a, localized predominantly in the nucleus, and E1b, localized in the cytoplasm. Previously we generated hemagglutinin (HA) epitope-tagged cDNA constructs, HA1-E1 (epitope tag placed after the first methionine) and HA2-E1 (epitope tag placed after the second methionine) (Handley-Gearhart, P. M., Stephen, A. G., Trausch-Azar, J. S., Ciechanover, A., and Schwartz, A. L. (1994) J. Biol. Chem.269, 33171–33178), which represent the native isoforms. HA1-E1 is exclusively nuclear, whereas HA2-E1 is found predominantly in the cytoplasm. Using high resolution isoelectric focusing and SDS-polyacrylamide gel electrophoresis, we confirm that these epitope-tagged constructs HA1-E1 and HA2-E1 represent the two isoforms E1a and E1b. HA1-E1/E1a exists as one non-phosphorylated and four phosphorylated forms, and HA2-E1/E1b exists as one predominant non-phosphorylated form and two minor phosphorylated forms. We demonstrate that the first 11 amino acids are essential for phosphorylation and exclusive nuclear localization of HA1-E1. Within this region are four serine residues and a putative nuclear localization sequence (NLS; 5PLSKKRR). Removal of these four serine residues reduced phosphorylation levels by 60% but had no effect on nuclear localization of HA1-E1. Each serine residue was independently mutated to an alanine and analyzed by two-dimensional electrophoresis; only serine 4 was phosphorylated. Disruption of the basic amino acids within the NLS resulted in loss of exclusive nuclear localization and a 90–95% decrease in the phosphorylation of HA1-E1. This putative NLS was able to confer nuclear import on a non-nuclear protein in digitonin-permeabilized cells in a temperature- and ATP-dependent manner. Thus the predominant requirement for efficient phosphorylation of HA1-E1/E1a is a functional NLS, suggesting that E1a may be phosphorylated within the nucleus. The ubiquitin-activating enzyme (E1) 1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-carrier enzyme; E3, ubiquitin-protein ligase; BSA, bovine serum albumin; Cy3, indocarbocyanine; HA, influenza hemagglutinin monoclonal antibody epitope; IEF, isoelectric focusing; NLS, nuclear localization sequence; Ub, ubiquitin; PCR, polymerase chain reaction; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; sulfo-SMCC, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1carboxylate. catalyzes the first reaction in the ubiquitin (Ub) conjugation pathway. Activation of Ub occurs by the formation of a high energy thiol-ester bond between E1 and the C-terminal glycine of Ub and the production of AMP and PPi. Activated Ub is then transferred to a ubiquitin-conjugating enzyme (ubiquitin-carrier enzyme, E2). E2 proteins conjugate Ub directly to a target substrate or, alternatively, transfer Ub to a ubiquitin-protein ligase (E3), which then conjugates Ub to a target protein (reviewed in Ref. 1Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1602) Google Scholar and 2Hochstrasser M. Curr. Opin. Cell Biol. 1995; 7: 215-223Crossref PubMed Scopus (785) Google Scholar). Multiple rounds of Ub conjugation result in the rapid degradation of the target protein by the 26 S proteasome (3Croux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2239) Google Scholar). Recent published results, however, suggest ubiquitination plays an indirect role in protein degradation as well (reviewed in Ref. 4Hochstrasser M. Cell. 1996; 84: 813-815Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Ubiquitination on cell surface receptors such as Ste2 (5Hicke L. Riezman H. Cell. 1996; 84: 277-287Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar), yeast α mating receptor (6Roth A.F. Davis N.G. J. Cell Biol. 1996; 134: 661-674Crossref PubMed Scopus (145) Google Scholar), and growth hormone receptor (7Strous G, J. van Kerkhof P. Govers R. Ciechanover A. Schwartz A.L. EMBO J. 1996; 15: 3806-3812Crossref PubMed Scopus (265) Google Scholar) triggers their endocytosis and degradation within the lysosome. Because Ub requires activation prior to participation in any subsequent reactions, E1 plays a key role in the Ub-conjugating pathway. E1 is localized in both the nucleus and the cytoplasm (8Schwartz A.L. Trausch J.S. Ciechanover A. Slot J.W. Geuze H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5542-5546Crossref PubMed Scopus (48) Google Scholar, 9Trausch J.S. Grenfell S.J. Handley-Gearhart P.M. Ciechanover A. Schwartz A.L. Am. J. Physiol. 1993; 264: C93-C102Crossref PubMed Google Scholar) and exists as two isoforms E1a (117 kDa) and E1b (110 kDa) (10Cook J.C. Chock P.B. J. Biol. Chem. 1992; 267: 24315-24321Abstract Full Text PDF PubMed Google Scholar, 11Handley-Gearhart P.M. Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1994; 269: 33171-33178Abstract Full Text PDF PubMed Google Scholar). To investigate the nature of these isoforms, epitope-tagged cDNA constructs of E1 were generated where the hemagglutinin (HA) epitope tag was placed after the first methionine (amino acid 1; HA1-E1) or after the second methionine (amino acid 40; HA2-E1) (11Handley-Gearhart P.M. Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1994; 269: 33171-33178Abstract Full Text PDF PubMed Google Scholar). HA1-E1 localized exclusively to the nucleus and displayed the same molecular weight as E1a, whereas HA2-E1 localized predominantly in the cytoplasm and displayed the same molecular weight as E1b (11Handley-Gearhart P.M. Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1994; 269: 33171-33178Abstract Full Text PDF PubMed Google Scholar). These observations are consistent with the hypothesis that the E1 isoforms are a result of alternate translational start sites. Of these isoforms, E1a/HA1-E1 is phosphorylated in vivo (11Handley-Gearhart P.M. Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1994; 269: 33171-33178Abstract Full Text PDF PubMed Google Scholar) on a serine residue (12Cook J.C. Boon Chock P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3454-3457Crossref PubMed Scopus (17) Google Scholar), whereas E1b/HA2-E1 is not phosphorylated. Phosphorylation of E1a occurs in a cell cycle-dependent manner (maximal in G2) and the resultant phosphorylated E1a was concentrated within nuclear extracts (13Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1996; 271: 15608-15614Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). On the basis of these observations, we proposed that an increase in phosphorylation of E1a functions to increase the import and/or retention of E1a in the nucleus (13Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1996; 271: 15608-15614Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The present study uses HA epitope-tagged cDNA constructs of E1 to identify amino acids that are important for nuclear localization and phosphorylation and whether phosphorylation of E1 is a prerequisite for its nuclear localization. We identify a specific serine residue that is phosphorylated in addition to a region of basic amino acids that is required for both nuclear localization and phosphorylation. Our data suggest that phosphorylation is not required for nuclear import, but that E1 may require a functional nuclear localization sequence for efficient phosphorylation. HeLa cells were cultured in Dulbecco's modified Eagle's medium and 10% fetal calf serum and maintained at 37 °C and 5% CO2 in a humidified chamber as described previously (9Trausch J.S. Grenfell S.J. Handley-Gearhart P.M. Ciechanover A. Schwartz A.L. Am. J. Physiol. 1993; 264: C93-C102Crossref PubMed Google Scholar). Transient transfections were performed using a calcium phosphate coprecipitation method as described by Chen and Okayama (14Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4823) Google Scholar); cells were processed 40–48 h following transfection. HA1-E1, HA2-E1, and HA1-E1-del-11 constructs were described previously (11Handley-Gearhart P.M. Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1994; 269: 33171-33178Abstract Full Text PDF PubMed Google Scholar). The N-terminal deletion constructs, HA1-E1-del-4, HA1-E1-del-22, and HA1-E1-del-30, were constructed by annealing a primer encoding the HA epitope tag (YPYDVPDYASG) and a 30-base overlap with the E1 sequence, beginning at amino acid 5, 23, or 31 (nucleotides 142, 196, or 220). The following point mutations (HA1-E1-S2A, HA1-E1-S3A, HA1-E1-S4A, and HA1-E1-S7A) were constructed using an HA-containing primer where serine 2, 3, 4, or 7 encoded within the 30-base overlap was replaced with an alanine (nucleotide 133, 136, 139, and 148). Using the same protocol, HA1-E1-R10A,R11A had lysines 10 and 11 replaced with alanines. HA1-E1-del-4,S7A, was constructed using an HA encoding primer with a 30-base pair overlap (which included a point mutation to change serine 7 to an alanine) beginning with amino acid 5. PCR products of the HA cDNA sequence attached to an 800-base fragment of E1 were generated using the HA-containing primers and a downstream E1 primer whose sequence included a unique NcoI site within the E1 sequence. PCR was performed using the Ericomp Twinblock thermocycler, andTaq DNA polymerase (Promega) with an annealing temperature of 55 °C. The human E1 cDNA in pGem7zf+ (E1pGem) was used as the template. PCR products were subcloned into the pCRII vector (TA cloning kit, Promega) and sequenced. Full-length E1 was constructed by replacement of a BamHI and NcoI fragment of E1pGem with the HA-E1 fragment generated by PCR. Full-length HA-E1 constructs were then subcloned into the mammalian expression vector pcDNA3 (Invitrogen). HA1-E1-del-8–11 was generated where the amino acids KKRR were removed using the Sculptor Mutagenesis kit (Amersham). All restriction enzymes were from Promega. HeLa cells were metabolically labeled with [32P]orthophosphoric acid (ICN) as described previously (13Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1996; 271: 15608-15614Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Cells were lysed in 20 mm Tris, pH 7.6, 0.25% Triton X-100, 0.2% DTT containing 0.2 mm phenylmethylsulfonyl fluoride, 2.5 μg/ml leupeptin, 1 μm pepstatin, 1 mm β-glycerophosphate, 1 mm sodium orthovanadate, and 5 mm sodium fluoride. The lysates were incubated on ice for 20 min, then centrifuged at 14,000 rpm for 15 min. Protein concentrations of cleared lysates were determined using the Bio-Rad protein assay with bovine serum albumin as standard. HA-tagged E1 was immunoprecipitated from radiolabeled extracts (200 μg of protein) using the 12CA5 monoclonal antibody raised against the HA epitope tag, as described previously (11Handley-Gearhart P.M. Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1994; 269: 33171-33178Abstract Full Text PDF PubMed Google Scholar). The immunoprecipitation buffer contained the following phosphatase inhibitors: 1 mmβ-glycerophosphate, 1 mm sodium orthovanadate, and 5 mm sodium fluoride. Samples were resolved on a 7.5% reducing gel and then transferred to nitrocellulose. HA-tagged E1 was visualized using the 12CA5 antibody, followed by a peroxidase-conjugated goat anti-mouse IgG (11Handley-Gearhart P.M. Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1994; 269: 33171-33178Abstract Full Text PDF PubMed Google Scholar). Immunoreactive proteins were detected using enhanced chemiluminescence (Amersham) and quantified using a Molecular Dynamics Densitometer. The immunoblot was then exposed in a Molecular Dynamics PhosphorImager cassette, and the radiolabeled proteins were quantified using a Molecular Dynamics Storm Optical Scanner. Immunofluorescence on transiently transfected HeLa cells was performed as described previously (11Handley-Gearhart P.M. Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1994; 269: 33171-33178Abstract Full Text PDF PubMed Google Scholar). HA-tagged E1 was detected using the 12CA5 monoclonal antibody followed by rhodamine-conjugated donkey anti-mouse IgG preabsorbed against bovine, goat, horse, human, rabbit, and sheep serum proteins (Jackson). Peptides were synthesized (Washington University) and cross-linked to the non-nuclear protein bovine serum albumin (BSA) (Calbiochem) using sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC, Pierce). This cross-links the N-terminal cysteine residue of the peptide to amino groups on the BSA. Sulfo-SMCC (1 mg/ml) was added at 20-fold molar excess to the BSA and incubated for 30 min at room temperature, excess sulfo-SMCC was removed using a Pharmacia PD10 column equilibrated in 100 mm sodium phosphate, pH 7.0. Peptides with a reduced cysteine residue are added in a 50-fold molar excess to the activated BSA and incubated overnight at 4 °C. Free peptide was then removed using a Sephadex G50 column equilibrated in 100 mm sodium phosphate, pH 7.0. The conjugated BSA was dialyzed overnight against 100 mm sodium carbonate, pH 9.2. The dialyzed peptide-BSA conjugate was labeled with Cy3 (indocarbocyanine) using the Fluorolink kit from Amersham. Free Cy3 was removed using a Sephadex G50 column equilibrated in phosphate-buffered saline, pH 7.2. The Cy3-BSA peptide was stored at −80 °C. Cell permeabilization and subsequent nuclear import were based on the methods of Adam et al.(15Adam S.A. Sterne-Marr R. Gerace L. J. Cell Biol. 1990; 111: 807-816Crossref PubMed Scopus (771) Google Scholar). HeLa cells were grown until they were subconfluent (18–36 h) on coverslips (15 mm × 15 mm). Cells were washed once with ice-cold import buffer (20 mm Hepes-KOH, pH 7.3, 110 mmpotassium acetate, 2 mm magnesium acetate, 1 mmEGTA, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin, 2 mm dithiothreitol). Cells were then permeabilized with import buffer containing 50 μg/ml digitonin (Sigma) for 5 min at 4 °C and washed three times with ice-cold import buffer. Coverslips were inverted on 50 μl of import mix (containing 50% (v/v) rabbit reticulocyte lysate (Promega), 2 μl of 20 mm ATP, 2 μl of 100 mm phosphocreatine, and 2 μl of 2 mg/ml creatine phosphokinase, 2 μl of 20 mg/ml ovalbumin, and 4 μl of Cy3-BSA peptide). Cells were incubated for 30 min at either 30 °C or 4 °C. For import in the absence of ATP, permeabilized cells were first depleted of ATP with 2 μl of 2000 units/ml apyrase for 1 h at 4 °C. Nuclear import was then performed as before except the ATP regenerating system in the import mix was replaced with apayrase. After import cells were rinsed twice with import buffer and fixed with 4% paraformaldehyde for 15 min at 4 °C and then washed with ice-cold import buffer. Samples were analyzed with an Olympus fluorescence microscope, and photographs were taken using a 50× oil objective and Tmax film (Eastman Kodak Co.). Isoelectric focusing (IEF) was performed using Immobiline Drystrips, pH 4–7 (Pharmacia), and electrophoresed using the Multiphor II system (Pharmacia). HeLa cells were metabolically labeled with [35S]methionine/cysteine Tran35S-label (ICN) or [32P]orthophosphoric acid as described previously (13Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1996; 271: 15608-15614Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Immunoprecipitated E1 or HA-tagged E1 was eluted from Protein A beads with IEF sample buffer (9 m urea, 0.5% Triton X-100, 2% ampholytes, 1% DTT), loaded onto the Drystrip and focused for 3 h at 300 V and for 13 h at 2200 V. After the IEF run, the Drystrips were equilibrated in SDS buffer (0.125 m Tris, 2% SDS, 10% glycerol, 4.9 mm DTT, pH 6.8) for 20 min. The dry strip was placed on a reducing 7.5% SDS-PAGE gel (18 × 16 cm) and electrophoresed for 5 h at 40 mA as the second dimension. The gel was fixed for 30 min and fluoroenhanced in Amplify (Amersham). The dried gel was then exposed to film for autoradiography at −80 °C. Two-dimensional gels were analyzed using a Molecular Dynamics Storm Optical Scanner. HeLa cells metabolically labeled with either [32P]orthophosphoric acid or Tran35S-label were lysed as described above but without the addition of phosphatase inhibitors. Lysate was then dialyzed overnight into 100 mm Tris/HCl, pH 7.0, 150 mm NaCl, 1 mm dithiothreitol. Dialyzed 32P- or35S-labeled lysate (300 μg of protein) was incubated with 1 unit of potato acid phosphatase (Sigma) in 100 mm sodium citrate, pH 5.8, 10 mm MgCl2 overnight at 4 °C (final volume of 80 μl). The following day another 1 unit of phosphatase was added and incubated for 2 h at ambient temperature. Parallel incubations were included without the addition of phosphatase. E1 was immunoprecipitated from the lysate as described previously (13Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1996; 271: 15608-15614Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). 32P-Labeled E1 was resolved by SDS-PAGE and transferred to nitrocellulose, and E1 was visualized by an anti-E1 polyclonal antibody. 35S-Labeled E1 was immunoprecipitated and resolved by two-dimensional gel electrophoresis (as described previously). E1 exists as two isoforms E1a (117 kDa) and E1b (110 kDa). Previously we prepared E1 cDNA constructs in which an HA-epitope tag was placed after the first methionine (HA1-E1) or the second methionine (HA2-E1) (Fig. 1 A; Ref. 11Handley-Gearhart P.M. Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1994; 269: 33171-33178Abstract Full Text PDF PubMed Google Scholar). HA1-E1 and HA2-E1 were similar in their molecular weight and phosphorylation state to E1a and E1b, respectively (11Handley-Gearhart P.M. Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1994; 269: 33171-33178Abstract Full Text PDF PubMed Google Scholar). Two-dimensional gel electrophoresis analysis of E1 from Chinese hamster (ts20) cells resolved E1 into the two isoforms E1a and E1b; E1a resolved further as three phosphorylated and one non-phosphorylated forms, whereas E1b resolved as one non-phosphorylated form (13Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1996; 271: 15608-15614Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). To further determine the relationship between human E1, HA1-E1, and HA2-E1, we analyzed these species by two-dimensional gel electrophoresis. HeLa cells were metabolically labeled with Tran35S-label or [32P]orthophosphoric acid, and human E1 was immunoprecipitated with a polyclonal antibody raised against human E1 (11Handley-Gearhart P.M. Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1994; 269: 33171-33178Abstract Full Text PDF PubMed Google Scholar). HeLa cells transiently transfected with HA1-E1 or HA2-E1 were metabolically labeled with Tran35S-label or [32P]orthophosphoric acid; HA-tagged proteins were immunoprecipitated with a monoclonal antibody recognizing the epitope tag (12CA5). Immunoprecipitated proteins were then resolved in the first dimension by isoelectric focusing, followed by SDS-PAGE in the second dimension (Fig. 1). 35S-Labeled human E1 migrated to its predicted isoelectric point of 5.7, but rather surprisingly E1a resolved as five spots and E1b resolved as three spots (Fig. 1 B). In addition to these predominant spots, other less abundant species which migrated toward the anode could be detected (Fig. 1 B). When human E1 was labeled with [32P]orthophosphoric acid, E1a resolved as four spots; however, no phosphorylation was observed for E1b (Fig.1 B). This is distinct from our previous observations withhamster E1 (13Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1996; 271: 15608-15614Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). We attribute these differences to the better resolving capabilities of our isoelectric focusing used in this study. Previously IEF was performed with 1.5-mm rod gels; however, in this study Pharmacia Immobiline Drystrips were used, in which the Drystrips contain a preformed immobilized and non-drifting pH gradient, which can be electrophoresed at much higher voltages and results in superior resolution. These differences were not due to cell type as separation of hamster E1 using this method generated the same pattern of spots (data not shown). The epitope-tagged forms of E1 migrated to the same isoelectric point as the wild type protein (Fig. 1 C). 35S-Labeled HA1-E1 resolved as five main spots (0, −1, −2, −3, and −4), consistent with our observations of E1a. 35S-Labeled HA2-E1 resolved as three predominant spots (−1, 0, and 1), this pattern was very similar to that of E1b. 32P-Labeled HA1-E1 resolved as four distinct spots (Fig. 1 C; spots −1, −2, −3, and −4) in a pattern similar to E1a. 32P-Labeled HA2-E1 resolved as two spots (−1 and −2); however, these were only visible with extended exposures (Fig. 1 C; approximately 8 times longer than for HA1-E1). These species are not very abundant; for instance spot 2 could not be detected when labeled with Tran35S-label (Fig.1 C) and spot −1 only represents 2.5% of the total HA2-E1. These phosphorylated forms were not detected in E1b because they are present at levels too low to detect. In addition to the phosphorylated forms of HA1-E1/E1a and HA2-E1/E1b, there are also some spots that migrate toward the anode (spots 1 and 2); these species probably represent alternative charged forms. The pattern of spots for both HA1-E1 and HA2-E1 is essentially identical to E1a and E1b and is consistent with our hypothesis that the two E1 isoforms result from alternate translational start sites at the first and second in-frame methionines in the E1 sequence. These results also demonstrate that addition of the HA epitope tag to E1 did not alter its phosphorylation state. To further confirm our observations, we dephosphorylated human E1 by treatment with potato acid phosphatase. Dephosphorylated35S-labeled E1a resolved as a pattern of spots similar to E1b; it was found predominantly as spot 0 with minor species migrating as spot −1, 1, and 2 (data not shown). There was very little change in the pattern of 35S-labeled E1b spots after dephosphorylation; only a slight decrease in spot −1 was observed. Taken together, these data suggest that HA1-E1/E1a exists predominantly as four phosphorylated and a non-phosphorylated form; HA2-E1/E1b exists as two minor phosphorylated forms and as a predominant non-phosphorylated form. Furthermore, in addition to the phosphorylated forms, both HA1-E1/E1a and HA2-E1/E1b can be resolved into other non-phosphorylated charged species. A relatively abundant species (spot 1) is detected in 35S-labeled HA2-E1/E1b preparations, although the precise nature of these charged variants is currently not known. We and others have previously demonstrated that E1a/HA1-E1 are phosphorylated, whereas no detectable phosphorylation has been demonstrated for E1b/HA2-E1 (11Handley-Gearhart P.M. Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1994; 269: 33171-33178Abstract Full Text PDF PubMed Google Scholar, 12Cook J.C. Boon Chock P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3454-3457Crossref PubMed Scopus (17) Google Scholar, 13Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1996; 271: 15608-15614Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). However, in our current study it appears that HA2-E1/E1b is phosphorylated but at levels approximately 100-fold less than HA1-E1/E1a (Fig. 1). Others have recently determined that E1 is only phosphorylated on serine residues (12Cook J.C. Boon Chock P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3454-3457Crossref PubMed Scopus (17) Google Scholar). As HA1-E1 is 40 amino acids longer than HA2-E1, it is tempting to speculate that this region at the N terminus contains the predominantly phosphorylated serine residues. Within this 40 amino acid region, there 10 serine residues. Thus, to identify which serines are involved in phosphorylation of HA1-E1, a series of N-terminal truncation mutants were generated (Fig. 2 A). Constructs were made where the HA epitope was attached to the N-terminal region of E1 to create proteins that were truncated in segments of about 10 amino acids (Fig. 2 A). These HA-tagged E1 truncation mutants were transiently expressed in HeLa cells and metabolically labeled with [32P]orthophosphoric acid. HA-tagged constructs were then immunoprecipitated using the 12CA5 monoclonal antibody and resolved by SDS-PAGE and transferred to nitrocellulose. The total amount of immunoreactive HA-tagged construct was determined by immunoblot using the 12CA5 antibody (Fig. 2 B). The nitrocellulose was subjected to autoradiography to determine the 32P incorporation (Fig. 2 B). Removal of the first 11 amino acids of the E1 sequence (HA1-E1-del-11) resulted in no detectable phosphorylation. Removal of 22 (HA1-E1-del-22), 30 (HA1-E1-del-30), and 40 amino acids (HA2-E1) at the N terminus also resulted in no detectable phosphorylation of the HA-tagged constructs. These data thus suggest the first 11 amino acids contain residues that are essential for efficient phosphorylation of HA1-E1/E1a. Phosphorylation of certain proteins correlates with their retention in either the nucleus or cytoplasm (reviewed in Ref. 16Jans D.A. Hubner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (389) Google Scholar). For example SWI5 is excluded from the nucleus when phosphorylated (17Jans D.A.. Moll T. Nasmyth K. Jans P. J. Biol. Chem. 1995; 270: 17064-17067Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), whereas STAT-3 is translocated to the nucleus after phosphorylation (18Ruff-Jamison S. Zhong Z. Wen Z. Chen K. Darnell J.E. Cohen S. J. Biol. Chem. 1994; 269: 21933-21935Abstract Full Text PDF PubMed Google Scholar). Indeed this is the case with E1; HA1-E1 is localized exclusively in the nucleus and phosphorylated, whereas HA2-E1 is found almost exclusively in the cytoplasm and is phosphorylated approximately 100-fold less than HA1-E1 (11Handley-Gearhart P.M. Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1994; 269: 33171-33178Abstract Full Text PDF PubMed Google Scholar). Previously we have determined the subcellular localization of HA1-E1-del-11 by immunofluorescence using the 12CA5 monoclonal antibody (11Handley-Gearhart P.M. Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1994; 269: 33171-33178Abstract Full Text PDF PubMed Google Scholar). Removal of the first 11 amino acids resulted in predominant cytoplasmic staining with both positive and negative nuclear staining, as was also observed with HA2-E1 (11Handley-Gearhart P.M. Stephen A.G. Trausch-Azar J.S. Ciechanover A. Schwartz A.L. J. Biol. Chem. 1994; 269: 33171-33178Abstract Full Text PDF PubMed Google Scholar). We thus determined the immunofluorescent localization of the HA-tagged E1 truncation mutants, HA1-E1-del-22 and HA1-E1-del-30. These constructs were localized with the same distribution as HA1-E1-del-11 (data not shown). Therefore, in addition to amino acids essential for phosphorylation, the first 11 amino acids also contains residues necessary for the exclusive nuclear localization of HA1-E1. The first 11 amino acids of the E1 sequence contains, in addition to serines 2, 3, 4, and 7, a putative nuclear localization sequence (NLS),5PLSKKRR11. To determine which residues are responsible for phosphorylation or for nuclear localization, two additional HA-tagged E1 constructs were prepared: HA1-E1-del-7, where the first 7 amino acids (including serines 2, 3, 4, and 7) were removed, and HA1-E1-del-8–11, where the basic region of t
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