(Received for publication, January 10, 1997)
From the Edward Mallinckrodt Departments of
Pediatrics and Molecular Biology and Pharmacology, Washington
University School of Medicine and Division of Pediatric
Hematology-Oncology, Children's Hospital, St. Louis, Missouri
63110 and the ¶ Unit of Biochemistry and Rappaport Institute for
Research in the Medical Sciences, Faculty of Medicine,
Technion-Israel Institute of Technology, Haifa 31096, Israel
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)1
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. 1 and 2). Multiple rounds of
Ub conjugation result in the rapid degradation of the target protein by
the 26 S proteasome (3). Recent published results, however, suggest
ubiquitination plays an indirect role in protein degradation as well
(reviewed in Ref. 4). Ubiquitination on cell surface receptors such as
Ste2 (5), yeast mating receptor (6), and growth hormone receptor
(7) 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 (8, 9) and exists as two isoforms E1a (117 kDa) and E1b (110 kDa) (10, 11). 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) (11). 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 (11). 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 (11) on a serine residue (12), 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 (13). 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 (13).
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 (9). Transient transfections were performed using a calcium phosphate coprecipitation method as described by Chen and Okayama (14); cells were processed 40-48 h following transfection.
Construction of HA-E1 MutantsHA1-E1, HA2-E1, and HA1-E1-del-11 constructs were described previously (11). 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, and Taq 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.
Metabolic Labeling, Immunoprecipitation, and Immunoblot Analysis of HA-E1 ConstructsHeLa cells were metabolically labeled with
[32P]orthophosphoric acid (ICN) as described previously
(13). 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 -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 (11). The immunoprecipitation
buffer contained the following phosphatase inhibitors: 1 mM
-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 (11). 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 (11). 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).
Conjugation of Peptides to BSA and Labeling with Cy3Peptides 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. (15). 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 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 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.).
Two-dimensional Gel ElectrophoresisIsoelectric 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
(13). 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- or 35S-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 (13). 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. 1A; Ref. 11).
HA1-E1 and HA2-E1 were similar in their molecular weight and
phosphorylation state to E1a and E1b, respectively (11).
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 (13). 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 (11). 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. 1B). In addition to these predominant spots, other less abundant species which migrated toward the anode could be detected (Fig. 1B). When human E1 was labeled with [32P]orthophosphoric acid, E1a resolved as four spots; however, no phosphorylation was observed for E1b (Fig. 1B). This is distinct from our previous observations with hamster E1 (13). 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. 1C). 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. 1C; 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. 1C; 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.
1C) 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. Dephosphorylated 35S-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 (11-13). 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
(12). 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. 2A). 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. 2A). 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. 2B). The nitrocellulose was
subjected to autoradiography to determine the 32P
incorporation (Fig. 2B). 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.
Immunolocalization of HA-tagged E1 N-terminal Truncation Mutants
Phosphorylation of certain proteins correlates with their retention in either the nucleus or cytoplasm (reviewed in Ref. 16). For example SWI5 is excluded from the nucleus when phosphorylated (17), whereas STAT-3 is translocated to the nucleus after phosphorylation (18). 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 (11). Previously we have determined the subcellular localization of HA1-E1-del-11 by immunofluorescence using the 12CA5 monoclonal antibody (11). 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 (11). 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.
Identification of Amino Acids Which Are Required for Phosphorylation and Nuclear LocalizationThe 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 the putative
NLS (8KKRR11) was deleted (Fig.
3A). These constructs along with HA1-E1 were transiently transfected into HeLa cells, labeled with
[32P]orthophosphoric acid, resolved by SDS-PAGE, and
transferred to nitrocellulose. The total amount of HA-tagged construct
was determined by immunoblot analysis, and 32P
incorporation was determined by autoradiography (Fig. 3B).
To determine the specific phosphorylation of the HA-tagged constructs, the total amount of HA-tagged E1 constructs was quantified by densitometry, and 32P incorporation was determined by
PhosphorImager analysis (Fig. 3B). Removal of the basic
amino acids (8KKRR11) in the putative NLS from
HA1-E1 drastically reduced the level of specific phosphorylation to 5%
of HA1-E1 (Fig. 3B). Additionally HA1-E1-del-8-11 was no
longer localized exclusively in the nucleus but found predominantly in
the cytoplasm (approximately 90% of the cells displayed completely
negatively stained nuclei) (Fig. 3C). This distribution is
identical to that observed with HA1-E1-del-11. This implies that the
basic region (8KKRR11) of the putative nuclear
localization sequence at the N terminus of E1 is required for the
exclusive nuclear localization of HA1-E1. The dramatic decrease in
phosphorylation of HA1-E1-del-8-11 and the predominant cytoplasmic
distribution strongly suggest a correlation between nuclear
localization and phosphorylation.
The specific phosphorylation of HA1-E1-del-7 (deletion including
serines 2, 3, 4, and 7) was approximately 50% that of HA1-E1 (Fig.
3B). These results suggest that this region contains serines which are phosphorylated within HA1-E1. HA1-E1-del-7 like the wild type
HA1-E1 was localized predominantly in the nucleus (Fig. 3C).
However, HA1-E1-del-7 showed cytoplasmic localization in a small
percentage of cells (approximately 3%, Fig. 3C). To
eliminate the possibility that serine phosphorylation was involved in
the nuclear targeting of HA1-E1, another construct was prepared
(HA1-E1-del-4,S7A, Fig. 4A). HA1-E1-del-4,S7A
was phosphorylated to the same extent as HA1-E1-del-7 (approximately
40% of HA1-E1), but no cytoplasmic localization was observed (Fig.
4A). This observation implies that phosphorylation of any of
the serine residues within the first 7 amino acids (including serines
2, 3, 4, and 7) is not required for exclusive nuclear
localization of HA1-E1.
The dramatic reduction in specific phosphorylation and predominant cytoplasmic localization of HA1-E1-del-8-11 suggests that phosphorylation occurs within the nucleus. An alternative explanation may be that the kinase which phosphorylates HA1-E1 on one or more of the serine 2, 3, 4, and 7 residues binds to the basic region of the NLS (8KKRR11). Thus removal of this region would disrupt both nuclear targeting and phosphorylation. In an attempt to reconcile these two possibilities a further HA-tagged construct was generated where arginine residues 10 and 11 were changed to alanines (HA1-E1-R10A,R11A; Fig. 4A). Removal of these two arginine residues also disrupts nuclear targeting (Fig. 4C) and substantially reduces the specific phosphorylation to levels comparable with HA1-E1-del-8-11 (Fig. 4B; 9% of HA1-E1). Although these results are not conclusive, they do suggest that phosphorylation of HA1-E1 may occur within the nucleus.
The E1 NLS Efficiently Imports a Non-nuclear Protein into the NucleusThe archetypal NLS is that of SV40 large T antigen, which
consists of a basic patch of amino acids on an -helix (19, 20). The
putative E1 NLS (5PLSKKRR11) shows considerable
sequence similarity to the SV40 NLS (71% homology over 7 amino acids)
(Fig. 5A). Digitonin-permeabilized cells have
been used to assay putative NLS motifs for their ability to import
non-nuclear proteins to the nucleus (15). This assay system was
employed to determine if the putative E1 NLS could import BSA into the
nucleus of digitonin-permeabilized HeLa cells. Peptides were
synthesized and cross-linked to BSA using sulfoSMCC via a terminal
cysteine residue. The BSA peptide was then labeled with the fluorescent
compound Cy3. Using reticulocyte lysate as a cytosol source, Cy3-BSA
peptides were then assayed for their ability to be imported into the
nucleus in digitonin-permeabilized HeLa cells (Fig. 5B).
Assays were carried out in the presence of ATP at 30 °C, the absence
of ATP at 30 °C, or the presence of ATP at 4 °C. Using this
system nuclear import of BSA-NLSSV40 was dependent upon
both ATP and temperature (data not shown), consistent with previously
published data (15). The E1 NLS imported BSA to the nucleus in the
presence of ATP and at 30 °C, but not in the absence of ATP nor at
4 °C (Fig. 5B). When KKRR were replaced with alanines
(BSA-NLSE1mutant), BSA was no longer imported to the
nucleus (data not shown).
Others have demonstrated that phosphorylation at or near a NLS may increase the rate of nuclear import or nuclear retention (16, 21). Within the putative E1 NLS (5PLSKKRRV12), there is one serine residue. We did not determine whether this serine residue was phosphorylated when this peptide was conjugated to BSA. However, a third BSA peptide was constructed where serine 7 was changed to an alanine (BSA-NLSE1-S7A; 5PLKKRRV12) to determine if this serine played a role in nuclear import. There was no substantial difference in nuclear import between BSA-NLSE1-S7A and BSA-NLSE1 (Fig. 5B). Thus, we conclude that the E1 sequence 5PLSKKRRV12 is able to confer nuclear import on a non-nuclear protein, and within this region KKRR is essential, whereas serine 7 is not.
Identification of Individual Serine Residues Which are PhosphorylatedTo address which serine residues are
phosphorylated within the first 7 amino acids of HA1-E1, a series of
serine point mutants were generated where serine residues 2, 3, 4, or 7 were individually changed to an alanine (Fig.
6A). When the specific phosphorylation of
each of these constructs was compared with HA1-E1 (Fig. 6B), HA1-E1-S4A was consistently the lowest (approximately 60%).
HA1-E1-S2A, HA1-E1-S3A, and HA1-E1-S7A generally had levels of specific
phosphorylation similar to the wild type HA1-E1. However, between
experiments the level of specific phosphorylation of each construct
compared with HA1-E1 varied somewhat. Therefore, to determine
definitively which residues are phosphorylated, each construct was
analyzed by IEF followed by SDS-PAGE. Each construct was transiently
transfected into HeLa cells and metabolically labeled with
[32P]orthophosphoric acid; HA-containing proteins were
immunoprecipitated and subjected to two-dimensional electrophoresis
(Fig. 6C). HA1-E1 migrates as four phosphorylated species
when analyzed by two-dimensional electrophoresis. Serine point mutants
HA1-E1-S2A, HA1-E1-S3A, and HA1-E1-S7A all migrated as four
phosphorylated species (Fig. 6C). However, HA1-E1-S4A
migrated as only three phosphorylated species indicating that changing
serine 4 to an alanine removed one phosphorylation site from HA1-E1.
Thus serine 4 is phosphorylated in HA1-E1/E1a.
Analysis of human E1, HA1-E1, and HA2-E1 by two-dimensional gel electrophoresis results in very similar electrophoretic patterns for both E1a/HA1-E1 and E1b/HA2-E1 (Fig. 1), respectively. This pattern differs from those previously reported for hamster E1 isoforms (13). We attribute these differences to the superior resolution of our isoelectric focusing gels used in this study. However, the data strongly suggest that E1a/HA1-E1 and E1b/HA2-E1 represent equivalent proteins, with E1a being translated from the first methionine and E1b from the second.
E1b/HA2-E1 exists as two minor phosphorylated forms, a predominant non-phosphorylated form, and several forms which migrated toward the anode (Fig. 1). Densitometric quantification of 35S-labeled E1b/HA2-E1 revealed that only 2.5-4% is found as the phosphorylated forms. Thus only a very small fraction of E1b is phosphorylated and may explain why previous studies by ourselves (11) and others (12, 22) were unable to detect it. E1a/HA1-E1 resolves as four phosphorylated and one non-phosphorylated forms (Fig. 1). Densitometric quantification of the 35S-labeled E1a/HA1-E1 spots revealed that 92-97% is found in the phosphorylated forms, respectively. Several minor positively charged species were also observed (as described previously for E1b/HA2-E1). Dephosphorylation of E1a changed the electrophoretic migration to one very similar to E1b. This suggests that in the basal non-phosphorylated state E1 exists as a heterogeneous mixture of charged forms. This pattern of charged forms was extremely reproducible and was also observed with hamster E1 (data not shown). This heterogeneous mixture of charged forms may arise from modifications such as deamination; however, further work will be required to resolve this.
In an attempt to identify which serine residues are phosphorylated in HA1-E1, N-terminal truncation mutants were generated (Fig. 2A). Removal of only the first 11 amino acids completely abolished phosphorylation of the HA-tagged E1 (Fig. 2B). Removal of further segments of the N terminus also resulted in no detectable phosphorylation (Fig. 2B). Thus the first 11 amino acids contain the necessary information for complete phosphorylation of HA1-E1/E1a.
HA1-E1 is localized exclusively in the nucleus; however, removal of the first 11 amino acids changes the subcellular localization to predominantly cytoplasmic (11). Consistent with this, HA1-E1-del-22, HA1-E1-del-30, and HA2-E1 are all found predominantly in the cytoplasm (data not shown and Ref. 11). Hence, the first 11 amino acids in the E1 sequence contain the necessary information for exclusive nuclear localization and complete phosphorylation.
For efficient nuclear transport, proteins larger than approximately 50 kDa require a NLS (23). The putative NLS (5PLSKKRR) at the extreme N terminus of E1 shares 71% homology with the SV40 NLS, which is regarded as the archetype. Deletion of the four basic residues from this putative NLS (HA1-E1-del-8-11) completely abrogated the exclusive nuclear localization of HA1-E1 (Fig. 3C). In addition, mutation of arginines 10 and 11 to alanines (HA1-E1-R10A,R11A) also abrogated the exclusive nuclear localization (Fig. 4C). Conjugation of this sequence (5PLSKKRRV12) to a non-nuclear protein (BSA) conferred import to the nucleus of digitonin-permeabilized cells (Fig. 5B). This import was both temperature- and ATP-dependent (Fig. 5B). Substitution of 8KKRR with alanines eliminated nuclear import of the substrate (BSA-NLSE1mutant, data not shown). Therefore by all criteria 5PLSKKRRV12 functions as an NLS in E1. However, despite the removal or disruption of this NLS, 5-10% of cells transfected with HA1-E1-del-8-11, HA1-E1-R10A,R11A, and HA2-E1 still show immunofluorescent staining in the nucleus (Ref. 11 and Fig. 4C). This suggests that there may be other functional NLS motifs within the E1 sequence. Indeed, within the C-terminal region there is a putative bipartite NLS (KRRKRK). Bipartite NLS motifs are characterized by two interdependent basic domains separated by 10 intervening "spacer" amino acids (24). Future studies will address the role of this region in nuclear localization of our HA-tagged constructs.
Several proteins have been identified whose rate of import to or retention in the nucleus is enhanced by phosphorylation near a NLS (16). For example, phosphorylation by casein kinase II near the SV40 large T antigen NLS increased the rate of nuclear import (21). Within the N-terminal 11 amino acids of the E1a sequence, there are serines 2, 3, 4, and 7. Serine 7 is a predicted protein kinase C site, and serine 4 can be phosphorylated by Cdc2 (22). Both of these residues are close to the NLS. Thus we defined the phosphorylation state of these serine residues and their potential role in nuclear targeting (Fig. 3). When the first 7 amino acids were deleted (HA1-E1-del-7, including the four serine residues), HA1-E1-del-7 was phosphorylated approximately 60% less than HA1-E1 (Fig. 3B) and was localized predominantly in the nucleus (Fig. 3C). However, approximately 3% of the cells also showed some cytoplasmic localization. One explanation for this observation may be that a certain level of phosphorylation is required for exclusive nuclear localization of HA1-E1. Alternatively, deletion of the first 7 residues includes 5PLS of the putative NLS, and this proline residue may be required for a secondary structural motif necessary for a functional NLS. To address this issue, another construct was generated where the first 4 amino acids were deleted and serine 7 was substituted with an alanine (HA1-E1-del-4,S7A). Phosphorylation of HA1-E1-del-4,S7A was approximately 20% that of HA1-E1 (Fig. 4B) and was localized exclusively in the nucleus (Fig. 4C). Additionally, in our in vitro nuclear import assays substitution of serine 7 for an alanine (BSA-NLSE1-S7A, Fig. 5) did not alter the efficiency of nuclear import of BSA. Hence, phosphorylation on any of the serine 2, 3, 4, or 7 residues is not essential for nuclear import, whereas it appears that the proline and leucine residues are required for exclusive nuclear targeting of HA1-E1.
The specific phosphorylation level of HA1-E1-del-7 is 60% less than the wild type HA1-E1. This suggests that one or more of the four serine residues within this 7-amino acid region are phosphorylated. Each of these serine residues was substituted individually to an alanine, and their specific phosphorylation compared with the wild type (Fig. 6). HA1-E1-S4A was approximately 50% lower than the wild type. However, the precise level of phosphorylation of each of the constructs varied from experiment. To delineate exactly which serine was phosphorylated, each of the 32P-labeled constructs were analyzed by two-dimensional gel electrophoresis (Fig. 6C). HA1-E1-S2A, HA1-E1-S3A, HA1-E1-S7A, and the wild type all resolved as four phosphorylated spots; however, HA1-E1-S4A resolved as only three spots (Fig. 6C). This suggests that serine 4 is phosphorylated in HA1-E1/E1a and is consistent with observations made by Nagai et al. (22). Our data also suggest that serine 4 is the site that is phosphorylated most predominantly as removal of this site reduces the level of phosphorylation by 50% of the wild type.
Disruption of the NLS by removal of the KKRR (HA1-E1-del-8-11) or substitution of the arginines for the alanines (HA-E1-R10A,R11A) results in loss of exclusive nuclear localization of HA1-E1 (Figs. 3C and 4C). However, between 5 and 10% of transfected cells still showed some detectable nuclear localization. In addition, HA1-E1-del-8-11 and HA1-E1-R10A,R11A were phosphorylated to less than 10% of HA1-E1 (Figs. 3B and 4B). This suggests that nuclear localization of HA1-E1 may be required for efficient phosphorylation. Only 3-5% of the total E1b is present in the nucleus (data not shown), and HA2-E1 is phosphorylated approximately 100-fold less than HA1-E1. HA2-E1/E1b also lacks serine 4, which is predominantly phosphorylated in HA1-E1. Thus it appears that the very low levels of phosphorylation of HA2-E1/E1b are due to the absence of serine 4 and its relative abundance outside the nucleus.
Despite these data, which suggest HA1-E1/E1a are phosphorylated in the nucleus, the precise function of E1 phosphorylation is not known. Previous work demonstrated that E1a is phosphorylated in a cell cycle-dependent manner; being maximally phosphorylated in G2 (13). However, no change in the enzymatic activity of E1a was observed with increased phosphorylation. Phosphorylation is not required for nuclear localization of HA1-E1 (Figs. 3 and 4), but may increase the rate of nuclear targeting and/or nuclear retention, neither of which was determined in our in vitro nuclear import assays (Fig. 5). Alternatively, phosphorylation of E1a may be required or important for stable interaction (or complex formation) with specific nuclear E2 proteins. E1 can be phosphorylated by p34cdc2 in vivo and in vitro (22). Pines and Hunter (25) have determined the localization of cyclins A and B, both of which associate with p34cdc2 to form the active kinase. Cyclin B is nuclear from the beginning of mitosis; however, cyclin A is nuclear from S phase onward and may well associate with p34cdc2 to phosphorylate substrates (including E1) in G2.
Many nuclear proteins including transcription factors such as p53,
c-Fos, N-Myc, c-Myc (26), and signal transducers such as STAT1 (27) are
substrates for the ubiquitin-proteasome pathway. This suggests the
requirement for a functional ubiquitin-proteasome system within the
nucleus. The proteasome is present in both the cytoplasm and the
nucleus (reviewed in Ref. 28), and recent work has identified the
functional NLS motifs present within the subunits (29, 30).
Interestingly, proteasomes purified from nuclear fractions were found
to contain the mammalian homolog to SUG1 (31). In our current study we
present evidence demonstrating that nuclear E1 is highly
phosphorylated, whereas the cytoplasmic form is not. The enzymes of the
ubiquitin-proteasome pathway present in the nucleus may have
modifications that distinguish them from their counterparts within the
cytoplasm, perhaps necessary for the different substrate specificities
of a nuclear ubiquitin-proteasome system.
We thank David Wilson and Jonathan Gitlin for constructive criticism.