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INTRODUCTION |
Among the numerous substructures of the cell nucleus (1), nuclear
dots containing PML1 and
Sp100 proteins (NDs or PML bodies) attracted the interest of many
researchers in recent years. NDs belong to the heterogeneous group of
nuclear bodies (2) and are distinct subnuclear organelles that do not
co-localize with any of the other known nuclear substructures (3-7).
Originally discovered as targets of autoantibodies in patients
suffering from the autoimmune disease primary biliary cirrhosis (8, 9),
NDs gained major attention when their disruption to a microgranular
form in the hematopoietic malignancy acute promyelocytic leukemia was
discovered (6, 7, 10, 11). In this disease, a natural constituent of
NDs, the PML protein, is expressed aberrantly as an oncogenic fusion
protein with the retinoic acid receptor
(RAR) (12). Expression of PML-RAR has been demonstrated to be sufficient to induce leukemias in vivo (13-15). Accordingly, a role of ND-associated
proteins in cell transformation and growth control or regulation of
differentiation (16-20) was postulated. In a recent report, NDs were
described to contain nascent RNA (21). On the other hand, NDs were
hypothesized to represent transcriptional repressing/regulating
complexes similar to the polycomb group complex (22). Interestingly,
numerous viral regulatory proteins target NDs and influence ND
structure and composition (23-29), and the expression of both known
major ND proteins, PML and Sp100, is induced by interferons (30-33)
(for review on NDs, see Ref. 34).
Most recent work on NDs focuses on the PML protein because of its
direct involvement in the development of acute promyelocytic leukemia.
However, the Sp100 protein was actually the first ND protein to be
characterized biochemically and through cloning of its cDNA using
sera from patients suffering from primary biliary cirrhosis, an
autoimmune liver disease (30, 35-37). Sp100 is an acidic protein with
a calculated molecular mass of 54 kDa and exerts a highly aberrant
electrophoretic mobility in SDS-polyacrylamide gel electrophoresis of
approximately 100 kDa (35, 36). The SP100 gene gives rise to
a number of alternatively spliced Sp100 variants (34, 38-41), some of
which contain an HNPP box and an HMG box (34, 38, 40, 41), the latter
representing a DNA-binding motif. This is of particular interest, as
Sp100 has been described to exhibit transcriptional modulatory effects
under certain experimental conditions (22, 39, 40). In recent
publications, the heterochromatin protein HP1 (22, 40) and hHMG2 (22)
were found to directly interact with Sp100. Finally, Sp100 is
covalently modified by the SUMO-1 protein (42), another feature shared
by Sp100 and PML (42-44). SUMO-1 is a small ubiquitin-related protein,
also known as PIC-1 (45), Sentrin (46), GMP-1 (47), or UBL-1 (48),
which is supposed to regulate protein targeting (43, 49, 50) or
inhibition of degradation (51). Modification of proteins by SUMO-1
resembles the ubiquitinylation pathway in that SUMO is attached to an
internal lysine residue but requires a unique set of activating and
conjugating enzymes (50, 52, 53). In the case of PML, modification by
SUMO-1 has very recently been reported to be essential for localization
of the protein to NDs (54). Interestingly, SUMOylation of PML is highly
enhanced after exposure of cells to arsenic trioxide (43), a pharmacon used in the therapy of acute promyelocytic leukemia (55-57).
Hyper-SUMOylation of the PML-RAR oncoprotein might be one of the
initial events of apoptosis induction in leukemic cells by
arsenic.2
In this study, we have performed a series of experiments in order to
characterize further the Sp100 protein regarding nuclear localization,
ND targeting, homomeric interaction, and the role of covalent
modification of Sp100 by SUMO-1. We have demonstrated that SUMO
modification of Sp100 strictly depends on nuclear import but does not
affect or require ND localization. Finally, we have performed molecular
modeling in order to generate a tertiary structure prediction for Sp100.
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EXPERIMENTAL PROCEDURES |
Recombinant DNA--
Subfragments of Sp100 were generated by
polymerase chain reaction amplification with specific oligonucleotides
corresponding to positions of the Sp100 cDNA given in the text,
which contained EcoRI (5'-oligonucleotides) and
SalI (3'-oligonucleotides) restriction sites at their
respective 5'- or 3'-ends. Sp100 fragments Sp100-(1-75), Sp100-(1-182), and Sp100-(1-334) were generated by restriction digestion using internal XhoI (Sp100-(1-75)),
SfuI (Sp100-(1-182)), or BamHI Sp100-(1-334)
restriction sites. Fragments were inserted into plasmids pM and pVP16
and used for two-hybrid experiments (see below) or into plasmids
pSG5-LINK and pSG5-FLAG for localization studies. These plasmids were
derived from plasmid pSG5 (58) by insertion of oligonucleotides
containing an artificial start-ATG codon followed by sequences encoding
the SV40 large T nuclear localization signal (pSG5-LINK) and the
FLAG-tag amino acid sequence (pSG5-LINK and pSG5-FLAG), followed by a
polylinker region in the same reading frame as pM or pVP16.
Plasmids pSG5-Sp100 and pSG5-SpAlt-C contain the entire cDNA
encoding Sp100 or the Sp100 splice variant SpAlt-C (41) inserted as
EcoRI fragments, respectively. Plasmid pSG5-LINK-EGFP was
constructed by an in-frame insertion of the cDNA encoding enhanced
green fluorescent protein (EGFP) derived from plasmid pEGFP-C1
(CLONTECH, Palo Alto, CA) into the EcoRI
restriction site of pSG5-LINK. The pSG5-SUMO construct contains the
SUMO-1 cDNA as a 1081-base pair SmaI/SspI
fragment inserted blunt-end into pSG5.
Site-directed mutagenesis was performed using the QuikChangeTM
site-directed mutagenesis protocol (Stratagene, La Jolla, CA) using the
following oligonucleotides: Mut297R(+),
5'-CTGGTGGATATAAGAAAGGAAAAGCC-3'; Mut297R(
),
5'-GGCTTTTCCTTTCTTATATCCACCAG-3'; Mut 298R(+),
5'-GTGGATATAAAAAGGGAAAAGCCATTTTC-3'; Mut298R(
),
5'-GAAAATGGCTTTTCCCTTTTTATATCCAC-3'; Mut300R(+),
5'-GATATAAAAAAGGAAAGGCCATTTTCTAATTC-3'; Mut300R(
),
5'-GAATTAGAAAATGGCCTTTCCTTTTTTATATC-3'; Mut447E(+), 5'-GAATACCCAGCAGGGAGAGACGTTTCAGC-3'; Mut447E(
),
5'-GCTGAAACGTCTCTCCCTGCTGGGTATTC-3'. Coding regions of all polymerase
chain reaction-derived inserts were sequenced using the
Taq-Cycle sequencing protocol with IRD-800 labeled primers
(MWG-Biotech, Ebersberg, Germany) and a Li-COR automated sequencing
device (MWG-Biotec, Ebersberg, Germany). Detailed sequence information
of all recombinant plasmids used in this study is available upon request.
Cell Extracts, Immunoblotting, and Antibodies--
Preparation
of total cellular protein extracts, SDS-polyacrylamide gel
electrophoresis (7.5 or 12.5% polyacrylamide), and protein transfer
onto nitrocellulose filters was carried out according to standard
protocols (59). Blots were blocked in 5% (w/v) dry milk in water and
incubated with primary antibodies (Abs) in the same solution. Washing
was carried out in phosphate-buffered saline/Tween (8 mM
Na2HPO4, 1.5 mM
KH2PO4, 140 mM NaCl, 2.6 mM KCl, pH 7.3, 0.1% Tween 20), and bound Abs were
detected with horseradish-conjugated secondary Abs and enhanced
chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).
Rat anti-PML and rat anti-Sp100 Abs were diluted 1:1000 and 1:2000,
respectively. The mAb anti-Gal4 was diluted 1:1000 in 5% dry milk (in
water). Secondary Abs were diluted 1:10,000. Anti-FLAG mAb M2 was
purchased from Sigma (Deisenhofen, Germany) and anti-Gal4 mAb from
Santa Cruz Biotechnology (Santa Cruz, CA). Production of the rabbit
anti-SpAB, rabbit anti-SpGH, rat anti-Sp26, and rat anti-PML-N Abs has
been described previously (33, 60).
Immunoprecipitation--
Cells were harvested from culture
dishes using a rubber policeman and washed once in phosphate-buffered
saline. The cell pellet was subsequently lysed in RIPA buffer (59)
supplemented with COMPLETETM proteinase inhibitor mixture (Roche
Molecular Biochemicals, Penzberg, Germany) according to the
manufacturer's recommendations. DNA and RNA were digested using 0.5 units/µl Benzonase (Merck, Darmstadt, Germany) at room temperature
for 10 min. After centrifugation at 10,000 × g, the
supernatant was removed, cleared by a second centrifugation at
10,000 × g, and used for immunoprecipitation. This
supernatant contained most of the Sp100 proteins expressed from the
transfected plasmids as determined by control experiments. Immunoprecipitation was carried out under RIPA conditions as described previously (42). For immunoprecipitation, rabbit anti-Sp100 (anti-SpGH)
or anti-Gal4 mAb (Santa Cruz Biotechnology, Santa Cruz, CA) was used.
Mammalian Two-hybrid Interaction Assay--
For in
vivo interaction studies, the CLONTECH
MatchmakerTM mammalian two-hybrid assay system
(CLONTECH, Palo Alto, CA) and HuH7 hepatoma cells
were used. The Sp100 cDNA and subfragments thereof were each cloned
in-frame into plasmids pM and pVP16 (see above) for expression of the
corresponding fusion proteins with the Gal4 DNA binding domain and the
VP16 transactivation domain, respectively. Reporter gene assays were
performed using plasmid pG5CAT according to the manufacturer's protocol
(CLONTECH, Palo Alto, CA). Cells of a 60-mm Petri
dish were transfected with 0.2 pmol of each pM and pVP16 constructs,
0.7 pmol of the reporter construct, and 1 µg of pCMV-
-Gal to
standardize transfection efficiencies. For measurement of CAT activity,
an equivalent of 2.0 A405 in the
-galactosidase assay was used. CAT activity was determined with a
commercial CAT enzyme-linked immunosorbent assay (Roche Molecular
Biochemicals, Penzberg, Germany).
Cells, Cytokines, Transfections, and Indirect Immunofluorescence
Microscopy--
Rat-1, HeLa S3, and HuH7 cells were maintained in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% fetal calf serum. Plasmid DNAs were introduced
into cells using the calcium phosphate precipitation procedure (59) or with FuGENETM 6 (Roche Molecular Biochemicals, Penzberg, Germany) according to the manufacturer's protocol. One to five micrograms of the
expression plasmids were precipitated per 6-cm dish. For indirect
immunofluorescence staining, cells were grown on coverslips and treated
as indicated. Cells were fixed at
20 °C for 5 min in methanol and
for 20 s in acetone. Rabbit or rat anti-Sp100 (Sp26) and rat
anti-PML (anti-PML-N) antibodies were diluted 1:400. The anti-FLAG mAb
was diluted 1:200. Cells were incubated with Abs for 30 min at room
temperature. For detection, dichlorotriazinylfluorescein- or lissamine
rhodamine-conjugated donkey anti-mouse, goat anti-rabbit, or donkey
anti-rat IgG Abs (Dianova, Hamburg, Germany) were diluted 1:200 in
phosphate-buffered saline.
Cell imaging was performed with a Zeiss Axiophot Microscope (Zeiss,
Oberkochen, Germany) and the SeescanTM video system by Intas
(Heidelberg, Germany). Images were processed on a 6500/275 Power
Macintosh computer (Apple, Cupertino, CA) using Adobe Photoshop 4.01 (Adobe Systems Inc.).
Molecular Modeling--
Molecular modeling was performed using
the SYBYL molecular modeling software (Tripos Inc., St. Louis) on an
Indigo workstation (Silicon Graphics, Mountain View, CA).
The Sp100 modeling was initiated by alignment of the Sp100 amino acid
sequence against 977 Protein Data Bank files of resolved three-dimensional structures within the SYBYL data base, using a cut
off of 19% sequence homology and a gap open penalty of 8 amino acids.
Three chains matching with sufficient homology (glycogen phosphorylase
b, trimethylamine dehydrogenase, and transketolase) were
selected for modeling.
Twenty four putative loops were detected and inserted into the model.
Disulfide bonds were found between cysteines 16-96, 209-238,
248-266, 270-289, and 309-373. Secondary structure prediction, debumping, and energy minimization of the raw model were done using
SYBYLs standard settings, followed by pair-wise root mean square fits
of the homologous areas of the model against the template chains. Final
refinement of the structure was done by subsequent steps of debumping
and energy minimization throughout the whole model, using 100 cycles
per step, until no forbidden values for bond length, angle, and torsion
were observed, and no further changes in the three-dimensional
structure could be detected.
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RESULTS |
Domains and Subfragments of the Sp100 Protein--
In order to
characterize functional domains of the Sp100 protein and to obtain
specific mutants for further functional studies, we have generated a
series of truncated versions of Sp100 by inserting fragments of the
Sp100 cDNA into the eukaryotic expression vectors pSG5-FLAG or
pSG5-LINK for localization studies and into vectors pM or pVP16 for
interaction studies (see "Experimental Procedures"). An overview of
the type of expressed Sp100 fragments, together with a schematic
representation of some putative Sp100 domains as determined by sequence
comparison or functional studies, is given in Fig.
1. At the N terminus, Sp100 contains a
short stretch of amino acids with significant sequence similarity to
MHC class I molecules (amino acids 9-49) (36) followed by a region,
which is the only Sp100 domain conserved in a truncated Sp100 protein predicted to be expressed in mice (amino acids 35-145). This murine Sp100 homologue or related protein is encoded in a highly amplified gene, Sp100-rs (61, 62). Since this amplified gene forms a homogeneously staining region (HSR) on murine chromosome 1 in several
wild mice strains (63, 64), the corresponding Sp100 protein domain was
denominated HSR domain (34) (see below). A Sp100 domain with
transcriptional transactivating properties is depicted according to Xie
et al. (39) as well as a region reported to mediate
interaction with the HP1 protein (22, 40).

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Fig. 1.
Schematic representation of domains of the
Sp100 protein and of the Sp100 subfragments used in this study.
Homology to MHC class 1 molecules is given according to Ref. 36, the
transactivating domain according to Ref. 39, and the HP1 binding site
according to Refs. 22 and 40. Also indicated is the Sp100 NLS (aa
444-450, light shading), the HSR domain (aa 40-147,
light shading in lower panel), as well as a short
-helical region within this domain (aa 40-56, dark
shading, helix 1).
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Identification of the Sp100 Nuclear Localization Sequence--
To
identify and characterize the sequence responsible for nuclear import
of Sp100 (NLS), the Sp100 fragments Sp100-(1-334) and Sp100-(326-480)
were expressed transiently in Rat-1 cells with an N-terminal FLAG
epitope tag and detected by immunostaining using a monoclonal antibody
specific for the FLAG epitope. Rat-1 cells were chosen in this
experiment because none of our polyclonal anti-Sp100 antisera
cross-reacts with the rat Sp100 protein. Therefore, also the
subcellular localization of untagged Sp100 proteins expressed from the
transfected plasmids could be unequivocally determined using these
anti-Sp100 antisera. As evident from Fig.
2A, Sp100-(1-334) exhibited a
cytoplasmic localization, whereas Sp100-(326-480) was detected mainly
in the nucleus (Fig. 2B), suggesting that the Sp100 NLS
resides between amino acids 334 and 480. In addition, the diffuse
nuclear distribution of Sp100-(326-480) indicates that this Sp100
region lacks the sequences necessary for ND targeting (see below).
Additional experiments using shorter N-terminal constructs confirmed
that the Sp100 NLS is located in the C terminus of the protein (data
not shown). A search for amino acid motifs in the NLS-containing region
that are similar to known NLS sequences revealed a single bona fide
Sp100 NLS between amino acids 444 and 450 (Fig. 1, top,
motif PSRKRRF). To test whether this sequence is essential and
sufficient for nuclear transport of Sp100, a single amino acid
substitution (K447E) was introduced into the untagged wild-type Sp100.
This changes the basic stretch of the NLS from PSRKRRF into
PSRERRF (underlined boldface letter indicates
the amino acid substitution). Transient expression of this Sp100 mutant
(Sp100(447E)) in Rat-1 cells revealed an almost complete abrogation of
nuclear transport (Fig. 2D), as evident by immunostaining
with polyclonal anti-Sp100 antibodies. To exclude that this K447E
mutation has a nonspecific effect on subcellular localization of Sp100
due to a change of the overall conformation of the protein, we have
inserted the cDNA encoding Sp100(447E) in frame also into
expression vector pSG5-LINK. This vector contains an artificial start
codon followed by SV40 large T-NLS and FLAG epitope encoding
sequences. The immunostaining pattern of the transiently
expressed NLS-FLAG-Sp100(447E) protein was indistinguishable from the
pattern obtained with transiently expressed wild-type Sp100 (Fig. 2,
E and C, respectively). These data demonstrate that nuclear localization of Sp100 can be almost completely abolished by the K447E amino acid substitution and implies that Sp100 has a
single NLS located between amino acids 444 and 450 that is essential and sufficient for nuclear import.

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Fig. 2.
Mapping of the Sp100 nuclear localization
signal. A and B, Rat-1 cells were
transiently transfected with plasmids pSG5-FLAG-Sp100(1-334)
(A) and pSG5-FLAG-Sp100(326-480). Cells were stained with
anti-FLAG mAb (red fluorescence). C-E, Rat-1
cells transiently transfected with pSG5-Sp100 (C),
pSG5-Sp100(447E) (D), or pSG5-LINK-Sp100(447E)
(E). Cells were stained with polyclonal rabbit anti-Sp100
antibodies (green fluorescence). Phase contrast pictures are
given below the corresponding immunofluorescence images.
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Determination of Sp100 Sequences Necessary for ND
Targeting--
For analysis of the ND targeting domain of Sp100, the
sequences encoding amino acids 1-253, 1-182, 1-75, 33-140,
208-480, 326-480, and 208-334 (compare Fig. 1) were inserted into
pSG5-LINK. Thus, the various Sp100 polypeptides were expressed with a
FLAG epitope tag and an additional NLS sequence to ensure nuclear
localization. After transfection of cells with the respective Sp100
constructs, the transiently expressed Sp100 polypeptides were
visualized using the anti-FLAG mAb (Fig.
3, red fluorescence).
Preformed nuclear dots were visualized by immunostaining of the PML
protein expressed from the endogenous PML gene by using a
polyclonal rat anti-PML antiserum (Fig. 3, green
fluorescence) on the same coverslips. HeLa S3 cells were used for
these transient transfection experiments because of their high level of
endogenous PML and Sp100 protein expression. The results of these
experiments are summarized in Fig. 3. All C-terminal Sp100 polypeptides
showed a diffuse nuclear distribution pattern and were not enriched at
nuclear dots stained by anti-PML antibodies (shown for the largest
C-terminal fragment Sp100-(208-480), Fig. 3, C and
D, respectively). N-terminal Sp100 fragments (red
label), in contrary, co-localized with the PML protein in NDs
(green label) (shown representatively for Sp100-(1-253) in
A and B or for Sp100-(33-149), in E
and F, respectively). As the shortest N-terminal fragment
Sp100-(1-75) was not detectable by immunofluorescence, Sp100-(33-149)
was the smallest Sp100 polypeptide which was targeted to NDs.
Interestingly, expression of Sp100-(208-480) led to a significant
redistribution of NDs resulting in fewer and more brightly staining
dots (Fig. 3, C and D). This is surprising because of its diffuse distribution, which suggests a lack of direct
interaction of this Sp100 polypeptide with NDs. In contrast, the
N-terminal fragments Sp100-(1-253) and Sp100-(33-149) did not show
such an effect when expressed at moderate levels (Fig. 3, A, B,
E, and F). A similar redistribution of NDs was also
observed with the Sp100 polypeptides Sp100-(326-480) and
Sp100-(208-334), which were likewise expressed with a diffuse pattern
(data not shown). This may be due to competition of the diffusely
distributed C-terminal fragment for cellular factors involved in
regulation of ND distribution and morphology, thereby indirectly
affecting ND structures. A control construct, expressing enhanced green fluorescent protein (EGFP) fused to the same FLAG epitope tag and NLS
sequences as the corresponding Sp100 constructs, did not trigger such
an effect (data not shown). Taken together, these data suggest that the
Sp100 nuclear dot targeting signal is located between amino acids 33 and 149 and that amino acids 208-480 might be involved in regulatory
processes.

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Fig. 3.
Mapping of the Sp100 ND-targeting
sequence. HeLa S3 cells were transiently transfected with plasmids
pSG5-LINK-Sp100(1-253) (A and B),
pSG5-LINK-Sp100(208-480) (C and D), or
pSG5-LINK-Sp100(33-149) (E and F), all
expressing FLAG epitope-tagged truncated Sp100 fragments. Cells were
double-stained with anti-FLAG mAb (red fluorescence,
left panels) and polyclonal rat anti-PML antiserum
(green fluorescence, right panels). PML is
expressed in all cells, whereas FLAG-tagged Sp100 is expressed only in
transfected cells.
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Mapping of a Dimerization Domain in the N terminus of the Sp100
Protein--
Investigation of the dimerization or multimerization
potential of the Sp100 protein is an important prerequisite for
understanding how NDs can be formed, as well as for construction of
dominant negative mutants of Sp100 needed for functional studies. To
test the homomeric interaction potential of Sp100, we have used the two-hybrid assay in mammalian HuH7 hepatoma cells and have performed co-immunoprecipitation experiments. Fig.
4 summarizes the results of the
two-hybrid studies and gives an overview on the type of Sp100 fragments
including the full-length Sp100 (aa 1-480), which were expressed as
fusion proteins with the Gal4 DNA binding domain or the
VP16-transactivating domain, respectively (Fig. 4A).
Measured were the amounts of CAT protein expression from the
co-transfected reporter plasmid. By using this assay, a strong signal
similar to that of the positive control (Gal4-p53/VP16-SV40 large T)
was detected with all N-terminal subconstructs containing the region between amino acids 33 and 149 (Fig. 4A, shaded),
indicating that this region can mediate homomeric interaction. The
shortest Sp100 polypeptide Sp100-(1-75), in contrast, completely
lacked dimerization activity according to this assay. Expression of all
Sp100 fusion proteins was confirmed by immunoblotting (data not shown).
Surprisingly, Gal4 and VP16 fusion proteins containing the region
between amino acids 334 and 480 resulted in a weaker or no CAT
expression (see combinations Gal4-(1-334) and VP16-(1-480) or
Gal4-(1-480) and Vp16-(1-480), respectively). This may be due to
inhibitory sequences in the C-terminal part of the protein or by steric
hindrance in formation of the transcriptional activating complex
driving the expression of the reporter plasmid.

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Fig. 4.
Analysis of Sp100 homomeric interaction using
the mammalian two-hybrid assay. A, CAT values for
co-transfection experiments with the Gal4-Sp100 constructs and
VP16-Sp100 constructs as given in the drawing. Levels of CAT expression
for a standardized 200-µl assay volume are converted into ranged
values according to C. The minimal homomeric interaction
domain observed in this experiment is shaded. B,
representative CAT values for two independent positive control
experiments. The heteromeric interaction between murine p53 and SV40
large T antigen and the homomeric interaction of the PML protein as
described previously (73) were measured using the corresponding Gal4-
and VP16 constructs. Three representative experimental results are
given as picogram of CAT protein per 200-µl assay volume.
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In order to confirm the data obtained by the two-hybrid analysis by an
independent assay and to investigate whether different splice variants
of Sp100 also heterodimerize, co-immunoprecipitation experiments were
performed (Fig. 5). HuH7 cells were
transiently transfected with plasmids pM-Sp100-(1-182) and
pSG5-Sp100-(1-480) or pM-Sp100-(1-480) and pSG5-SpAlt-C. The latter
plasmid expresses the Sp100 splice variant SpAlt-C (41), which differs
from Sp100 by the lack of C-terminal 32 amino acids that are replaced
by 24 amino acids. For co-immunoprecipitation, a monoclonal antibody specific for the Gal4 domain of the Sp100 fusion proteins expressed from the pM constructs was used. Precipitates and
supernatants were subjected to immunoblotting using polyclonal
anti-Sp100 antibodies for detection (rabbit anti-SpAB, specific for aa
1-253 of the Sp100 protein). As evident from Fig. 5, left
panel, co-expression of Gal4-Sp100-(1-182) and wild-type
Sp100-(1-480) followed by immunoprecipitation with anti-Gal4 mAb
resulted in efficient co-precipitation of the untagged full-length
Sp100-(1-480) (left panel, lane 2). In contrast, without
co-expression of Gal4-Sp100-(1-182), Sp100-(1-480) was not
precipitated (lane 1) and remained in the supernatant (lane 3). The intensity of the signal for the
co-precipitated Sp100-(1-480) was much higher than that obtained for
the Gal4-Sp100-(1-182) precipitated directly by the mAb, which could
be taken as evidence for multimerization of both Sp100 polypeptides.
However, the SpAB antiserum used for detection in these experiments was
raised against Sp100-(1-253), implying that there are probably more
potential epitopes present on the Sp100-(1-480) protein than on the
Sp100-(1-182) fragment. Therefore, the signal intensities of the two
bands do not necessarily reflect the relative molar amounts of the two polypeptides. The reciprocal immunoprecipitation experiment using an
antiserum raised against the C-terminal region of Sp100 (anti-SpGH, corresponding to Sp100 amino acids 383-474) confirmed these data in
that Sp100-(1-182) was co-precipitated with Sp100-(1-480) using anti-SpGH (data not shown).

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Fig. 5.
Analysis of Sp100 homomeric interaction using
immunoprecipitation. HuH7 cells were transiently transfected with
plasmids pM-Sp100(1-182) and pSG5-Sp100 as indicated (left
panel) or with pM-Sp100 and pSG5-SpAlt-C as indicated (right
panel). Immunoprecipitation (IP) was carried out using
an anti-Gal4 mAb. Pellets (pell.) and supernatants
(s.n.) of the precipitation reaction were used for
immunoblotting. The immunoprecipitated proteins were detected with the
polyclonal rabbit anti-Sp100 antiserum (produced with Sp100 amino acids
1-253). Schematic representations of the expressed proteins are given
below the blots. Indicated is the Gal4-domain recognized by
the anti-Gal4 mAb used for immunoprecipitation. Positions of the
corresponding proteins are marked by arrows.
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To be able to compare directly the relative amounts of co-precipitated
proteins and in order to test for heterodimerization of two variant
Sp100 proteins, we have co-expressed Gal4-Sp100-(1-480) and the Sp100
variant SpAlt-C. When immunoprecipitated with anti-Gal4 mAb, SpAlt-C
was co-precipitated with Gal4-Sp100-(1-480) (lane 6) but
remained in the supernatant when expressed alone (lanes 7 and 10, respectively). Gal4-Sp100-(1-480), when expressed
alone, was precipitated as expected (lane 5). Since in this
experiment both proteins contain the complete Sp100-(1-253) region,
which is recognized by the SpAB antiserum, the signal intensities
directly correlate with the relative amounts of the co-precipitated
proteins. In this experiment, equivalent amounts of SpAlt-C and
Gal4-Sp100-(1-480) were precipitated (lane 6). A comparison
of the signal intensities of the SpAlt-C and Gal4-Sp100-(1-480) bands
in the corresponding supernatants (lane 9) demonstrates that
most of the Gal4-Sp100-(1-480) was precipitated, whereas an excess of
SpAlt-C remained in the supernatant. If the two proteins would form
multimers, the relative amounts of the proteins in the precipitates
should reflect the relative total amounts of the expressed proteins.
Accordingly, in this experiment an excess of SpAlt-C over
Gal4-Sp100-(1-480) should be expected in the precipitate, which is not
the case. Therefore, these data provide indirect evidence that Sp100
might form dimers rather than multimers. As, however, Sp100 multimers might be disassembled under the buffer conditions used, additional experiments are required to clarify whether Sp100 is capable of multimer formation. Consistent with our study, homomeric interaction of
Sp100 has been reported very recently also by another group (40).
Taken together, our data imply that Sp100 can form homodimers and that
splice variants of Sp100 can heterodimerize. The region mediating this
interaction resides within the same domain responsible for ND
targeting. It covers Sp100 amino acids 33-149, which are essentially
encoded by the Sp100 exons three and four as we have shown previously
by analysis of the gene organization (62).
The Sp100 ND-targeting and Dimerization Domain Defines a Protein
Family--
The Sp100 region between amino acids 33 and 149, which is
important for ND targeting as well as for dimerization, is conserved in
a murine-truncated Sp100 homologue, which is expressed from a highly
amplified gene, designated Sp100-rs (61-63). In some mice strains, the amplification has progressed to such extremely high copy
numbers that the amplified gene locus is visible as a homogeneously staining region (HSR) on the corresponding murine chromosome (64, 65).
In addition, the same region is also conserved in a human Sp100-related
protein, the leukocyte-specific LYSP100/Sp140 (20, 38). We compared the
sequences of these proteins and performed similarity searches in the
GenBankTM data base in order to examine whether the Sp100
dimerization and ND-targeting domain represents a motif also found in
other proteins. Fig. 6 gives an alignment
of the corresponding Sp100 domain, as well as the corresponding domains
of the murine HSR-encoded Sp100-rs protein and the LYSP100/Sp140
protein. In addition, we found significant homology to this region in
two additional proteins, the AIRE autoimmune regulator protein and a
putative human protein (AA431918, WashU-Merck EST Project). Amino acid
residues at several positions are fully conserved (Ile-33, Ala-36,
Phe-41, Pro-42, Leu-48, Asp-50, Pro-72, Leu-82, Leu-97, Phe-98,
Asn-102, Tyr-106, Leu-109, and Phe-116) between the different proteins. Therefore, the region corresponding to Sp100 amino acids 40-147 indeed
defines a novel protein motif. As this domain comprises most of the
sequence of the truncated murine Sp100-rs protein, it was termed HSR
domain (34).

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Fig. 6.
Alignment of HSR domains from the proteins
Sp100, Sp100-rs (HSR), LYSP100B, AIRE, and a putative human protein
derived from the EST sequence AA431918. Identities are in
dark shading and similarities are in light
shading.
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Analysis of Sequences Necessary for Sp100 SUMOylation--
As we
have shown previously, the Sp100 protein undergoes post-translational
modification by covalent attachment of the small ubiquitin-related
protein SUMO-1 (42). For PML, it has been reported that modification by
SUMO-1 regulates targeting of PML to NDs (43) and that PML lacking the
functional SUMOylation sites fails to localize to NDs (54). It is
likely that only one SUMO molecule is attached to Sp100 (42), which is
different from PML which is modified by at least three molecules of
SUMO-1 (54). In order to understand the role of SUMO-1 modification of
Sp100, we have performed a series of experiments to map the corresponding lysine residue where SUMO-1 is attached in
vivo and to examine the effects of mutation of this site from
Sp100. To ensure correct nuclear localization irrespective of the
presence of the Sp100 NLS, we have expressed a number of Sp100
fragments fused to the Gal4-domain (Fig.
7A). For analysis of
SUMOylation, we have developed a co-transfection assay in HuH7 cells.
HuH7 cells were used because endogenous SUMOylation of transiently expressed Sp100 is low as determined by immunoblotting (shown representatively for Sp100 expressed without Gal4 sequences, Fig. 7B, left panel). Sp100, expressed transiently from the
transfected plasmid pSG5-Sp100, was detected by Sp100 antibodies as a
broad band at approximately 100 kDa (lane 1), which, at
lower exposure times, resolves at least into four distinct bands
probably representing different phosphorylated forms of Sp100 (not
shown). A weak additional band reactive with anti-Sp100 antibodies is
visible at 100 kDa (Fig. 7B, asterisk). As we
have shown previously, this band represents Sp100 modified by SUMO
(42). In order to increase SUMOylation, we co-expressed SUMO-1 by
co-transfection of 1 or 5 µg of the respective expression vector
pSG5-SUMO. This resulted in a strong increase of the intensity of the
upper band corresponding to increased modification of the transiently
expressed Sp100 (Fig. 7B, lanes 2 and 3,
respectively). Interestingly, no difference in modification was
observed between the co-transfection of 1 or 5 µg of pSG5-SUMO, indicating saturation of the modifying enzymatic machinery. In further
experiments, co-transfection of 1 µg of pSG5-SUMO was performed with
5 µg of Sp100 expression vectors and compared with transient
transfection with the corresponding Sp100 expression vector alone. An
increase in intensity of an additional higher migrating band after
co-transfection was taken as evidence for SUMOylation of the
corresponding Sp100 polypeptide. The results of the corresponding
co-transfection experiments with the Gal4-Sp100 subfragments are
summarized in Fig. 7A. Only the full-length
Gal4-Sp100-(1-480) and the large C-terminal fragment
Gal4-Sp100-(208-480) showed a strongly increased signal intensity for
the higher migrating band after co-expression of SUMO-1. These data
suggest that sequences necessary for SUMOylation are located
C-terminally of amino acid 253 and N-terminally of amino acid 326.

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Fig. 7.
Mapping of the lysine residue necessary for
SUMOylation of Sp100. A, schematic overview of the Gal4
fusion proteins of Sp100 that were expressed with and without
co-expression of SUMO-1. Emergence of an additional band indicating
modification by SUMO-1 is indicated for each construct on the
right side of the graph. The minimal region required for
SUMOylation as determined in this experiment is shaded and
the corresponding amino acid sequence is given below. Amino
acid changes introduced by mutagenesis are indicated by
arrows and numbers. B, HuH7 cells were
transiently transfected with plasmids pSG5-Sp100 (left panel,
lanes 1-3), pSG5-Sp100(297R) (lanes 4 and
5), pSG5-Sp100(298R) (lanes 6 and 7),
pSG5-Sp100(300R) (lanes 8 and 9),
pSG5-Sp100(447E) lacking a functional NLS (lanes 10 and
11) and pSG5-LINK-Sp100(447E), re-adding an NLS to the 447E
mutant (lanes 12 and 13) without (lanes 1, 4, 6, 8, 10, and 12) or with co-expression of SUMO-1
(lanes 2, 3, 7, 9, 11, and 13). Mock control
(pUC-19) is given in lane 14. The Sp100 protein with the
K297R substitution completely lacks SUMO modification when expressed
alone (lane 4) or after co-expression of SUMO-1 (lane
5)
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Since SUMO modification occurs at lysine residues of proteins (49, 50,
66), we have changed three of the four lysines in the corresponding
Sp100 region into arginine (K297R, K298R, and K300R) In order to
exclude any influence of these sequences on the efficiency of the
modification, the constructs express Sp100 without heterologous
sequences. We have also included the Sp100(447E) mutant in this
analysis in order to investigate the role of nuclear transport for
SUMOylation. In this assay, the Sp100 mutant Sp100(297R) was completely
deficient in SUMO modification (Fig. 7, lanes 4 and
5), whereas the mutants Sp100(298R) and Sp100(300R) (Fig. 7,
lanes 6-9) were efficiently modified after co-expression of
SUMO-1 (Fig. 7, lanes 7 and 9, respectively) and
also exhibited some modification without co-expression of SUMO-1 (Fig.
7, lanes 6 and 8). According to these data,
lysine 297 is most likely the definite and only residue where SUMO-1 is
attached in vivo. Also the Sp100(447E) mutant with the
non-functional NLS was severely compromised in SUMO modification in
this assay (Fig. 7, lanes 10 and 11). Only a very
faint band was visible after co-expression of SUMO-1 (Fig. 7,
lane 11), and without co-expression this band was completely
absent (Fig. 7, lane 10). As this could be due either to
lack of SUMO modification in the cytoplasm or to direct involvement of
the lysine, which is mutated in the Sp100 K447E polypeptide in the
modification process, we have inserted the Sp100(447E) cDNA into
plasmid pSG5-LINK, thereby providing the mutant with an active
heterologous NLS. As evident from Fig. 7, lanes 12 and
13, Sp100(447E) when expressed with a functional NLS again
becomes efficiently modified by SUMO-1, arguing against lysine 447 as
being directly involved in the SUMOylation reaction. The altered
electrophoretic mobility of this protein is caused by 25 heterologous
amino acids derived from the pSG5-LINK vector. These data indicate that
modification of Sp100 by SUMO-1 is tightly linked to nuclear
localization. In order to determine whether the weak SUMO-modified
Sp100(447E) band visible after co-transfection might be due to some
weak modifying activity also present in the cytoplasm or whether this
faint band is due to the small amount of Sp100 present in the nucleus
after transient expression, which is then normally modified, we
performed a co-expression experiment of Sp100(447E) and SUMO-1 in Rat-1
cells. Rat-1 cells were chosen in this experiment because of the lack
of reactivity of the anti-Sp100 antisera with the rat Sp100 homologue.
In Fig. 8, left panels, the
result of such a co-expression experiment is documented. Cells were
double-labeled with polyclonal rabbit anti-Sp100 (green) and
the anti-SUMO-1 mAb (red). Again, Sp100(447E) is expressed predominantly in the cytoplasm with some minor amount still localizing in the nucleus where it is targeted to NDs (Fig. 8, panel
Sp100(447E)). The same cells, when stained with the anti-SUMO mAb,
showed predominantly diffuse nuclear distribution with some additional
dot-like staining (Fig. 8, SUMO-1). This pattern was typical
for transient expression of SUMO-1 in all cell lines tested so far
(data not shown). An overlay of both pictures clearly demonstrates that
the dot-like structures in the nucleus show almost perfect
co-localization. The large amount of cytoplasmic Sp100, in contrast, is
not stained by the anti-SUMO mAb indicating that cytoplasmic Sp100 is
not modified. The co-immunostaining of nuclear Sp100 with SUMO-1 is in
agreement with the interpretation of the immunoblot data, indicating that the minor amount of Sp100(447E) localized in the nucleus becomes
efficiently modified by SUMO-1.

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Fig. 8.
Left panel, co-expression of
Sp100(447E) and SUMO-1 in Rat-1 cells. Cells were transiently
transfected with pSG5-Sp100(447E) and pSG5-SUMO followed by double
immunostaining using polyclonal rat anti-Sp100 (green
fluorescence) and a monoclonal anti-SUMO Ab (red
fluorescence). For better co-localization, the images were
electronically merged (merge). Co-localization of the
signals is observed only in the nucleus. Right panel,
expression of Sp100 mutants in Rat-1 cells. Rat-1 cells were
transiently transfected with plasmids pSG5-Sp100, pSG5-Sp100(297R), or
pSG5-Sp100(300R), followed by immunostaining using polyclonal rabbit
anti-Sp100 (red fluorescence). The intracellular
distribution pattern of all three Sp100 proteins is practically
indistinguishable.
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For PML, it has been reported that mutagenesis of SUMO modification
sites resulted in poor nuclear dot formation with diffuse distribution
of the transiently expressed protein. In order to analyze whether
SUMOylation of Sp100 has a similar role for ND targeting, we have
performed immunofluorescence analyses using Rat-1 cells transiently
transfected with plasmids pSG5-Sp100(297R), pSG5-Sp100(298R), and
pSG5-Sp100(300R). As evident from Fig. 8, right panels, the
Sp100(297R) mutant exhibited a nuclear speckled distribution
indistinguishable from the mutants Sp100(298R) (not shown),
Sp100(300R), and wild-type Sp100 (Fig. 8, Sp100(297R), Sp100(300R), and Sp100, respectively). Therefore,
SUMOylation of Sp100 is not necessary for ND targeting and accumulation
of Sp100 at NDs. In summary, these data provide strong evidence that Sp100 becomes SUMOylated at lysine residue 297 and, furthermore, indicate that SUMOylation of Sp100 depends on a functional nuclear import signal but is not required for ND targeting.
By using our data of the specific modification site of Sp100, we
compared the lysine motifs of all so far known SUMOylation sites to
find similarities that would allow us to predict a consensus sequence
for SUMOylation motifs. The result of this comparison shows that the
SUMOylation sites of Sp100 (this report), IkappaB (51), RanGAP (49,
50), and two out of three lysines described for PML (54) fit very well
into a consensus sequence for SUMOylation (Fig.
9A) similar to the prediction
made from the comparison of the SUMOylation sites in the human RanGAP-1
protein and I
B carried out by Desterro et al. (51). The
core motif of this consensus sequence is (I/L)KXE, but a
contribution of additional amino acids, such as (R/H) at position
4-5 or (K/R) at position +1-2, is certainly conceivable. In Fig.
9B, we have used this consensus sequence to predict
additional SUMOylation sites in other ND proteins or proteins related
to NDs. A potential SUMOylation site is present in the non-amplified
murine homologue of Sp100 (63), in the LYSP100/Sp140 protein (20, 38),
as well as in a human nuclear phosphoprotein (HNPP) (67), which
contains a protein motif also present in Sp100 splice variants (34).
Finally, in an Sp100 splice variant (40, 41), there is a second bona
fide SUMOylation site in the HMG domain of the protein, which is
therefore also present in HMG-1.

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Fig. 9.
Consensus sequence for modification by
SUMO-1. A, alignment of all SUMOylation sites known to date.
The lysine residue, which is covalently modified, is indicated by the
arrow. Identical amino acids are in the dark
shading and similarities are in the light shading.
B, predicted SUMOylation sites in other related
proteins.
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This consensus sequence is a useful basis for studies on SUMOylation of
additional proteins.
A Three-dimensional Structure Modeling for Sp100 and Localization
of the Functional Domains--
Functional domains of proteins, which
are involved in protein-protein interaction, post-translational
modification, and subcellular localization, should reside on readily
accessible regions on the surface of the protein molecule. In order to
evaluate if the experimentally defined functional domains of Sp100
fulfill this criterion, we have generated a model of the Sp100 protein
using the SYBYL® molecular modeling software (for parameters see
"Experimental Procedures"). The result of this molecular modeling
is depicted in Fig. 10. Three spatial
views of the molecule are given at the top of Fig. 10. Functional domains and residues are colorized as indicated. In this
model, the Sp100 molecule has an asymmetric shape consisting of two
subdomains. One domain comprises the lower protruding part, which also
contains the dimerization and ND-targeting domain (HSR domain, colored
in cyan). Within the HSR domain, there is an exposed
-helix (amino acids 40-56), for which it is very tempting to speculate that it mediates protein-protein interaction (Figs. 1 and 10,
Helix 1). However, this predicted helix is also present in
the Sp100-(1-75) construct, which did not show any dimerization in our
experiments. Therefore, it cannot be sufficient for dimerization of
Sp100. The upper part of Sp100 consists of a large, wing-shaped domain,
which contains the Sp100 NLS exposed on the molecule in a flexible arm
(colored in green) and therefore is predicted to be readily
accessible for the nuclear import machinery. A subdomain of the upper
Sp100 domain with a high helical content is in part equivalent to the
region of Sp100 with transactivating properties. Lysine 297 likewise is
also located at the surface of the molecule (depicted in
red) and, therefore, accessible for the modifying enzymes.
We have attached the model for ubiquitin to lysine 297 as an
illustration for the relative size of the Sp100 protein and the SUMO-1
molecule. Despite its limited sequence homology, the structure of
SUMO-1 has been reported to resemble that of ubiquitin as determined by
NMR (68). Taken together, the experimentally defined functional regions
of Sp100 and the computer-generated structure prediction are in
excellent agreement and together can be used for the construction of
specific mutants for the elucidation of the cellular function of
Sp100.

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Fig. 10.
Three-dimensional structural prediction of
the Sp100 protein as obtained by molecular modeling. At the
top of the panel, three spatial views of the model are
given. At the bottom of the panel, to demonstrate the
relative sizes of the proteins, a model of ubiquitin, which is reported
to be almost identical in structure to SUMO-1 (68), was graphically
attached to Lys-297 of the Sp100 protein (depicted in red as
space-fill representation). Experimentally defined regions are colored
as indicated. Note the helical stretch (Helix 1) at the
outside of the protein.
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DISCUSSION |
In our study, we have performed a detailed characterization of
structural and functional sequence features of the Sp100 protein regarding subcellular localization, dimerization, and covalent modification by SUMO-1. We have mapped the Sp100 NLS, as well as a
ND-targeting and dimerization sequence which defines a new protein
motif, termed HSR domain, also present in other proteins. Furthermore,
we have identified the lysine residue necessary for modification of
Sp100 by SUMO-1 and demonstrated that a functional NLS is essential for
modification of Sp100 by SUMO-1. We have shown that SUMOylation of
Sp100 is not essential for ND targeting of Sp100 and, vice
versa, that ND targeting is not necessary for SUMOylation.
Finally, we propose a consensus sequence for SUMOylation of proteins
based on the comparison of the known SUMOylation motifs on other
proteins. A three-dimensional structural prediction of the Sp100
protein was generated by molecular modeling. Consistent with our
mapping data in this model all functional sites of the Sp100 protein
were found in surface-exposed regions.
So far, separation of the ND-targeting and dimerization motif was not
possible, because the shortest Sp100 fragment Sp-(1-75) could not be
detected by immunofluorescence staining. Therefore, we cannot exclude
that the ND targeting observed for the transiently expressed Sp100
polypeptides may be in part or even in toto be due to
dimerization of the protein with endogenous Sp100 located at NDs.
However, since our data provide indirect evidence that Sp100 can
dimerize, but probably not multimerize, the ND targeting has to be, at
least in part, due to a targeting toward the Sp100 docking site at NDs,
independently of homodimerization. Another possibility is that the
Sp100 multimerization capability is restricted to the fraction of Sp100
localized at NDs, possibly due to a changed conformation when compared
with Sp100 in solution, as it is present in the cytoplasm of cells (42)
and in the cellular extracts used for our immunoprecipitation
experiments. The fact that Sp100 with a mutated NLS exhibits a diffuse
cytoplasmic distribution also argues against multimer formation of
Sp100 alone. Accordingly, using Sp100 from solubilized cellular protein
extracts, only the dimeric form would be observed. However, a definite
answer as to whether Sp100 is capable of multimer formation requires
additional experiments, like sucrose gradient analysis. Analysis of
further Sp100 protein subfragments is required in order to separate
homomeric interaction from ND targeting in order to confine the ND
targeting signal of Sp100. As this signal would be expected to interact with novel ND components, the identification of the sequences necessary
is of major interest for the isolation of these components. One has to
keep in mind, however, that it is very well possible that
homodimerization of the corresponding Sp100 domain might be required in
order to generate a functional ND-targeting signal.
An interesting finding in these studies was the discovery of a novel
protein motif, the HSR domain, within this ND targeting/dimerization domain. It will be interesting to investigate whether the corresponding domains in the other members of the HSR domain family proteins have the
same function. In fact, at least the LYSP100/Sp140 protein shows
partial (38) or even complete (20) co-localization with NDs in
lymphocytes, and also for the murine Sp100-rs protein an ND
localization is likely (62). It could be hypothesized also that the
AIRE protein and the AA431918 protein are novel ND proteins.
Remarkably, three members of the HSR family share more extensive
sequence similarity. The Sp100 splice variant Sp100-B (SpAlt-212), the
LYSP100/Sp140 protein, and the AIRE protein share the N-terminal HSR
domain and an additional protein motif toward the C terminus called the
HNPP box (34, 41), also recently termed the SAND domain (69). Recent
data from our laboratory argue that the HNPP box/SAND domain might
direct proteins to nuclear domains different from NDs and therefore
represent a targeting signal antagonistic to the HSR domain on the same
molecule (41). One could speculate that the genes encoding these
proteins represent a family derived from a common ancestor gene and
might fulfill similar functions in the cell. The AIRE protein is of
special interest in this regard, as it is mutated in a monogenic
hereditary autoimmune disease and appears to play a major role in
control of the immune response (70).
Another interesting observation in our studies is the redistribution of
NDs caused by expression of the C-terminal Sp100 fragments Sp100-(208-480), Sp100-(326-480), and Sp100-(208-334). As these Sp100 fragments are diffusely distributed in the nucleoplasm, redistribution of NDs is most likely due to an indirect effect, such as
competition of the overexpressed Sp100 fragment for cellular factors.
As this redistribution was also observed when a Sp100-(208-480) K297R
mutant was expressed, it is most likely not due to competition for
cellular SUMO-1 (data not shown). However, it is also conceivable that
the Sp100 fragments compete for the corresponding SUMO-modifying enzyme, thereby leading to hypomodification of ND-located cellular Sp100 and/or PML, which may result in redistribution of NDs.
Alternatively, competition for other cellular factors, such as kinases,
could play a role. In fact, the region present on all three Sp100
constructs tested (aa 326-334) contains a consensus casein kinase 2 phosphorylation site.
SUMO modification of proteins has recently been shown to be a novel
cellular mechanism of general importance for a multitude of biological
processes (reviewed in Ref. 66). Therefore, the identification and
mutational analysis of the Sp100 SUMOylation site as shown here is of
general relevance. The strict dependence of SUMO modification of Sp100
on nuclear targeting raises the question whether SUMO modification is
restricted to the nuclear compartment in general. In fact, most of the
proteins for which covalent SUMOylation has been demonstrated so far,
i.e. Sp100, PML, and I
B can be found, at least in part,
inside the nucleus. For I
B translocation into the nucleus of the
newly synthesized protein was demonstrated (71). For RanGAP-1, staining
at the mitotic spindle was reported (47). Taking this into account, one
could hypothesize that SUMOylation of proteins could involve nuclear
import as an obligatory step. Whether the modification reaction itself
occurs in the nucleoplasm, during nuclear import, or after docking at
the nuclear pore at the outside of the nuclear pore in the cytoplasm
remains to be shown. Since in our experiments the Sp100-(208-480)
polypeptide with a diffuse nuclear localization still becomes
SUMOylated, ND targeting is most likely not a prerequisite for
SUMOylation. Another open question is whether the proteins (described
in previous works) to interact with SUMO-1, such as the tumor necrosis
factor receptor, Fas, Rad51, and Rad52, are indeed modified by SUMO-1
or only interact with the SUMO-1 modification of another protein, which
in turn again could be modified in the nucleus. If so, SUMO-1
modification could very well be part of a signaling cascade.
In our study, we found no effect of removal of SUMO modification of
Sp100 by mutagenesis on nuclear import or ND targeting of the protein.
In this regard, SUMO modification of Sp100 differs obviously from that
of the PML protein, which was reported to be severely compromised in ND
targeting when SUMO modification was abolished by mutagenesis (54).
However, since one of the known functions of SUMO modification is
protein targeting, as shown for the RanGAP1 protein, which is bound to
the nuclear pore when modified by SUMO-1 (49, 50), one may hypothesize
that SUMOylation regulates protein-protein interaction also in the case
of Sp100. A good candidate for such a SUMO-regulated association is the
HP1 protein, which has been reported to interact directly with Sp100
(22, 40). In these experiments, the interaction domain was mapped to
amino acids 287-333. Given the relative size of SUMO-1 modification,
which is similar to that of ubiquitin (shown in Fig. 10), one would
expect that SUMO modification of Sp100 at amino acid position 297 will
interfere with HP1 binding. This could either enhance binding, as in
the case of RanGAP1, or on the other hand, prevent HP1 association
through steric hindrance. Co-precipitation experiments in order to
elucidate a possible role of SUMO-1 modification in regulation of HP1
binding to Sp100 are currently underway. On the other hand, SUMOylation
was reported to act as a stabilizing factor by counteracting
ubiquitin-mediated degradation (51, 72). However, we currently have no
evidence that Sp100 lacking the SUMOylation site is less stable than
SUMO-modified Sp100 when transiently expressed. We also do not know
whether the unmodified form of Sp100 is more rapidly degraded than the modified form. Another hypothesis is that SUMO modification plays a
role in certain cell cycle stages, as we have provided evidence that
during mitosis PML becomes demodified and, by this mechanism, might
regulate ND disassembly, which occurs in mitosis (42). Likewise,
SUMOylation of Sp100 might regulate a cell cycle-specific function of Sp100.
Collectively, our data provide important information and new tools for
the analysis of the function of Sp100 and NDs. Mutants consisting only
of the HSR domain may act in a dominant negative manner, as they are
capable of dimerization with Sp100, but lack the entire C terminus
including the SUMOylation site and the HP1 binding domain. Mutants
lacking the N-terminal part of the protein, which induced changes in ND
distribution when expressed transiently, likewise might act in a
transdominant fashion. Moreover, expression of Sp100 without
SUMOylation site in a stable system should give new clues to the role
of this modification during the cell cycle, differentiation, or during
virus infection.