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Journal of Virology, December 1999, p. 9789-9795, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Latent Nuclear Antigen of Kaposi's
Sarcoma-Associated Herpesvirus Interacts with RING3, a Homolog of the
Drosophila Female Sterile Homeotic (fsh)
Gene
Georgina M.
Platt,1
Guy R.
Simpson,1
Sibylle
Mittnacht,2 and
Thomas
F.
Schulz1,*
Molecular Virology Group, Department of
Medical Microbiology and Genitourinary Medicine, The University of
Liverpool, Liverpool L69 3GA,1 and Cell
and Molecular Biology Section, The Institute of Cancer Research,
Chester Beatty Laboratories, London SW3 6JB,2
United Kingdom
Received 28 May 1999/Accepted 10 August 1999
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ABSTRACT |
Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) is the likely
infectious cause of Kaposi's sarcoma, primary effusion lymphoma, and
some cases of multicentric Castleman's disease. Its latent nuclear
antigen (LANA) is expressed in the nuclei of latently infected cells
and may play a role in the persistence of episomal viral DNA in
dividing cells. Here we report that LANA interacts with RING3, a
nuclear protein and member of the Drosophila fsh (female
sterile homeotic) family of proteins, some of which have previously
been implicated in controlling gene expression. Binding of RING3 to
LANA involves the ET domain, characteristic of fsh-related proteins,
suggesting that this highly conserved region is involved in
protein-protein interactions. The interaction between RING3 and LANA
results in phosphorylation of serine and threonine residues located
between amino acids 951 and 1107 in the carboxy-terminal region of
LANA. However, RING3 is not itself a kinase but appears to recruit an
as yet unidentified serine/threonine protein kinase into the complex
which it forms with LANA.
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INTRODUCTION |
Kaposi's sarcoma-associated virus
(KSHV) or human herpesvirus 8 (HHV-8) (6) is a type 2 gammaherpesvirus found in all forms of Kaposi's sarcoma (KS), in
primary effusion lymphoma (PEL/BCBL) (5), and in some cases
of multicentric Castleman's disease (30). KSHV is present
in the endothelial and spindle (tumor) cells of KS lesions (4,
31), where it persists in a latent form with limited viral gene
expression. Among the latent viral genes expressed in KS spindle cells
and PEL cells is the latent nuclear antigen (LANA) encoded by the open
reading frame 73 (orf73) (18, 19, 25). LANA is a
large nuclear protein (>200 kDa) of 1,162 amino acids (in the case of
the prototypic BC-1/HBL-6 sequence) with three distinct domains: a
proline rich N-terminal domain, a long acidic internal repeat region
which includes an extended leucine zipper motif, and a carboxy-terminal
end containing a putative nuclear localization signal (27).
In the nuclei of latently KSHV/HHV-8 infected PEL and KS spindle cells,
LANA is located in subnuclear bodies of unresolved identity (12,
17, 25).
LANA may be required for the episomal replication of viral genomes
(2) by tethering viral DNA to mitotic chromosomes, and some
of its sequence features could suggest that LANA may also act as a
viral transactivator (27). Several other gammaherpesviruses have positional homologs of the orf73 gene, although their
overall sequence homology is low and an internal repeat region is not always present (1, 9, 20, 28, 37).
Here we report that LANA binds to RING3. Originally identified as an
open reading frame located within the major histocompatibility complex
class II region (3), the RING3 protein belongs to the Drosophila female sterile homeotic (fsh) family of proteins,
which are related by the presence of two bromodomains, thought to be involved in protein-protein interactions, and an ET domain (extra terminal) of unknown function (16, 23). Three further human homologs of RING3, orfX, brdt, and HUNKI, have been identified (16, 23) [GenBank accession no. Y12059], and homologs of fsh have also been found in chickens, mice, rats, toads,
Caenorhabditis elegans, and Saccharomyces
cerevisiae (21, 26, 35, 36, 38). Whether RING3 is a
nuclear mitogen-activated kinase with autophosphorylating properties
(8, 24) is controversial (26). Drosophila (fsh)
and yeast (BDF-1) members of this family have been implicated in
controlling gene expression or modulating chromatin structure (7,
10, 22, 33, 34).
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MATERIALS AND METHODS |
Yeast two-hybrid screening.
Yeast two-hybrid screening was
performed with the Matchmaker II system plasmids (Clontech), the
reporter strain PJ69-4A (14), and the Y187 strain for
-galactosidase plate assays. A 636-bp PCR fragment encoding amino
acids 951 to 1162 of LANA was generated from the BCP-1 cell line by PCR
with primers BD1
(5'-GACAGAATTCGATTACCCTGTTGTTAGCACA-3') and BD3
(5'-GTGTGGATCCTTATGTCATTTCCTGTGGAGAGTC-3')
(restriction sites are underlined) and cloned into the polylinker
of the GAL4 binding domain vector, pAS2-1 (Clontech), yielding plasmid
pAS2-73c. A trp+ transformant in PJ69-4A,
expressing the LANA/GAL4 BD fusion protein, was transformed with a
human leukocyte cDNA library in the yeast vector pGAD10 (Clontech).
Library plasmids were isolated (39) and cotransformed with
pAS73C into Y187 (Clontech) to confirm the interaction in a
-galactosidase colony lift assay with
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal)
as a substrate. A liquid assay to measure -galactosidase activity
was performed with
o-nitrophenyl--D-galactopyranoside (ONPG)
(Clontech) as a substrate from three independent transformants, each
performed in duplicate.
Recombinant baculoviruses.
Spodoptera frugiperda (Sf9)
cells were maintained in Grace's insect medium (Gibco-BRL) containing
10% fetal calf serum. The complete RING3 cDNA (2,262 bp), generated by
reverse transcription-PCR with HEK 293 cell RNA and the primers R3.1
(5'-CTCAGATCTGGCTTCGGTGCCTGCTTTG-3') and R3.2
(5'-CTCGGATCCTTAGCCTGAGTCTGAATCACT-3'), and the
EcoRI-BamHI insert from pAS-73C (encodes the
C-terminal 212 amino acids) were cloned into the baculovirus transfer
vector pVLH6 (a gift from Richard Marais, Institute of
Cancer Research), yielding constructs pVLH6-R3 and
pVLH6-73C, respectively. A baculovirus expressing full-length LANA (bp 127296 to 123808) (27) was generated by using PCR and BCP-1 DNA and primers Gs10
(5'-GAGAGATCTATTTCCCGAGGATGGCGCCC-3') and 73p3a
(5-CTCGAATTCTTATGTCATTTCCTGTGGAGA-3') and the
transfer vector pVL1392 (PharMingen). To avoid PCR-induced errors
within the internal repeat region, this region and the
carboxyl-terminal end of the orf73 gene was replaced with
the corresponding sequence subcloned from a cosmid (GenBank accession
no. AF148805) by using BamHI sites within (bp 11214) and
downstream of (bp 88370) the orf73 gene. Recombinant
baculoviruses were produced by using the BaculoGold transfection system
(Pharmingen) and amplified by infection of Sf9 cells. For routine
production of recombinant proteins, 3 × 106 cells
were infected with 1 to 10 infectious units per cell of each
baculovirus and harvested at 48 h postinfection.
GST fusion proteins and pull-down assays.
Glutathione
S-transferase (GST) fusion proteins (29)
containing different regions of LANA and RING3 (see Fig. 2 and 3) were produced by using pGEX-1 (Pharmacia) and appropriate PCR primers (details available from the authors on request). Sf9 cells infected with the appropriate recombinant baculovirus or uninfected cells were
harvested, washed in phosphate-buffered saline, lysed in 150 µl of
lysis buffer (50 mM Tris [pH 7.8], 150 mM NaCl, 1 mM EDTA, 1%
Nonidet P-40 [NP-40], 50 µM leupeptin, 1.0 µM pepstatin A, 200 µM benzamidine, 1.0 mM phenylmethylsulfonyl fluoride [PMSF]), incubated on ice for 10 min, and centrifuged (10 min at
13,000 × g and 4°C). Cleared lysates were diluted in
1 ml of binding buffer (lysis buffer with 0.1% NP-40), and equal
volumes of lysates were mixed with equal amounts of GST-, GST-LANAC-,
or GST-RING3-coated glutathione beads. Samples were incubated for
1 h at room temperature and washed three times in 1 ml of binding
buffer before being resuspended in loading buffer and analyzed by
Western blot analysis.
Site-directed Mutagenesis.
A 1,315-bp
SphI-EcoRI fragment was subcloned from
pVLH6-R3 into pUC18 and mutagenized by using the Quick
Change site-directed mutagenesis kit (Stratagene) and the following
primers: for K574A, 5'-CCCAAAAAGGCCA
CAGCGACAG CCCCACCTGCC-3' and
5'-GGCAGGTGGGGCTGTCGCTGTGGCCTTTTTGGG-3' (mutations are underlined); and for E599A,
5'-CCCATGAGTTACGATGCGAAGCGGCAGCTGAGC-3' and 5'-GCTCAGCTGCC
GCTTCGCATCGTAACTCATGGG-3'. Mutations were
confirmed by sequencing. A mutated 1,081-bp
SacI-BamHI fragment was substituted for the
wild-type sequence in pVLH6-R3. To construct an in-frame deletion
within the ATP binding domain, two PCRs were performed with
pVLH6-R3 as a template and the following primers: reaction
1, R3.1 (5'-CTCAGATCTGGCTTCGGTGCCTGCTTTG-3') and
R3ATPRev (5'-GAGCTTGGTGCCACTTCCTC CTAAAGCAGCACTGCCACC-3'),
and reaction 2, R3ATPFor
(5'-GGTGGCAGTGCTGCTTTAGGAGGAAGTGGCACCAAGCTC-3') and R3.2
(5'-CTCGGATCCTTAGC CTGAGTCTGAATCACT-3'). A 50-ng
portion of the 600-bp product from reaction 1 and 50 ng of the 1,700-bp product from reaction 2 were mixed and used in a second round of PCR
with primers R3.1 and R3.2. The resulting 2,238-bp
BglII-BamHI fragment was cloned into
pVLH6. The presence of the in-frame deletion was confirmed
by sequence analysis.
Immunoprecipitation of RING3 LANA complexes.
BCP-1 cells (a
KSHV/HHV-8-infected body cavity-based B-cell lymphoma cell line) were
lysed (2 × 107 cells per ml) in phosphate-buffered
saline containing 0.5% NP-40, 0.25% sodium deoxycholate, 0.05%
sodium dodecyl sulfate (SDS), 50 µM leupeptin, 1.0 µM pepstatin A,
200 µM benzamidine, and 1.0 mM PMSF, incubated on ice for 10 min, and
centrifuged at 13,000 × g for 5 min. Lysates were
mixed with either rabbit anti-RING3 polyclonal antiserum or prebleed
rabbit serum and incubated on ice for 1 h. The protein-antibody
complexes were recovered with protein G beads (incubation at 4°C for
3 h), washed five times with 1 ml of PBS-0.1% NP-40, and
resuspended in loading buffer. Samples were analyzed on Western blots.
Protein kinase assays.
Uninfected Sf9 cells and Sf9 cells
infected with the recombinant RING3 baculovirus were washed twice in
PBS, lysed in 150 µl of lysis buffer (1% NP-40, 50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA [pH 7.8], 25 mM -glycerophosphate, 1 mM
sodium vandate, 50 µM leupeptin, 1.0 µM pepstatin A, 200 µM
benzamidine, 1.0 mM PMSF), incubated on ice for 10 min, and centrifuged
(10 min at 13,000 × g and 4°C). Preimmune beads were
prepared by incubating 100 µl of prewashed protein G beads with 10 µl of prebleed serum in 1 ml of binding buffer (lysis buffer with
0.2% NP-40 substituted) overnight and washing three times with binding
buffer. A 15-µl volume of lysate was diluted in 500 µl of binding
buffer and precleared by incubation with 20 µl of preimmune beads for
1 h. The beads were removed from the lysate by centrifugation
(3,000 × g for 1 min twice). The cleared lysates were
incubated with rabbit anti-RING3 antiserum or rabbit prebleed serum (45 min at 4°C) and then with protein G beads (2 h at 4°C). The beads
were washed five times in 1 ml of wash buffer (lysis buffer with 0.4%
NP-40 and 0.05% SDS substituted) and once in 1 ml of kinase buffer (50 mM HEPES [pH 7.4], 10 mM MgCl2, 10 mM
-glycerophosphate, 1 mM sodium vandate). The beads were resuspended
in 20 µl of kinase buffer, and 10 µl was added to a kinase reaction
mixture containing the above kinase buffer, 2 mM dithiothreitol, 10 µM ATP, 4 µCi of [
-32P]ATP and 500 ng of GST or
GST-LANAC fusion protein as substrates. The reaction mixtures were
incubated at 30°C for 30 min before being quenched with SDS loading
buffer. The reaction products were resolved on SDS-10% polyacrylamide
gels, and the gels were dried and exposed to autoradiography film.
Phosphoaminoacid analysis was performed on the GST-LANAC-excised gel
slice essentially as previously described (40).
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RESULTS |
Identification of cellular proteins which interact with LANA.
To identify cellular proteins which interact with LANA, a yeast
two-hybrid screen was performed with a carboxy-terminal fragment of
LANA (amino acids 951 to 1162) fused to the DNA binding domain of GAL4
as bait. This region of LANA, located carboxy-terminally to the
internal repeat region, contains a putative nuclear localization signal
and shows some homology to the C-terminal end of orf73 positional
homologs in other rhadinoviruses, suggesting a degree of functional
conservation. We screened a human leukocyte cDNA library that expressed
proteins fused to the GAL4 activation domain. From 3 × 106 transformants, 3 clones which activated reporter gene
expression in the presence of the LANA fusion protein (but not in the
presence of the unrelated fusion proteins, murine
p5372-390 and human lamin C) were isolated.
Sequence analysis showed that one of the clones encoded amino acids 384 to 754 of the RING3 protein. RING3 was originally identified as an open
reading frame located within the major histocompatibility complex class
II region (3), although it does not have any obvious
function within the immune response. The RING3 protein belongs to the
Drosophila fsh subclass of proteins, related by the presence
of two bromodomains and an ET domain of unknown function (21). The bromodomain is evolutionarily conserved, and
although the function remains to be elucidated, it has been suggested
that it may mediate protein-protein interactions (13, 15).
The interaction was further confirmed by cotransformation of the Y187
yeast strain with the above RING3 and LANA yeast two-hybrid plasmids
and testing for activation of the -galactosidase reporter gene. A
colony lift assay showed that the colonies turned blue in the presence
of both plasmids but not when single plasmids were present (data not
shown). We attempted to quantify the strength of this interaction in a
liquid -galactosidase assay. The LANA-RING3 interaction resulted in
9.35% of the -galactosidase activation obtained with a strong
positive control (simian virus 40 [SV40]-p53 interaction), compared
with a negative control (pAS2-1/pACT-2) value of 0.57% of the SV40-p53 interaction.
LANA binds to RING3.
To confirm the interaction biochemically,
a GST binding assay was performed. As shown in Fig.
1a, the full-length 105-kDa RING3
protein, expressed in Sf9 insect cells, binds to a recombinant GST-LANA fusion protein (GST-LANAC) containing amino
acids 951 to 1162 (lane 2), but not to GST alone (lane 4). A smaller
product of about 70 kDa, which reacted with an affinity-purified
antibody to RING3 and could be a degradation product or an
alternatively processed form of RING3, did not bind to GST-LANAC (Fig.
1a). The complete LANA protein from BCP-1 cells also binds to a
recombinant GST RING3 protein (GST-R3B), containing amino acids 554 to
754 of RING3, including the ET domain (amino acids 588 to 754) (Fig. 1b, lane 1), but not to GST alone (lane 2). To investigate whether the
RING3-LANA complexes can be detected in vivo, coimmunoprecipitation from BCP-1 cells was performed. Lysates prepared from BCP-1 cells were
incubated with either a rabbit RING3 antibody or prebleed rabbit serum.
Figure 1c shows that immunoprecipitation with the RING3 antiserum
results in the detection of both RING3 (lane 2) and LANA (lane 3). This
data indicates that an interaction between RING3 and LANA occurs in the
KSHV/HHV-8-infected BCP-1 cell line in vivo.
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FIG. 1.
Interaction of RING3 and LANA. (a) RING3 binds to the
C-terminal 212 amino acids of LANA. Lysates of uninfected Sf9 cells
(lanes 1 and 3) or Sf9 cells infected with the recombinant RING3
baculovirus (lanes 2 and 4) were incubated with either GST-LANAC (lanes
1 and 2) or GST alone (lanes 3 and 4) immobilized on glutathione beads.
Binding of RING3 was detected by Western blot analysis with an
affinity-purified rabbit antibody raised against the R3B fusion protein
(Fig. 3). (b) The complete LANA protein from the BCP-1 cell line binds
to RING3. Cellular lysates of BCP-1 cells were incubated with a GST
RING3 fusion protein (amino acids 554 to 754) (lane 1) or GST alone
(lane 2). Binding of LANA was detected on Western blots with serum from
a KS patient. (c) Detection of LANA-RING3 complexes in BCP-1 cells by
coimmunoprecipitation. Lysates of BCP-1 cells were incubated with
either the RING3 antibody (lanes 2 and 3) or prebleed (pb) rabbit serum
(lanes 1 and 4). Immunoprecipitation (IP) with a rabbit antibody raised
against the C-terminal LANA region was used as a positive control for
the presence of LANA (lane 5). The protein-antibody complexes were
isolated by using protein G beads and analyzed by Western blotting with
either a rat anti-RING3 peptide antibody (a gift from K. Thorpe,
Cambridge University, Cambridge, United Kingdom) (lanes 1 and 2) or
human serum from a KS patient (lanes 3 to 5). The positions of the
RING3 and LANA proteins are indicated. The sizes of the protein markers
are shown in kilodaltons.
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Taken together, the results from the yeast two-hybrid screening, GST
binding assays and coimmunoprecipitation strongly support
the
conclusion that LANA and the RING3 protein interact specifically
both
in vitro and in
vivo.
Mapping of binding domains in LANA and RING3.
To define the
domains within LANA which bind to RING3, we investigated the ability of
a series of GST fusion proteins containing deletions within the LANA
carboxy terminus (illustrated in Fig. 2a)
to interact with the full-length RING3 protein expressed in insect
cells. We detected binding of RING3 to deletions C1, C4, and C5 (Fig.
2b, lanes 2, 4, and 5, respectively) but not to GST (lane 1) or
deletions C2 and C6 (lanes 3 and 6, respectively). RING3 binds to both
C1 and C5 (amino acids 1048 to 1162 and 951 to 1055 respectively),
which span the complete C-terminal region of LANA. The binding domain
within C5 was further mapped to C8 (amino acids 1007 to 1055 [Fig. 2c,
lane 5]), but the binding domain within C1 could not be further mapped
by using C7 (amino acids 1048 to 1107 [lane 4). C1 and C5 overlap by 8 amino acids (RDPKCQWK); however, a GST fusion containing this peptide
did not bind RING3 (lane 3), suggesting that the motif (RDPKCQWK) does
not represent a minimal region for binding.
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FIG. 2.
Deletion mapping of the LANA domains which interact with
RING3. (a) Schematic diagram of the LANA fragments expressed as fusion
proteins. The ability to bind to RING3 is summarized on the right. The
positions of the internal repeat region (repeat), the nuclear
localization signal (NLS), and the leucine zipper motif within LANA are
indicated. LANA is numbered 1 to 1162 as in a previous study (25,
27). (b and c) Lysates prepared from Sf9 cells infected with the
RING3 recombinant baculovirus were incubated with different GST-LANA
deletion proteins, and binding was detected by using the
affinity-purified antibody to RING3. (d and e) The GST-LANA fusion
proteins used in the binding on a Coomassie blue-stained
SDS-polyacrylamide gel.
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Taken together, these results suggest that C1 and C8, corresponding to
amino acids 1048 to 1162 and 1007 to 1055, respectively,
may represent
two nonoverlapping binding domains within LANA which
contribute to the
interaction with
RING3.
A similar approach was used to map the LANA binding site in the
C-terminal region of RING3 (Fig.
3). A
series of fragments
of RING3 were expressed as GST fusion proteins
(illustrated in
Fig.
3a), and their ability to bind to both the
full-length LANA
and its C-terminal fragment (amino acids 951 to 1162)
expressed
in insect cells was determined. LANA (Fig.
3c) and its
C-terminal
212 amino acids (Fig.
3b) bound to R3D (amino acids 592 to
754),
corresponding to nearly the complete ET domain (amino acids 588
to 754), and a truncated ET domain (R3F, amino acids 640 to 754),
but
not the last 60 amino acids of the ET domain (R3G). A region
upstream
of the ET domain (amino acids 492 to 587; R3E) also contributed
to this
interaction.
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FIG. 3.
Deletion mapping of RING3 domains which interact with
LANA. (a) Schematic diagram of the RING3 regions used in the
construction of the GST-RING3 deletion proteins. The RING3 protein is
numbered 1 to 754 as in a previous study (3). The positions
of the two extended bromodomains (15), the ET domain
(21), the nuclear localization signal (NLS), the postulated
ATP binding motif (G554PSGFGPSG561), the
catalytic lysine (K574), and the catalytic glutamic acid
(E599) (8) are indicated. The ability of the GST
RING3 fusion proteins to bind to LANA is summarized on the right. (b
and c) Binding of LANA to the RING3 deletion proteins. Lysates of Sf9
cells expressing the recombinant c-terminal LANA fragment (LANAC) (b)
or complete LANA (c) were incubated with the GST-RING3 deletion
proteins immobilized on glutathione-Sepharose beads, and binding was
detected on Western blots with serum from a patient with KS. The sizes
of the protein markers are indicated in kilodaltons on the left. (d)
The GST-RING3 fusion proteins used in the binding assays on a Coomassie
blue-stained SDS-polyacrylamide gel.
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Involvement of RING3 in phosphorylation of LANA.
Two recent
reports (8, 24) had suggested that RING3 might be a
mitogen-activated nuclear serine/threonine kinase. We therefore
investigated whether RING3 could phosphorylate LANA and thus perhaps be
involved in the regulation of LANA function. Recombinant RING3 protein
immunoprecipitated from insect cells was included in an in vitro kinase
assay with GST or GST-LANAC as substrates. A 105-kDa phosphorylated
protein, corresponding to the immunoprecipitated RING3 protein, was
detected on autoradiographs (Fig. 4a,
lanes 1 and 2), and a 47-kDa protein, corresponding to GST-LANAC, was
phosphorylated strongly in the presence of the immunoprecipitated RING3
protein (lane 2). Weak background phosphorylation of GST-LANAC was
observed in the absence of recombinant RING3 (lane 6), but this
phosphorylation was greatly increased in the presence of
immunoprecipitated RING3. To further confirm that additional sites of
GST-LANAC became phosphorylated in the presence of RING3,
two-dimensional phosphoamino acid and tryptic phosphoamino acid maps
were generated. The phosphoamino acid map (Fig.
5a) indicates that there is increased
phosphorylation of the C-terminal LANA fragment on serine and threonine
residues in the presence of RING3, and the tryptic peptide map (Fig.
5b) confirms that LANA becomes phosphorylated on specific sites in the
presence of RING3. The serine and threonine residues targeted by this
RING3-mediated phosphorylation were further investigated by using the
GST-LANA fusion proteins (Fig. 2a) as substrates. Figure 4b shows that C4 (amino acids 951 to 1107) is efficiently phosphorylated and C1
(amino acids 1048-1162) becomes weakly phosphorylated; however, C5
(amino acids 951 to 1055) is not phosphorylated. This suggests that the
phosphorylation sites of the C-terminal LANA fragment are located
between amino acids 951 and 1107 and possibly between amino acids 1055 and 1107. Figure 4c shows that neither C7 (amino acids 1048 to 1107)
nor C8 (amino acids 1007 to 1055) becomes phosphorylated. However,
since C7 does not bind to RING3 (Fig. 2c) and since binding may be
required for phosphorylation, the possibility that the phosphorylation
sites do lie between 1055 and 1107 cannot be ruled out.
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FIG. 4.
The C-terminal domain of LANA becomes phosphorylated in
the presence of RING3. (a) Cellular lysates of SF9 cells infected with
the RING3 baculovirus (lanes 1 to 4) or uninfected SF9 cells (lanes 5 to 8) were immunoprecipitated (IP) with either a rabbit antibody to
RING3 (lanes 1, 2, 5, and 6) or prebleed (pb) rabbit serum (lanes 3, 4, 7, and 8). The immunoprecipitated proteins were added to an in vitro
kinase assay mixture containing either 500 ng of GST-LANAC (lanes 2, 4, 6, and 8) or 500 ng of GST alone (lanes 1, 3, 5, and 7) as substrates
and [-32P]ATP. Phosphorylated proteins were resolved
by SDS-polyacrylamide gel electrophoresis and visualized by
autoradiography. The positions of the phosphorylated RING3 and
GST-LANAC proteins are indicated, and the sizes of the protein markers
are indicated in kilodaltons on the right. (b) A 500-ng portion of each
of the GST LANA deletion proteins C1 (lane 3), C2 (lane 4), C4 (lane
5), and C5 (lane 6) (Fig. 2) was used as a substrate in an in vitro
kinase assay in the presence of the recombinant RING3 protein. GST
(lane 1) or GST-LANAC (lane 2) were used as negative and positive
controls, respectively. (c) A 500-ng portion of each of the GST-LANA
deletion proteins C7 (lane 3) and C8 (lane 4) were used as substrates
in an in vitro kinase assay.
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FIG. 5.
Phosphorylation of LANA in the presence of RING3 occurs
on specific sites. GST-LANAC was phosphorylated by an in vitro kinase
assay in the presence or absence of immunoprecipitated recombinant
RING3, as in Fig. 4, and eluted from a gel slice (38). (a)
Phosphoamino acid analysis by two-dimensional electrophoresis on
cellulose-acetate sheets. The positions of the phosphoserine (S) and
phosphothreonine (T) and the direction of the first (pH 1.9) and second
(pH 3.5) electrophoretic dimensions are indicated. (b) Tryptic
phosphopeptide map. Tryptic peptides were separated on thin-layer
chromatography plates. The directions of the first (electrophoresis)
and second (chromatography) dimensions are indicated. The numbers 1 to
3 denote the specific peptides observed only in the presence of
immunoprecipitated RING3.
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To investigate whether, as suggested previously (
8,
24), the
phosphorylated 105-kDa RING3 protein observed in our experiments
may be
due to an autophosphorylating activity of RING3, which
in turn could
have phosphorylated LANA, we mutagenized RING3 at
predicted
functionally important sites (
8). A postulated catalytic
lysine (K
574) residue in the predicted protein kinase
subdomain
II was reported to be essential for RING3-mediated
phosphorylation
of three common substrates, MBP, kemptide, and MLCK
(
8). Similarly,
deletion of a large section of the carboxy
terminus, including
the putative catalytic glutamine residue
(E
599) in the putative
subdomain III, also eliminated the
renaturable autophosphorylation
activity of RING3. In addition, an ATP
binding motif, G
554PSGFGPSG
561,
had been
predicted in the putative subdomain I (
8) (Fig.
3a).
Recombinant RING3-expressing baculoviruses containing either a
single
mutation (K
574A), a double mutation
(K
574A/E
599A), or an
in-frame deletion within
the putative ATP binding motif (G
554PSGFGPS
561)
were constructed. Phosphorylation of the three mutant RING3 proteins
(Fig.
6, lanes 2, 4, and 6) and the
47-kDa GST-LANAC fusion protein
was still observed. These results
strongly imply that binding
of RING3 to LANA mediates phosphorylation
of the latter on specific
sites but that RING3 is not itself a nuclear
kinase with autophosphorylation
properties. Our findings suggest that
an insect protein kinase
interacting with RING3 is coimmunoprecipitated
with RING3 and
is responsible for the phosphorylation of RING3 and
LANA.
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|
FIG. 6.
Mutational analysis of the putative protein kinase
subdomains of RING3. Three mutant, insect cell-expressed,
immunoprecipitated RING3 proteins were added to an in vitro kinase
assay mixture in the presence of either GST (lanes 1, 3, 5, and 7) or
GST-LANAC (lanes 2, 4, 6, and 8), as in Fig. 4. Wild-type RING3 protein
(WT) (lanes 1 and 2), mutation of the postulated catalytic lysine
residue to alanine (K574A) (lanes 3 and 4), mutation of the
two postulated catalytic residues K574 and E599
to alanine (K574/E599) (lanes 5 and 6),
deletion of 8 amino acids within the postulated ATP binding motif
( G554PSGFGPS561) (lanes 7 and 8), and
uninfected Sf9 lysates (lanes 9 and 10) are shown.
|
|
| |
DISCUSSION |
In this report we demonstrate that the KSHV LANA protein, encoded
by orf73 and a major component of LANA of KSHV-infected cell
lines, interacts with the nuclear RING3 protein. This interaction involves two domains in LANA, amino acids 1007 to 1055 and 1049 to
1162. LANA binds to RING3 within its ET domain and to an additional upstream region. The ET domain is characteristic of members of the fsh
subclass of proteins (21). Our findings suggest that the ET
domain may play a role in protein-protein interactions and that binding
of LANA might substitute for the binding of a physiological ligand. To
our knowledge, this is the first function attributed to the
evolutionarily conserved ET domain. Among the four human fsh homologs,
homology is particularly high in the region between amino acids 588 and
671 (72% amino acid identity). It is therefore possible that LANA also
binds to the other human fsh homologues, and this is currently under investigation.
We also show that the interaction between RING3 and LANA results in the
phosphorylation of LANA but that RING3 is unlikely to be a nuclear
kinase, as postulated previously (8, 24). The first of these
studies (8) identified RING3 as a 90-kDa autophosphorylated
mitogen-inducible nuclear kinase in HeLa cell nuclear extracts. In the
second study (24) an 85-kDa renaturable autophosphorylation
activity was detected in mouse tissue extracts immunoprecipitated with
a RING3 antibody, and the study concluded that RING3 and p85 are
identical. However, a more recent study (26) did not find
RING3 to be autophosphorylated but reported a 90-kDa band with
autophosphorylating properties which was distinct from RING3. In the
same study, RING3 had an apparent molecular mass of 110 kDa, similar to
our observations.
The presence of several sequence motifs in RING3 with homology to the
catalytic domain of protein kinases has suggested that RING3 might be a
protein kinase (8). Mutation of the catalytic lysine
(K574) in subdomain II had been reported to eliminate the ability of RING3 to phosphorylate the substrates MBP, kemptide and MLCK
(8). The same authors reported that deletion of the C-terminal ET domain, including a putative catalytic glutamic acid
(E599) residue in the predicted subdomain III, eliminated autophosphorylation activity. In our hands, point mutation of these two
residues had no effect on the phosphorylation of RING3 or LANA. In
addition, an in-frame deletion of part of a potential ATP binding motif
(GXGXXG) did not affect the ability of immunoprecipitated RING3 to
become phosphorylated or to mediate LANA phosphorylation. These results
support the findings of Rhee et al. (26) that RING3 itself
may not be a kinase. The fact that RING3 and the various mutants still
become efficiently phosphorylated in our kinase assay suggests that a
protein kinase present in insect cells associated with RING3 is
coimmunoprecipited with the RING3 protein and consequently
phosphorylates both LANA and RING3. As mentioned above, an 85-kDa
phosphorylated protein was observed when mouse tissue extracts
immunoprecipitated with RING3 antisera were used in an assay to detect
autophosphorylation activity (24). It is possible that this
phosphorylated protein is the mouse homolog of the insect kinase which
forms a complex with RING3. In binding to LANA, the RING3 protein may
act as a scaffold between the kinase and the viral protein, allowing
the LANA protein to become phosphorylated by the kinase and thus
modulating its activity. The identity of this kinase is currently under investigation.
Many of the known bromodomain-containing proteins are involved in
controlling signal-dependent but not basal transcription (15, 33,
34). The Drosophila, human (hBrg1), and mouse (mBrg1) brahma proteins form part of the SWI/SNF class of transcription factors
which exist within large multiprotein complexes and play a role in
chromatin remodelling (10). Very little is known about the
precise function of the fsh subclass of proteins, although it has been
speculated that they may also play a role in transcriptional regulation
(15, 26). Maternal expression of the Drosophila gene female sterile homeotic (fsh) is required
for normal embryonic development (11, 13). Genetic studies
in Drosophila provide evidence that the fsh protein behaves
as a transacting activator of Trithorax (22), which may act
by modulating the chromatin structure to alter transcriptional control
(reviewed in reference 33). Another member of the
fsh subclass of proteins is the product of the Saccharomyces
cerevisiae BDF1 gene, which may be required for the transcription
of many genes through modulating chromatin structure and for
progression through the meiotic cell cycle (7, 22).
Recent evidence suggests that LANA is required for KSHV episome
persistence, most probably by binding to an oriP-like region in KSHV DNA and tethering it to mitotic chromosomes (2). It is conceivable that this interaction could involve binding to RING3.
Similar to LANA, the Epstein-Barr virus EBNA-1 protein is required for
the propagation of viral episomes but also acts as a transcriptional
activator (32). Whether LANA has similar properties that
might involve an interaction with RING3 is unknown.
| |
ACKNOWLEDGMENTS |
This work was supported by the Medical Research Council of Great
Britain and the North West Cancer Research Fund.
We thank John Trowsdale for helpful discussions, K. Thorpe for the
antibody to a RING3 peptide, and Frederic Aurade for comments about the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Molecular
Virology Group, Department of Medical Microbiology and Genitourinary
Medicine, The University of Liverpool, Liverpool L69 3GA, United
Kingdom. Phone: 44 151 706 4382. Fax: 44 151 706 5805. E-mail:
tschulz{at}liv.ac.uk.
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Journal of Virology, December 1999, p. 9789-9795, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
