From the Center for Retrovirus Research and
Department of Veterinary Biosciences, ¶ Comprehensive Cancer
Center, The Arthur G. James Cancer Hospital and Solove Research
Institute, and the
Department of Molecular Virology, Immunology,
and Medical Genetics, Ohio State University,
Columbus, Ohio 43210-1093
Received for publication, October 4, 2002, and in revised form, February 12, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The PXIXIT
calcineurin binding motif or highly related sequences are found in a
variety of calcineurin-binding proteins in yeast, mammalian cells, and
viruses. The accessory protein p12I encoded in the HTLV-1
pX ORF I promotes T cell activation during the early stages of HTLV-1
infection by activating nuclear factor of activated T cells (NFAT)
through calcium release from the endoplasmic reticulum. We identified
in p12I, a conserved motif, which is highly homologous with
the PXIXIT calcineurin-binding motif of NFAT.
Both immunoprecipitation and calmodulin agarose bead pull-down assays
indicated that wild type p12I and mutants of
p12I that contained the motif-bound calcineurin. In
addition, an alanine substitution p12I mutant
(p12I AXAXAA) had greatly reduced
binding affinity for calcineurin. We then tested whether
p12I binding to calcineurin affected NFAT activity.
p12I competed with NFAT for calcineurin binding in
calmodulin bead pull-down experiments. Furthermore, the
p12I AXAXAA mutant enhanced NFAT
nuclear translocation compared with wild type p12I and
increased NFAT transcriptional activity 2-fold greater than wild type
p12I. Similar to NFAT, endogenous calcineurin phosphatase
activity was increased in Jurkat T cells expressing p12I
independent of its calcineurin binding property. Thus, the reduced binding of p12I to calcineurin allows enhanced nuclear
translocation and transcription mediated by NFAT. Herein, we are the
first to identify a retroviral protein that binds calcineurin. Our data
suggest that HTLV-1 p12I modulates NFAT activation to
promote early virus infection of T lymphocytes, providing a novel
mechanism for retrovirus-mediated cell activation.
As a complex retrovirus, human T lymphotropic virus type 1 (HTLV-1)1 encodes common
retrovirus structural and enzymatic proteins as well as regulatory (Tax
and Rex) and accessory (p12I, p27I,
p13II, and p30II) proteins from four open
reading frames (ORFs I-IV) in its unique pX region (1-4). The
understanding of the molecular pathogenesis of HTLV-1 infection,
immortalization, and transformation has been primarily focused on the
role of Tax and Rex (5, 6); however, emerging evidence also indicates
the important roles of accessory proteins in establishment of HTLV-1
infection (7).
The ORF I-encoded protein HTLV-1 p12I is highly conserved
among different isolates, and its messenger RNA can be detected in infected cell lines and directly from infected patient cells (2-4, 8,
9). Humoral antibody and cytotoxic T lymphocyte responses against ORF
I-encoded viral proteins are elicited in infected carriers and diseased
patients, indicating the expression of the protein during the natural
infection (10, 11). p12I localizes in the endoplasmic
reticulum and cis-Golgi compartment (12-14) and associates with the
H+ vacuolar ATPase (15), the interleukin-2 receptor We have reported that p12I is required for early
establishment of HTLV-1 infection (19, 20). When compared with the wild type infectious molecular clone (ACH), ORF I-mutated ACH-immortalized T
cell lines (ACH·p12I) failed to establish infection in
rabbits (21, 22). ACH·p12I also exhibited reduced
infectivity in quiescent peripheral blood mononuclear cells but not in
activated peripheral blood mononuclear cells, suggesting a role for
p12I in T cell activation during the early stages of
infection (20). Recently, we have demonstrated that p12I
localizes to the ER and associates with an ER luminal protein, calreticulin (12), and selectively activates the nuclear factor of
activated T cells (NFAT) in a calcium-dependent manner (12, 23). Importantly, p12I expression in lymphocytes directly
increases cytoplasmic calcium from ER calcium stores (12, 24), which
serves to explain how the protein activates the NFAT signaling pathway.
The transcriptional activation of NFAT is regulated by the
calcium/calmodulin-dependent serine/threonine phosphatase,
calcineurin (25, 26). Through protein-protein interaction, calcineurin triggers the dephosphorylation and subsequent nuclear translocation of
NFAT, which results in transactivation of NFAT-inducible cytokine genes
including interleukin-2 (25, 26). A conserved calcineurin-binding motif
(PXIXIT) has been identified in the N-terminal
regulatory domain of NFAT, which serves as a major binding site with
both inactivated and activated calcineurin (27, 28). When this motif is
mutated, NFAT is functionally impaired (27, 28). In addition, secondary
calcineurin binding sites in NFAT have been identified that contribute
to higher affinity binding with calcineurin (29, 30).
The NFAT SPRIEIT calcineurin binding sequence and synthetic peptides
containing the critical binding amino acids
(PXIXIT) inhibit binding between calcineurin and
NFAT without affecting calcineurin phosphatase activity (27, 28). A
number of calcineurin-binding proteins inhibit either calcineurin
phosphatase activity or its substrate NFAT transcriptional activity.
These include the antiapoptotic protein Bcl-2 (31, 32),
calcineurin B homologous protein (33), AKAP79 (A
kinase anchoring protein) (34), and
myocyte-enriched calcineurin-interacting protein 1 (35). Interestingly,
the African swine fever virus immunosuppressive protein A238L inhibits
NFAT activation and has a calcineurin binding sequence resembling that of NFAT (36, 37).
Herein, we identified a conserved sequence (PSLP(I/L)T) in
p12I, which is highly homologous to the
PXIXIT calcineurin-binding motif of NFAT.
Full-length p12I and the PSLP(I/L)T motif-containing
mutants bound calcineurin in both immunoprecipitation and calmodulin
bead pull-down assays. In contrast, serial mutations of
p12I that lacked PSLP(I/L)T motif or had selective alanine
substitutions of the motif (p12I
AXAXAA) exhibited abolished or decreased binding
affinity with calcineurin. Furthermore, p12I competed with
NFAT for calcineurin binding in calmodulin bead pull-down experiments.
Interestingly, the p12I AXAXAA mutant
induced more NFAT nuclear translocation than wild type and increased
NFAT transcriptional activity (~2-fold) in a reporter gene assay when
compared with wild type p12I. The existence of a
calcineurin binding motif in p12I suggests that there may
be at least two regulatory actions for p12I that modulate
NFAT activation: 1) to cause calcium release from ER stores and 2)
through calcineurin binding. Collectively, our data provide new insight
into the role of a retroviral accessory protein in mediating early
events in T cell infection and activation.
Cell Lines--
The 293T HEK (American Type Culture Collection
catalog no. 1573) cells and HeLa-Tat cells (AIDS Research and Reference
Reagent Program, National Institutes of Health, HLtat, catalog no.
1293) were maintained in Dulbecco's modified Eagle's medium
(supplemented with 10% fetal bovine serum, 100 µg of streptomycin
plus penicillin/ml, and 2 mM L-glutamine
(Invitrogen). Jurkat T cells (clone E6-1; American Type Culture
Collection catalog no. TIB-152) were maintained in RPMI medium
(Invitrogen) supplemented with 15% fetal bovine serum, 100 µg of
streptomycin plus penicillin/ml, 2 mM
L-glutamine, and 10 mM HEPES (Invitrogen)
(complete RPMI).
Plasmids and Site-directed Mutagenesis--
pME18S and
pMEp12I plasmids were kindly provided by G. Franchini
(National Institute of Health) (14). pMEp12I expresses an
influenza hemagglutinin (HA1)-tagged HTLV-1 p12I fusion
protein. HA-NFAT1c-GFP expression vector (28) (from A. Rao, Harvard
Medical School) was used for NFAT expression in 293T and HeLa-Tat cell
lines. The serial deletion mutants of p12I (1-47, 48-99,
15-69, and 15-86) were previously described (12). Point mutants of
p12I were generated by PCR-based site-directed mutagenesis
using a QuikChangeTM site-directed mutagenesis kit
(Stratagene, La Jolla, CA). Point mutant L74I was made by substituting
the 74th leucine residue to isoleucine within the 99-amino acid
sequence of p12I. Point mutant p12I
AXAXAA was made by multiple alanine substitution
of the PXLXLT motif (e.g. 70th proline
to alanine, 71st leucine to alanine, 73rd leucine to alanine,
and 75th threonine to alanine) motif. To construct the L74I
mutant, 5'-CCTCAGCCCGTCGCTGCCGATCACGATGCGTTTCCCC-3' and
5'-GGGGAAACGCATCGTGATCGGCAGCGACGGGCTGAGG-3' primers were
used. To construct the p12I AXAXAA
mutant,
5'-CCTCTTCTCCTCAGCGCGTCGGCGCCGGCAGCGATGCGTTTCCCC-3' and
5'-GGGGAAACGCATCGCTGCCGGCGCCGACGCGCTGAGGAGAAGAGG-3' primers were used. Sanger sequencing was used to confirm that the
sequences of each p12I mutant plasmids were correct and in
frame. An NFAT-luciferase construct, pNFAT-luc, which has a trimerized
human distal interleukin-2 NFAT site inserted into the minimal
interleukin-2 promoter, was used for the reporter gene assay (38).
Transient Transfection and Immunoprecipitation Assay--
293T
cells were seeded at 50% confluence in 10-cm-diameter tissue culture
dishes 24 h before transfection. Cells were transfected with 10 µg of pME18S, wild type pMEp12I, and mutants of
p12I using LipofectAMINETM reagent (12)
(Invitrogen). For Jurkat T cell transfection, 107 cells
were electroporated in complete RPMI (350 V, 975 microfarads; Bio-Rad
Gene Pulser II) with 30 µg of pME18S, wild type pMEp12I
and mutant expression vectors. To test the binding between
p12I and calcineurin, 293T cells or Jurkat T cells were
transfected with wild type pMEp12I or mutated
p12I expression vectors. Transfected 293T cells (1-2 × 107) or Jurkat T cells (1 × 107) were
lysed with Triton X-100 buffer (1% deoxycholic acid, 0.1% SDS, 1%
Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH
7.5), with Complete® Protease Inhibitor (Roche Molecular
Biochemicals)). CaCl2 (2 mM), EDTA (5 mM), or EGTA (5 mM) were added to test the
calcium chelation effect in p12I and calcineurin binding.
Cell lysates were incubated with 1:200 diluted rabbit polyclonal
calcineurin antibody (Chemicon International, Inc., Temecular, CA)
overnight. The immune complex mixture was then incubated with 50 µl
of protein A/G plus agarose beads (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) for 4 h. Beads were washed three times in Triton
X-100 buffer and boiled in SDS sample buffer, and supernatants were
analyzed by Western immunoblotting. To test pharmacological inhibitors
in a p12I and calcineurin binding assay, cyclosporin A
(Sigma) (10 µM) or glycine,
N,N'-1,2-ethanediylbis(oxy-2,1-phenylene)-bis-N-2-(acetyloxy)methoxy-2-oxoethyl-, bis(acetyloxy)methyl ester (BAPTA-AM; Molecular Probes, Inc., Eugene, OR) (1 µM) were added in culture 30 min
before cell lysis.
Calmodulin-Agarose Bead Pull-down Assay--
To test
p12I binding with calcineurin and to test for competition
between p12I and NFAT for calcineurin binding, a calmodulin
bead assay was performed as previously described (39). Briefly, 293T
cells or Jurkat T cells were transfected with wild type
pMEp12I or mutated p12I expression vectors or
an NFAT expression vector. Transfected 293T (2 × 107)
or Jurkat T cell (1 × 107) were lysed in Triton X-100
buffer (1% deoxycholic acid, 0.1% SDS, 1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), with Complete® protease inhibitor (Roche Molecular
Biochemicals)) with CaCl2 (2 mM).
Calmodulin-agarose (phosphodiesterase 3',5'-cyclic nucleotide activator
from bovine brain) beads (Sigma) were washed three times in 1 ml of
preactivation buffer (10 mM Tris, pH 8.0, 150 mM NaCl, and 2 mM CaCl2) to
activate calmodulin. Lysates were then incubated with 60 µl of packed
beads for 3 h at 4 °C. To remove unbound proteins, the beads
were washed three times with washing buffer (20 mM Hepes,
pH 7.4, 150 mM NaCl, 5 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-100, and protease inhibitors supplemented with CaCl2 (2 mM)). The beads were boiled in
SDS sample buffer, and supernatants were analyzed by Western immunoblotting.
Immunoblotting--
Western immunoblotting was used to analyze
immunoprecipitated and calmodulin bead pull-down products. Protein
concentrations in all lysates were determined by BCA assay (micro-BCA
Protein Assay; Pierce). Cell lysates were separated by SDS-PAGE
followed by transfer to nitrocellulose membranes. Membranes were
blocked with 5% nonfat dry milk for 2 h, incubated overnight at
4 °C with monoclonal anti-HA antibody (1:1000) (clone 16B-12;
Covance Research Products), with polyclonal calcineurin antibody
(1:1000) (Chemicon International) or with monoclonal NFAT1c antibody
(1:500) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for detection
of p12I, calcineurin, and NFAT2 (23), respectively. The
blots were developed using horseradish peroxidase-labeled secondary
antibody and enhanced chemiluminescence (Cell Signaling Technologies,
Beverly, MA). The density of each band was measured using a commercial software package (Gel-pro Analyzer software; Media Cybernetic, Inc.,
Silver Spring, MD).
NFAT Reporter Gene Assay--
For analysis of NFAT
transcriptional activity in wild type pMEp12I and
alanine-substituted mutant (p12I
AXAXAA) transfected Jurkat T cells, NFAT-driven
luciferase activity assay was performed as previously described (23).
Briefly, Jurkat T cells (107) were electroporated as
described above with 30 µg of expression vectors (pME18S,
pMEp12I, or p12I AXAXAA
mutant), 10 µg of reporter plasmid (NFAT-luc), and 1 µg of
pCMV·SPORT- NFAT-GFP Localization--
HeLa-Tat cells were seeded into
chamber slides (Fisher) and were cotransfected with 2 µg of
HA-NFAT1c-GFP expression vector (28) and 4 µg pME18S,
pMEp12I, or p12I AXAXAA
mutant expression vectors. Two days post-transfection, cells were fixed
for 15 min with 2% paraformaldehyde and were visualized by an Olympus
BH-2 fluorescence microscope or Zeiss LSM510 confocal microscope. To
quantify the HA-NFAT1c-GFP nuclear localization, cells that had sole or
predominant nuclear fluorescence were counted as positive cells. Values
were obtained from two wells of a single representative experiment
(~600 cells). To measure GFP intensity in cytoplasmic and nuclear
regions, MetaMorph® Imaging software (Universal Imaging Corp.,
Downingtown, PA) was used. Values represent nuclear/cytoplasmic ratio
of GFP intensity of up to 25 cells.
Calcineurin Phosphatase Activity--
Endogenous calcineurin
phosphatase activity of Jurkat cells was measured by using the
calcineurin assay kit (Biomol; Plymouth Meeting, PA) according to the
manufacturer's instructions. 107 Jurkat cells were
transfected with 30 µg of expression vectors (pME18S,
pMEp12I, or p12I AXAXAA
mutant). At 24 h post-transfection, phosphatase activity was
measured using phosphopeptide substrate (RII peptide) in the presence
or absence of EGTA. The released free PO4 was measured colorimetrically by using the Biomol Green reagent.
The Putative Calcineurin-binding Motif in p12I Is
Highly Conserved--
The peptide alignment of p12I
sequences with the calcineurin binding peptide sequence from NFAT
(SPRIEIT) was carried out using a commercial software package (Align X
software of the Vector NTI suite; InforMax Inc., Bethesda, MD). We
compared p12I sequences from pMEp12I plasmid,
ACH molecular proviral clone, and five different p12I
sequences from the Entrez protein data base (available on the World
Wide Web at www.ncbi.nlm.nih.gov). We identified a highly conserved
70PSLP(I/L)T75 sequence in p12I
(Fig. 1). p12I sequences from
various HTLV-1 strains had nearly identical sequences compared with the
consensus calcineurin binding PXIXIT sequence of
NFAT except the 72nd leucine residue. The pMEp12I plasmid
was the only clone that had a leucine residue at amino acid 74. This
putative calcineurin-binding motif is also conserved in the closely
related simian T cell leukemia virus type 1 (STLV-1) p12I
(40). However, the accessory proteins R3 and G4 of bovine leukemia virus, also a member of the Deltaretrovirus group, did not share these
consensus sequences (41).
The C-terminal Half (Residues 48--
99) of p12I
Containing PSLP(I/L)T Binds Calcineurin
To define the regions in p12I that are responsible for
binding with calcineurin, we tested serially deleted p12I
mutants for calcineurin binding (Fig. 2A). The mutants
p12I 48-99, p12I 15-86, and p12I
15-69 bound calcineurin in immunoprecipitation and calmodulin bead
pull-down assays (Fig. 2B). However, the mutant
p12I 1-47 (the N-terminal half of p12I) failed
to bind calcineurin in either assay (Fig. 2B). These data
indicated that amino acids 48-99 of p12I, which contains
the putative calcineurin-binding
(70PSLP(I/L)T75) motif, was probably
responsible for calcineurin binding. The mutant p12I
15-69, which did not contain the putative calcineurin binding motif,
weakly bound to calcineurin (Fig. 2B). This finding was not
unexpected, because secondary calcineurin binding sites are predicted
from the p12I amino acid sequence when compared with
homologous alignment of secondary binding sequences of NFAT (data not shown).
The PSLP(I/L)T Motif Is Responsible for Interaction
between p12I and Calcineurin--
To more specifically
test whether the calcineurin-binding region in p12I was due
to the PSLP(I/L)T motif, we constructed an alanine substitution mutant
of p12I (p12I AXAXAA) and
carried out immunoprecipitation and calmodulin bead pull-down assays
(Fig. 3). These substitutions were
designed to replace critical proline, leucine, isoleucine, and
threonine amino residues with alanine residues (Fig. 3A).
The p12I AXAXAA mutant had
significantly reduced binding affinity for calcineurin (Fig.
3B). This result indicated that the PSLP(I/L)T motif in
p12I is largely responsible for calcineurin binding. Our
data, however, did not exclude the possibility of secondary binding
sites in p12I as implicated by our serially deletion
mutant results. In addition, we constructed a mutant L74I that
converted a leucine from pMEp12I to an isoleucine more
common at this position in p12I sequences from most HTLV-1
strains including the ACH molecular clone (PSLPLT
versus PSLPIT) (Fig. 1). There was no significant
difference in binding affinity between these two sequences (Fig. 3).
The other single substitution mutants in the putative
calcineurin-binding (70PSLP(I/L)T75) motif,
P70A, S71A, P73A, and T75A, did not result in significant reduction in
binding affinity (data not shown).
p12I and Calcineurin Binding Is
Calcium-dependent but Is Not Affected by Cyclosporin
A--
Calcineurin is activated via cooperative interactions between
calcium and calmodulin (42). Previous reports indicated that calcium
was required for binding of NFAT to calcineurin as a complex with
calmodulin-Sepharose beads (39). To test the calcium requirement for
the association between p12I and calcineurin, we treated
cell lysates with the calcium chelators, EDTA and EGTA, or used
BAPTA-AM-treated cells prior to conducting immunoprecipitation
experiments. The strong calcium chelator EGTA eliminated
p12I binding to calcineurin (Fig.
4, lane 4), whereas
the weaker calcium chelator EDTA and BAPTA-AM reduced the binding
affinity in immunoprecipitation assays (Fig. 4, lanes
3 and 5). p12I binding was decreased
when cell lysates were treated with a decreased concentration of
CaCl2 or when treated with increasing concentration of EDTA
(data not shown). These data indicated that the binding of
p12I to calcineurin required calcium, which is similar to
the requirement of calcium for effective NFAT binding to calcineurin
(39). In parallel, because the binding of NFAT to calcineurin was not
affected by calcineurin inhibitor cyclosoporin A (39), we tested the affect of cyclosporin A in the p12I binding to calcineurin.
As expected, p12I binding was not inhibited by cyclosporin
A (Fig. 4, lane 6). This result suggested that
the cyclosporin A-cyclophilin complex site is distinct from sites
required for the calcium/calmodulin activation and NFAT and
p12I binding (43). Collectively, our data indicated that
the calcineurin binding properties of NFAT and p12I were
similar in requiring calcium for calmodulin binding, and each did not
compete with binding of the cyclosporin A-cyclophilin complex. The
results suggested that both proteins might have similar binding
mechanism or share an identical binding site in calcineurin.
p12I Competes with NFAT for Calcineurin
Binding--
Because synthetic peptides or proteins, which contain the
calcineurin-binding motif, compete with NFAT for calcineurin binding (27, 28, 37), we tested whether the p12I and calcineurin
binding inhibits NFAT and calcineurin binding. We transiently
cotransfected 293T cells with a constant amount of NFAT expression
plasmid with increasing amounts of the wild type pMEp12I
expression plasmid (Fig. 5A)
as well as plasmids expressing p12I mutants
(p12I 1-47, 48-99, 15-69, or
AXAXAA mutants) (Fig. 5B). We then
performed a calmodulin bead pull-down assay to detect the amount of
bound NFAT and wild type p12I or its mutant proteins
in the calcineurin A subunit. As expected, wild type
p12I and the calcineurin binding mutants, p12I
48-99, p12I 15-69, and p12I
AXAXAA, were bound in a
concentration-dependent manner, but the noncalcineurin
binding mutant p12I 1-47 expressed in a parallel manner
failed to bind calcineurin (Fig. 5B). The amount of
calcineurin (A subunit) from pull-down experiments remained constant in
all assays (Fig. 5A). Both wild type p12I (Fig.
5A) and p12I 48-99 (Fig. 5B)
effectively competed against NFAT for calcineurin binding. In the
noncalcineurin binding mutant p12I 1-47 transfection, the
amount of pulled-down NFAT was relatively constant regardless of the
amount of mutant p12I 1-47 transfection (Fig.
5B). In addition, the weakly calcineurin binding p12I 15-69
mutant, which did not contain calcineurin binding sequence, did not
compete with NFAT for calcineurin binding (Fig. 5B). As
expected, mutation in calcineurin binding sequence (p12I
AXAXAA mutant) resulted in reduction of
competition between p12I and NFAT (Fig. 5B).
Together, these results indicated that the PSLP(I/L)T calcineurin
binding sequence is critical for p12I competition with NFAT
for calcineurin binding.
The PSLP(I/L)T Calcineurin Binding Sequence in
p12I Is an Inhibitory Motif for NFAT Transcriptional
Activity--
p12I activates NFAT transcriptional activity
in T cells in a calcium-dependent manner (25). In contrast,
the NFAT SPRIEIT calcineurin binding sequence and synthetic peptides
based on this motif inhibit NFAT transcriptional activation by
competing with NFAT for calcineurin binding (28). To further
investigate whether p12I binding to calcineurin affected
NFAT transcriptional activity, we performed an NFAT-driven reporter
gene assay using the p12I alanine substitution mutant,
p12I AXAXAA, which exhibited lowered
binding affinity for calcineurin in the immunoprecipitation and
calmodulin bead pull-down assay. Jurkat T cells were transiently
transfected with wild type pMEp12I or p12I
AXAXAA expression plasmids and then measured NFAT
transcriptional activities. Whereas the wild type pMEp12I
showed typical ~20-fold induction in the presence of phorbol 12-myristate 13-acetate stimulation (23) compared with the pME empty
vector, the p12I AXAXAA mutant
induced an ~40-fold enhancement of luciferase activity compared with
the pME control vector (Fig. 6). These
data suggested that the lowered binding between calcineurin and the
p12I AXAXAA mutant allows more NFAT
to be available for the reporter gene assay. Alternatively, the
calcineurin-binding motif in p12I may function as a
negative modulator motif for NFAT activation. In addition,
co-transfection of wild type pMEp12I and p12I
AXAXAA mutant resulted in ~25-fold induction
over the pME control vector, which indicated that pMEp12I
and p12I AXAXAA did not compete for
NFAT activation.
Lowered p12I Binding Affinity to Calcineurin Induces
More NFAT Nuclear Translocation--
To test whether p12I
binding to calcineurin affected NFAT nuclear translocation, we
performed NFAT-GFP fusion protein localization assay when co-expressed
with wild type p12I or p12I
AXAXAA mutant in HeLa-Tat cells. As expected,
both wild type p12I and p12I
AXAXAA mutant expression induced NFAT-GFP nuclear
translocation, whereas NFAT-GFP was retained in the cytoplasm in
HeLa-Tat cells transfected with pME vector control (Fig.
7, A-C). Nuclear
translocation of NFAT-GFP was activated by ionomycin and was inhibited
by calcineurin inhibitor cyclosporin A (Fig. 7D). When
untreated, cells transfected with wild type p12I or
p12I AXAXAA mutant had ~80%
nuclear NFAT-GFP translocation, which was 5-6-fold greater than in pME
control-transfected cells (Fig. 7D). We then measured GFP
intensity in nuclear and cytoplasmic regions of NFAT-GFP-expressing
cells to quantify NFAT nuclear translocation. As demonstrated in Fig.
7E, The nuclear/cytoplasmic ratio of NFAT-GFP was less than
1 in the control pME-transfected cells, ~2 in pMEp12I,
and 2.5 in p12I AXAXAA mutant
transfection. Thus, p12I AXAXAA, the
lowered binding mutant to calcineurin, induced more NFAT translocation
than wild type p12I.
p12I Binding to Calcineurin Does Not Affect Phosphatase
Activity--
Because many calcineurin binding proteins inhibit
calcineurin phosphatase activity (42), we tested whether
p12I binding to calcineurin affected calcineurin
phosphatase activity. We measured endogenous calcineurin phosphatase
activity in Jurkat T cells expressing pME, pMEp12I, and the
p12I AXAXAA mutant. As expected, wild
type p12I stimulated calcineurin phosphatase activity
~3-fold over pME empty vector (Fig. 8).
However, the difference between phosphatase activity induced by wild
type p12I and the p12I
AXAXAA mutant was not statistically significant
(p > 0.5). Wild type p12I, which binds
calcineurin with higher affinity compared with the p12I
AXAXAA mutant, did not show significant reduction
in phosphatase activity (Fig. 8). Similar to NFAT, our data indicated
that p12I binding to calcineurin via PSLP(I/L)T calcineurin
binding sequence did not inhibit calcineurin phosphatase activity.
These data indicate that the increased NFAT transcription and nuclear
translocation induced by the p12I
AXAXAA mutant was independent of calcineurin
binding and was more likely due to the known calcium mobilization
properties of the viral protein.
Herein, we are the first to demonstrate that the HTLV-1 accessory
protein p12I binds to calcineurin and modulates NFAT
activation. We identified in p12I a highly conserved
calcineurin-binding motif, which is homologous to a
PXIXIT calcineurin-binding motif of NFAT.
p12I was consistently co-precipitated with calcineurin in
both immunoprecipitation and calmodulin bead pull-down assays. We found
that the PSLP(I/L)T motif in p12I was largely responsible
for calcineurin binding through the use of serial deletion and alanine
substitution mutants. Like NFAT, p12I binding required
calcium but was not inhibited by cyclosporin A. Thus, these two
proteins appear to bind calcineurin via similar motifs.
The PXIXIT calcineurin binding motif or highly
related sequences are found in a variety of calcineurin binding
proteins of yeast, mammalian cells, and viruses (44). These proteins
are either calcineurin substrates or inhibitors of calcineurin-mediated signaling. For example, the yeast transcription factor Crz1p has a
PIISIQ motif that mediates calcineurin interaction (35), and its
pattern of regulation is similar to that of NFAT. Therefore, the
dephosphorylation, nuclear accumulation, and transcriptional activity
of Crz1p depend on this calcineurin-binding motif (44). Like NFAT or
Crz1p, p12I itself may be a substrate of calcineurin. We
are currently investigating whether p12I is regulated by
calcineurin-mediated dephosphorylation. Also, calcineurin binding
motifs are found in calcineurin inhibitor proteins. Cellular protein
myocyte-enriched calcineurin-interacting protein 1 and cain/cabin1
(calcineurin inhibitor) are endogenous calcineurin inhibitors.
Myocyte-enriched calcineurin-interacting protein 1, which contains a
PKIIQT calcineurin-binding motif, is expressed abundantly in striated
muscle and inhibits calcineurin dependent signaling pathways when
overexpressed (35). Cain/cabin1 has a PEITVT motif in a 38-amino acid
putative calcineurin-binding domain (45). A viral protein A238L of
African swine fever virus, which inhibits NFAT transcriptional
activity, interacts with calcineurin via the PKIIIT sequence (37).
Our data indicate that PSLP(I/L)T calcineurin binding sequence of
p12I modulates NFAT nuclear translocation and transcription
activity. This sequence of p12I is critical for competition
with NFAT for calcineurin binding. This result is consistent with our
previous study that tested NFAT-driven reporter gene assay using
truncation mutants and alanine substitution mutants (AXXA)
of putative PXXP motifs (Src homology 3 domain binding
motifs).2 There were two
positive (aa 33-47 and 87-99) and two negative regions (aa 1-14 and
70-86) for NFAT transcription in p12I, and the mutation in
the third PXXP motif
(70PXXP73) resulted in increased
NFAT activation about 2-fold more than wild type. The
70PSLP(I/L)T75 calcineurin binding site
corresponds to the aa 70-86 negative region and third PXXP
motif. This NFAT-inhibitory function of the PSLP(I/L)T motif was not
from the inhibition of calcineurin phosphatase activity.
Calcineurin-binding proteins such as AKAP79, calcineurin B homologous
protein, cain/cabin1, and A238L inhibit calcineurin phosphatase
activity presumably by affecting calcineurin active sites (42).
However, PXIXIT motif-mediated binding itself does not inhibit the catalytic activity of calcineurin, because NFAT
activation requires enzymatic activity of calcineurin as well as
binding via this motif. Synthetic peptides based on the PXIXIT motif inhibit NFAT activation without
affecting calcineurin catalytic activity (27, 28). Likewise,
p12I binding to calcineurin via a similar motif does not
inhibit calcineurin catalytic activity but instead influences NFAT and
calcineurin interaction by competing for binding with NFAT similar to
artificial peptides representing this motif (27, 28).
It is unclear why p12I has two regulatory functions for
NFAT transcriptional activity: positive modulation by increasing
cytosolic calcium concentration from ER stores (12) and negative
modulation by calcineurin binding. Interestingly, Bcl-2 has these
similar properties with p12I, and the functional
relationship between calcium release from the ER and calcineurin
binding of Bcl-2 is also still unresolved. Bcl-2 maintains calcium
homeostasis and prevents apoptosis by localizing not only at the
mitochondrial membrane but also in the ER membranes (46, 47). When
Bcl-2 is at the ER membrane, its function appears to increase ER
calcium permeability, like p12I (46). Although its function
at the ER membrane is still poorly understood, it has been suggested
that Bcl-2 functions as an ion channel protein (46). On the other hand,
Bcl-2 binds calcineurin via its BH4 domain and inhibits NFAT activity
by sequestering calcineurin from NFAT binding without affecting
calcineurin catalytic activity (31, 32). By inhibiting NFAT activation,
Bcl-2 may prevent Fas ligand (FasL) expression and further apoptosis.
Like Bcl-2, p12I may affect apoptosis in HTLV-1-infected T
cells. Another ER membrane protein, CAML (calcium
modulator and cyclophilin ligand), also has
parallel properties to p12I. It activates NFAT by
increasing calcium flux when overexpressed in Jurkat T cells (48, 49).
Also, CAML binds with calcineurin indirectly, through its association
with cyclophilin (50).
In summary, we demonstrate that the HTLV-1 accessory protein
p12I interacts with calcineurin, an important regulator of
NFAT signaling, via a highly conserved PSLP(I/L)T motif. Thus, HTLV-1,
a retrovirus associated with lymphoproliferative disease, expresses a
conserved accessory protein to further T cell activation, an important
antecedent to effective viral infection, via a calcium/calcineurin/NFAT pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
and
-chain (16), and major histocompatibility complex class I heavy
chain (17). A small hydrophobic protein, p12I, contains
several potential functional motifs, including two transmembrane
domains, leucine zipper motifs, and four Src homology 3 domain-binding
motifs (PXXP) (2, 15, 18). However, defined motifs of
p12I that may alter key T cell signaling pathways that
thereby influence cell activation have not been identified.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-gal to normalize for transfection efficiency. The
transfected cells were plated in six-well plates at a density of 5 × 105/ml. Cells were stimulated with 20 ng/ml phorbol
12-myristate 13-acetate (Sigma) at 6 h post-transfection. After
18 h of stimulation, cells were lysed (Cell Culture Lysis Reagent;
Promega, Madison, WI) and analyzed for luciferase activity according to
the manufacturer's protocol (Promega, Madison, WI). Values were
normalized for transfection efficiency based on
-galactosidase
activity. Data expressed in reporter gene assays were the mean of at
least two independent experiments conducted in triplicate. Statistical
analysis was performed using Student's t test.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (45K):
[in a new window]
Fig. 1.
HTLV-1 p12I contains a highly
conserved putative calcineurin-binding motif in p12I.
Sequences from HTLV-1 and STLV-1 were aligned with calcineurin binding
sequence (SPRIEIT) of NFAT using a commercial software program (Align
X, Vector NTI version 7.0). ACH (22), pMEp12I (16), HTLV-1A
(Entrez Protein AAA45390), HTLV-1B (AAA85329), HTLV-1C (AAB23360),
HTLV-1D (B46181), HTLV-1E (C61547), and STLV-1 isolate number 14 (40)
were compared. The conserved SPSLP(I/L)T sequence is indicated in a
shaded box, and black boxes
indicate amino acid residues that have similar properties. The
consensus motif is indicated in the bottom
row.
To test
whether p12I binds calcineurin, we transiently transfected
p12I into both 293T and Jurkat T cells and performed both
immunoprecipitation and calmodulin bead pull-down experiments (Fig.
2). In calcium-containing buffer,
calmodulin beads precipitated both calcineurin and its binding protein
NFAT (39). p12I was consistently pulled down in
calmodulin-agarose bead and immunoprecipitation assays (Fig.
2B). Thus, p12I, as a full-length protein, had
the capacity to bind endogenously expressed calcineurin in both 293T
and Jurkat T cells. As expected, using the pMEp12I vector,
the detected p12I in higher percentage SDS gels (15%)
showed typical doublet bands as described (18). The two forms of
p12I may be due to post-translational modification of
p12I, such as phosphorylation or ubiquitylation (18).
View larger version (26K):
[in a new window]
Fig. 2.
The C-terminal half of p12I
containing the PSLP(I/L)T motif is required for calcineurin
binding. A, schematic representation of wild type
pMEp12I and serial p12I deletion mutants. The
shaded boxes indicate PXXP motifs, and
the bar represents the location of the PSLP(I/L)T conserved
sequence. B, co-precipitation of wild type p12I
and serial deletion p12I mutants with calcineurin in
immunoprecipitation (IP) and calmodulin agarose bead
pull-down (CaM bead) assays. Jurkat T or 293T
cells transfected with pME empty vector wild type pMEp12I
or mutant p12I plasmids were lysed and analyzed by Western
blot. Polyclonal calcineurin antibody was used for immunoprecipitation
assay. Monoclonal HA-1 antibody was used to detect wild type and mutant
p12I.
View larger version (34K):
[in a new window]
Fig. 3.
Alanine substitution mutant of
p12I (AXAXAA) decreases
binding affinity for calcineurin. A, schematic
representation of wild type pMEp12I, alanine substitution
mutant p12I AXAXAA, and L74I plasmid.
Gray boxes, PXXP motifs; bar, the
PSLP(I/L)T conserved sequence. B, 293T cells were
transfected with wild type pMEp12I, p12I
AXAXAA, and L74I plasmid. Binding was tested by
immunoprecipitation (IP) and calmodulin-agarose bead
pull-down (CaM bead) assays.
View larger version (30K):
[in a new window]
Fig. 4.
p12I and calcineurin binding is
inhibited by calcium chelators but not inhibited by cyclosporin
A. CaCl2 (2 mM) or calcium chelator
EDTA (5 mM) (lane 3) or EGTA (5 mM) (lane 4) was added in Triton
X-100 cell lysis buffer, and calcium chelator BAPTA-AM (1 µM) (lane 5) or calcineurin
inhibitor cyclosporin A (10 µM) (lane
6) was added in 293T cells 30 min before cell lysis and
following immunoprecipitation (IP) assay.
View larger version (36K):
[in a new window]
Fig. 5.
p12I binding to calcineurin
decreases the amount of NFAT binding to calcineurin. A,
a constant amount of HA-NFAT1c-GFP (7.5 µg) and increasing amounts of
pMEp12I (0-15 µg) were cotransfected into 293T cells.
The amount of NFAT and wild type p12I proteins bound
to calcineurin A subunit was detected by calmodulin pull-down assay.
B, 293T cells were cotransfected with increasing amounts
(0-15 µg) of wild type pMEp12I and mutant
(p12I 1-47, 48-99, or 15-69 or
AXAXAA mutants) plasmids and a constant amount of
HA-NFAT1c-GFP (7.5 µg). Then wild type p12I or
p12I mutants, NFAT, and calcineurin A subunit were
precipitated by calmodulin beads. Optical density of NFAT, indicating
the amount of calcineurin binding, was measured. Optical density of
each NFAT band was normalized by corresponding calcineurin A
bands.
View larger version (15K):
[in a new window]
Fig. 6.
The substitution mutation in calcineurin
binding sequence in p12I induces increased NFAT
transcription activity and does not compete against wild type
p12I for NFAT transcription activity. Jurkat T cells
were co-transfected with 30 µg of effector plasmid (pME control
vector, pMEp12I, p12I
AXAXAA mutant, or both pMEp12I and
p12I AXAXAA mutant (15 µg each))
and 10 µg of NFAT reporter plasmid and then were left unstimulated or
stimulated with phorbol 12-myristate 13-acetate (PMA). Each
bar represents the -fold induction of NFAT-luciferase
activity in wild type pMEp12I, p12I
AXAXAA mutant, or both plasmid-transfected
cells over luciferase activity of the pME empty vector-transfected
cells. Values were the means of quadruplicate samples in two
independent experiments and were statistically significant (*,
p < 0.01, pMEp12I compared with
p12I AXAXAA alone).
View larger version (24K):
[in a new window]
Fig. 7.
Reduced p12I binding to
calcineurin induces increased NFAT nuclear translocation. Confocal
microscopic image of HeLa-Tat cell cotransfected with HA-NFAT1c-GFP
expression vector and pME empty vector (A),
pMEp12I (B), or p12I
AXAXAA mutant expression vectors (C).
D, the percentage of cells that had nuclear predominant
fluorescence. Cells were untreated, stimulated with ionomycin (3 µM, 10 min before fixation), or inhibited with
cyclosporin A (1 µM, 16 h before fixation).
E, nuclear/cytoplasmic ratio of NFAT-GFP intensity of cells
transfected with pME, pMEp12I, or p12I
AXAXAA vector (*, p < 0.05, pME
vector control compared with pMEp12I. **, p < 0.05, p12I AXAXAA compared with
pMEp12I).
View larger version (11K):
[in a new window]
Fig. 8.
Calcineurin phosphatase activity is not
affected by p12I binding to calcineurin. Endogenous
calcineurin activity from Jurkat T cells transfected with pME,
pMEp12I or p12I AXAXAA
mutant was tested by measuring free phosphate (nmol) released from
phosphopeptide substrate (RII peptide) (*, p < 0.01 versus pMEp12I and p12I
AXAXAA).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank A. Phipps for review of the manuscript, T. Vojt for preparation of figures, and G. Franchini and A. Rao for sharing valuable reagents.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants RR-14324, AI-01474, and CA-92009 (to M. D. L.) and NCI, National Institutes of Health, Grant CA-70529 (to the Ohio State University Comprehensive Cancer Center).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Howard Hughes Medical Institute, Molecular Pathogenesis Program, The Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York, NY 10016.
** To whom correspondence should be addressed: Center for Retrovirus Research and Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210-1093. Tel.: 614-292-4489; Fax: 614-292-6473; E-mail: Lairmore.1@osu.edu.
Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M210210200
2 W. Ding, S.-J. Kim, A. M. Nair, B. Michael, K. Boris-Lawrie, A. Tripp, G. Feuer, and M. D. Lairmore, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HTLV-1, human T lymphotropic virus type 1; ORF, open reading frame; NFAT, nuclear factor of activated T cells; GFP, green fluorescent protein; STLV-1, simian T cell leukemia virus type 1; HA, hemagglutinin; BAPTA-AM, glycine N,N'-1,2-ethanediylbis(oxy-2,1-phenylene)-bis-N-2-(acetyloxy)methoxy-2-oxoethyl-, bis(ace-tyloxy)methyl ester.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Seiki, M., Hikikoshi, A., Taniguchi, T., and Yoshida, M. (1985) Science 228, 1532-1534[Medline] [Order article via Infotrieve] |
2. | Koralnik, I. J., Gessain, A., Klotman, M. E., Lo Monico, A., Berneman, Z. N., and Franchini, G. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8813-8817[Abstract] |
3. | Furukawa, K., Furukawa, K., and Shiku, H. (1991) FEBS Lett. 295, 141-145[CrossRef][Medline] [Order article via Infotrieve] |
4. | Berneman, Z. N., Gartenhaus, R. B., Reitz, M. S., Blattner, W. A., Manns, A., Hanchard, B., Ikehara, O., Gallo, R. C., and Klotman, M. E. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3005-3009[Abstract] |
5. | Mesnard, J. M., and Devaux, C. (1999) Virology 257, 277-284[CrossRef][Medline] [Order article via Infotrieve] |
6. | Yoshida, M. (2001) Annu. Rev. Immunol. 19, 475-496[CrossRef][Medline] [Order article via Infotrieve] |
7. | Lairmore, M. D., Albrecht, B., D'Souza, C., Nisbet, J. W., Ding, W., Bartoe, J. T., Green, P. L., and Zhang, W. (2000) AIDS Res. Hum. Retroviruses 16, 1757-1764[CrossRef][Medline] [Order article via Infotrieve] |
8. | Cereseto, A., Berneman, Z., Koralnik, I., Vaughn, J., Franchini, G., and Klotman, M. E. (1997) Leukemia 11, 866-870[CrossRef][Medline] [Order article via Infotrieve] |
9. | Ciminale, V., Pavlakis, G. N., Derse, D., Cunningham, C. P., and Felber, B. K. (1992) J. Virol. 66, 1737-1745[Abstract] |
10. | Dekaban, G. A., Peters, A. A., Mulloy, J. C., Johnson, J. M., Trovato, R., Rivadeneira, E., and Franchini, G. (2000) Virology 274, 86-93[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Pique, C.,
Uretavidal, A.,
Gessain, A.,
Chancerel, B.,
Gout, O.,
Tamouza, R.,
Agis, F.,
and Dokhelar, M. C.
(2000)
J. Exp. Med.
191,
567-572 |
12. |
Ding, W.,
Albrecht, B.,
Luo, R.,
Zhang, W.,
Stanley, J. R.,
Newbound, G. C.,
and Lairmore, M. D.
(2001)
J. Virol.
75,
7672-7682 |
13. | Koralnik, I. J., Mulloy, J. C., Andresson, T., Fullen, J., and Franchini, G. (1995) J. Gen. Virol. 76, 1909-1916[Abstract] |
14. | Koralnik, I. J., Fullen, J., and Franchini, G. (1993) J. Virol. 67, 2360-2366[Abstract] |
15. |
Franchini, G.
(1995)
Blood
86,
3619-3639 |
16. | Mulloy, J. C., Crowley, R. W., Fullen, J., Leonard, W. J., and Franchini, G. (1996) J. Virol. 70, 3599-3605[Abstract] |
17. |
Johnson, J. M.,
Nicot, C.,
Fullen, J.,
Ciminale, V.,
Casareto, L.,
Mulloy, J. C.,
Jacobson, S.,
and Franchini, G.
(2001)
J. Virol.
75,
6086-6094 |
18. |
Trovato, R.,
Mulloy, J. C.,
Johnson, J. M.,
Takemoto, S.,
Deoliveira, M. P.,
and Franchini, G.
(1999)
J. Virol.
73,
6460-6467 |
19. |
Collins, N. D.,
Newbound, G. C.,
Albrecht, B.,
Beard, J. L.,
Ratner, L.,
and Lairmore, M. D.
(1998)
Blood
91,
4701-4707 |
20. |
Albrecht, B.,
Collins, N. D.,
Burniston, M. T.,
Nisbet, J. W.,
Ratner, L.,
Green, P. L.,
and Lairmore, M. D.
(2000)
J. Virol.
74,
9828-9835 |
21. | Collins, N. D., Newbound, G. C., Ratner, L., and Lairmore, M. D. (1996) J. Virol. 70, 7241-7246[Abstract] |
22. | Kimata, J. T., Wong, F., Wang, J., and Ratner, L. (1994) Virology 204, 656-664[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Albrecht, B.,
D'Souza, C. D.,
Ding, W.,
Tridandapani, S.,
Coggeshall, K. M.,
and Lairmore, M. D.
(2002)
J. Virol.
76,
3493-3501 |
24. |
Ding, W.,
Albrecht, B.,
Kelley, R. E.,
Muthusamy, N.,
Kim, S.,
Altschuld, R. A.,
and Lairmore, M. D.
(2002)
J. Virol.
76,
10374-10382 |
25. | Masuda, E. S., Imamura, R., Amasaki, Y., Arai, K., and Arai, N. (1998) Cell Signal. 10, 599-611[CrossRef][Medline] [Order article via Infotrieve] |
26. | Rao, A., Luo, C., and Hogan, P. G. (1997) Annu. Rev. Immunol. 15, 707-747[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Aramburu, J.,
Yaffe, M. B.,
Lopez-Rodriguez, C.,
Cantley, L. C.,
Hogan, P. G.,
and Rao, A.
(1999)
Science
285,
2129-2133 |
28. | Aramburu, J., Garcia-Cozar, F., Raghavan, A., Okamura, H., Rao, A., and Hogan, P. G. (1998) Mol. Cell 1, 627-637[Medline] [Order article via Infotrieve] |
29. |
Garcia-Cozar, F. J.,
Okamura, H.,
Aramburu, J. F.,
Shaw, K. T.,
Pelletier, L.,
Showalter, R.,
Villafranca, E.,
and Rao, A.
(1998)
J. Biol. Chem.
273,
23877-23883 |
30. |
Park, S.,
Uesugi, M.,
and Verdine, G. L.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
7130-7135 |
31. | Shibasaki, F., Kondo, E., Akagi, T., and Mckeon, F. (1997) Nature 386, 728-731[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Srivastava, R. K.,
Sasaki, C. Y.,
Hardwick, J. M.,
and Longo, D. L.
(1999)
J. Exp. Med.
190,
253-265 |
33. |
Lin, X.,
Sikkink, R. A.,
Rusnak, F.,
and Barber, D. L.
(1999)
J. Biol. Chem.
274,
36125-36131 |
34. |
Kashishian, A.,
Howard, M.,
Loh, C.,
Gallatin, W. M.,
Hoekstra, M. F.,
and Lai, Y.
(1998)
J. Biol. Chem.
273,
27412-27419 |
35. |
Rothermel, B.,
Vega, R. B.,
Yang, J.,
Wu, H.,
Bassel-Duby, R.,
and Williams, R. S.
(2000)
J. Biol. Chem.
275,
8719-8725 |
36. |
Miskin, J. E.,
Abrams, C. C.,
Goatley, L. C.,
and Dixon, L. K.
(1998)
Science
281,
562-565 |
37. |
Miskin, J. E.,
Abrams, C. C.,
and Dixon, L. K.
(2000)
J. Virol.
74,
9412-9420 |
38. |
Northrop, J. P.,
Ullman, K. S.,
and Crabtree, G. R.
(1993)
J. Biol. Chem.
268,
2917-2923 |
39. |
Loh, C.,
Shaw, K. T.,
Carew, J.,
Viola, J. P.,
Luo, C.,
Perrino, B. A.,
and Rao, A.
(1996)
J. Biol. Chem.
271,
10884-10891 |
40. | Saksena, N. K., Srinivasan, A., Ge, Y. C., Xiang, S. H., Azad, A., Bolton, W., Herve, V., Reddy, S., Diop, O., Mirandasaksena, M., Rawlinson, W. D., Vandamme, A. M., and Barresinoussi, F. (1997) AIDS Res. Hum. Retroviruses 13, 425-432[Medline] [Order article via Infotrieve] |
41. |
Lefebvre, L.,
Ciminale, V.,
Vanderplasschen, A.,
D'Agostino, D.,
Burny, A.,
Willems, L.,
and Kettmann, R.
(2002)
J. Virol.
76,
7843-7854 |
42. |
Rusnak, F.,
and Mertz, P.
(2000)
Physiol. Rev.
80,
1483-1521 |
43. | Griffith, J. P., Kim, J. L., Kim, E. E., Sintchak, M. D., Thomson, J. A., Fitzgibbon, M. J., Fleming, M. A., Caron, P. R., Hsiao, K., and Navia, M. A. (1995) Cell 82, 507-522[Medline] [Order article via Infotrieve] |
44. |
Boustany, L. M.,
and Cyert, M. S.
(2002)
Genes Dev.
16,
608-619 |
45. |
Lai, M. M.,
Burnett, P. E.,
Wolosker, H.,
Blackshaw, S.,
and Snyder, S. H.
(1998)
J. Biol. Chem.
273,
18325-18331 |
46. |
Foyouzi-Youssefi, R.,
Arnaudeau, S.,
Borner, C.,
Kelley, W. L.,
Tschopp, J.,
Lew, D. P.,
Demaurex, N.,
and Krause, K. H.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5723-5728 |
47. |
He, H.,
Lam, M.,
McCormick, T. S.,
and Distelhorst, C. W.
(1997)
J. Cell Biol.
138,
1219-1228 |
48. |
Holloway, M. P.,
and Bram, R. J.
(1996)
J. Biol. Chem.
271,
8549-8552 |
49. |
von Bulow, G. U.,
and Bram, R. J.
(1997)
Science
278,
138-141 |
50. |
Holloway, M. P.,
and Bram, R. J.
(1998)
J. Biol. Chem.
273,
16346-16350 |