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J. Biol. Chem., Vol. 276, Issue 28, 25804-25812, July 13, 2001
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From the ¶ Department of Internal Medicine, Division of
Infectious Diseases and the
Received for publication, July 19, 2000, and in revised form, April 19, 2001
Human immunodeficiency virus type 2 (HIV-2) gene
expression is regulated by upstream promoter elements, including the
peri-Ets (pets) site, which mediate enhancer stimulation
following treatment with the phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA). We previously
showed that the oncoprotein DEK binds to the pets site in a
site-specific manner. In this report, we show that binding to the HIV-2
pets site is modulated by treatment of U937 monocytic cells with TPA,
an activator of protein kinase C. TPA treatment resulted in a
reduction in the levels of DEK and the formation of a faster migrating
pets complex in gel shift assays. We show further that the actions of
TPA on pets binding can be duplicated by phosphatase treatment of
nuclear proteins and is blocked with okadaic acid, a protein
phospatase-2A (PP2A) inhibitor. Finally, we demonstrate that ectopic
expression of the catalytic domain of PP2A can activate the HIV-2
enhancer/promoter alone or in synergy with TPA, an effect mediated in
part through the pets site. These results suggest that, through an
interaction with the protein kinase C pathway, PP2A is strongly
involved in regulating HIV-2 enhancer-mediated transcription. This is a
consequence of its effects on DEK expression and binding to the pets
site, as well as its effects on other promoter elements. These findings
have implications not only for HIV-2 transcription but also for
multiple cellular processes involving DEK or PP2A.
Transcriptional regulation of the genome of human immunodeficiency
virus type-2 (HIV-2)1 is
mediated by cellular factors acting via response elements located in
the 5' long terminal repeat (LTR) (1-5). One such response element in
the HIV-2 LTR, denoted pets
(peri-Ets), is a TG-rich site found
between two Elf-1 binding sites (PuB1 and PuB2) (2, 4, 5). Recently,
our laboratory demonstrated that the oncoprotein DEK binds to the HIV-2
pets response element in a site-specific manner (6). DEK was first
identified as part of a fusion protein with the nucleoporin CAN
(Nup214) seen in a subtype of acute myelogenous leukemia (7, 8). The
ubiquitously expressed dek gene encodes a 50-kDa nuclear
phosphoprotein and is transcribed at high levels, especially in
hematopoietic tissues (9, 10). Since its initial cloning, DEK or the
immune response to DEK has been associated with several different
cellular and pathologic processes. These include ataxia telangiectasia
(11), pauciarticular onset juvenile rheumatoid arthritis (12), systemic lupus erythematosus (13-16), sarcoidosis (13, 15), idiopathic uveitis
(15), and Kikuchi's disease (18).
The pets site mediates transcriptional activation in response to
12-O-tetradecanoylphorbol-13-acetate/ phorbol 12-myristate 13-acetate (TPA/PMA), phytohemagglutinin, PMA + phytohemagglutinin, T-cell receptor activation by soluble or
immobilized antibodies, and antigen (4, 5). Although the roles of
various kinases activated by these agents, such as protein kinase C
(PKC) and I Additional evidence that PP2A functions in regulating HIV LTR activity
is provided by studies with okadaic acid (OKA), a marine sponge toxin
that specifically inhibits protein phosphatase-1 (PP1) and PP2A. OKA
has been reported to activate the HIV-1 LTR by inducing NF- Despite the observation by our laboratory that the HIV-2 pets site
mediates activation of the promoter in response to several mitogenic
and differentiating agents, DEK binding appears to be constitutive.
This leads to questions of how activation through this HIV-2 element is
regulated. In this paper, we demonstrate that TPA activation of the
monocytic cell line U937 alters DEK protein levels and binding to the
pets response element through a signaling cascade involving PKC and
PP2A. We also show that ectopic expression of the catalytic domain of
PP2A can increase the basal activity of the HIV-2 enhancer and enhances
TPA-mediated activation of the promoter, acting largely through the
pets response element. Finally, we show that the effects of TPA or
PP2Ac on the HIV-2 enhancer can be blocked by treatment with the PP2A
inhibitor OKA. Thus, PP2A plays an integral role in modulating
transcription of the AIDS-causing retrovirus HIV-2.
Cell Culture--
Human U937 monocytic cells were maintained in
RPMI 1640 medium containing 10% fetal bovine serum (Life Technologies,
Inc.), penicillin (50 units/ml), streptomycin (50 µg/ml), and
L-glutamine (2 mM).
Nuclear Protein Extraction--
Nuclear protein extracts from
U937 monocytic cells were prepared using a modification of the method
of Dignam (28). All steps were performed at 4 °C. Cells were washed
once with 10 ml of phosphate-buffered saline and twice with 2 ml of
buffer A (10 mM Hepes, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM DTT, and
1× Complete protease inhibitor solution (Roche Molecular
Biochemicals)). The cells were resuspended in lysis buffer (15 µl of
buffer A/107 cells + 0.01% Nonidet P-40) for 5 min. The
nuclei were pelleted and the supernatant discarded. The nuclear
membrane was disrupted by resuspension of the pellet in 10 µl of
buffer C/107 cells (20 mM Hepes, pH 7.9, 25%
glycerol, 420 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 0.5 mM DTT, 1× Complete
mini protease inhibitor (Roche Molecular Biochemicals)) and gentle
rocking of the tubes for 10 min followed by centrifugation at 12,000 rpm for 10 min. The supernatant containing the nuclear proteins was
removed and diluted by the addition of 5 µl of modified buffer D (20 mM Hepes, pH 7.9, 20% glycerol, 50 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 1× Complete mini protease
inhibitor)/µl of buffer C. Protein concentration was quantitated
using the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA).
Gel Shift Assay/Electrophoretic Mobility Shift Assay
(EMSA)--
For EMSA, 200 ng of probe DNA (sense 5'-TAT ACT TGG
TCA GGG CGA ATT CTA ACT AAC AGA-3') with a pets site (bold
letters) and mutated PuB2 site was end-labeled using T4 polynucleotide kinase (New England BioLabs). Nuclear extract (5-10 µg) was
incubated with 2 × 104 cpm of pets probe in
reaction buffer containing 10 mM Tris-HCl (pH 7.5), 4%
glycerol, 1 mM EDTA, 100 mM KCl, 5 mM DTT, 2 µg of bovine serum albumin, and 1.5 µg of
poly dI·dC in a final reaction volume of 20 µl at room temperature
for 15 min. For competitive binding reactions, 200 ng of cold pets
probe or an oligonucleotide (5'-TAT ACT AGA
TCT GGG CGA ATT CTA ACT AAC AGA-3') containing a mutated pets site (mutated bases are underlined) was added. Material
from the binding reactions was separated by electrophoresis at room
temperature in a 4% native acrylamide gel at 116 V for 3 h in TGE
buffer (50 mM Tris base, 380 mM glycine, 2 mM EDTA, 50 µM Potato Acid Phosphatase Treatment of Nuclear Extract--
Potato
acid phosphatase (PAP) assays were performed as described by Pongubala
(29). Briefly, 1 unit of PAP Type II (Sigma) was mixed with 10 µg of
nuclear extract, 0.2 mM EDTA, 0.5 mM DTT, 100 mM KCl in a final reaction volume of 20 µl. PAP,
heat-inactivated by boiling for 5-10 min, was used in control
reactions. The PAP reactions were incubated at 30 °C for 15-60 min,
and 6 µl was used in binding reactions with 5 × 104
cpm of pets probe labeled using Klenow polymerase. EMSA was
performed as described above.
DEK Western Blot--
40 µg of nuclear extract was mixed with
SDS-polyacrylamide gel electrophoresis loading dye, denatured by
boiling for 5 min, and separated by electrophoresis on a 10% SDS
polyacrylamide gel. The proteins were transferred to polyvinylidene
difluoride membrane (Amersham Pharmacia Biotech) by semi-dry blot
transfer (FisherBiotech, FB-SDB-2020) at 250 mA for 1.5 h. The
membrane was blocked with TBS-T (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.02% Tween 20) + 5% dry milk for 1 h at
room temperature. The blot was then incubated with a 1:1000 dilution of
rabbit polyclonal DEK antiserum (gift of Gerard Grosveld) in TBS-T + 5% dry milk for 1 h at room temperature followed by three 15-min
washes with TBS-T. Secondary antibody, a 1:10,000 dilution of goat
anti-rabbit peroxidase-conjugated IgG (H+L) (Life Technologies, Inc.),
was added and the blot incubated for 45 min in TBS-T + 5% milk at room
temperature. After the membrane was washed as described above, the
secondary antibody was detected using Supersignal chemiluminescent
substrate (Pierce) as instructed. For some Western blots, the anti-DEK
primary antibody was detected using a 1:8000 dilution of goat
anti-rabbit alkaline phosphatase-conjugated IgG (Sigma) and
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT)
alkaline phosphatase substrate (Sigma).
Shift-Western Blotting--
The shift-Western assay was
performed as described previously (30) with modifications. Briefly, 30 µg of nuclear extract was used in binding reactions followed by EMSA
on a 5% native acrylamide gel. Following separation of the
nucleoprotein complexes, the gel and nitrocellulose membrane were
soaked in transfer buffer (39 mM glycine, 48 mM
Tris base, 20% (v/v) methanol) with 0.1% SDS for 30 min at room
temperature. The gel, Whatman 3MM filter papers, and membranes were
stacked as described (30). Proteins were transferred to nitrocellulose
membrane (Schleicher & Schuell), and probe was transferred to DE-81
paper (Amersham Pharmacia Biotech) using a semi-dry blotting unit
(FisherBiotech, FB-SDB-2020) at 300 mA for 2.5-3 h. The nitrocellulose
membrane was subsequently immunoblotted using a rabbit polyclonal
Phosphatase Inhibition--
U937 cells were treated with 5 or 50 nM OKA (Calbiochem) only or pretreated for 30-45
min with OKA prior to stimulation with 32 nM TPA (Sigma).
EMSA and DEK Western blots were performed 24 h post-treatment.
PKC Inhibition--
U937 cells were treated with only 2 µM staurosporine or 10 µM Gö6983
(Calbiochem) or pretreated for 30-45 min prior to stimulation with 32 nM TPA. 24 h post-treatment, nuclear extract was
prepared and EMSA was performed.
Plasmids--
HIV-2-chloramphenicol acetyltransferase
(HIV-2-CAT) and HIV-2-luciferase (HIV-2-luc) plasmids contain
enhancer/promoter sequences ( Transfections--
5 µg of reporter plasmid, alone or in
combination with PP2Ac expression vector, was combined with empty pCMV5
vector for a total of 25 µg of total DNA. U937 undergoing logarithmic
growth were harvested at 5 × 105 cells/ml, washed
once with 50 ml of serum-free RPMI 1640 medium, and resuspended to a
concentration of 25 × 106 cells/ml in serum-free RPMI
medium. 107 cells (0.4 ml) were aliquotted to 0.4-cm
electroporation cuvettes (Invitrogen) and incubated for 5 min at room
temperature with the DNA mixture to be transfected. The cells were
electroporated using an Invitrogen Electroporator II at a setting of
330 V, 1000 microfarads, and infinite resistance with an input voltage
of ~325 V. Following electroporation, the cells from each
cotransfection were resuspended in 10 ml of RPMI + 10% fetal bovine
serum and split evenly (5 × 107 cells/plate) into
control and treatment groups. 12-16 h post-transfection, the treatment
group was stimulated with 32 nM TPA. Any pretreatment with
phosphatase inhibitors occurred 30 min prior to TPA stimulation. 24 h post-stimulation, the cells were harvested, pelleted, washed once with 10 ml of phosphate-buffered saline, and resuspended in 100 µl of 0.25 M Tris-HCl (pH 7.5) (CAT assays) or 1×
passive lysis buffer (luciferase assays; Promega). The protein
concentration of the lysates was determined by the Bradford reagent
(Bio-Rad) method.
Chloramphenicol Acetyltransferase (CAT) Assay--
For CAT
assays, 5-30 µl of cell lysate was combined with 20 µl of 100 mM Tris-HCl (pH 7.5). The final volume was increased to 50 µl with lysis buffer, if necessary, and the mixture was incubated for
15 min at 65 °C to inactivate endogenous acetyltransferases. The
lysates were then transferred to 7-ml scintillation vials, and 200 µl
of CAT reaction mixture (125 mM Tris-HCl, 1.25 mM chloramphenicol, 0.1 µCi of
[3H]acetyl-CoA) was added. 5 ml of Econofluor-2
(PerkinElmer Life Sciences) scintillation fluid was overlaid and
the reaction incubated for 15-90 min at 37 °C before counting.
Background and total counts were determined by mixing 200 µl of CAT
reaction mixture with 5 ml of Econfluor-2 or Scintiverse (Fisher
Scientific), respectively. CAT activity was background-subtracted and
normalized for the total amount of protein used.
Luciferase Assays--
Luciferase assays were performed using
the Promega luciferase assay system according to the manufacturer's instructions.
Phosphatase Activity--
PP2A enzymatic activity in whole cell
lysates from U-937 cells transfected with 2.5, 5, or 10 µg of pCMV5
PP2Ac expression vector was determined using the PP2A assay kit from
Life Technologies according to the manufacturer's instructions.
Stimulation of U937 Cells Alters the Pattern of Binding to the pets
Site and Reduces DEK Protein Expression--
TPA is a biologically
active diterpene with pleiotropic effects that binds to and
activates PKC. Although TPA has been shown to activate the HIV-2
promoter, an effect mediated in part by the pets site (Fig.
1A), the effect of TPA
treatment on binding to the pets site has not been investigated. To
determine whether binding to the pets site could be modulated by cell
activation, we treated U937 monocytic cells for at least 24 h with
32 nM TPA or the carrier dimethyl sulfoxide
(control). Nuclear extracts were prepared and EMSA performed using a
radiolabeled probe containing a pets response element. As seen in Fig.
1B, TPA treatment resulted in an increase in the
electrophoretic mobility of the pets complex on gel shift assays. The
effect of TPA on pets binding was also observed using other monocytic
cell lines, including HL-60 and THP-1 (data not shown). Time course
experiments with U937, HL-60, and THP-1 monocytic cell lines indicated
that the complex transition begins as early as 30 min and is complete
between 24 and 48 h post-treatment (data not shown).
To confirm that the change in mobility of the pets complex was indeed
due to phorbol ester treatment, U937 cells were treated with dimethyl
sulfoxide, 50 nM TPA, 50 nM 4
Our laboratory has previously shown that the oncoprotein DEK binds the
pets response element in a site-specific manner (6). Because DEK is a
nuclear phosphoprotein (9), we postulated that the TPA-induced changes
in the pets complex seen on EMSA might be because of changes in the DEK
phosphorylation state or intracellular levels. To examine the latter
hypothesis, we performed DEK Western blots using the nuclear extract
used for EMSA in Fig. 1C. Treatment of the cells with either
TPA or 4 Shift-Western Assay Demonstrates That DEK Is Present in the Upper
but Not Lower pets-specific Band Seen on EMSA--
As available
antibodies against DEK do not consistently work in "shift-shift"
assays, the shift-Western assay, which enables the localization of
known DNA-binding proteins to specific EMSA bands (30), was used to
assess the presence or absence of DEK in specific complexes on EMSA. In
this assay, nucleoprotein complexes within the gel matrix are
electroblotted onto nitrocellulose membrane and DE-81 anion exchange
paper, which capture the protein and radiolabeled probe, respectively.
The nitrocellulose membrane is subsequently blotted for the protein of
interest and the DE-81 paper autoradiographed. The overlapping of bands
on both membranes suggests that the specific nuclear factor detected by
immunoblot is localized to the gel shift band. A gel shift assay using
nuclear extract from untreated or 32 nM TPA-treated U937
was performed using a radiolabeled pets probe followed by shift-Western
assay using The PKC Inhibitors Staurosporine and Gö6983 Block the Effect
of TPA--
To determine whether PKC activation was required for the
effect of TPA on pets site binding, we utilized the PKC inhibitors staurosporine and Gö6983. Gö6983 is a staurosporine
derivative capable of inhibiting the Potato Acid Phosphatase Treatment of Nuclear Extract from
U937 Cells Leads to an Increase in the Electrophoretic Mobility of the
pets Complex--
Our observation that PKC activation by TPA results
in the formation of the faster migrating complex within minutes
post-treatment (data not shown) suggests that post-translational
modifications such as phosphorylation/dephosphorylation may be
responsible, in part, for the alterations seen in the electrophoretic
mobility of the pets complex. To further address this hypothesis, we
treated nuclear extract from unstimulated U937 cells with active or
heat-inactivated Type II PAP for 20 min at room temperature and then
performed gel shift assays using a 32P-labeled pets probe.
Active PAP treatment led to the formation of a new band on gel shift at
the same position as the band seen with TPA treatment of U937 (Fig.
3A, lane 4). The formation of this new band was specific to PAP treatment, as heat-inactivated PAP
had no effect (Fig. 3A, lane 1). PAP treatment also had no effect on the binding pattern when nuclear extract from TPA-treated U937 cells was used (Fig. 3A, lane 5). Because 20 min of PAP
treatment resulted in a partial transition to the TPA-induced pets
band, U937 nuclear extract was subjected to longer PAP treatment (15, 30, or 60 min) followed by gel shift assay. Extended PAP treatment effectively led to the loss of much of the uninduced pets complex (Fig.
3B, lane 1) and the formation of a band (Fig.
3B, lanes 2-4) with identical mobility in gel
shift assay to the TPA-induced complex (Fig. 3B, lane
5). These results suggest that the TPA-induced complex is the
result of dephosphorylation of the protein(s) bound to the pets
site.
OKA Blocks the TPA-induced Loss of DEK and Formation of the
TPA-induced pets Band in Gel Shift Assays--
OKA is a potent and
specific inhibitor of the catalytic activity of PP1 and PP2A (19, 40,
41). To determine whether the change in mobility of the pets complex in
response to TPA treatment of U937 was due to PP1/PP2A activity, U937
cells were pretreated for 30-45 min with 5 or 50 nM OKA
prior to treatment with TPA for 24 h. EMSA was performed using
either a radiolabeled pets probe or, as a positive control, an NF-
DEK Western blots were performed with equal amounts of the nuclear
extract used for EMSA in Fig. 4A. Concomitant with the TPA-induced change in pets site binding, a loss of DEK protein was also
observed (Fig. 4B, lane 2). The loss of DEK due
to TPA treatment could be blocked by 50 nM of OKA (Fig.
4B, lane 6), whereas 5 nM OKA had no
effect (Fig. 4B, lane 5). The DEK band also
appeared to be more heavily stained when 50 nM OKA was used (Fig. 4B, lanes 4 and 6) as compared
with 5 nM OKA alone (Fig. 4B, lane 3)
or no treatment (Fig. 4B, lane 1). Taken together these data suggest that TPA treatment of U937 cells results in activation of a phosphatase, likely PP2A, that dephosphorylates the DEK
protein bound to the HIV-2 LTR pets site, with subsequent loss of DEK
from the nucleus and binding of a second, unknown factor to the pets site.
PP2Ac Activates the HIV-2 Enhancer--
The above findings
suggested the involvement of PP2A in the activation of the HIV-2 LTR
and the possibility that PP2A may act on the HIV-2 LTR, at least in
part, through the pets site. To test this intriguing possibility, we
transfected U937 cells with an HIV-2 enhancer-CAT reporter and
pCMV5 PP2Ac, a plasmid encoding the catalytic domain of PP2A (gift of
Marc Mumby) or empty pCMV5 vector. Transfected cells from each
condition received 32 nM TPA treatment or went untreated
for 24 h. Increasing levels of ectopic PP2Ac enhanced both the
base-line (Figs. 5A and
6B) and TPA-induced (Fig. 5A) activity of the
HIV-2-CAT reporter significantly over no treatment or TPA alone,
respectively. To further assure that the effects that we observed were
indeed due to increased levels of PP2A activity, we performed PP2A
enzymatic activity assays (Life Technologies). As seen in Fig.
5B, ectopic expression of PP2A resulted in an increase in
intracellular PP2A activity over base line, which was further augmented
by the addition of TPA. Similar results were obtained with a Promega
PP2A activity assay (data not shown). Furthermore, the increased
HIV-2-reporter activity seen in U937 cells transfected with pCMV5 PP2Ac
and treated with TPA (750-fold over base line) could be reduced in a
dose-responsive manner by increasing levels of OKA (Fig.
5C). Although OKA had a similar effect on U937 cells
treated with TPA alone, much greater levels of OKA were required
to achieve the same degree of CAT activity reduction in cells
expressing ectopic PP2Ac. This antagonistic action of OKA on TPA
activation of the HIV-2-CAT reporter has also been observed for the
HIV-1 enhancer in Jurkat, a T-lymphocyte cell line (25).
In contrast to the antagonistic effect of OKA on TPA and PP2A activity,
OKA alone enhanced CAT reporter activity in a
dose-dependent fashion, with the maximal activity (a
150-fold increase) occurring at 200 nM (data not shown).
This is most likely because of activation of NF- Activation of the HIV-2 Promoter by PP2A Is Mediated by the pets,
PuB2, and Sp1 Sites--
Because binding to the pets site of HIV-2 is
modulated by phosphatase activity, we sought to determine whether the
activation of the HIV-2 promoter by PP2A is mediated in part by the
pets response element. Various site-directed deletion or mutant
HIV-2-reporter gene constructs were used in these experiments (Fig.
6A). Because PP2Ac increases
the activity of the HIV-2-CAT reporter construct (Fig. 5A),
we assessed the role of the pets site in the response of the HIV-2
promoter to ectopic PP2Ac. Mutation of the pets site led to a
significant decrease in the PP2Ac activation of the HIV-2 LTR
(p
PP2A has been shown previously to regulate expression through Sp1 and
In this report, we have made the following observations. 1) TPA
treatment of U937 monocytic cells results in a marked reduction in the
levels of the DEK oncoprotein, with a concomitant change in the
mobility of the pets complex in gel shift assays. 2) The change in the
electrophoretic mobility of the pets complex is phorbol
ester-dependent and can be blocked with PKC inhibitors. 3)
Dephosphorylation of the nuclear extract from U937 cells resulted in a
pattern of binding to the pets site similar to that seen with TPA
treatment, implicating a phosphatase in the signaling pathway. 4)
Okadaic acid, a potent inhibitor of PP2A/PP1, is able to block the
effects of TPA treatment on pets binding and DEK protein levels. OKA
also suppresses HIV-2 LTR activation in response to TPA. 5) The
catalytic domain of PP2A synergizes with TPA to activate the HIV-2 LTR,
an effect mediated in part by the pets response element, which can be
antagonized by treatment with OKA. These finding are summarized in Fig.
7 and demonstrate that PP2A plays a
significant role in one or more of the pathways activated by PMA,
culminating at the HIV-2 promoter. By overexpressing the catalytic
domain of PP2A, the transcriptional activity induced by these pathways
is enhanced. Additionally, as PP2A can also activate the promoter in
the absence of PMA (Fig. 5A), PP2A overexpression may be acting on
signaling pathways not normally activated by TPA, which in concert with
PMA-induced signaling pathways lead to synergistic activation of the
HIV-2 enhancer.
We have demonstrated in this paper that binding to the pets site is
responsive to signaling by the phorbol ester TPA (an agent known to
activate the HIV-2 LTR). TPA activation of U937 cells appears to
trigger an exchange of DEK for another pets factor, because DEK protein
levels are significantly reduced post-TPA treatment and the DEK protein
is not observed in the TPA-induced pets complex by shift-Western assay.
The reduction in DEK levels following TPA treatment has also been
observed in other monocytic cell lines (HL60 and THP-1), suggesting
that this is not a cell line-specific phenomenon (data not shown).
These observations, and the fact that DEK is expressed constitutively
at high levels, suggest that post-translational modifications to DEK
and alteration of its intracellular levels following induction of
specific cellular signal transduction pathways allow for greater
control of the DEK effect on transcription versus only
modulating basal expression levels.
The identity of the second pets factor remains unknown, but in the
course of purifying pets-binding proteins, we noted that a 65-kDa
nuclear factor also appears to bind to the pets site specifically (42).
However, as it was not the dominant pets-binding protein under the
conditions used, we did not pursue its identification further. In light
of the current data, we are now attempting to identify this second
factor, as it may be the protein that activates the HIV-2 LTR through
the pets site in response to antigenic stimulation.
Although TPA activates PKC (Fig. 2, A and B),
further observations implicate a phosphatase, likely PP2A (Figs. 3,
A and B and 4A), in the mediation of
the transition from the slower to faster mobility complex seen on EMSA;
this suggests that PKC potentially up-regulates, directly or
indirectly, intracellular PP2A activity. If this is the case, then
ectopic PP2Ac expression may serve to accelerate the rate at which the
transition from one pets complex to another takes place in the presence
of TPA stimulation. We have observed that the pets complex transition
in response to TPA stimulation of various monocytic cell lines (U937,
HL60, and THP-1) begins as early as 30 min post-TPA treatment and is
completed within 24-48 h (data not shown). Recent studies suggest that
PP2A exists in complexes with various kinases, allowing for rapid
modifications of the phosphorylation state of the kinases or substrates
(19, 43). Although no direct association has been shown between PP2Ac and PKC, the alpha isoform of PKC (PKC Several published reports implicate PP2A as potentially regulating
HIV-1 replication, generally suggesting that this cellular enzyme
suppresses HIV-1 transcription or replication (23, 25-27). Okadaic
acid inhibition of PP2A has been shown to activate the HIV-1 LTR,
(25-27). In contrast, OKA suppresses the activation of the HIV-1 LTR
by TPA in the Jurkat T-lymphocyte cell line (25). In the present study,
where both OKA and TPA independently activate the HIV-2 LTR, each agent
antagonizes the effects of the other. Although the exact mechanism by
which these agents antagonize each other remains unknown, it is clear
from these observations and published reports that OKA and TPA are
pleiotropic in their actions and may stimulate different and
opposing pathways.
As inhibitors often target more than one enzyme, more direct evidence
of the involvement of a particular phosphatase can be provided by
overexpressing the gene encoding the phosphatase. In our study, we
utilized a constitutively driven PP2Ac expression vector to achieve
increased expression of the catalytic domain despite the autoregulatory
mechanisms that often serve to keep the intracellular levels of the
enzyme constant (45). We found that overexpression of the C subunit of
PP2A increases basal HIV-2 enhancer activity and synergizes with
TPA-mediated activation of the enhancer. The involvement of PP2Ac in
the activation of the HIV-2 enhancer is strengthened further by the
inability of mutant C-subunits (gift of David Pallas) to activate the
HIV-2 promoter (data not shown). The stimulation of the HIV-2 promoter is due in part to the pets response element, but the PuB2 and Sp1 sites
also play a role. Our studies are the first indicating that ectopic
expression of the catalytic domain of PP2A activates the HIV-2 LTR. The
observations presented here are also the first showing that the
intracellular levels of DEK can be modulated in response to cell
stimulation and differentiation by agents such as TPA and OKA. This may
well have implications for the pathogenesis of cancer, as both DEK and
PP2A have been implicated in oncogenic processes.
DEK may be a tumor suppressor protein under some circumstances, a
finding that fits observations by Meyn et al. (11) that DEK
can complement the radiation-sensitive phenotype of fibroblasts from
ataxia telangiectasia patients. PP2A, a regulator of cell growth, metabolism, and intracellular signaling, is generally viewed as
a tumor suppressor, because its inhibition by OKA promotes tumor
formation (19). Our studies suggest that PP2A may directly or
indirectly affect intracellular levels of the DEK protein, as OKA
treatment blocks DEK loss. Because phosphatase activity triggers the
transition seen with TPA treatment, inhibition of PP2A may allow DEK to
remain in its phosphorylated state and stay bound to the pets response
element. This would have the effect of suppressing TPA-induced HIV-2
LTR activity and that of expression driven by cellular promoters
containing a binding site for DEK. In addition to cancer, DEK
regulation may also have implications for autoimmune disease, as
DEK binds to the Y-box of the HLA DQA-1 promoter, an enhancer element
for which allelic variation is associated with a propensity to develop
autoimmune disease.2
Although HIV-1 and HIV-2 show many similar characteristics, individuals
generally have a much longer asymptomatic period following infection
with HIV-2 than with HIV-1 (46). A recent study by Popper et
al. (17) suggested that differences in the pathogenicity of HIV-2
and HIV-1 may be due to the lower viral load of HIV-2, a consequence of
lower rates of HIV-2 replication in infected cells. Given this finding,
it is important to understand how HIV-2 transcription and replication
are regulated and to compare it with HIV-1. Our results implicate the
interaction of DEK and PP2A in the activation of HIV-2. In addition,
this study also suggests that PP2A may act synergistically with other
activating signals to boost transcription regulated by the HIV-2 LTR.
This finding was unexpected, in view of the observations of
several groups that OKA activates HIV-1 transcription, leading to the
assumption that PP2A suppresses HIV-1 transcription (25, 26, 27). Thus, to ascertain whether this represents one reason for the difference in
pathogenic potential or a shared mechanism of activation between the
two viruses, our future studies will directly compare the effect of PP2A on HIV-1 and HIV-2 transcription and replication.
We thank Marc Mumby for the gift of pCMV5
PP2Ac vector, Gerard Grosveld for antibodies to DEK, and David Pallas
for the PP2Ac mutant expression vectors.
*
This work was supported by Grant 36685 from NIAID, National
Institutes of Health (to D. M. M.) and an American Cancer Society grant.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.
¶
§ Supported by a Rackham merit fellowship from
the University of Michigan.
**
To whom correspondence should be addressed: Dept. of Internal
Medicine, Division of Infectious Diseases, 5220 MSRB3, 1150 W. Medical
Center Dr., Ann Arbor, MI 48109-0640. Tel.: 734-936-3844; Fax:
734-764-0101; E-mail: dmarkov@umich.edu.
Published, JBC Papers in Press, April 24, 2001, DOI 10.1074/jbc.M006454200
2
B. Adams and D. Markovitz, manuscript in preparation.
The abbreviations used are:
HIV, human
immunodeficiency virus;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
pets, peri-Ets;
OKA, okadaic acid;
PP2A, protein phosphatase-2A;
PP2Ac, catalytic subunit of
PP2A;
PKC, protein kinase C;
LTR, long terminal repeat;
OKA, okadaic
acid;
PMA, phorbol 12-myristate 13-acetate;
PP1, protein phosphatase-1;
DTT, dithiothreitol;
EMSA, electrophoretic mobility shift assay;
PAP, potato acid phosphatase;
CAT, chloramphenicol acetyltransferase;
PDD, phorbol 12,13-didecanoate.
Protein Phosphatase 2A Activates the HIV-2 Promoter through
Enhancer Elements That Include the pets Site*
,
, and¶**
Cellular & Molecular Biology
Program, University of Michigan Medical Center, Ann Arbor, Michigan
48109-0640 and Therapeutic Systems Research Laboratories,
Ann Arbor, Michigan 48108
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B kinase, in regulation of the HIV-1 LTR have been well
documented, little is known about the actions of specific phosphatases
on LTR activity. A significant portion of the intracellular
serine/threonine-specific protein phosphatase activity of many cells is
due to phosphoprotein phosphatase-2A (PP2A) activity (19). The PP2A
holoenzyme is composed of three subunits denoted A (65-kDa scaffold
subunit), B (55-kDa regulatory subunit), and C (37-kDa catalytic
subunit) (19, 20). These subunits associate to form an AC dimer (the core enzyme) or an ABC trimer (holoenzyme), each of which has different
substrate specificities. Direct regulation of PP2A activity occurs in
the replication cycle of some DNA tumor viruses such as simian virus 40 (SV40) and polyoma virus, which both encode antigens, small-t and
middle-T, respectively, that form complexes with PP2A core enzyme (21).
These complexes, although not transforming on their own, facilitate or
enhance the cellular transformation processes caused by these viruses.
Recently, a report by Reudiger et al. (22) provided direct
evidence for the involvement of PP2A in HIV-1 LTR regulation. In these
experiments, increasing the ratio of PP2A core enzyme to holoenzyme by
using an N-terminal mutant of the A subunit of PP2A inhibited
Tat-stimulated HIV-1 transcription and virus production.
B nuclear
translocation and binding to its cognate sites (23-26) or via an
NF-B-independent mechanism involving the Sp1 response elements (26).
OKA-induced Sp1-mediated transcription is further enhanced by the
presence of Tat (26, 27). These studies with OKA suggest the
involvement of one or more phosphatases (PP2A or PP1) in regulating
transcriptional activation of the HIV-1 LTR.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol). The gel
was then dried and autoradiographed.
-DEK primary antibody detected by ECL after staining with a
horseradish peroxidase-conjugated secondary antibody. The DE-81 paper
was dried and autoradiographed. The blot and autoradiograph were
aligned, and the positions of the bands in each were compared.
556/+156) from the HIV-2 ROD strain
(31). 
pets (2), pets/PuB2 (2),107 (2), Sp1, and B
HIV-2 (1)-reporter plasmids have been described previously. All HIV-2
site-specific mutant reporter plasmids were generated by the
gap-heteroduplex method from wild-type HIV-2 enhancer (2). pCMV5 PP2Ac
(gift of Marc Mumby) constitutively expresses the catalytic domain of
PP2A under the control of the CMV promoter. All plasmids were prepared
using the Concert Maxiprep kit (Life Technologies, Inc.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (52K):
[in a new window]
Fig. 1.
Effect of phorbol ester treatment on binding
to the HIV-2 pets response element and DEK levels. A,
diagram of the HIV-2 enhancer. The sequences of the pets, PuB1, and
PuB2 elements along with the transcription factors known to bind these
elements are shown. B, TPA treatment of U937
monocytic cells results in a change in the electrophoretic mobility of
the pets complex in EMSA. U937 monocytic cells cultured at a density of
5 × 105/ml in RPMI 1640 + 10% fetal bovine serum
were treated with dimethyl sulfoxide (DMSO) carrier
(control) or 32 nM TPA for 24 h. Following treatment,
nuclear extract was prepared and EMSA performed using a radiolabeled
pets probe. The specificity of the EMSA bands was determined using 200 ng of competitor oligonucleotide in the binding reactions. The
competitor oligonucleotides used are as follows: lanes 1 and
4, no competitor ( ); lanes 2 and 5, mutated pets site (mut); lanes 3 and
6, cold probe (pets). The arrowheads
in panels B, C, and E denote the location of the
specific bands seen in the absence (open) or presence
(solid) of TPA, respectively. C, TPA and
4-PDD, but not the inactive phorbol ester 4-PDD, alter binding to
the pets site of HIV-2. Binding reactions and EMSA were carried out
using nuclear extract from U937 cells treated for 24 h with the
carrier dimethyl sulfoxide (lane 1), 50 nM
4-PDD (lane 2), 50 nM TPA (lane
3), or 50 nM 4-PDD (lane 4).
D, TPA and 4-PDD but not 4-PDD treatment of U937 cells
leads to a marked reduction of DEK levels. 40 µg of lysate from U937
cells treated with 50 nM 4-PDD, 50 nM TPA,
or 50 nM 4-PDD was used for DEK Western blot.
E, shift-Western blotting localizes DEK to the upper but not
lower TPA-induced pets band seen on EMSA. 30 µg of nuclear extract
from untreated (lanes 1 and 3) or 32 nM TPA-treated (lanes 2 and 4) U937
cells was used in binding reactions and EMSA. The protein-DNA complexes
were transferred from the native gel to membranes by electroblotting.
The protein was immobilized on nitrocellulose and a DEK Western blot
performed (lanes 3 and 4). pets probe was
transferred to DE-81 paper (lanes 1 and 2), which
was then dried and autoradiographed. Both membranes were aligned, and
the bands on each were compared.
-PDD, or 50 nM 4-PDD. Like TPA, 4-PDD is capable of activating
PKC, whereas 4-PDD (an isomer of 4-PDD) is devoid of phorbol
activity. 4-PDD but not 4-PDD elicited the same increase in the
mobility of the pets complex as TPA did in EMSA (Fig. 1C),
thus confirming that phorbol activity was necessary.
-PDD, but not 4-PDD, also led to a marked reduction in
DEK protein levels (Fig. 1D). The loss of DEK correlated
with the change in the electrophoretic mobility of the pets complex
seen in the gel shift assay (Fig. 1C). DEK protein levels
were also reduced significantly in the HL-60 and THP-1 monocytic cell
lines treated with TPA for at least 24 h (data not shown). The
inability to detect DEK protein by Western blot post-TPA treatment of
U937 cells implies that the change in electrophoretic mobility of the
pets band may, in fact, be due to a loss of DEK from the complex and
binding of a new cellular factor.
-DEK antiserum (Fig. 1E). An overlap can be
clearly seen between the DEK protein band on the Western blot (Fig.
1E, lane 3) and the band on the DE-81 paper
autoradiograph from the samples that received no treatment (Fig.
1E, lane 1). This finding was not unexpected
given that DEK has already been shown capable of binding to the pets
site (6). However, no overlap between the DEK protein bands and the
TPA-induced pets band was observed (Fig. 1E, lanes
2 and 4). This observation correlates with the loss of
DEK seen with TPA treatment (Fig. 1D) and suggests the
possibility of a second pets factor. Another interpretation of these
results is that there are alternatively spliced or post-translationally modified forms of DEK induced by TPA that the anti-DEK antiserum could not detect. To test this possibility, we performed Western blots
using total cell lysates from U937 or 293 cells transfected with a
FLAG-DEK expression vector. Both the FLAG monoclonal and DEK rabbit
polyclonal antibodies detected identical sets of bands on Western blots
(data not shown).
, , 
, 
, 
, and µ PKC isoforms (32) without affecting other intracellular kinases. The
, , , and isoforms of PKC have all been implicated in the
TPA-induced signaling cascade leading to the differentiation of
monocytes into macrophages (33-39). As is seen in Fig.
2, A and B,
pretreatment of U937 cells with 2 µM staurosporine or 10 µM Gö6983 for 30-45 min prior to the addition of
32 nM TPA is able to block the TPA-elicited changes in pets
site binding. Gö6983 completely blocked the TPA-induced changes,
and staurosporine clearly diminished but did not completely block the
TPA-induced changes in pets site binding (Fig. 2, A and
B). Staurosporine also appears capable of inducing the
complex seen with TPA treatment (Fig. 2A, lane
7), perhaps due in part to the pleiotropic inhibitory
actions of staurosporine, which block a variety of kinases in addition
to PKC. The more specific PKC inhibitor, Gö6983, does not induce
the faster migrating complex either alone or in combination with TPA
(Fig. 2B). Both staurosporine and Gö6983 were also
able to partially block the effects of TPA at lower concentrations in a
dose-dependent manner (data not shown). Additional specific
bands besides those indicated by arrowheads are observed in
Fig. 2, A and B. The identity of the proteins in
these bands is unknown. However, these bands are not always observed on
binding gels using the pets probe (compare Fig. 1, B and
E with Fig. 2, A and B) and may
represent intermediates in the transition from one complex to another.
Another possibility is that the observed bands may also result from the
partial degradation of one or more of the factors in the specific
complexes seen in the binding gels. Given the sporadic appearances of
these additional bands, we have chosen not to pursue identification of
the DNA-binding factors within them.
View larger version (60K):
[in a new window]
Fig. 2.
The PKC inhibitors staurosporine or
Gö6983 block the TPA-induced changes in pets site binding.
U937 cells were pretreated for 30 min with PKC inhibitor before 32 nM TPA was added for 24 h and EMSA performed.
Treatment conditions are as follows. A, lanes
1-3, no treatment; lanes 4-6, 32 nM TPA,
lane 7, 2 µM staurosporine; and lane
8, TPA + staurosporine. B, lane 1, no
treatment; lane 2, TPA; lane 3, 10 µM Gö6983; and lane 4, Gö6983 + TPA.
View larger version (25K):
[in a new window]
Fig. 3.
PAP treatment of nuclear extract from U937
cells induces a transition from the upper to lower pets complex.
A, potato acid phosphatase type II (1 unit) was used to
treat 10 µg of nuclear extract (NE) from control or
TPA-treated U937 cells for 20 min at 25 °C prior to EMSA with a pets
probe labeled using Klenow polymerase. Untreated U937 nuclear extract
was treated with: lane 1, heat-inactivated PAP
(INA); lane 2, PAP buffer; or lane 4,
PAP. 24-h TPA-treated U937 nuclear extract was treated with: lane
3, PAP buffer; or lane 5, PAP. A band can be seen at
the level of the TPA-induced complex (lane 5) with active
(lane 4) but not heat-inactivated (lane 1) PAP
treatment of U937 nuclear extract. B, nuclear extract from
untreated U937 cells was incubated with 1 unit of PAP Type II for 15, 30, or 60 min (lanes 2-4) at 25 °C prior to EMSA.
Binding reactions using nuclear extract from untreated (lane
1) or 32 nM TPA-treated U937 cells (lane 5)
were also included as controls. The arrowheads in
panels A and B indicate the positions of the
specific bands.
B
probe, because OKA-mediated inhibition of PP2A induces NF-B binding.
50 nM OKA was able to both induce NF-B binding (Fig.
4A, lane 18) and block the
effects of TPA (Fig. 4A, lane 12) on the mobility of the
pets complex. 5 nM OKA proved to be a sub-optimal dose,
unable to elicit activation of NF-B (Fig. 4A, lane 17) or
to block the effect of TPA (Fig. 4A, lane 9). Neither 5 nor
50 nM OKA (Fig. 4A, lanes 3 and 6)
alone had any effect on binding to the pets site when compared with
untreated controls.
View larger version (41K):
[in a new window]
Fig. 4.
OKA treatment of U937 cells blocks the
TPA-induced loss of DEK and changes pets site binding on EMSA.
A, EMSA was performed using nuclear extract from U937 cells
subjected to the following treatments for 24 h: lane 1,
no treatment; lane 2, 32 nM TPA; lanes
3-5, 5 nM OKA; lane 6-8, 50 nM OKA; lanes 9-11, 5 nM OKA + TPA;
lanes 12-14, 50 nM OKA + TPA. Competitive
binding reactions utilized 200 ng of cold probe (S) or an
oligonucleotide with a mutated pets site (M). Because OKA
has been shown to induce NF- B binding, the same nuclear extract was
used in binding reactions with a NF-B probe (lanes
15-20) as a positive control. The arrowheads indicate
the positions of specific bands with the pets probe. B,
okadaic acid treatment prevents the TPA-induced loss of DEK. A DEK
Western blot of the nuclear extract preparations used for EMSA is
shown. Equal amounts of nuclear extract (40 µg) were used in each
lane as confirmed by Ponceau S staining. The positions of full-length
DEK (50 kDa) and the DEK breakdown product (35 kDa) are indicated. OKA
at levels capable of inducing NF-B binding (50 nM) but
not at a sub-optimal dose (5 nM) is capable of blocking the
TPA-induced loss of DEK and the concomitant loss of the specific upper
band seen on EMSA with pets probe.
View larger version (34K):
[in a new window]
Fig. 5.
PP2A activates the HIV-2 promoter.
A, U937 cells were cotransfected with HIV-2-CAT reporter and
1, 5, or 10 µg of pCMV5 PP2Ac. Cells were treated with 32 nM TPA 24 h post-transfection or left untreated.
B, PP2A enzymatic assay was performed using whole cell
lysates from U937 cells transfected with 2.5, 5, or 10 µg of pCMV5
PP2Ac and treated with 50 nM TPA (black bars) or
left untreated (white bars) for at least 24 h.
C, U937 cells were cotransfected with HIV-2-CAT reporter and
5 µg of pCMV5 PP2Ac. 24 h post-transfection, the cells received
no treatment, 32 nM TPA only, or 50, 100, 200, or 400 nM OKA (30-45 min pretreatment) + TPA. For all
transfections, the total amount of transfected DNA was normalized using
pCMV5 vector. CAT assays were performed 18-24 h later. The CAT
activity values shown in panel C are normalized relative to
the CAT activity in the absence of TPA and pCMV5 PP2Ac.
B and Sp1 by OKA as
has been reported previously (23, 25-27). These data support and
extend the observations that PP2A phosphatase activity appears to be
important in the signaling cascade culminating at the HIV-2 LTR.
0.05) and a further decrease (p 0.02) could be observed with an additional mutation in the PuB2 site
(Fig. 6B). To address concerns that the deletions in the
enhancer region of the CAT reporters had a nonspecific adverse effect
on the promoters, we cotransfected U937 cells with a plasmid expressing
the Tat transactivating gene from HIV-2 (Tat-2) and
the mutant constructs. As seen in Fig. 6C, Tat-2 results in
a marked increase in CAT activity over base line for all of the
constructs. This demonstrates that the mutant reporters are still
transcriptionally active in U937 cells, consistent with previously
published reports from our laboratory utilizing these reporter
constructs (2, 4, 5).
View larger version (41K):
[in a new window]
Fig. 6.
PP2Ac Activation of the HIV-2 promoter is
mediated by the pets, PuB2, and Sp1 sites. A, diagram
of wild-type and mutant HIV-2 reporters used for transfections.
B, U937 cells were cotransfected with 5 µg of wild-type or
mutant HIV-2-CAT reporter and 0, 2.5, or 5 µg of pCMV5 PP2Ac. CAT
values have been normalized for the total amount of protein used in the
assay. The average values relative to a base-line activity for each
plasmid construct of 1 unit and the standard error of the mean from
three separate experiments are shown. The absolute CAT activity values
are 4.2, 28.4, 36.1, 2.2, 5.1, 8.4, 3, 2.7, and 2.9 starting from the
first bar. C, 5 µg of the HIV-2-CAT, pets
HIV-2-CAT, or pets/PuB2 HIV-2-CAT reporters and 2 µg of HIV-2
Tat-2 where indicated were transfected into U937 cells. 48 h
post-transfection, CAT assays were performed. The increased CAT
activity of pets HIV-2-CAT or pets/PuB2 HIV-2-CAT seen with
Tat-2 cotransfection indicates that the reporter plasmids are still
highly responsive. A representative experiment from three separate
transfections is shown. D, U937 cells were cotransfected
with 5 µg of wild-type, pets, Sp1, B, or 107 HIV-2-luc
reporters and 7.5 µg of pCMV5 PP2Ac (solid bars) or empty
pCMV5 (open bars). Cells were harvested, and the luciferase
assay was performed 48 h post-transfection, with the values
normalized relative to the amount of protein used. Luciferase values
are expressed as relative luciferase units (RLU) along with
the standard deviation and represent the average of four separate
experiments. The differences in luciferase activity in the absence or
presence of ectopic PP2Ac are statistically significant for the
wild-type (p 0.01), Sp1 (p 0.001), and B (p 0.001) HIV-2 reporters. Note
that for wild-type and B, a significant increase is seen with
PP2A, whereas the significant change for Sp1 is a decrease. The
inset shows the results with the B mutant on a scale that
more clearly demonstrates the differences.
B sites (23, 25-27). To test whether these sites play a role in the
response of the HIV-2 promoter to PP2A, we transfected U937 cells with
plasmids in which the reporter gene (luciferase) was under control of
the intact HIV-2 promoter or promoters in which the pets sequence, B
site, all upstream enhancer elements (107), or the 3' Sp1 site were
altered or deleted. Although mutation of the B site had no effect on
the response to PP2A, loss of the pets site, all upstream enhancer
elements, or the 3' Sp1 site diminished the PP2A-induced activation
(Fig. 6D). The Sp1 mutant actually showed decreased activity
in response to PP2A (Fig. 6D). Taken together, the data from
Fig. 6 show that the pets, PuB2, and Sp1 sites contribute to the
activation of the HIV-2 promoter by PP2A and that the B site is not
necessary for this effect.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (33K):
[in a new window]
Fig. 7.
Signaling overview. Summary diagram and
potential model of the signaling cascade leading to HIV-2 activation by
PP2A and TPA.
) phosphorylates PP2Ac in vitro and is dephosphorylated by PP2A (44). The
consequences of the PKC-mediated phosphorylation of PP2Ac are unknown.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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