(Received for publication, January 31, 1997)
From the Human immunodeficiency virus-1 (HIV-1) Tat, a
nuclear transcription factor, has been shown to function
extracellularly, implying that some Tat molecules escape nuclear import
and are secreted. This raises the question of what regulates, in
HIV-1-infected cells, the nuclear targeting of the polypeptide. Here we
show that cytosolic components activated by Ca2+ ions
are required to reveal the karyophilic properties of Tat: in
vitro translated Tat molecules do not associate with isolated nuclei unless preincubated with Ca2+. Moreover,
Ca2+ ions induce karyophilicity of chemically synthesized
Tat molecules only upon addition of cytosolic extracts. The
Ca2+-induced karyophilicity is prevented by inhibitors of
either tyrosine kinases (herbimycin A and genistein) or tyrosine
phosphatases (vanadate), suggesting the involvement of
Ca2+-dependent
phosphorylation/dephosphorylation events. In line with these
observations, the transcriptional activity of Tat is inhibited by
treatment with either vanadate or genistein. The same occurs with Tat
mutants lacking either one or both the two tyrosine residues (positions
26 and 47). Hence, Ca2+-dependent tyrosine
kinase(s) and phosphatase(s) act on accessory cellular protein(s),
which in turn are responsible of Tat karyophilicity.
In eukaryotic cells, gene expression is often regulated by
post-translational modifications such as phosphorylation (1) of the
relevant transcription factors or associated proteins. These
modifications may modulate the cellular localization of the
transcription factors, their DNA binding capacity, or their transactivating activity. Examples of transcription factors able to
undergo inducible nuclear import include steroid receptors (2), the
v-jun oncogenic counterpart of the AP-1 complex member c-jun (3), the yeast SW15 (4), and NF- In addition to this group of transcription factors, which have a
different intracellular localization depending on their functional state, many examples of proteins with dual intracellular targeting have
been reported (7). Among HIV1 products, the
matrix protein MA contains two subcellular localization signals with
competing effects: a myristoylated N terminus that targets the protein
to the plasma membrane and a nuclear localization sequence.
Myristoylation is the dominant signal. However, a small subset of MA
molecules undergo tyrosine phosphorylation, and this modification is
sufficient to reveal their karyophilic properties (8). The case of
human immunodeficiency virus-1 (HIV-1) Tat is still more complex,
as in addition to its transactivating function, necessary for viral
replication (9, 10), it can also be secreted by transfected or
virus-infected cells (11) and exert several extracellular
activities that interfere with growth regulation of different cells
(12, 13). While much effort has been spent on elucidating the molecular
details of transactivation of HIV-1, the mechanisms regulating nuclear
import versus secretion of Tat protein are largely
unknown.
In a previous study, we have shown that Tat molecules, synthesized
in vitro in wheat germ extracts, do not associate with nuclei when added to lysed cells (14); however, we noted that after a
period of incubation in culture medium, some Tat molecules became
capable of associating postlytically with the nuclear fraction, suggesting that some components of the culture medium might induce modification(s) on Tat molecules that reveal its karyophilicity.
Here we exploit in vitro and in vivo assays to
investigate the effects of Ca2+ and of cytosolic factors on
the nuclear targeting of Tat.
PMA, A23187, ionomycin, sodium orthovanadate, and
cyclosporin A (CsA) were obtained from Sigma (Milano, Italy);
herbimycin A, bisindolylmaleimide I, okadaic acid, genistein, KN-62,
staurosporin from Calbiochem (Milano, Italy); and alkaline phosphatase
from Boehringer (Mannheim, Germany).
Plasmids used in this study are: pcDNA1-Tat
(in which the Tat coding region from pSV2Tat (14) was subcloned in
pcDNA-1neo, Invitrogen, San Diego, CA), pJK2 (Ref. 15, coding for
The cDNAs were
transcribed in vitro with either SP6 or T7 polymerase
(Promega, Firenze, Italy) and the transcripts translated in a wheat
germ extract (Promega) or in a rabbit reticulocyte lysate (Amersham,
Milano, Italy) as specified by the supplier. [35S]Methionine or [35S]cysteine (both
>1000 Ci/mmol, Amersham) were employed for labeling of HoxD9 and Tat
or Tat mutants, respectively. Free radioactive amino acids were removed
by dialysis with Centricon-10 microconcentrators (Amicon, Beverly,
MA).
108 HeLa cells were
washed three times in PBS, once in 25 mM Hepes, 125 mM KC2H3O2, resuspended
in 2.5 volumes of the same buffer containing a mixture of protease
inhibitors, and homogenized in a glass potter. Homogenate was spun at
800 × g for 10 min and the supernatant was collected
and spun again at 100,000 × g for 1 h. Aliquots
of supernatant were frozen in liquid nitrogen and stored at Aliquots of radiolabeled Tat, Tat
mutants, or HoxD9 proteins synthesized in translation mix (normalized
so as to contain 5 × 104 trichloroacetic
acid-precipitable counts/min) or 500 ng of chemically synthesized Tat
or 200 ng of biotinylated chemically synthesized Tat (in which a biotin
was added at the N terminus) (both provided by G. Fassina, Tecnogen,
Piana di Monteverna (CE), Italy) were incubated for various periods of
time, at different temperatures (from 4 to 37 °C) in 70 µl of
buffer containing 0.1 M NaCl, 40 mM
NaHCO3, 1 mM NaH2PO4, 1 mM MgSO4, 5 mM KCl supplemented
with different ions or drugs as indicated in the figure legends. When indicated, aliquots of wheat germ lysates or of cytosol prepared as
above were added at a final concentration of 1 mg/ml.
At the end of the incubation, the proteins were added to 1.5 × 106 HeLa cells lysed in 50 µl of PBS, 1% Triton X-100
containing 10 mM EGTA, 10 mM NaF, 1 mM N-ethylmaleimide and protease inhibitors phenylmethylsulfonyl fluoride aprotinin, leupeptin) and kept on ice for
20 min as described (14). Samples were spun at 800 × g
for 10 min, and the supernatant was collected as the cytoplasmic fraction. Nuclei were prepared as in Ref. 14. Briefly, the nuclear pellet was washed twice in the same buffer containing sucrose 0.4 M, loaded onto a cushion of PBS, 0.7 M sucrose
and spun 15 min at 800 × g. Nuclei were treated with
50 µg/ml of DNase for 10 min at 37 °C and boiled in Laemmli sample
buffer (18).
Cytoplasmic and
nuclear fractions were then resolved on 12% SDS-PAGE and processed for
fluorography, when 35S-labeled proteins were used, or
transferred to nitrocellulose filters (Hybond C extra, Amersham) when
synthetic or biotinylated Tat was used (14). Blots were probed with
anti-Tat antibodies (Ref. 19, kind gift of B. Ensoli, Roma, Italy)
followed by peroxidase-conjugated secondary antiserum (Dako, Milano,
Italy) or with peroxidase-conjugated streptavidin (Amersham) and
developed by chemiluminescence (Amersham).
Different exposures of the same film were scanned at a Ultrascan
densitometer (Molecular Dynamics, Milano, Italy). The purity of the
nuclear and cytoplasmic fractions was determined by probing the blots
with anti-Erp 72 and anti-calnexin antibodies or by localizing histones
(14).
Jurkat and
COS7 cells were cultured in RPMI 1640 medium and Dulbecco's modified
Eagle's medium, respectively, supplemented with 10% fetal calf serum
and 1 mM L-glutamine (all from Life Technologies, Inc., Milano, Italy) in 5% CO2 in humidified
atmosphere. 2 × 106 COS7 cells in PBS/glucose (1 mg/ml) were transfected with 10 µg of CMV- 6 × 105 Jurkat cells were transfected by 30 µl of
Lipofectin (Life Technologies, Inc.) with 5 µg of
CMV- Transfected cells were then washed twice with saline solution and lysed
with 50 µl of lysis buffer containing 100 mM
2-mercaptoethanol, 9 mM MgCl2, 0.125% Nonidet
P-40, and 1 mg/ml ONPG. The reaction was stopped after 4-6 h with 50 µl of Na2C03, and As an in vitro assay to follow
karyophilicity, in vitro translated Tat is added to
detergent-lysed cells on ice, as such or after incubation at 37 °C
with Ca2+ or other divalent ions, and the presence of
radioactive Tat is evaluated in the cytoplasmic or in the nuclear
fraction. As shown in Fig. 1 (panel A and
inset), 35S-labeled Tat molecules, translated in
wheat germ lysates, accumulate in the cytoplasm but are absent from the
nuclear fraction. Upon treatment with CaCl2, Tat becomes
able to associate with nuclei. Nuclear association is not observed
following incubation with other divalent ions, such as
Zn2+, Mg2+, or Sr2+ (panel
A). Similar results are obtained with Tat translated in rabbit
reticulocyte lysates and using nuclei isolated after mechanic disruption rather than detergent lysis of the cell (not shown). As
expected, the effects of Ca2+ are prevented by the
simultaneous addition of EGTA (Fig. 1, panel B). However,
once Tat molecules have been rendered karyophilic by incubation with
Ca2+ ions at 37 °C, they remain so even if returned at
4 °C or treated with EGTA. The Ca2+-induced Tat
karyophilicity is dependent on time, temperature, and concentration
(Fig. 1, panels C-E): it is not observed at temperatures
lower than 18 °C (panel C), and it requires at least 15 min at 37 °C (panel D). Karyophilicity is still induced
with concentrations of Ca2+ as low as 30 µM
(panel E).
The effects of Ca2+ ions are specific for Tat. In the same
assay, the amount of the transcription factor HoxD9 that accumulates within the nuclear fraction is not modified by Ca2+
treatment (Fig. 1, panel A).
To
investigate whether Ca2+ ions act directly on Tat molecules
or rather through modifications of accessory cytosolic molecules, a
full-length chemically synthesized Tat, modified by the addition of a
biotin to the N-terminal (biotinyl-Tat), was incubated in the presence
or absence of Ca2+ ions as above and added to Triton
X-100-lysed cells. Its association with nuclear or cytoplasmic
fractions was investigated by Western blotting with
streptavidin-conjugated peroxidase. As shown in Fig. 2,
panels A and B, there is only a small increase in
the amount of biotinyl-Tat that associates with nuclei following
Ca2+ treatment. In the absence of Ca2+ the
addition of wheat germ extracts or HeLa cells cytosol does not promote
significantly nuclear association; however, when CaCl2 is
added to the mix of biotinyl-Tat and cell extracts, the karyophilicity of biotinyl-Tat is unveiled. EGTA completely prevents Tat nuclear association. Similar results were obtained when unmodified synthetic Tat was revealed by anti-Tat antibodies (data not show).
Taken together, these results suggest that cytosolic components play a
role in modifying the fate of Tat.
The possible involvement of
Ca2+-dependent phosphatases or kinases on
Ca2+-induced Tat karyophilicity was investigated by
studying the effects of drugs affecting either phosphorylation or
dephosphorylation. When added to in vitro translated Tat
together with Ca2+, staurosporin, KN-62 (Fig.
3, panel A) and bisindolylmaleimide (not
shown), which inhibit, with different specificity, serine/threonine kinases such as calmodulin, protein kinase A, and protein kinase C, do
not affect significantly the nuclear association of Tat. Only a slight
decrease is observed with the Ser-Thr phosphatase inhibitor okadaic
acid. However, no effect is detected by treating with alkaline
phosphatase either in the presence or absence of Ca2+. In
contrast, an almost complete abrogation of Tat nuclear targeting is
obtained with herbimycin A and genistein, two TK inhibitors, and with
sodium orthovanadate (Na3VO4), that inhibits
PTPases.
The nuclear association of Hox D9 is not influenced by
Na3VO4 or herbimycin A (Fig. 3, panel
B).
To investigate whether
Ca2+-mediated phosphorylation/dephosphorylation events play
a role also on the transcriptional activity of Tat, COS7 cells were
co-transfected with cDNAs encoding Tat (pcDNA-Tat) and its
reporter gene HIV-1-LTR
The two tyrosine residues present in Tat
(positions 26 and 47) were replaced so as to determine whether they are
the targets of the Ca2+-dependent
phosphorylation/dephosphorylation events leading to karyophilicity. The
three mutants obtained (Tat26F, Tat47F, or TatDF, in which both
tyrosine have been replaced) were analyzed for their acquisition of
karyophilicity in response to Ca2+ treatment as well as for
their transactivation activity.
Fig. 5, panel A, shows that the
karyophilicity of the three in vitro translated
35S-labeled Tat mutants is unveiled by Ca2+ and
inhibited by vanadate as in the case of wild type Tat. Moreover, when
co-transfected with the reporter gene pJK2, Tat mutants are able to
induce
In this paper we describe a novel mechanism of control of HIV-1
Tat activity, involving tyrosine phosphorylation/dephosphorylation of
cytosolic component(s), whose activation, dependent on
Ca2+, unveils the karyophilicity of Tat.
The kinetics and the irreversibility of the Ca2+-induced
karyophilicity (Fig. 1) suggest that Ca2+ ions do not act
directly on Tat itself; this notion is further supported by the
observation that chemically synthesized Tat does not associate to
nuclei after treatement with Ca2+ unless cytosolic factors
are added. Ca2+ may thus activate cellular protein(s) which
in turn confer karyophilicity to Tat. As drugs blocking either TK or
PTPase inhibit karyophilicity, Ca2+-dependent
tyrosine phosphorylation/dephosphorylation events seem to be involved
in controlling the nuclear association of Tat. The similar inhibitory
effects of drugs acting on targets with opposite functions (TK and
PTPase) is not surprising as kinases may be activated by
dephosphorylation and vice versa (1, 20). The nature of the TK(s) and
PTPase(s) involved, as well as the order of their activation, remain to
be investigated. Whatever their nature, the factors are highly
conserved through evolution, as demonstrated by the similar activity of
wheat germ and HeLa cell cytosolic extracts. Several examples of
Ca2+-induced serine/threonine protein kinases or
phosphatases involved in activation of transcription factors have been
provided (1, 5, 21); in contrast, while the importance of TKs and
PTPases in the early steps of signal transduction is well established, their direct involvement in regulating the activity of transcription factors is less defined. Tyrosine phosphorylation of the three subunits
of the interferon-stimulated gene factor 3 appears to be responsible
for their translocation into the nucleus and hence their activation
(22); the regulatory enzyme involved is TYK-2, a cytoplasmic,
Ca2+-independent TK induced by interferon- In the case of Tat, substitution of either one or both Tyr-26 or Tyr-47
results in mutant Tat molecules that undergo the same control of
nuclear association as wild type Tat. These observations confirm that
the Ca2+-activated TK/PTPases involved do not act directly
on Tat molecules. Rather, these data suggest that the target of the
phosphorylation is accessory protein(s), perhaps involved in
chaperoning Tat molecules to the nucleus. In agreement with this, the
functional activity is only slightly inhibited when Tyr-26 and/or
Tyr-47 are replaced: these mutants are able to transactivate a reporter
gene at approximately the same extent as wild type Tat. Three Tat
mutants bearing single substitution of Tyr-26 with Ala or Tyr-47 with
Ala or histidine have been reported previously (24, 25); in these
mutants substitution of either tyrosine did not abolish Tat
transcriptional activity, although Ala-26 had a weaker activity in HeLa
cells. The maintainance of the full activity by the Tat mutants
described here may be due to the more conservative substitution of Tyr
with Phe.
The TK/PTPases-mediated induction of Tat karyophilicity has a
functional correspondence in the finding that genistein and vanadate
ihibit HIV-1 LTR transactivation in transfected cells, of either
lymphoid or non-lymphoid origin. Genistein and vanadate also inhibit
transactivating activity of the three Tyr mutants, confirming that the
TK/PTPases involved in Tat nuclear targeting and transcriptional
activity act on accessory protein(s).
Our observations of a more effective transactivation of LTR- Altogether, these findings suggest that, depending on their activation
state, HIV-1-infected cells have the possibility of modifying the
intracellular fate and the transcriptional activity of Tat by
modulating its karyophilicity through activation of TK/PTPase, with
obvious influence on viral replication and on the development of
HIV-associated syndromes. Compounds that block the activity of these
enzymes may lead to novel strategies to slow the spread of the virus in
HIV-infected individuals.
We thank Drs. F. Blasi, B. Ensoli, M. Emerman, and V. Zappavigna for reagents and advice and Dr. G. Fassina
for generously supplying chemically synthesized Tat and biotinylated
Tat. We also thank Dr. C. E. Grossi for support and suggestions.
National Institute of Cancer Research,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
B, which is
imported to the nucleus following dissociation from the cytosolic
anchoring protein I
B-a (5, 6).
Drugs
-galactosidase under the control of HIV-LTR, obtained from M. Emerman, Seattle, WA), CMV-
-gal (Ref. 16, coding for
-galactosidase under the control of CMV promoter, obtained from F. Blasi, Milano, Italy), HoxD9 (Ref. 17, obtained from V. Zappavigna,
Milano, Italy). The three Tat mutants, Tat26F, Tat47F, and TatDF, were
generated by in vitro site-directed mutagenesis (QuickChange
site-directed mutagenesis kit, Stratagene), using oligonucleotide
primers that replace the two tyrosines into phenylalanines
(5
-CCAATTG-3
and the
complementary for Tat26F and TatDF);
5
GCATCTCTGGCAGGAAG3
and the complementary
for Tat 47F and Tat DF). The mutations were checked by DNA
sequencing.
100 °C
until used.
-gal or 10 µg of pJK2
alone or together with 10 µg of pcDNA1-Tat, pcDNA1-Tat26F,
pcDNA1-Tat47F, or pcDNA1-TatDF by electroporation (250 mV, 960 microfarads) in a Bio-Rad gene pulser apparatus (Bio-Rad, Milano,
Italy) and cultured in complete medium at 104/well in
96-well plates for 48 h. When indicated, herbimycin A (2 µM) or genistein (180 µM) was added for the
last 4 h of incubation, while orthovanadate (100 µM) was
added for the last 18 h.
-galactosidase or with 5 µg of pJK2 alone or together with 2 µg of pcDNA1-Tat in six-well plates. After 20 h, cells were
transferred to 96-well plate at 104/well and incubated for
additional 24 h in the absence or presence of 30 ng/ml PMA and 2 µg/ml ionomycin. Herbimycin A (2 µM) was added to the
cultures for the last 4 h, orthovanadate (100 µM) or
CsA (12 µM) was added for the last 18 h.
-galactosidase activity
was determined by reading A at 415 nm (16).
In Vitro Translated Tat Molecules Become Karyophilic upon Treatment
with Ca2+
Fig. 1.
Ca2+ specifically induces
association of in vitro translated Tat to isolated nuclei. Panel
A, 5 × 104 cpm of in vitro translated
Tat (tat, first to sixth columns) or
of in vitro translated HoxD9 (hoxD9,
seventh and eighth columns) were treated 1 h
at 37 °C without () or with 1 mM CaCl2
(Ca2+), MgCl2
(Mg2+), CaCl2 + MgCl2
(Ca2+ + Mg2+),
ZnCl2 (Zn2+), or SrCl2
(Sr2+) and then incubated 20 min on ice with
106 HeLa cells lysed in 1% Triton X-100. Nuclear and
cytosolic fractions were isolated and resolved by SDS-PAGE. The
percentage of nuclear Tat was calculated from densitometric analyses of
autoradiograms. Means of three different experiments and standard
deviations are shown. The inset shows a representative
fluorogram. N, nuclear fraction; C, cytosolic
fraction. Panel B, 5 × 104 cpm of in
vitro translated Tat was incubated 60 min at 37 °C without (
)
or with 1 mM CaCl2
(Ca2+) or 1 mM CaCl2 + 10 mM EGTA (EGTA + Ca2+) or 1 mM CaCl2 for 60 min followed by 10 mM EGTA for other 60 min (Ca2+
1h+EGTA) or 1 mM CaCl2
followed by 60 min at 4 °C. The rate of Tat nuclear association was
determined as above. Panels C and D, 5 × 104 cpm of in vitro translated Tat were
incubated with 1 mM CaCl2 or for 1 h at
different temperatures (panel C) or for different periods of
time at 37 °C (panel D) and the percentage of Tat nuclear association was determined. Panel E, in vitro translated Tat
was treated as above with different concentrations of CaCl2
and nuclear association was determined.
[View Larger Version of this Image (27K GIF file)]
Fig. 2.
Cytosolic extracts are required to reveal the
karyophilicity of Tat. Panel A, 200 ng of chemically
synthesized Tat biotinylated at the N terminus was incubated with or
without Ca2+ and/or cytosolic extracts, either from HeLa
cells (lanes 6-8) or from wheat germ (WG, lanes
4-5), as indicated. After 60 min at 37 °C, samples were added
to 106 HeLa cells lysed in Triton X-100 on ice. Nuclear
pellets and cytosolic soluble material were isolated, resolved by
SDS-PAGE, blotted, and revealed by streptavidin-conjugated peroxidase.
Panel B, the percentage of nuclear Tat was calculated from
densitometric analyses of autoradiograms. Means of three different
experiments and standard deviations are shown.
[View Larger Version of this Image (28K GIF file)]
Fig. 3.
The Ca2+-dependent
Tat association to nuclei is inhibited by orthovanadate and herbimycin
A. 5 × 104 cpm of in vitro translated
Tat (panel A) or of Hox D9 (panel B) were
incubated for 1 h at 37 °C without () or with 2 mM CaCl2 (Ca2+), in the absence or
presence of staurosporin (stauro, 1 µM), KN-62
(50 mM), herbimycin A (Herb A, 100 µM), orthovanadate
(Na3VO4, 50 mM), okadaic acid (Okad. ac, 1 µM), or 2 units of alkaline phosphatase (CIP).
At the end of incubation with the different drugs, Tat was added on ice
to 106 HeLa cells lysed in 1% Triton X-100 and incubated
for 20 min. The percentage of Tat nuclear association was determined as
above.
[View Larger Version of this Image (20K GIF file)]
-galactosidase (pJK2), and the levels of
transactivation were evaluated after culture in the absence or presence
of Na3VO4 or genistein. Both drugs inhibit the
transactivation of the reporter gene (Fig. 4,
panel A). As a control, neither drug affects the expression
of
-galactosidase under the control of a CMV promoter in the cells
(Fig. 4, panel A). Fig. 4, panel B, shows that in
a T cell line (Jurkat) co-transfected with Tat and pJK2, activation by
PMA/ionomycin results in an increase in LTR transactivation.
Na3VO4 and genistein inhibit transactivation both in resting and in activated cells, while the immunosuppressant CsA
has an inhibitory effect on activated cells only (panel B). As a control, panel C shows that
Na3VO4, genistein, or CsA does not alter the
expression of CMV-
-gal in resting or PMA/ionomycin-activated Jurkat
cells. No detectable
-galactosidase activity was observed in resting
or activated Jurkat cells transfected with the reporter gene pJK2 alone
(not shown).
Fig. 4.
The transactivating activity of Tat is
increased in stimulated Jurkat cells and is inhibited by
Na3VO4 and herbimycin A. Panel A,
COS cells transfected with CMV -gal (open bars) or
co-transfected with pJK2 and pcDNA Tat (closed bars)
were cultured for 48 h in the absence (
) or presence of
Na3VO4 for the last 18 h or genistein
(gen) for the last 4 h prior to lysis in the presence
of ONPG.
-Galactosidase activity was determined by colorimetric assays (16). Panel B, Jurkat cells co-transfected with pJK2 and pcDNA Tat, resting (closed bars), or activated with
PMA/ionomycin (open bars), were cultured for 48 h in
the absence (
) or presence of Na3VO4 for the
last 18 h or genistein (gen) or CsA for the last 4 h and
-gal activity was determined as above. Panel C, Jurkat cells transfected with CMV
-gal, resting (closed
bars) or activated with PMA/ionomycin (open bars) were
cultured for 48 h with or without Na3VO4
for the last 18 h or genistein (gen) or CsA for the
last 4 h. The
-galactosidase activity was determined as
above.
[View Larger Version of this Image (24K GIF file)]
-galactosidase expression at approximately the same extent as
wild type Tat (Fig. 5, panel B) and are similarly inhibited
by Na3VO4 and genistein.
Fig. 5.
Tyr residues are dispensable for Tat nuclear
association and transcriptional activity. Panel A, in vitro
translated Tat mutants in which Tyr-26, Tyr-47, or both were
substituted with phenylalanine residues (26F, 47F, and
DF, respectively) were treated with Ca2+ in the
absence (lane 2) or presence (lane 3) of
Na3VO4 as in Fig. 3, and their nuclear
association was evaluated. Panel B, COS cells co-transfected
with pJK2 and the three Tat mutants were cultured in the absence
(closed bars) or presence of genistein (GEN, open
bars) or Na3VO4 (gray bars) as
in Fig. 4, and -galactosidase activity was determined.
[View Larger Version of this Image (24K GIF file)]
(23). Another
example of differential targeting modulated by tyrosine phosphorylation is HIV-1 MA protein (8).
galactosidase in stimulated rather than in resting Jurkat T cells are
in line with previous reports (26, 27) and support the hypothesis of a
role for intracellular [Ca2+] in regulating Tat activity.
The immunosuppressive drug CsA was shown to block the Tat-mediated
increase of IL-2 promoter activity in activated T cells (28).
Similarly, we show that CsA inhibits LTR transactivation in stimulated,
but not resting, T cells, suggesting that, in activated cells,
additional, CsA-sensitive, cellular factors act in concert with Tat
with resulting activation of HIV-1 gene expression.
*
This work was supported in part by grants from CNR (PF
ACRO), Associazione Italiana Ricerca sul Cancro (AIRC), and Ministero Sanità (special project AIDS).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: Laboratory of Cell Biology, Howard Hughes
Medical Inst., The Rockefeller University, New York, NY 10021.
Recipient of a fellowship from Fondazione S. Raffaele.
**
To whom correspondence should be addressed: Laboratory of Clinical
Pathology, National Institute of Cancer Research, Largo Rosanna Benzi,
10, 16132 Genova, Italy. Tel.: 39-10-5600204; Fax: 39-10-5600210;
E-mail: annarub{at}hp380.ist.unige.it.
1
The abbreviations used are: HIV, human
immunodeficiency virus; HIV-1, human immunodeficiency virus-1; CsA,
cyclosporin A; LTR, long terminal repeat; CMV, cytomegalovirus;
-gal,
-galactosidase; PBS, phosphate-buffered saline; PAGE,
polyacrylamide gel electrophoresis; ONPG,
o-nitrophenyl-
-D-galactopyranoside; TK,
tyrosine kinase; PTPase, protein tyrosine phosphatase; PMA,
phorbol 12-myristate 13-acetate.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.