Study of the roles of the Src family kinases p56
and p59
in T cell
receptor (TcR) (
)function has been hampered by a lack of
specific pharmacological inhibitors. Here, we describe and utilize a
novel, potent, and Src family-selective small molecule inhibitor to
further study the role of these kinases in T cell activation.
The lck gene, which encodes a lymphocyte-specific,
membrane-associated protein-tyrosine kinase of the nonreceptor
type(1, 2) , was first identified in retrovirally
induced murine T cell leukemias(2, 3, 4) .
Lck contains a unique N-terminal sequence (5) that directs its
specific interaction with the cytoplasmic domains of the CD4 and CD8
glycoproteins(6, 7, 8, 9, 10) .
This interaction is required for antigen-specific responses of several
different T cell hybridomas(11, 12) . Lck has also
been reported to associate with the IL-2R
-chain via a distinct
interaction(13) , although its role in IL-2R function remains
unclear. Several reports have demonstrated that Lck plays an important
role in both T cell maturation and activation. Loss of Lck expression
in the human Jurkat T cell line abrogates its response to anti-TcR
antibodies(14) , and inactivation of the lck gene or
overexpression of a dominant negative lck transgene in mice
leads to an arrest of thymocyte development at a stage prior to the
expression of CD4, CD8. and TcR(15, 16) .
Whereas
the role of Lck in T cell development and function is well established,
the role of Fyn is less well defined. Small amounts of Fyn are found
associated with the TcR complex following mild detergent extraction of
T lymphocytes(17) . Furthermore, activation of T cells through
the TcR results in increased enzymatic activity of Fyn(18) .
Additional evidence that Fyn is involved in lymphocyte activation comes
from experiments in transgenic animals. Twenty-fold overexpression of
FynT in transgenic thymocytes results in enhanced responsiveness to
anti-CD3 antibody as measured by the stimulation of tyrosine
phosphorylation in whole cells, Ca
accumulation, and
proliferation(19) . Furthermore, overexpression of a
catalytically inactive form of FynT in the thymocytes of transgenic
mice substantially inhibited TcR-mediated T cell
activation(19) . Finally, gene-knockout mice that lack either
p59
or
p59
demonstrate defects in TcR/CD3-
or alloantigen-mediated signaling(20, 21) , suggesting
that Fyn plays a role in TcR-mediated signaling in vivo.
Here, we show that unlike previously described protein-tyrosine
kinase inhibitors, PP1 inhibits Lck and FynT in vitro at
concentrations significantly lower than those required to inhibit
ZAP-70, JAK2, the EGF-R kinase, and protein kinase A. It inhibits whole
cell tyrosine phosphorylation and proliferation in T cells stimulated
with anti-CD3 and mitogens. Finally, it selectively inhibits IL-2 gene
expression over GM-CSF and IL-2R gene induction in human T cells. Thus,
this compound appears to dissect a component of TcR signaling not
distinguished by other inhibitors of TcR signaling such as FK506. This
compound is a powerful new tool to study the role of Src family
protein-tyrosine kinases in lymphocyte function.
EXPERIMENTAL PROCEDURES
Cells and Culture Conditions
Human peripheral
blood lymphocytes (PBL) were obtained from healthy donors by gradient
separation of whole blood using Histopaque (22) and were
cultured in RPMI 1640 medium containing 10% fetal calf serum.
Anti-CD3-induced proliferation experiments were carried out using 1
10
PBL cells/well in 96-well assay plates (Costar
Corp.) that were precoated with 20 µg/ml rabbit anti-mouse
antiserum (Jackson Laboratories, Bar Harbor, MA) in PBS for 4 h. PBL
were treated with varying concentrations of compound and either
anti-CD3 (50 ng/ml; Becton Dickinson) or PMA (10 ng/ml; Sigma) plus PHA
(2 µg/ml; Wellcome). Proliferation was assessed by the addition of
1 µCi/well [
H]thymidine (DuPont NEN) at 48 h,
followed by harvesting the cells at 72 h using a Skatron harvester.
Results are reported as an average IC
, determined from a
plot of the percent inhibition of proliferation from media control,
derived from separate experiments run in triplicate (see Table 2). Influenza-induced T cell proliferation (see Table 2, Ag-Sp (which represents specific antigen)) was assessed
by combining 1
10
PBL in triplicate with 160 µl
of RPMI 1640 medium containing 10% fetal calf serum with 20 µl of
either diluted test compound or media alone in a 96-well microtiter
plate (Costar Corp.). Antigen (influenza virus vaccine Fluzone,
Connaught Laboratories) was prepared by centrifuging and washing (three
times) 2 ml of vaccine through a Centricon-3 concentrator (Amicon,
Inc.) to remove preservative and diluting the remaining material to 40
ml (1:20). Twenty microliters of antigen was then added to each well,
and the plates were incubated for 72 h at 37 °C in 5%
CO
. [
H]Thymidine was then added (0.5
µCi/well), and the plates were incubated for an additional 18 h at
37 °C. Cells were harvested with a 96-well harvester (Tomtec), and
the amount of incorporated [
H]thymidine was
determined using a Pharmacia Biotech
-plate counter.
Concentrations that cause 50% inhibition of proliferation
(IC
) were determined from a plot of the percent inhibition
of proliferation from media control versus concentration of
test compound added. Results are presented as the mean IC
from repeated experiments (see Table 2). T cell
proliferation in the one-way mixed lymphocyte reaction was assessed by
combining in triplicate 5
10
fresh PBL, 5
10
irradiated (5000 rads) pooled stimulator PBL, and
diluted test compound in RPMI 1640 medium in each well of a 96-well
assay plate. After 18 h of incubation at 37 °C in 5%
CO
, 0.5 µCi of [
H]thymidine was
added to each well, and the cells were incubated for another 18 h. The
cells were then harvested using a Pharmacia Biotech
-plate system.
Percent inhibition was determined by the following equation: %
inhibition = 1 - (mean cpm of drug-treated cells/mean cpm
of control stimulated cells)
100. IC
values
represent the concentration of drug that caused 50% inhibition of the
control response. Results are presented as the mean IC
from repeated triplicate experiments (see Table 2). Purified
human T cells, used in the whole cell phosphotyrosine analyses, were
isolated by the T-Kwik method (One Lambda, Canoga Park, CA). T cell
purity, as determined by flow cytometric analysis, was generally
>90% CD3
T cells, with the major contaminant being
CD16
cells (natural killer cells). Jurkat T cells
(American Type Culture Collection) were maintained in RPMI 1640 medium,
10% fetal calf serum. All cells were maintained at 37 °C in a
humidified 5% CO
atmosphere.
Immune Complex Enzyme Assays
The enolase substrate
(Sigma) used for measuring Lck and FynT catalytic activity (see Fig. 2and Table 1) was prepared as described(23) .
The acid-treated enolase was diluted 1:20 with 1
PBS before
aliquoting 100 µl/well into a Nunc 96-well high protein binding
assay plate. Assay wells were then aspirated; blocked with 0.5% bovine
serum, 1
PBS for 1 h at 37 °C; and then washed five times
with 300 µl of 1
PBS/well. The source of Lck was either
LSTRA cells or Lck expressed in HeLa cells using a vaccinia expression
system(8, 24) . FynT (a gift of R. Perlmutter,
University of Washington, Seattle) was expressed in HeLa cells using
the vaccinia system(24, 25) . Cells (12.5
10
/ml) were lysed in lysis buffer (20 mM Tris, pH
8.0, 150 mM NaCl, 0.5% Nonidet P-40, and 23 trypsin inhibitory
units/ml aprotinin), and the lysates were clarified by centrifugation
at 14,000 cpm for 15 min at 4 °C in an Eppendorf tube. The Lck
antibody was produced by immunizing rabbits with a synthetic peptide
containing residues 41-54 of the N-terminal domain of Lck. The
anti-Fyn antibody was obtained from Upstate Biotechnology, Inc. The
clarified lysates were then incubated with the appropriate anti-kinase
antibody at 10 µg/ml for 2 h at 4 °C. Protein A-Sepharose beads
(Pharmacia Biotech Inc.; prepared as a 50% (w/v) suspension) were added
to the antibody/lysate mixture at 250 µl/ml and allowed to incubate
for 30 min at 4 °C. The beads were then washed twice in 1 ml of
lysis buffer and twice in 1 ml of kinase buffer (25 mM HEPES,
3 mM MnCl
, 5 mM MgCl
, and 100
µM sodium orthovanadate) and resuspended to 50% (w/v) in
kinase buffer. Twenty-five microliters of the bead suspension was added
to each well of the enolase-coated 96-well high protein binding plate
together with an appropriate concentration of compound and
[
-
P]ATP (DuPont NEN; 25 µl/well of a
200 µCi/ml solution in kinase buffer). After incubation for 20 min
at 20 °C, 60 µl of boiling 2
solubilization buffer (26) containing 10 mM ATP was added to the assay wells
to terminate the reactions. Thirty microliters of the samples was
removed from the wells, boiled for 5 min, and run on a 7.5%
SDS-polyacrylamide gel. The gels were subsequently dried and exposed to
Kodak X-AR film (see Fig. 2A). For quantitation, films
were scanned using a Molecular Dynamics laser scanner, and the optical
density of the major substrate band, enolase p46, was determined.
Concentrations of compound that caused 50% inhibition of enolase
phosphorylation (IC
) were determined from a plot of the
density versus concentration of compound (see Fig. 2B). In companion experiments for measuring the
activity of compounds against Lck (see Fig. 2C), the
assay plate was washed with two wash cycles on a Skatron harvester
using 50 mM EDTA, 1 mM ATP. Scintillation fluid (100
µl) was then added to the wells, and
P incorporation
was measured using a Pharmacia Biotech micro-
-counter.
Concentrations of compound that caused 50% inhibition of enzyme
activity (IC
) were determined from a plot of the percent
inhibition of enzyme activity versus concentration of
compound. Since there was good correlation between the gel and plate
assays, subsequent repeat experiments for both Lck and FynT were
performed using scintillation counting (see Table 1). EGF-R
activity was measured by immunoprecipitation of EGF-R from A-431 cells
obtained from the American Type Culture Collection. Cell lysates were
prepared by adding 4 ml of lysis buffer to a T-75 flask that contained
a confluent layer of cells. The lysates were clarified by
centrifugation as described above and then incubated with 10 µg/ml
anti-EGF-R (Upstate Biotechnology, Inc.) for 2 h at 4 °C. Protein
A-Sepharose beads were added to the antibody/lysate mixture at 250
µl/ml and allowed to incubate for 30 min at 4 °C. The beads
were then washed twice in 1.0 ml of lysis buffer and twice in 1.0 ml of
kinase buffer (as described above) and finally resuspended to 50% (w/v)
in kinase buffer. To each 1.5-ml assay tube was aliquoted 50 µl of
bead suspension, which was then spun for 15 s at 14,000 rpm in an
Eppendorf microcentrifuge, and the supernatant was discarded. To the
bead pellet was then added 5 µl of the appropriate compound
dilution, 5 µl of EGF (Upstate Biotechnology, Inc.) to a final
concentration of 100 pM, and 5 µl of a 33 µCi
[
-
P]ATP/ml solution in kinase buffer. After
incubation for 20 min at 20 °C, the beads were washed once with 1.0
ml of lysis buffer and once with 1.0 ml of 1
PBS. To the bead
pellet was added 60 µl of boiling 2
solubilization buffer (26) containing 10 mM ATP. Samples were run on 7.5%
SDS-polyacrylamide gels, which were subsequently dried and exposed
using BAS-III imaging plates. Labeled EGF-R protein bands were
visualized, and
P incorporation was quantitated using the
BAS-2000 BioImaging analyzer (Fuji Medical Systems USA, Stamford, CT).
Concentrations of compound that caused 50% inhibition of enzyme
activity (IC
) were determined from a plot of the percent
inhibition of enzyme activity by different concentrations of compound.
Murine JAK2 was produced in baculovirus and supplied as an immune
complex bound to protein A-Sepharose beads (Upstate Biotechnology,
Inc.). JAK2 beads (2.5 µl) were resuspended in 20 µl of kinase
buffer (10 mM HEPES, pH 7.4, 50 mM NaCl, 5
mM MgCl
, 5 mM MnCl
, 0.1
mM Na
VO
, 0.25 mCi/ml
[
-
P]ATP) for 10 min at room temperature.
The beads were then washed, and JAK2 autophosphorylation was measured
by eluting the labeled proteins into SDS-PAGE buffer and was analyzed
on a 7.5% polyacrylamide gel. Bands corresponding to JAK2 were
quantitated using the Fuji BAS-1000 phosphoimager. IC
values were determined as described above. Full-length ZAP-70
kinase (27, 28, 29) was produced using
baculovirus expression(30) . Lysates from Sf9 cells infected 48
h previously with a human ZAP-70 recombinant virus were prepared as
described above for Lck, and a 1:100 dilution was used in a soluble
kinase assay. Briefly, kinase activity was quantitated by measuring the
incorporation of
-
P into the substrate
p62(31) , using SDS-PAGE to resolve phosphorylated p62 and a
phosphoimager to quantitate radioactivity. ZAP-70-specific activity was
assessed by subtracting p62 phosphorylation obtained using Sf9 cell
lysates infected with nonrecombinant baculovirus. IC
values were determined as described above.
Figure 2:
Inhibition of Lck catalytic activity in vitro. A, Lck phosphorylation of the enolase
substrate was analyzed in the presence of increasing concentrations of
PP1 using SDS-PAGE as described under ``Experimental
Procedures.'' Protein standards (in kilodaltons) are shown on the
right. B, phosphorylation of the enolase substrate in A was quantitated using a Molecular Dynamics laser scanner, and the
results are plotted for determination of an IC
value as
described under ``Experimental Procedures.'' C,
duplicate reactions run in parallel with A were quantitated
using scintillation counting as described under ``Experimental
Procedures,'' and the results are plotted to determine an
IC
value.
Whole Cell Phosphotyrosine Measurements
Inhibition
of anti-CD3-stimulated tyrosine phosphorylation in purified human
peripheral blood T cells was measured as follows. All incubations were
carried out at 37 °C in an Eppendorf Thermomixer 5436 at a mixing
setting of 11. Cells (1
10
in 100 µl of RPMI
1640 medium) were incubated for 15 min with drug prior to a 6-min
incubation with 1 µg of anti-CD3/ml (anti-leu4, 100 µg/ml;
Becton Dickinson). The final volume of the reaction was 115 µl.
Reactions were terminated by the addition of 57.5 µl of 3
solubilization buffer (26) incubated at 100 °C prior to its
addition. Samples were mixed, boiled for 5 min, and stored at -70
°C. Western blots of these cell lysates, run on 10%
SDS-polyacrylamide gels, were probed with a polyclonal
anti-phosphotyrosine antibody, and immune complexes were detected with
I-labeled protein A (ICN) (26) . For
quantitation, films were scanned using a Molecular Dynamics laser
scanner, and the optical densities of the major substrate band, p70,
were quantitated in the presence of anti-CD3 (in the presence and
absence of drug). Percent inhibition was calculated as follows: (1
- (p70 optical density units in presence of drug/p70 units in
absence of drug))
100. IC
equals the concentration
of compound at which 50% inhibition was measured.
cDNA Probes
Plasmid DNAs were prepared as
described (32) . cDNA inserts were isolated by digestion with
the appropriate restriction enzymes, preparative agarose gel
electrophoresis, electroelution, and passage over a G-50 spin
column(33) . The cDNA probes were labeled using a DNA labeling
kit (Pharmacia Biotech Inc.). The human cDNAs for IL-2, GM-CSF,
IL-2R
, and glyceraldehyde-3-phosphate dehydrogenase were obtained
from the American Type Culture Collection.
Northern Blots
Total RNA was isolated from 1
10
fresh isolated human PBL using acid
guanidinium/phenol/chloroform(34) . Ten to fifteen micrograms
of total RNA/lane was electrophoresed on a 6% formaldehyde, 1.5%
agarose gel in buffer containing 5 mM NaHPO
, pH
4.45, 15 mM Na
HPO
, pH 8.85, and 1.5%
formaldehyde. The RNA was capillary-blotted to GeneScreen (DuPont NEN)
and UV-cross-linked using a UV Stratalinker 8000. The immobilized RNA
was hybridized to probe DNA (4
105 dpm/ml) in 50% deionized
formamide, 1% SDS, 1 M NaCl, and 10% dextran sulfate. The
blots were incubated, with agitation, overnight at 42 °C. The blots
were subsequently washed with constant agitation, twice in 2
SSC (1
SSC: 0.15 M NaCl, 0.015 M sodium
citrate) for 5 min at room temperature, twice for 30 min in 2
SSC containing 1% SDS at 60 °C, and then twice for 30 min each in
0.1
SSC at room temperature prior to autoradiography. For each
probe, a single blot was stripped and hybridized to cDNA probes
specific for each mRNA.
Promoter-Reporter Plasmid Transfections
The
promoter-luciferase reporter plasmids were constructed as described
previously(32) . Briefly, the promoters from the human IL-2
(positions -327 to +51) and IL-2R
(positions -479
to +109) genes were cloned into pUC13 upstream of the firefly
luciferase gene and SV40 polyadenylation signal. The human Jurkat T
cell line was transfected as described previously, and luminescence was
measured using a Dynatech ML1000 Luminometer. All experiments were run
in triplicate, and the data are presented as mean light units. Standard
deviations greater than 0.1 are shown.
RESULTS
Selective Inhibition of Src Family Kinases in
Vitro
The pyrazolopyrimidine PP1 (Fig. 1) was synthesized
as one of a series of compounds used for inhibition of p56
and p60
, based on a parent compound
first discovered in tyrosine kinase inhibitor screens. When PP1 was
first examined in vitro for its ability to inhibit tyrosine
phosphorylation of enolase by p56
, it was found to be a
potent inhibitor of this enzyme (Fig. 2). p56
isolated from LSTRA was incubated with enolase and various
concentrations of PP1 in a 96-well plate. Solubilization buffer was
then added, and the reactions were run out on an SDS-polyacrylamide
gel. As shown in Fig. 2A, PP1 inhibited phosphorylation
of the 46-kDa enolase protein in a dose-dependent manner. The 46-kDa
enolase band was quantitated using a Molecular Dynamics laser scanner,
and the IC
for inhibition of enolase was
5 nM (Fig. 2B). Duplicate reactions were run in
triplicate in the 96-well plate in the presence of various
concentrations of PP1 and quantitated using a Pharmacia Biotech
micro-
-plate reader (Fig. 2C). The IC
for inhibition of Lck in these duplicate reactions using the
plate reader was 20 nM. A repeat experiment comparing the gel
assay with the plate reader yielded IC
determinations of 8
and 4 nM, respectively (data not shown). Similar results were
obtained using a peptide substrate containing the ITAM sequence derived
from the human TcR
-chain (data not shown). Since the gel and
plate assays provided similar results and the plate reader was more
facile for quantitating enolase phosphorylation, subsequent repeat
experiments were performed using the plate reader to obtain average
IC
values for inhibition of Lck and FynT. In three
separate subsequent experiments, PP1 inhibited p56
activity with an average IC
of 5 nM (Table 1). Similar results were obtained with p56
expressed in HeLa cells using a vaccinia virus expression system
(data not shown)(8, 24) . The compound was also a
potent inhibitor of a second lymphocyte Src family kinase,
p59
(IC
= 6 nM),
expressed using the vaccinia system (Table 1). A closely related
pyrazolopyrimidine, PP2 (Fig. 1), was similarly effective in the
inhibition of Lck and FynT (Table 1). In further selectivity
tests using other Src family protein-tyrosine kinases, PP1 also
inhibited Src (170 nM) and Hck (20 nM), while PP2
demonstrated potent inhibition of Hck (5 nM) (data not shown).
In contrast, PP1 and PP2 were both 50-100-fold less active in the
inhibition of A-431 epidermal growth factor receptor
autophosphorylation (IC
= 0.25 and 0.48
µM, respectively). Further specificity for inhibition of
Lck and FynT was demonstrated when it was found that PP1 and PP2 were
essentially inactive for inhibition of ZAP-70 and JAK2 (Table 1)
and protein kinase A (data not shown). Since the activity of the ZAP-70
enzyme may be enhanced following phosphorylation at residue 493 by
Lck(35) , we also examined whether inhibition of ZAP-70 by PP1
was altered when coexpressed in insect cells together with Lck as
described previously (35) . Although coexpression of ZAP-70
with the catalytic domain of Lck consistently led to a 3-4-fold
elevation in the specific activity of ZAP-70 for the p62 substrate, PP1
was still unable to inhibit this enzyme up to concentrations of 100
µM (data not shown). For comparison purposes, we also
examined the activity of staurosporine and genistein, two previously
described tyrosine kinase inhibitors. The fermentation product,
staurosporine, has previously been demonstrated to be a potent but
nonselective protein kinase inhibitor(36, 37) . In the
experiments reported here (Table 1), staurosporine was found to
be a nanomolar inhibitor of p56
and
p59
as well as a low micromolar inhibitor of
the EGF-R kinase. However, unlike PP1 and PP2, it was also a potent
inhibitor of the ZAP-70 and JAK2 tyrosine kinases. For further
comparison, the naturally occurring isoflavone genistein (36) was tested for its ability to inhibit the four tyrosine
kinases. As expected, it was the least potent inhibitor (Table 1). Thus, relative to other reported tyrosine kinase
inhibitors, the novel compounds PP1 and PP2 demonstrated potent and
selective inhibition of the Src family kinases, such as p56
and p59
.
Figure 1:
Structures of
PP1
(4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine)
and PP2
(4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine).
Both compounds are related to the previously reported
pyrrolo[2,3-d]pyrimidines(50) .
Repression of Early Signaling Events in Human T
Cells
One of the earliest events in TcR/CD3 triggering of T
cells is the stimulation of the tyrosine phosphorylation of multiple
substrates(38) . Previous studies have demonstrated that
tyrosine kinase inhibitors are capable of inhibiting TcR-induced T cell
activation(39, 40) . Therefore, PP1 was tested for its
ability to inhibit anti-CD3-stimulated tyrosine phosphorylation in
purified human T cells (Fig. 3). Human T cells were left
untreated (lane 1) or were treated with 1 µg/ml anti-CD3
alone (lane 2) or with anti-CD3 after a 15-min preincubation
with 1 µM (lane 3), 10 µM (lane
4), or 100 µM (lane 5) PP1. Following
termination of the reactions, anti-phosphotyrosine Western blotting was
used to measure stimulation of tyrosine phosphorylation(26) . A
representative phosphotyrosine blot is shown in Fig. 3. Anti-CD3
induced the tyrosine phosphorylation of a number of proteins in human T
cells (compare lanes 1 and 2). These substrates
included proteins of approximately 100, 70, 50-60, and 35 kDa.
The phosphorylation of all substrates was significantly reduced by 1,
10, and 100 µM PP1 (Fig. 3, lanes
3-5, respectively), with 50% inhibition occurring at
0.5 µM PP1 as measured by quantitating the p70 band
from two separate experiments (data not shown). PP2 was equipotent with
PP1 for inhibition of anti-CD3-induced tyrosine phosphorylation (data
not shown). Thus, treatment of human T cells with this potent Lck and
FynT inhibitor blocks the increase in whole cell tyrosine
phosphorylation observed following treatment with anti-CD3. These
results are consistent with participation of these kinases in early T
cell signal transduction; however, they do not rule out a role for
other Src family kinases that may be present in T cells.
Figure 3:
PP1 is a potent inhibitor (IC
= 0.55 µM) of T cell tyrosine
phosphorylation. Human peripheral blood T cells were purified as
described under ``Experimental Procedures.'' Cells (1
10
) were incubated for 15 min at 37 °C in either the
absence (lanes 1 and 2) or presence of a 1 µM (lane 3), 10 µM (lane 4), or 100
µM (lane 5) concentration of the tyrosine kinase
inhibitor PP1 prior to the addition of 1 µg of anti-leu4/ml (lanes 2-5) or media (lane 1). Following an
additional 6-min incubation with anti-leu4 (CD3), the reactions were
terminated, and the samples were analyzed for tyrosine phosphorylation
by Western blotting with polyclonal anti-phosphotyrosine antibody (26) as described under ``Experimental Procedures.''
Protein standards (in kilodaltons) are shown on the
right.
Inhibition of TcR-induced T Cell Proliferation by
PP1
Since PP1 blocked anti-CD3-induced tyrosine phosphorylation,
we next examined whether it could inhibit the proliferation of human T
cells in response to different stimuli in repeated experiments using
different donors (Table 2). Human PBL were isolated using
Histopaque and plated in the presence of the appropriate compound and
stimulus. Proliferation was assessed using
[
H]thymidine incorporation. The results are
presented as an average of individual experiments performed using
different human PBL donors. An examination of the activity of PP1
yielded an average IC
of 0.5 µM when PBL were
treated with anti-CD3 in 10 different donors and 26 µM when proliferation was induced by PMA/IL-2 in seven donors (Table 2). Similar results were obtained using purified T cells
(data not shown). An examination of other T cell proliferative signals
revealed somewhat reduced potency for PP1 (Table 2). Fresh human
T cells derived from five donors stimulated in a one-way mixed
leukocyte reaction were also inhibited with an average IC
of 3.9 µM. T cells derived from healthy donors and
stimulated with influenza virus vaccine (Ag-Sp) were inhibited with an
average IC
of 5.2 µM (Table 2). A
similar profile was observed with PP2. Thus, PP1 and PP2 demonstrated
inhibition of tyrosine kinase-dependent TcR-induced T cell
proliferation. However, they were less effective inhibitors of
proliferative signals that by-pass the T cell receptor complex (e.g. PMA/IL-2). As a control, staurosporine potently
inhibited T cell proliferation in response to both alloantigen (MLR)
and specific antigen (Ag-Sp) (Table 2), in line with its reported
potent broad inhibitory properties(36) . Genistein, a
previously characterized tyrosine kinase
inhibitor(36, 37) , demonstrated less potent
inhibition of T cell proliferation using all stimuli examined (Table 2).
Selective Inhibition of IL-2 Gene Induction by
PP1
Since PP1 selectively depressed TcR-dependent proliferation
of T cells, we tested whether this compound would demonstrate
specificity for inhibition of lymphokine genes involved in T cell
proliferation. Human PBL were isolated from a normal donor and were
either left untreated (Fig. 4, lane 1) or treated with
PHA (2 µg/ml) and PMA (10 ng/ml) for 18 h in the absence (lane
2) or presence of either 1 µM (lane 3) or 5
µM (lane 4) PP1. FK506 was used at 0.1 µM as a control (Fig. 4, lane 5). IL-2 mRNA induction
was almost completely inhibited by both 1 and 5 µM PP1 (Fig. 4, lanes 3 and 4, respectively).
However, the GM-CSF, IL-2R, and glyceraldehyde-3-phosphate
dehydrogenase mRNA levels were not significantly affected by these
concentrations of PP1. Interestingly, IL-2R mRNA levels were slightly
enhanced in the presence of PP1. In contrast, FK506, a powerful
immunosuppressive that is an inhibitor of the
Ca
-dependent phosphatase calcineurin(41) ,
potently suppressed IL-2 and GM-CSF mRNA induction (Fig. 4, lane 5), as previously reported(42) . IL-2R mRNA
induction was also partially repressed by this concentration of FK506.
These results suggest that PP1 may selectively inhibit signaling events
required for the activation of the IL-2 gene and that the signaling
events leading to GM-CSF and IL-2R induction may be distinct.
Figure 4:
Northern analysis of the effects of PP1 on
gene expression in human PBL. Fresh human PBL were either left
untreated (lane 1) or treated for 18 h in RPMI 1640 medium
containing 10% fetal bovine serum with PHA (2 µg/ml) and PMA (10
ng/ml) in the absence (lane 2) or presence of 1 µM PP1 (lane 3), 5 µM PP1 (lane 4), or
0.1 µM FK506 (lane 5). Cells were harvested and
washed, and total RNA was extracted for Northern analysis as described
under ``Experimental Procedures.'' GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
To
further test this hypothesis, we examined whether PP1 could selectively
inhibit IL-2 over IL-2R gene expression at the level of transcription
using reporter plasmids containing either the IL-2 or IL-2R promoter
linked to the firefly luciferase gene (Fig. 5). Human Jurkat T
cell lines were transfected with these reporter constructs, and the
cells were stimulated with PHA (2 µg/ml) and PMA (10 ng/ml) for 18
h prior to assay for luciferase activity as described
previously(32) . As shown in Fig. 5A, both the
IL-2 and IL-2R promoter constructs were induced to express luciferase
in response to PHA + PMA (compare uninduced (U) and
stimulated (S) bars for each reporter construct). PP1
demonstrated dose-dependent inhibition of the IL-2 reporter construct (Fig. 5B, squares) with an IC
of
1 µM. In contrast, the IL-2R reporter was not
inhibited by PP1 up to concentrations of 35 µM (Fig. 5B, circles). These results are
consistent with the Northern data and suggest that compound PP1 is
capable of selectively inhibiting signaling events required for IL-2
gene induction.
Figure 5:
Inhibition of IL-2 and IL-2R reporter
activity by PP1. A, Jurkat T cells were transfected with
either IL-2 or IL-2R promoter-luciferase constructs and then cultured
in RPMI 1640 medium containing 10% fetal bovine serum for 18 h in the
absence (unstimulated (U)) or presence (stimulated (S)) of PHA (2 µg/ml) and PMA (10 ng/ml). Cells were then
harvested and examined for luciferase activity as described under
``Experimental Procedures.'' B, PP1 was added to
cells transfected with either the IL-2 (squares) or IL-2R (circles) reporter constructs for 18 h in the presence of PHA
+ PMA prior to harvesting the cells and testing for luciferase
activity as described under ``Experimental Procedures.'' All
values represent the average of three test wells, with standard
deviations greater than 0.1 light units shown. The data are
representative of numerous repeat
experiments.
DISCUSSION
In this study, we have disclosed the structure and activity
of a novel tyrosine kinase inhibitor (PP1) that potently inhibits Lck
and FynT, anti-CD3-induced protein tyrosine phosphorylation, and
subsequent IL-2 gene activation in T lymphocytes. Moreover, this
compound shows selectivity for the Src family over other families of
tyrosine kinases including ZAP-70, JAK2, and the EGF-R kinase.
Several properties distinguish PP1 from previously identified
tyrosine kinase inhibitors. Its combination of potency (nanomolar in vitro and low micromolar in intact cells) and selectivity
for members of the Src family of tyrosine kinases is unprecedented.
Earlier studies have reported the ability of other tyrosine kinase
inhibitors to inhibit signal transduction in T lymphocytes. Prolonged
treatment (12-16 h) with 1 µM herbimycin A, a
benzoquinoid ansamycin antibiotic, was shown to inhibit TcR-mediated
tyrosine phosphorylation, inositol phospholipid hydrolysis, and calcium
elevation(40) . The lengthy incubation required to observe
inhibition and the covalent interaction of herbimycin A with sulfhydryl
groups on protein-tyrosine kinases (37) have limited its use.
The isoflavone genistein (43) has been also shown to block T
cell receptor signal transduction and early activation
events(44) . In those studies, incubation with 40 µM genistein prior to receptor cross-linking with anti-CD3
significantly inhibited Lck activity and TcR-
phosphorylation as
well as activation of the IL-2 gene, but was unable to inhibit IL-2
secretion induced by Ca
ionophore and PMA, agents
that by-pass the TcR. The high concentrations of genistein required to
observe inhibition in vitro and in whole cells and its lack of
specificity in protein kinase inhibition (36) contrast with the
potency and specificity we have observed with PP1. A styryl-based
protein-tyrosine kinase inhibitor, 67B-83-A(45) , was
previously shown to be selective for Lck as compared with EGF-R
(>100-fold), and it was also selective for inhibition of Lck when
compared with other Src family kinases (from >10-fold to
>200-fold). However, the compound had an IC
of 7
µM for inhibition of Lck in vitro, several orders
of magnitude higher than that which we have observed for PP1 using
similar protocols for kinase inhibition. Staurosporine, a member of the
indolecarbazole group of antibiotics, is also a potent inhibitor of Src
family tyrosine kinases in vitro. However, in our hands,
staurosporine demonstrated less specificity than PP1 and potently
inhibited Lck, FynT, ZAP-70, and JAK2 (Table 2). Other
investigators have similarly reported that staurosporine is a potent
inhibitor of Src family kinases (IC
= 90-200
nM) and was also effective for inhibition of nonreceptor
tyrosine and serine/threonine protein
kinases(36, 37) . A new quinolone derivative, WIN
61651, has also been described as an inhibitor of p56
(46) . This compound is significantly less potent than
PP1 for inhibition of Lck (18-24 µM) and appears to
be less selective since it demonstrates equal potency for the
platelet-derived growth factor receptor. Finally, the tyrphostins have
been extensively studied for their ability to inhibit various classes
of protein-tyrosine kinases(47, 48) . Tyrphostins
generally show selectivity for protein-tyrosine kinases over other
classes of kinases such as protein kinase A, protein kinase C, or
Ca
/calmodulin-dependent kinases. However, less
information is available concerning the activities of these compounds
on T cell kinases and function. Although studies of tyrosine kinase
inhibitors are confounded by the lack of standardized systems for
comparison of specificity and potency of protein kinase inhibition, our
data support the conclusion that PP1 is the most potent and selective
inhibitor of Src family tyrosine kinases such as Lck and FynT reported
to date.
The ability of PP1 to dissect signaling pathways was most
evident in two of our experiments. First, PP1 was effective at blocking
anti-CD3-induced T cell activation events, while it was less effective
at inhibiting the TcR-independent proliferation induced by PMA and
IL-2. Second, using Northern and reporter assays, we found that PP1 was
more effective at inhibiting IL-2 gene expression than either GM-CSF or
IL-2R gene induction. These results suggest that Lck and FynT may play
a specific role in IL-2 gene expression required for TcR-induced T cell
proliferation, but not in the induction of the GM-CSF or IL-2R genes.
In contrast, FK506 inhibited anti-CD3-induced expression of IL-2 and
GM-CSF mRNAs. Thus, the inhibitory effects of PP1 appear to be more
specific than those of FK506. FK506 forms a complex with FKBP, and the
FK506-FKBP complex competitively binds to and inhibits the
Ca
/calmodulin-dependent phosphatase
calcineurin(41) . This results in the inhibition of genes that
respond to TcR Ca
-dependent signaling such as IL-2
and GM-CSF(42) . Since PP1 inhibits Ca
flux
in anti-CD3-stimulated T cells (data not shown), it was anticipated
that FK506 and PP1 would show similar patterns for inhibition of
TcR-induced genes in T cells. However, FK506 and PP1 differentially
affected GM-CSF mRNA induction. This suggests that a
Ca
-independent signal may emanate from the TcR to
induce GM-CSF expression and that FK506, but not PP1, inhibits this
signal. The nature of this other signal and the mechanism by which
FK506 may inhibit this other pathway remain to be defined.
The
potency of PP1 in inhibiting intracellular activities (e.g. T
cell tyrosine phosphorylation and proliferation) was considerably
reduced relative to inhibition of isolated kinase activity. This
difference may be attributed in part to permeability of the compound
and its distribution within the cell, but may also be a reflection of
the kinetic characteristics of the inhibitor itself. Preliminary
studies with PP1 (data not shown) show complex kinetics for inhibition
of Lck; however, at certain concentrations of ATP, the compound appears
to be competitive with ATP. It therefore is possible that the
intracellular millimolar concentrations of ATP found within the cell (49) would act to decrease the potency of the inhibitor in
vivo. Studies are underway to carefully define the kinetics of Lck
inhibition by PP1 and related compounds. Although this property of PP1
and its related compounds appears to limit their usefulness as
pharmacological agents in the treatment of T cell-mediated disease,
their discovery represents a significant advance in the use of tyrosine
kinase inhibitors as tools to study the role of Lck and FynT in T cell
signaling.