lnterleukin-2
signal
phosphorylation Kregg
Abstract: natural
killer
of
Interleukin-2 (NK) cell
in human
and activation
J. Einspahr
Departments
transduction Robert
*Immunology
(IL-2) proliferation
of the tyrosine
T. Abraham,”
and
tPharmacology,
Christopher Mayo
potently stimulates and cytotoxic func-
tion. However, the molecular mechanisms by which IL-2 delivers activation signals from the IL-2 receptor to the NK cell interior are incompletely understood. Previous studies demonstrated that IL-2 stimulation induced the tyrosine phosphorylation of multiple proteins in NK cells, together with a prominent reduction in the electrophoretic mobility of p5fiIck The present studies mdicate that IL-2 induces a rapid ( 1 mm) increase in the catalytic activity of p5fiIck, as measured by increases in protein tyrosine kinase activity in vitro. Furthermore, in response to IL-2, p56’ itself undergoes complex alterations in serine and tyrosine phosphorylation. Cyanogen bromide cleavage maps indicate that IL-2 stimulates a pronounced increase in the phosphorylation of the NH2-terminal region of p56’ containing multiple known sites of serine phosphorylation. In addition, IL-2 induced a marked increase in the phosphorylation of a COOH-terminal peptide containing the regulatory Tyr-505 residue of p5filck. These results suggest that p56w’ serves as a substrate for both protein serine and tyrosine kinases activated during stimulation of this cell type with IL-2. Furthermore, these results indicate that the pleiotropic effects of IL-2 on NK cell physiology are initiated and regulated by a complex and multitiered interaction of different protein kinases including p56u(. J. Leukoc. Biol. 52: 565-571; 1992. Key Words: . protein tyrosine
interleu/cin-2
.
NK
cells
.
signal
transduction
kinases
INTRODUCTION Intenleukin-2 pleiotropic
(IL-2) effects on
is a T the growth,
cell-derived differentiation,
tion ofnatural killer (NK) cells and T cells. IL-2 receptor (Kd 10 pM) consists subunits: a 55-kd transmembnane protein 75-kd transmembrane protein (IL-2R/3)
cytokine and
with activa-
The high-affinity of at least two (IL-2Ra), and a [1-3]. When cx-
pressed independently, the IL-2Ra and IL-2R/3 subunits each bind IL-2 with low (Kd 10 nM) and intermediate (Kd 1 nM) affinities, respectively [1-3]. The IL-2Rj3 subunit is necessary and sufficient for transmission of activating signals, and therefore only the intermediateon high-affinity receptors for IL-2 can initiate cellular activation [1-3]. The intermediate-affinity receptor is constitutively cxpressed on the majority of circulating CDl6, CD56 NK cells [4, 5], and the high-affinity form is expressed on a subpopulation CD56 [6, IL-2 as well
of
NK
cells
that
are
phenotypically
CD16,
7]
stimulation as increases
Clinic
and
J. Dick
kinase and
Foundation,
multisite
Paul
Rochester,
p56ICk
J. Leibson*
Minnesota
production [8-10]. However, the molecular mechanisms undenlying the transduction of regulatory signals from the IL-2 receptor at the cell surface to the cell interior are not cornpletely defined. Previous studies have demonstrated that IL-2 induces a rapid increase in the tyrosine phosphorylation of multiple proteins in NK cells [11-14] and in T cells [13, 15-17], but the IL-2 receptor itself lacks intrinsic tynosine kinase activity [1-3]. It is therefore likely that other nonreceptor, membrane-associated tynosine kinases, such as the src-family tyrosine kinases, may associate with the IL-2 receptor and thereby relay information from the receptor to the cell interior. The lymphocyte-specific, src-family kinase p56” is a candidate molecule because this tyrosine kinase has been reported to associate physically with IL-2Rf3 [18] and be catalytically activated upon IL-2 stimulation in other lymphocyte populations [18, 19]. Furthermore, our group and others have shown that IL-2 stimulation leads to physical modification of a subpopulation of 561ck molecules to isoforms exhibiting decreased electrophoretic mobility on polyacrylamide gels [11, 19]. In spite of these initial observations, the chemical nature of the IL-2-induced 56k modification remains unclear. Without such information the relationship of these IL-2-induced alterations to the regulation of 56lck functional activity and to proposed models of src-family tyrosine kinase activation is unknown. Therefore, the purpose of the present study was to charactenize directly the specific biochemical and functional changes in p561 during IL-2 activation of NK cells. We now demonstrate directly that p56” is tyrosine and senine phosphorylated after IL-2 stimulation. Furthermore, we define the sites of phosphorylation to be in the COOHterminal region of the molecule containing Tyr-505 and in the NH2-terminal region of the 56k molecule, which was previously identified as the major site of senine phosphorylation. These phosphorylation events are paralleled by a rapid increase in the catalytic activity of p56”. Our results mdicate that 56lck is a substrate kinases following IL-2 receptor likely that complex interactions kinases,
IL-2-elicited
including 56kk regulatory
physiological NK cells.
difluoride; trophoresis; Clinic,
killer;
L,
TLC,
of Leukocyte
dodecyl
generation initiate
of the
activation
of
IL-2
IL-2, saline;
PVDF,
interleukin-2; polyvinylidene
sulfate-polyacrylamide
gel
dcc-
chromatography.
J. Leibson,
Paul 7,
with
chemiluminescence;
sodium
MN
to the ultimately
phosphate-buffered
thin-layer
requests: April
associated
enhanced
PBS,
SDS-PAGE,
Rochester,
Received
Journal
EC
natural
for both senine and tyrosine engagement. It is therefore among different protein
contribute signals that
responses
Abbreviations: NK,
Reprint
of NK cells induces cellular proliferation, in cytotoxic activity and lymphokine
NK cells:
Department
of Immunology,
Mayo
55905. 1992;
Biology
accepted
July
Volume
1, 1992.
52,
November
1992
565
MATERIALS
AND
Chemicals
and
METHODS
fetal bovine serum, 2 mM L-glutamine, and 20 g/rnl gentamicin. Cells were then resuspended at a concentration of 2 x 10’ cells/ml and incubated for 3 h at 37#{176}Cwith 0.5 mCi/mi [32P]orthophosphate. Cell samples (1 ml) were then stimulated as indicated, the reaction was terminated, and cell extracts were prepared as previously described [11, 12]. For immunoprecipitation, antip56tk3964 antibody was bound to protein A-Sepharose in lysis buffer [11, 12] containing 0.1% bovine serum albumin. Prior to immunoprecipitation, cell extracts were precleared with protein A-Sepharose for 1 h at 4#{176}C.The extracts were then incubated for 3 h at 4#{176}C with antip56k394 antibodies bound to protein A-Sepharose. After extensive washing with lysis buffer, bound protein-antibody complexes were eluted from the protein A-Sepharose beads by heating in 2 x sodium dodecyl sulfate-polyacrylarnide gel electrophonesis (SDS-PAGE) sample buffer at 100#{176}Cfor 8 mm. Eluted proteins were separated by discontinuous SDS-PAGE and electrophoretically transferred to polyvinylidene difluonide (PVDF) membranes as previously described [11]. Radiolabeled proteins were
cytokines
Human Hoffman
recombinant IL-2 was generously provided by LaRoche, Nutley, NJ. Purchased reagents included ‘251-labeled protein A, [32P]orthophosphate, and [‘y-32P]ATP from ICN Radiochemicals, Irvine, CA.
Antibodies Fluonesceinand phycoerythnin-conjugated monoclonal antibodies were used for NK cell phenotyping. Antibodies specific for CD3, CD16, CD56, and HLA-DR were obtained from Becton-Dickinson Monoclonal Center (Mountain View, CA). Antibodies specific for CD3, CD4, CD8, CD2,
and
CD11b
(Rariton, specific munization
were NJ).
obtained
limpet hemocyanin of the munine tively, antiserum a
Isolation, lines
Ortho
Diagnostics
(anti-p5639-64)
protein tyrosine of rabbits
against
from
Antiserum
with
to
kinase pS6 a peptide
System
the
lymphocyte-
was prepared (conjugated
by imto keyhole
protein) corresponding to residues 39-64 amino acid sequence [20]. Alternato p56” (antip561TPE) was also generated
visualized by autoradiography. For phosphoamino acid analysis, the region of the membrane containing S6kk was excised, and the membrane was cut into 2 x 2 mm pieces. The membrane fragments were wetted with methanol and then washed with distilled water. The bound proteins were hydrolyzed in 5.7 N HC1 for 1 h at 100#{176}C [24]. Samples were lyophilized in a Speed Vac microcentnifuge (Savant Instruments, Hicksville, NY) and resuspended in 4 z1 of an aqueous solution (H20, ethanol, methanol, 10:5:1). Phosphoamino acid content of the reconstituted hydrolysates was analyzed by thin-layer chromatography (TLC) (silica gel 60A plates, 20 x 20 cm, Whatman Chemical Separation, Inc.) as previously described [25]. 32p labeled phosphoamino acids were visualized by autoradiography of the TLC plate.
56tck
fusion
56Ick.trE
passage,
protein
as described
and characterization
[21].
of clonal
NK cell
Cloned human CD16 NK cell lines were isolated and maintamed in culture as described previously [11]. Phenotypes of the clonal NK cell lines were monitored by flow cytofluonimetry [22]. These cells were uniformly negative for expression of the CD3 complex and all remaining surface markers were consistent with the previously reported phenotype of activated human NK cells [23]: CD16, CD56, CD11b, CD2, CD4, and HLA-DR.
NK cell stimulation
and immunoblot
analysis
Phosphopeptide
Prior to experimental stimulation, NK cells were resuspended at a concentration of 5 x 106 cells/ml in RPMI 1640 medium supplemented with 4% fetal bovine serum, 2 mM L-glutamine, and 20 g/ml gentamicin, and 1-ml aliquots were stimulated as indicated. Experiments were terminated and cell extracts were prepared as previously described [ii, 12]. Unless indicated otherwise, immunoblot analyses of 56k utilizing antip561TPE antiserum and 1251-labeled protein A for detection purposes were performed as described previously
[ii].
In
certain experiments, detection of antibodywas accomplished by first incubating the blots for 1 h in a blocking solution containing 10% nonfat dry milk, 0.05% Tween in phosphate-buffered saline (PBS). The blot was then incubated for 2-3 h with anti-p56” antiserum (diluted 1:1000) in blocking solution and subsequently washed six times with 0.1% Tween in PBS. After incubation for 1 h with horseradish penoxi-
In vitro kinase
dase-labeled
32p labeling, acid NK ing
566
immunoprecipitation,
and phosphoamino
analysis cells with
were prepared phosphate-free
Journal
for metabolic RPMI 1640
of Leukocyte
Biology
32
medium
labeling by containing
Volume
52,
wash4%
November
of p56
To determine the sites of S61ck phosphorylation induced by IL-2 stimulation, phosphopeptide mapping experiments were performed by cleavage of immunopnecipitated p56” with cyanogen bromide. For these studies, NK cells were metabolically labeled with [32P]orthophosphate (1 mCi/ml) and stimulated and 56k was immunoprecipitated from cellular lysates as described above. Immunoprecipitated proteins were separated by SDS-PAGE and transferred electrophoretically to nitrocellulose membranes, and p56’ bands were visualized by autoradiognaphy. The portions of the membrane containing 56k were excised, and the protein was subjected to cyanogen bromide cleavage [26). Peptides generated by cyanogen bromide cleavage of p56 ek were analyzed by electrophoresis on 15% SDS-polyacrylamide gels. Autoradiography was performed on the dried gel to visualize phosphopeptides.
bound p56/ck nitrocellulose
protein A (Amersham; 1:1000 in PBS with 10% dry milk and 40 xg/rnl gentamicin sulfate), the blot was washed extensively and labeled proteins were detected using the enhanced chemiluminescence (ECL)-Western blotting detection system (Amersharn).
analysis
assay of p56
activity
After stimulation of whole cells with IL-2, the catalytic activity of immunoprecipitated p56 was measured in vitro. For these experiments, NK cells were stimulated and lysed and immunoprecipitates of 561tk were prepared as described above. The immunoprecipitates were washed with assay buffer containing 50 mM 4-(2-hydroxyethyl)-1-pipenazineethanesulfonic acid (HEPES), 1 0 mM KC1, 5 mM MgCl2, 0.03 % t-octylphenoxypolyethoxyethanol (Triton X-100), 5 mM MnCl2, 10 g/ml leupeptin, S g/ml aprotinin, and 100 iM NaVO4. After the samples were washed, assay buffer (30
1992
jl) containing added to each
1 M sample.
at 25#{176}Cand The samples pernatant
then were
was
sample
each
eluted
8.5%
washed centrifuged
removed.
buffer,
The
ATP The
and 260 samples with
proteins
visualized
gels
by
addition
of
at
IL-2
and
15
on
radiolabeled
the
PMA ii
mm.
electrophoresis
of
U/mi)
(1
I
SDS-PAGE
65#{176}C for
by
autoradiography
was 5 mm
lysis buffer [11, 12]. for 30 s and the su-
heated
resolved
SDS-polyacrylamide
were
the
was
were
[y-32P]ATP incubated for
ice-cold at lO,000g
After
sample
Ci/ml were
proteins
dried
sdck
>
gels.
0’
2’
10’
60’
120’
30’
RESULTS IL-2 treatment of human activity of p561’ Previous
studies
of
bility
of
a
tyrosine
NK
of
(Fig.
1A
strate
IL-2
U/mi)
(10
PMA
of
induces
in
p5fiuCC
tyrosine
human
in the
NK
cells.
electrophoretic
molecules.
11
mo-
In
this
study,
)sdck
>
performed to determine whether the modification of p56 correlated with an alcatalytic activity of this src-family protein
the
Our
leads
tivity
the catalytic
were
kinase.
cells
IL-2
proteins
by a decrease
subpopulation
experiments IL-2-dependent teration in
that
multiple
is accompanied
stimulates
I
demonstrated
phosphorylation
This
NK cells
results
as
p56lck
and
B)
enolase
that
increase
assessed
and
by
lB).
The
IL-2
in the in
stimulation
tyrosine
vitro
of the amount
of
2’
ac-
subimmunoassessed by
was constant between samples as analyses for 5fiuck. Following IL-2 stimulation p56lck autophosphorylation increased 3.4-fold experiments) and enolase phosphorylation in-
0’
5dck
Fig.
1’
2. Time-
1
kDa
and
of
were
stimulated
IL-2
or
were
stopped
The
60’
proteins gels
to
the (100
were
with
the
on cells
membranes was
performed
and
dcc-
(5
x 106 human
indicated.
Reac-
containing
by were
the of
times
a buffer
proteins
of p56”6
IL-2
NK concentrations
for
analyzed
these
nitrocellulose
visualization
indicated
lysed
of
human
ng/ml)
were
and
120’
effects Cloned
with PMA
cells
lysate
SDS-polyacrylamide electrophoretically Detection and
p561(k.
with
and
cell
10’
concentration-dependent
mobility
recombinant X-l00.
{
2’
cells/sample) tions
5dck
30’
>
trophoretic
-69
120’
I
0’
A.
60’
(100 U/mi)
IL-2
activity
exogenous 56hk
10’
of
kinase
autokinase
phosphorylation
(Fig.
precipitated immunoblot for 2 mm, ( mean ofthree
indicate
to a rapid
0’
1%
electrophoresis
on
subsequently
probed
8.5%
transferred
with
with
Triton
the
antip56. ECL
method
of Amersham. -46
o,
B.
1’
creased
4.9-fold
the
clone
NK
activity
kDa
-69
5kk Enoiase
Fig.
1.
ment were
of human stimulated
Stimulation
Reactions
of
terminated,
munoprecipitated
Kinase enolase
polyacrylamide radioactive
kinase
these
lysates
[y32PJATP
Proteins gels. phosphate
were After were
(A)
[y-32PATP
separated drying visualized
the
by gel, by
following
IL-2
and
p56’
antip56k3S64
were and
then the
was
in buffer
exogenous
containing
im-
antibodies.
performed
electrophoresis proteins
treat-
(5 x 106 cells/sample) indicated in minutes.
prepared,
using 56hk
or
activity
NK cells the times
were
lysates
of immunoprecipitated
either
(B).
tyrosine
Cloned human U/mI IL-2 for cell
from
assays
containing
p56’
NK cells. with 100
were
44
>
on
substrate
8.5%
SDS-
incorporated
autoradiography.
Einspahr
et al.
(mean and
persisted
of two
its
state
between
experiments).
Depending
of activation, 10
and
60
the
mm
elevated (data not
on kinase shown).
IL-2 stimulates the rapid phosphorylation of p561’ time- and concentration-dependent manner
in a
As
cata-
described
above,
IL-2
stimulation
leads
to
altered
of 561ck as measured in vitro. In addition, previously demonstrated that stimulation ofhuman NK with IL-2 leads to the appearance of isoforms of p561” lectively termed ps6kk+) exhibiting decreased electrophoretic mobility under denaturing conditions [11]. We therefore lytic
activity
ther
investigated
the
nature
of
these
IL-2-induced
we cells (colfur-
modifica-
tions of 561ck The results in Figure 2 show that the decreased electrophoretic mobility of p56’ is dependent on both the IL-2 concentration and the time of IL-2 stimulation. Although an IL-2 concentration of 1 U/ml fails to induce the appearance of more slowly migrating p56 isoforms, a concentration of 10 U/ml elicits this response (Fig. 2). Furthermore, an IL-2 concentration of 100 U/ml elicits
IL-2-induced
phosphorylation
and
activation
of
p56Ick
567
the maximal degree of mobility shift and also elicits a more prolonged appearance of these slower-migrating 561ck+ isoforms (Fig. 2). This graded increase in the extent of S61ck modification induced by increasing IL-2 concentrations parallels the concentration range (10 to 100 U/ml) oven which IL-2 stimulates proliferative and other biological effects in these human NK cell clones [23, 27]. We further investigated the nature of this IL-2-induced modification to determine whether the phosphorylation state
IL-2
IL.2
0’
60’
0’
60’
69 kDa -1
of this protein changes concurrently trophoretic migration of labeled NK cells rapidly induces
with the retarded elecIL-2 treatment of 32p the phosphorylation of 561k in a manner that precisely reflects the appearance of the slower-migrating p56” isoforms (Fig. 3). Isofonms of p56” are cleanly discernible within 2 mm and appear maximally within 60 mm ofstimulation (Figs. 2 and 3). The time course of 56kk modification and phosphonylation also resembles the time course of IL-2-induced tynosine phosphorylation of intracellular proteins in intact NK cells [11].
I
signal
phosphorylation Kregg
Abstract: natural
killer
of
Interleukin-2 (NK) cell
in human
and activation
J. Einspahr
Departments
transduction Robert
*Immunology
(IL-2) proliferation
of the tyrosine
T. Abraham,”
and
tPharmacology,
Christopher Mayo
potently stimulates and cytotoxic func-
tion. However, the molecular mechanisms by which IL-2 delivers activation signals from the IL-2 receptor to the NK cell interior are incompletely understood. Previous studies demonstrated that IL-2 stimulation induced the tyrosine phosphorylation of multiple proteins in NK cells, together with a prominent reduction in the electrophoretic mobility of p5fiIck The present studies mdicate that IL-2 induces a rapid ( 1 mm) increase in the catalytic activity of p5fiIck, as measured by increases in protein tyrosine kinase activity in vitro. Furthermore, in response to IL-2, p56’ itself undergoes complex alterations in serine and tyrosine phosphorylation. Cyanogen bromide cleavage maps indicate that IL-2 stimulates a pronounced increase in the phosphorylation of the NH2-terminal region of p56’ containing multiple known sites of serine phosphorylation. In addition, IL-2 induced a marked increase in the phosphorylation of a COOH-terminal peptide containing the regulatory Tyr-505 residue of p5filck. These results suggest that p56w’ serves as a substrate for both protein serine and tyrosine kinases activated during stimulation of this cell type with IL-2. Furthermore, these results indicate that the pleiotropic effects of IL-2 on NK cell physiology are initiated and regulated by a complex and multitiered interaction of different protein kinases including p56u(. J. Leukoc. Biol. 52: 565-571; 1992. Key Words: . protein tyrosine
interleu/cin-2
.
NK
cells
.
signal
transduction
kinases
INTRODUCTION Intenleukin-2 pleiotropic
(IL-2) effects on
is a T the growth,
cell-derived differentiation,
tion ofnatural killer (NK) cells and T cells. IL-2 receptor (Kd 10 pM) consists subunits: a 55-kd transmembnane protein 75-kd transmembrane protein (IL-2R/3)
cytokine and
with activa-
The high-affinity of at least two (IL-2Ra), and a [1-3]. When cx-
pressed independently, the IL-2Ra and IL-2R/3 subunits each bind IL-2 with low (Kd 10 nM) and intermediate (Kd 1 nM) affinities, respectively [1-3]. The IL-2Rj3 subunit is necessary and sufficient for transmission of activating signals, and therefore only the intermediateon high-affinity receptors for IL-2 can initiate cellular activation [1-3]. The intermediate-affinity receptor is constitutively cxpressed on the majority of circulating CDl6, CD56 NK cells [4, 5], and the high-affinity form is expressed on a subpopulation CD56 [6, IL-2 as well
of
NK
cells
that
are
phenotypically
CD16,
7]
stimulation as increases
Clinic
and
J. Dick
kinase and
Foundation,
multisite
Paul
Rochester,
p56ICk
J. Leibson*
Minnesota
production [8-10]. However, the molecular mechanisms undenlying the transduction of regulatory signals from the IL-2 receptor at the cell surface to the cell interior are not cornpletely defined. Previous studies have demonstrated that IL-2 induces a rapid increase in the tyrosine phosphorylation of multiple proteins in NK cells [11-14] and in T cells [13, 15-17], but the IL-2 receptor itself lacks intrinsic tynosine kinase activity [1-3]. It is therefore likely that other nonreceptor, membrane-associated tynosine kinases, such as the src-family tyrosine kinases, may associate with the IL-2 receptor and thereby relay information from the receptor to the cell interior. The lymphocyte-specific, src-family kinase p56” is a candidate molecule because this tyrosine kinase has been reported to associate physically with IL-2Rf3 [18] and be catalytically activated upon IL-2 stimulation in other lymphocyte populations [18, 19]. Furthermore, our group and others have shown that IL-2 stimulation leads to physical modification of a subpopulation of 561ck molecules to isoforms exhibiting decreased electrophoretic mobility on polyacrylamide gels [11, 19]. In spite of these initial observations, the chemical nature of the IL-2-induced 56k modification remains unclear. Without such information the relationship of these IL-2-induced alterations to the regulation of 56lck functional activity and to proposed models of src-family tyrosine kinase activation is unknown. Therefore, the purpose of the present study was to charactenize directly the specific biochemical and functional changes in p561 during IL-2 activation of NK cells. We now demonstrate directly that p56” is tyrosine and senine phosphorylated after IL-2 stimulation. Furthermore, we define the sites of phosphorylation to be in the COOHterminal region of the molecule containing Tyr-505 and in the NH2-terminal region of the 56k molecule, which was previously identified as the major site of senine phosphorylation. These phosphorylation events are paralleled by a rapid increase in the catalytic activity of p56”. Our results mdicate that 56lck is a substrate kinases following IL-2 receptor likely that complex interactions kinases,
IL-2-elicited
including 56kk regulatory
physiological NK cells.
difluoride; trophoresis; Clinic,
killer;
L,
TLC,
of Leukocyte
dodecyl
generation initiate
of the
activation
of
IL-2
IL-2, saline;
PVDF,
interleukin-2; polyvinylidene
sulfate-polyacrylamide
gel
dcc-
chromatography.
J. Leibson,
Paul 7,
with
chemiluminescence;
sodium
MN
to the ultimately
phosphate-buffered
thin-layer
requests: April
associated
enhanced
PBS,
SDS-PAGE,
Rochester,
Received
Journal
EC
natural
for both senine and tyrosine engagement. It is therefore among different protein
contribute signals that
responses
Abbreviations: NK,
Reprint
of NK cells induces cellular proliferation, in cytotoxic activity and lymphokine
NK cells:
Department
of Immunology,
Mayo
55905. 1992;
Biology
accepted
July
Volume
1, 1992.
52,
November
1992
565
MATERIALS
AND
Chemicals
and
METHODS
fetal bovine serum, 2 mM L-glutamine, and 20 g/rnl gentamicin. Cells were then resuspended at a concentration of 2 x 10’ cells/ml and incubated for 3 h at 37#{176}Cwith 0.5 mCi/mi [32P]orthophosphate. Cell samples (1 ml) were then stimulated as indicated, the reaction was terminated, and cell extracts were prepared as previously described [11, 12]. For immunoprecipitation, antip56tk3964 antibody was bound to protein A-Sepharose in lysis buffer [11, 12] containing 0.1% bovine serum albumin. Prior to immunoprecipitation, cell extracts were precleared with protein A-Sepharose for 1 h at 4#{176}C.The extracts were then incubated for 3 h at 4#{176}C with antip56k394 antibodies bound to protein A-Sepharose. After extensive washing with lysis buffer, bound protein-antibody complexes were eluted from the protein A-Sepharose beads by heating in 2 x sodium dodecyl sulfate-polyacrylarnide gel electrophonesis (SDS-PAGE) sample buffer at 100#{176}Cfor 8 mm. Eluted proteins were separated by discontinuous SDS-PAGE and electrophoretically transferred to polyvinylidene difluonide (PVDF) membranes as previously described [11]. Radiolabeled proteins were
cytokines
Human Hoffman
recombinant IL-2 was generously provided by LaRoche, Nutley, NJ. Purchased reagents included ‘251-labeled protein A, [32P]orthophosphate, and [‘y-32P]ATP from ICN Radiochemicals, Irvine, CA.
Antibodies Fluonesceinand phycoerythnin-conjugated monoclonal antibodies were used for NK cell phenotyping. Antibodies specific for CD3, CD16, CD56, and HLA-DR were obtained from Becton-Dickinson Monoclonal Center (Mountain View, CA). Antibodies specific for CD3, CD4, CD8, CD2,
and
CD11b
(Rariton, specific munization
were NJ).
obtained
limpet hemocyanin of the munine tively, antiserum a
Isolation, lines
Ortho
Diagnostics
(anti-p5639-64)
protein tyrosine of rabbits
against
from
Antiserum
with
to
kinase pS6 a peptide
System
the
lymphocyte-
was prepared (conjugated
by imto keyhole
protein) corresponding to residues 39-64 amino acid sequence [20]. Alternato p56” (antip561TPE) was also generated
visualized by autoradiography. For phosphoamino acid analysis, the region of the membrane containing S6kk was excised, and the membrane was cut into 2 x 2 mm pieces. The membrane fragments were wetted with methanol and then washed with distilled water. The bound proteins were hydrolyzed in 5.7 N HC1 for 1 h at 100#{176}C [24]. Samples were lyophilized in a Speed Vac microcentnifuge (Savant Instruments, Hicksville, NY) and resuspended in 4 z1 of an aqueous solution (H20, ethanol, methanol, 10:5:1). Phosphoamino acid content of the reconstituted hydrolysates was analyzed by thin-layer chromatography (TLC) (silica gel 60A plates, 20 x 20 cm, Whatman Chemical Separation, Inc.) as previously described [25]. 32p labeled phosphoamino acids were visualized by autoradiography of the TLC plate.
56tck
fusion
56Ick.trE
passage,
protein
as described
and characterization
[21].
of clonal
NK cell
Cloned human CD16 NK cell lines were isolated and maintamed in culture as described previously [11]. Phenotypes of the clonal NK cell lines were monitored by flow cytofluonimetry [22]. These cells were uniformly negative for expression of the CD3 complex and all remaining surface markers were consistent with the previously reported phenotype of activated human NK cells [23]: CD16, CD56, CD11b, CD2, CD4, and HLA-DR.
NK cell stimulation
and immunoblot
analysis
Phosphopeptide
Prior to experimental stimulation, NK cells were resuspended at a concentration of 5 x 106 cells/ml in RPMI 1640 medium supplemented with 4% fetal bovine serum, 2 mM L-glutamine, and 20 g/ml gentamicin, and 1-ml aliquots were stimulated as indicated. Experiments were terminated and cell extracts were prepared as previously described [ii, 12]. Unless indicated otherwise, immunoblot analyses of 56k utilizing antip561TPE antiserum and 1251-labeled protein A for detection purposes were performed as described previously
[ii].
In
certain experiments, detection of antibodywas accomplished by first incubating the blots for 1 h in a blocking solution containing 10% nonfat dry milk, 0.05% Tween in phosphate-buffered saline (PBS). The blot was then incubated for 2-3 h with anti-p56” antiserum (diluted 1:1000) in blocking solution and subsequently washed six times with 0.1% Tween in PBS. After incubation for 1 h with horseradish penoxi-
In vitro kinase
dase-labeled
32p labeling, acid NK ing
566
immunoprecipitation,
and phosphoamino
analysis cells with
were prepared phosphate-free
Journal
for metabolic RPMI 1640
of Leukocyte
Biology
32
medium
labeling by containing
Volume
52,
wash4%
November
of p56
To determine the sites of S61ck phosphorylation induced by IL-2 stimulation, phosphopeptide mapping experiments were performed by cleavage of immunopnecipitated p56” with cyanogen bromide. For these studies, NK cells were metabolically labeled with [32P]orthophosphate (1 mCi/ml) and stimulated and 56k was immunoprecipitated from cellular lysates as described above. Immunoprecipitated proteins were separated by SDS-PAGE and transferred electrophoretically to nitrocellulose membranes, and p56’ bands were visualized by autoradiognaphy. The portions of the membrane containing 56k were excised, and the protein was subjected to cyanogen bromide cleavage [26). Peptides generated by cyanogen bromide cleavage of p56 ek were analyzed by electrophoresis on 15% SDS-polyacrylamide gels. Autoradiography was performed on the dried gel to visualize phosphopeptides.
bound p56/ck nitrocellulose
protein A (Amersham; 1:1000 in PBS with 10% dry milk and 40 xg/rnl gentamicin sulfate), the blot was washed extensively and labeled proteins were detected using the enhanced chemiluminescence (ECL)-Western blotting detection system (Amersharn).
analysis
assay of p56
activity
After stimulation of whole cells with IL-2, the catalytic activity of immunoprecipitated p56 was measured in vitro. For these experiments, NK cells were stimulated and lysed and immunoprecipitates of 561tk were prepared as described above. The immunoprecipitates were washed with assay buffer containing 50 mM 4-(2-hydroxyethyl)-1-pipenazineethanesulfonic acid (HEPES), 1 0 mM KC1, 5 mM MgCl2, 0.03 % t-octylphenoxypolyethoxyethanol (Triton X-100), 5 mM MnCl2, 10 g/ml leupeptin, S g/ml aprotinin, and 100 iM NaVO4. After the samples were washed, assay buffer (30
1992
jl) containing added to each
1 M sample.
at 25#{176}Cand The samples pernatant
then were
was
sample
each
eluted
8.5%
washed centrifuged
removed.
buffer,
The
ATP The
and 260 samples with
proteins
visualized
gels
by
addition
of
at
IL-2
and
15
on
radiolabeled
the
PMA ii
mm.
electrophoresis
of
U/mi)
(1
I
SDS-PAGE
65#{176}C for
by
autoradiography
was 5 mm
lysis buffer [11, 12]. for 30 s and the su-
heated
resolved
SDS-polyacrylamide
were
the
was
were
[y-32P]ATP incubated for
ice-cold at lO,000g
After
sample
Ci/ml were
proteins
dried
sdck
>
gels.
0’
2’
10’
60’
120’
30’
RESULTS IL-2 treatment of human activity of p561’ Previous
studies
of
bility
of
a
tyrosine
NK
of
(Fig.
1A
strate
IL-2
U/mi)
(10
PMA
of
induces
in
p5fiuCC
tyrosine
human
in the
NK
cells.
electrophoretic
molecules.
11
mo-
In
this
study,
)sdck
>
performed to determine whether the modification of p56 correlated with an alcatalytic activity of this src-family protein
the
Our
leads
tivity
the catalytic
were
kinase.
cells
IL-2
proteins
by a decrease
subpopulation
experiments IL-2-dependent teration in
that
multiple
is accompanied
stimulates
I
demonstrated
phosphorylation
This
NK cells
results
as
p56lck
and
B)
enolase
that
increase
assessed
and
by
lB).
The
IL-2
in the in
stimulation
tyrosine
vitro
of the amount
of
2’
ac-
subimmunoassessed by
was constant between samples as analyses for 5fiuck. Following IL-2 stimulation p56lck autophosphorylation increased 3.4-fold experiments) and enolase phosphorylation in-
0’
5dck
Fig.
1’
2. Time-
1
kDa
and
of
were
stimulated
IL-2
or
were
stopped
The
60’
proteins gels
to
the (100
were
with
the
on cells
membranes was
performed
and
dcc-
(5
x 106 human
indicated.
Reac-
containing
by were
the of
times
a buffer
proteins
of p56”6
IL-2
NK concentrations
for
analyzed
these
nitrocellulose
visualization
indicated
lysed
of
human
ng/ml)
were
and
120’
effects Cloned
with PMA
cells
lysate
SDS-polyacrylamide electrophoretically Detection and
p561(k.
with
and
cell
10’
concentration-dependent
mobility
recombinant X-l00.
{
2’
cells/sample) tions
5dck
30’
>
trophoretic
-69
120’
I
0’
A.
60’
(100 U/mi)
IL-2
activity
exogenous 56hk
10’
of
kinase
autokinase
phosphorylation
(Fig.
precipitated immunoblot for 2 mm, ( mean ofthree
indicate
to a rapid
0’
1%
electrophoresis
on
subsequently
probed
8.5%
transferred
with
with
Triton
the
antip56. ECL
method
of Amersham. -46
o,
B.
1’
creased
4.9-fold
the
clone
NK
activity
kDa
-69
5kk Enoiase
Fig.
1.
ment were
of human stimulated
Stimulation
Reactions
of
terminated,
munoprecipitated
Kinase enolase
polyacrylamide radioactive
kinase
these
lysates
[y32PJATP
Proteins gels. phosphate
were After were
(A)
[y-32PATP
separated drying visualized
the
by gel, by
following
IL-2
and
p56’
antip56k3S64
were and
then the
was
in buffer
exogenous
containing
im-
antibodies.
performed
electrophoresis proteins
treat-
(5 x 106 cells/sample) indicated in minutes.
prepared,
using 56hk
or
activity
NK cells the times
were
lysates
of immunoprecipitated
either
(B).
tyrosine
Cloned human U/mI IL-2 for cell
from
assays
containing
p56’
NK cells. with 100
were
44
>
on
substrate
8.5%
SDS-
incorporated
autoradiography.
Einspahr
et al.
(mean and
persisted
of two
its
state
between
experiments).
Depending
of activation, 10
and
60
the
mm
elevated (data not
on kinase shown).
IL-2 stimulates the rapid phosphorylation of p561’ time- and concentration-dependent manner
in a
As
cata-
described
above,
IL-2
stimulation
leads
to
altered
of 561ck as measured in vitro. In addition, previously demonstrated that stimulation ofhuman NK with IL-2 leads to the appearance of isoforms of p561” lectively termed ps6kk+) exhibiting decreased electrophoretic mobility under denaturing conditions [11]. We therefore lytic
activity
ther
investigated
the
nature
of
these
IL-2-induced
we cells (colfur-
modifica-
tions of 561ck The results in Figure 2 show that the decreased electrophoretic mobility of p56’ is dependent on both the IL-2 concentration and the time of IL-2 stimulation. Although an IL-2 concentration of 1 U/ml fails to induce the appearance of more slowly migrating p56 isoforms, a concentration of 10 U/ml elicits this response (Fig. 2). Furthermore, an IL-2 concentration of 100 U/ml elicits
IL-2-induced
phosphorylation
and
activation
of
p56Ick
567
the maximal degree of mobility shift and also elicits a more prolonged appearance of these slower-migrating 561ck+ isoforms (Fig. 2). This graded increase in the extent of S61ck modification induced by increasing IL-2 concentrations parallels the concentration range (10 to 100 U/ml) oven which IL-2 stimulates proliferative and other biological effects in these human NK cell clones [23, 27]. We further investigated the nature of this IL-2-induced modification to determine whether the phosphorylation state
IL-2
IL.2
0’
60’
0’
60’
69 kDa -1
of this protein changes concurrently trophoretic migration of labeled NK cells rapidly induces
with the retarded elecIL-2 treatment of 32p the phosphorylation of 561k in a manner that precisely reflects the appearance of the slower-migrating p56” isoforms (Fig. 3). Isofonms of p56” are cleanly discernible within 2 mm and appear maximally within 60 mm ofstimulation (Figs. 2 and 3). The time course of 56kk modification and phosphonylation also resembles the time course of IL-2-induced tynosine phosphorylation of intracellular proteins in intact NK cells [11].
I
