J Biol Chem, Vol. 274, Issue 46, 33131-33142, November 12, 1999
A Catalytically Active Jak2 Is Required for the Angiotensin
II-dependent Activation of Fyn*
Peter P.
Sayeski
§,
M. Showkat
Ali¶,
Afshin
Safavi,
Michelle
Lyles,
Sung-Oh
Kim
,
Stuart J.
Frank, and
Kenneth E.
Bernstein**
From the Department of Pathology and Laboratory
Medicine, Emory University School of Medicine, Atlanta, Georgia 30322 and the Department of Medicine, Division of Endocrinology and
Metabolism, University of Alabama, Birmingham, Alabama 35294
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ABSTRACT |
Recent work with interleukins has shown a
convergence of tyrosine phosphorylation signal transduction cascades at
the level of the Janus and Src families of tyrosine kinases. Here we
demonstrate that activation of the seven-transmembrane
AT1 receptor by angiotensin II induces a physical
association between Jak2 and Fyn, in vivo. This association
requires the catalytic activity of Jak2 but not Fyn. Deletion studies
indicate that the region of Jak2 that binds Fyn is located between
amino acids 1 and 240. Studies of the Fyn SH2 and SH3 domains
demonstrate that the SH2 domain plays the primary role in Jak2/Fyn
association. Not surprisingly, this domain shows a marked preference
for tyrosine-phosphorylated Jak2. Surface plasmon resonance estimated
the dissociation equilibrium constant (Kd) of this
association to be 2.36 nM. Last, in vivo studies in vascular smooth muscle cells show that, in response to
angiotensin II, Jak2 activation is required for Fyn activation and
induction of the c-fos gene. The significance of these data is that Jak2, in addition to serving as a critical angiotensin II
activated signal transduction kinase, also functions as a docking protein and participates in the activation of Fyn by providing phosphotyrosine residues that bind the SH2 domain of Fyn.
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INTRODUCTION |
The Jak family of nonreceptor tyrosine kinases includes Jak1,
Jak2, Jak3, and Tyk2. Each protein is approximately 130 kDa and
contains seven conserved Jak homology domains (JH1 to JH7) (1, 2). The
Jak kinases induce gene regulation through the signal transducers and
activators of transcription. Unlike almost all other protein-tyrosine
kinases, members of the Jak family bear no
SH21 or SH3 domains. In
contrast to the Jaks, members of the Src family of protein-tyrosine
kinases are approximately 55-62 kDa in mass and do possess SH2 and SH3
domains. There are nine known members of the Src kinase family. While
the expression of most members is restricted to hematopoietic cells,
Fyn and Src are widely expressed by many cell types. There appears to
be some functional redundancy of these two proteins, since knockout
mice lacking either gene are born alive, while disruption of both the
fyn and src alleles results in embryonic
lethality (3-5). This redundancy is also seen in the activation of
similar signaling pathways by Fyn and Src (6).
Recent studies have demonstrated various levels of cross-talk between
Jak2 and other signaling pathways. For example, activation of gp130 by
the hematopoietic cytokine, interleukin-11, induces protein complex
formation between the Jak and Src family tyrosine kinases.
Specifically, treatment of 3T3-L1 cells with interleukin-11 leads to a
transient complex of Grb2, Jak2, and Fyn (7). Subsequent studies by
Yang et al. demonstrated an
interleukin-11-dependent association of Jak2 with other
signaling molecules including protein phosphatase 2A,
phosphatidylinositol-3-kinase, and the Src family kinase Yes (8). To
date, the regions that mediate these interactions, as well as the
hierarchy of the signal transduction cascades, have not been established.
Angiotensin II is the effector molecule of the renin angiotensin
system. It is vital for maintaining a wide variety of physiological responses including salt and water balance, blood pressure, and vascular tone. These effects are transduced through a
seven-transmembrane surface receptor called AT1 (9). We
have previously demonstrated that treatment of vascular smooth muscle
cells (VSMC) with angiotensin II results in Jak2 autophosphorylation
and activation (10). The angiotensin II-dependent
activation of Jak2 requires the intracellular amino acids 319-322
(YIPP) found in the AT1 receptor carboxyl terminus (11).
Angiotensin II has also been shown to activate the kinase activity of
Fyn and Src as measured by both autophosphorylation and phosphorylation
of synthetic substrates (12, 13). However, neither the mechanism of
activation nor the region of the AT1 receptor that mediates
Fyn and Src activation is known.
Here we investigate whether angiotensin II, acting through the
AT1 receptor, can cause convergence of the Jak and Src
tyrosine kinase signal transduction cascades. This is an important
question because angiotensin II acts through a seven-transmembrane
receptor in contrast to the interleukins, which activate the cytokine
superfamily of receptors. There are several differences between these
receptor families. In the absence of ligand, an appreciable amount of
Jak2 appears bound to the cytoplasmic tail of the cytokine receptor (14). Ligand binding to the extracellular surface of the cytokine receptor results in receptor aggregation and Jak2 activation. In
contrast, little Jak2 co-precipitates with the AT1 receptor in the absence of angiotensin II; ligand binding appears to trigger Jak2 autophosphorylation and concurrent binding with the
AT1 receptor (10, 11). Once bound to the AT1
receptor, the catalytically active Jak2 activates the signal
transducers and activators of transcription in a manner that is similar
to the cytokine receptors and thus transduces signals to the nucleus
(15-17).
In this report, we demonstrate that treatment of VSMC with angiotensin
II leads to a physical association of Jak2 and Fyn. More importantly,
we have characterized the molecular interactions that mediate this
association. Specifically, the binding of Jak2 and Fyn requires a
catalytically active Jak2 molecule and is mediated by the association
of a Jak2 phosphotyrosine and the SH2 domain of Fyn. The functional
consequence of this interaction is that, in response to angiotensin II,
a tyrosine-phosphorylated Jak2 serves as docking site not only for Fyn
binding but for Fyn activation.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
All cells were cultured at 37 °C in a 5%
CO2 humidified atmosphere. VSMC were grown in DMEM plus
10% fetal calf serum and used between passages 7-22. COS-7 cells were
cultured in the same medium. BSC-40 cells were cultured in DMEM plus
10% newborn calf serum. 100-mm dishes of VSMC, at approximately 75%
confluence, were growth-arrested by incubation in serum-free DMEM for
48 h before use. Cell culture reagents were obtained from Life
Technologies, Inc. Tyrosine kinase inhibitors were purchased from
Calbiochem. All other reagents were purchased from Sigma.
Plasmid and GST Fusion Protein Constructs--
pBOSwtJk2 (Jak2
WT) and pBODJK2
VIII (Jak2 DN) were a generous gift from Dr. D. M. Wojchowski and were previously described (18). Construction of the
pRC-Jak2-WT and pRC-Jak2-ATD plasmids was described elsewhere (19). The
vector expressing the wild type AT1 receptor cDNA
(pZeo/WT) was previously reported (11). The c-fos/luciferase
construct (p2FTL) was a generous gift from Dr. W. S. Chen and was
described previously (20). Construction of the GST/Jak2 fusion protein
(amino acids 1-294) was also reported (21). The Fyn fusion proteins
GST/SH2, GST/SH3, and GST/SH2 + SH3 were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA).
Transient Cell Transfection--
COS-7 cells were transfected
exactly as described (11). In order to maintain equal protein
expression, we used 20% more of the Jak2 DN plasmid when compared with
Jak2 WT. For BSC-40 cell transfection, cells were seeded in 100-mm
dishes and transfected at near confluency with either 4 µg of
pRC-Jak2-WT or 16 µg of pRC-Jak2-ATD in the presence of 20 µl of
Lipofectin. For all negative controls, plates were transfected with an
equal amount of empty vector.
Generation of Stably Transfected VSMC/Neo and VSMC/Jak2 DN Cell
Lines--
Passage three VSMC cells were transfected with 20 µg of
pBODJK2VIII (Jak2 DN) and 2 µg of pRC-CMV (neomycin cassette) in 20 µl of Lipofectin to generate the VSMC/Jak2 DN cell lines. Control cells were transfected with only pRC-CMV (VSMC/Neo). Stably transfected clones were generated by growing the cells for 3 weeks in medium containing 600 µg/ml G418. Visible colonies were ring-cloned, yielding four independent VSMC/Neo cell lines and 17 VSMC/Jak2 DN cell
lines. Expression of the Jak2 DN protein was confirmed by both Western
and Northern blot analysis. For these studies, VSMC/Neo and VSMC/Jak2
DN cells were used from passages 7-17.
Assay of Luciferase Activity--
100-mm dishes of 70%
confluent VSMC/Neo and VSMC/Jak2 DN cell lines were transfected with 10 µg of c-fos/luciferase in 20 µl of Lipofectin for 5 h. The cells were then trypsinized and seeded into six-well plates at
4.5 × 105 cells/well and allowed to attach overnight
in serum-containing medium. The following morning, the cells were
washed and placed into serum-free DMEM. Two days later, the cells were
stimulated with angiotensin II, and luciferase activity was measured
from detergent extracts in the presence of ATP and luciferin using the
Reporter Lysis Buffer System (Promega) and a luminometer (Turner Designs model 20/20). We confirmed that both cell lines had a similar
transfection efficiency (15%) by transfecting them with a cDNA
encoding green fluorescent protein downstream of an SV40 promoter and
counting cells under both fluorescent and phase contrast light.
Starting luciferase values between the two cell lines were within 20%
of one another and were always on the linear aspect of the detection curve.
Northern Blot Analysis--
RNA was isolated by the guanidine
thiocyanate/phenol/chloroform method of extraction (22). The RNA was
quantitated, and 25 µg of total RNA from each sample was
electrophoresed through a 1% agarose gel containing 6% formaldehyde.
The RNA was transferred and UV-cross-linked onto GeneScreen nylon
membranes (NEN Life Science Products). The membranes were probed with
radiolabeled cDNAs encoding either the mouse c-fos or
human GAPDH genes. [32P]dCTP (>6000 Ci/mmol) was
purchased from Amersham Pharmacia Biotech.
Immunoprecipitation and GST Pull Down Assays--
To prepare
lysates, cells were washed with 2 volumes of ice-cold
phosphate-buffered saline containing 1 mM
Na3VO4 and lysed in 1.0 ml of ice-cold gentle
lysis buffer (25 mM Tris, pH 7.5, 10% glycerol, 1%
Nonidet P-40, 140 mM NaCl, 4 mM benzamidine, 1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride,
2 mM Na3VO4, and 10 µg/ml
aprotinin). Samples were transferred to microcentrifuge tubes, gently
sonicated, and incubated on ice for 1 h. Samples were spun at
12,000 × g for 5 min at 4 °C, and supernatants were
normalized using the Dc Protein Assay (Bio-Rad). Normalized
lysates (~400 µg/ml) were immunoprecipitated with 1 µg of
antibody and 20 µl of a 50% slurry of Protein A/G-agarose beads
(Santa Cruz Biotechnology) for 6-16 h at 4 °C. The
immunoprecipitating anti-Fyn (FYN3) and anti-Jak2 (HR758) polyclonal
antibodies and the anti-Fyn (15) monoclonal antibody were purchased
from Santa Cruz Biotechnology. The anti-p130cas mAb (P27820)
and anti-Tyr(P) mAb (PY20) were purchased from Transduction Laboratories. Immune complexes were washed three times with wash buffer
(25 mM Tris, pH 7.5, 150 mM NaCl, and 0.1%
Triton X-100) and resuspended in SDS sample buffer. For GST/Fyn pull
down assays, COS-7 cell lysates were precleared with 7.5 µg of
Sepharose-bound GST for 1 h at 4 °C. To each sample, 0.2 µg
of soluble GST or GST/Fyn fusion protein was added along with 20 µl
of a glutathione-Sepharose 4B slurry and incubated for 15-20 min at
4 °C. Beads were washed four or five times with wash buffer
containing 1 M NaCl and resuspended in sample buffer. All
sample buffers containing proteins were separated by SDS-PAGE (National
Diagnostics) and transferred onto nitrocellulose membranes (Schleicher
and Scheull).
Western Blotting--
After blocking for 1 h in 5% dry
milk/TBST (100 mM Tris, pH 7.5, 0.9% NaCl, and 0.05%
Tween 20) at 23 °C, nitrocellulose membranes were probed with
primary antibody for 1-2 h at 23 °C in 5% milk/TBST. Blots were
washed with TBST, and proteins were visualized with ECL following the
manufacturer's instructions (Amersham Pharmacia Biotech). Blotting
antibodies purchased from Santa Cruz Biotechnology were anti-Fyn mAb
(15) and anti-GST mAb (B14). Antibodies purchased from Upstate
Biotechnology, Inc. (Lake Placid, NY) were anti-Tyr(P) mAb (4G10) and
anti-Jak2 pAb (758). Antibodies from Transduction Laboratories were
anti-Fyn mAb (F19720), anti-Tyr(P) mAb (PY20), and anti-Stat1 mAb
(S21120). All markers are kilodaltons.
In Vitro Binding Assays--
Jak2 was synthesized in
vitro using the TnT T7 Quick Coupled Transcription/Translation
System (Promega) in the presence of [35S]methionine
(Amersham Pharmacia Biotech). Yields were estimated to be 200 ng of
Jak2/50-µl reaction as determined by internal controls provided by
the manufacturer. For binding assays, 0.2 µg of Sepharose-bound
GST/Fyn fusion protein was combined with 7.5 µl of
35S-labeled Jak2 (about 30 ng) in 1 ml of Gentle Lysis
Buffer. Samples were incubated at 4 °C for 2-3 h, washed three
times with wash buffer, resuspended in sample buffer, and separated by
SDS-PAGE. Gels were fixed for 0.5 h in isopropyl
alcohol/H2O/acetic acid (25:65:10), washed twice with
H2O, and soaked in Amplify (Amersham Pharmacia Biotech).
Gels were dried under vacuum and exposed to film for 2-4 weeks at
80 °C.
Vaccinia Virus-mediated Jak2 Overexpression--
Jak2 was
overexpressed using the vaccinia virus-mediated transfection/infection
protocol (23). Briefly, 100-mm dishes of nearly confluent BSC-40 cells
were transfected with 20 µg of pRC-Jak2-WT and 20 µl of Lipofectin.
After 4 h, vTF7-3 (24) was added at a multiplicity of infection
of 1.0 and incubated for 1 h. The medium was removed, and cells
were incubated overnight in Dulbecco's modified Eagle's medium (DMEM)
plus 10% NCS. At 18-20 h postinfection, lysates were prepared and
loaded onto a Q Sepharose ion exchange column (Amersham Pharmacia
Biotech). The column was washed extensively with 0.2 M
NaCl, and Jak2 was eluted with 0.8 M NaCl. The sample was
desalted, and proteins under 100 kDa in mass were removed by a
Centricon 100 concentrator using a solution containing 25 mM Tris, pH 7.4, 150 mM NaCl. Purity and
quantity were determined by Ponceau staining and immunoblotting the sample.
Surface Plasmon Resonance Measurements--
Kinetic parameters
for the interaction between GST/Fyn (GST/SH2 + SH3) and partially
purified Jak2 were estimated by surface plasmon resonance using a
BIAcoreTM instrument (Biacore AB). In this study, an
anti-GST monoclonal antibody (Biacore) was covalently linked to CM5
research grade sensor chips using an amine coupling kit (Biacore).
Briefly, carboxylate moieties on CM5 dextran surfaces were activated
with a 30-µl injection of a 1:1 mixture of
N-hydroxysuccinimide and
N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride at a flow rate of 5 µl/min. Immediately following activation, 35 µl of 30 µg/ml of anti-GST in 10 mM
sodium acetate, pH 5.5, was injected across each surface at a flow rate
of 5 µl/min. Unreacted N-hydroxysuccinimide esters were
blocked with a 35-µl injection of 1 M ethanolamine, pH
8.5, at a flow rate of 5 µl/min. This procedure typically yielded
4,000-5,000 resonance units of immobilized anti-GST. Either the fusion
protein, GST/Fyn, or GST alone (as a control) was captured on anti-GST
surfaces by injecting 5 µg/ml of protein in TBS buffer (25 mM Tris-HCl, 150 mM NaCl, 3.4 mM
EDTA, 0.005% P20, pH 7.4) at a flow rate of 5 µl/min, yielding an
increase of 830-550 resonance units. Captured GST/Fyn and GST dissociated from immobilized anti-GST at a rate of <3.0 resonance units/min. Binding interactions were conducted by injecting either 60 µl of partially purified Jak2 (~45 nM) in TBS buffer or
TBS alone (as a control) over captured GST/Fyn or GST surfaces at a
flow rate of 20 µl/min. Following each Jak2 injection, anti-GST surfaces were regenerated with 10 µl of 10 mM glycine, pH
2.2, followed by 10 µl of 0.05% SDS at a flow rate of 5 µl/min.
Sensograms representing the interaction between captured GST/Fyn and
partially purified Jak2 were corrected by subtracting the response
yielded by injecting partially purified Jak2 over a captured GST
surface. Association (ka) and dissociation
(kd) rate constants were determined from corrected
sensograms using a global fitting routine provided by Biacore
(BIAevaluation 3.0).
Jak2 Transphosphorylation Kinase Assays--
COS-7 cells were
transfected as described above to express either no Jak2, wild type
Jak2, or dominant negative Jak2. Two days later, normalized lysates
were prepared. To each lysate, 0.1 µg of Sepharose-bound GST/Jak2
(amino acids 1-294) or GST alone was added, and samples were incubated
at 23 °C for 25-30 min. Samples were pelleted by centrifugation,
and beads were washed four or five times with radioimmune precipitation
buffer (23). Samples were placed in sample buffer, separated on
SDS-PAGE, transferred onto nitrocellulose, and Western blotted with
anti-Tyr(P) mAbs.
In Vitro Kinase Assays--
To determine if Jak2 was sufficient
for Fyn activation, COS-7 cells were transfected to overexpress either
no Jak2, wild type Jak2, or dominant negative Jak2. To determine if
Jak2 was required for Fyn activation, COS-7 cells were transfected with
2 µg of the wild type AT1 receptor cDNA (pZeo/WT) and
either 2.0 µg of wild type Jak2 or 2.4 µg of dominant negative
Jak2. Two days later, cells were treated as described in the figure
legends, and normalized lysates were immunoprecipitated with anti-Fyn
pAb. The immunoprecipitates were washed twice with wash buffer and
twice with kinase buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 10 mM MgCl2, and 0.5 mM dithiothreitol). The precipitates were resuspended in 40 µl of the same kinase buffer containing 50 µM ATP, 2.5 µCi of [
-32P]ATP (Amersham Pharmacia Biotech), and 4 µg of GAP p62 tyrosine kinase substrate (Santa Cruz Biotechnology).
Samples were incubated for 25 min at 30 °C, and reactions were
terminated by adding SDS sample buffer. Radiolabeled proteins were
separated by SDS-PAGE, transferred onto nitrocellulose, and exposed to film.
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RESULTS |
Angiotensin II-dependent Association of Jak2 and
Fyn--
Angiotensin II, acting through the seven-transmembrane
AT1 receptor, has been shown to activate Jak2 and Fyn as
measured by autophosphorylation or phosphorylation of synthetic
substrates (10, 11, 13). We hypothesized that the AT1
receptor might act mechanistically similar to the cytokine superfamily
of receptors and cause association of the Jak and Src family kinases.
To investigate this possibility, VSMC were stimulated with angiotensin
II, and the resulting lysates were immunoprecipitated with an anti-Fyn pAb (FYN3) that does not cross-react with other Src tyrosine kinases. The immunoprecipitates were then blotted with anti-phosphotyrosine antibody. As shown in Fig. 1A,
angiotensin II induced the association of several
phosphotyrosine-containing proteins with Fyn. Maximal protein
association was seen at about 5 min after the addition of angiotensin
II. Even at 1 h after the ligand addition, the level of associated
proteins containing phosphotyrosine was substantially above base line.
Jak2 is 130 kDa in mass and is tyrosine-phosphorylated in response to
angiotensin II (10, 11). Fig. 1A demonstrated that several
phosphotyrosine-containing proteins in the 130-kDa size range, as well
as the 68-72-kDa range, associated with Fyn in an angiotensin
II-dependent manner. Equal loading of all lanes was
verified by reprobing the blot with anti-Fyn mAbs (Fig.
1A).
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Fig. 1.
Angiotensin II-dependent
association of Jak2 and Fyn. A, quiescent VSMC were
stimulated with 10 7 M angiotensin II as
indicated, and the anti-Fyn pAb immunoprecipitates were blotted with
anti-Tyr(P) mAbs. The membrane was then stripped and blotted with
anti-Fyn mAbs. Shown is one of two representative blots. B,
quiescent VSMC were stimulated with 107 M
angiotensin II as indicated, and the resulting lysates were
immunoprecipitated with anti-Fyn pAb (lanes 1-7) or with
rabbit IgG (lanes 8-10). In lanes
5-7, the immunoprecipitating anti-Fyn pAb was preabsorbed
with 5 µg of FYN3-immunizing peptide. The immunoprecipitates were
blotted with anti-Jak2 pAb. The membrane was then blotted with anti-Fyn
mAbs to demonstrate equivalent loading. Similar results were seen in
six separate experiments. C, quiescent VSMC were stimulated
with 107 M angiotensin II as indicated, and
the anti-Fyn mAb immunoprecipitates were blotted with anti-Jak2 pAb.
The membrane was then blotted with anti-Fyn pAb. Similar results were
seen in two separate experiments. D, quiescent VSMC were
stimulated with 107 M angiotensin II as
indicated, and the anti-Jak2 pAb immunoprecipitates were blotted with
anti-Fyn mAb. The membrane was then blotted with anti-Jak2 pAb. Similar
results were seen in four separate experiments.
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To specifically investigate if Jak2 associates with Fyn in an
angiotensin II-dependent manner, we now blotted the
anti-Fyn pAb immunoprecipitates with anti-Jak2 pAb (Fig. 1B,
lanes 1-4). This study showed an increased
association of Jak2 and Fyn as early as 1 min after the addition of
angiotensin II. In multiple time course studies, the Jak2 signal
returned to near basal levels 2 h after angiotensin II treatment
(data not shown). The specificity of Jak2/Fyn association was confirmed
by five separate approaches. First, Fig. 1B,
lanes 5-7, shows the result of preabsorbing the anti-Fyn pAb with the FYN3-immunizing peptide. As expected, the peptide
competes with and prevents the immunoprecipitation of Fyn and the
associated Jak2 protein. Second, Fig. 1B, lanes
8-10, shows that if pooled rabbit IgG is substituted for
the immunoprecipitating anti-Fyn antibody, neither Fyn nor Jak2 is
immunoprecipitated. Third, blotting anti-Fyn pAb immunoprecipitates
with an anti-Jak1 mAb did not produce a ligand-dependent
association, indicating that the association between Jak2 and Fyn is
specific for these two molecules (data not shown). Fourth, we switched
our immunoprecipitating anti-Fyn pAb to an anti-Fyn mAb that was raised
against a different region of Fyn. Like the polyclonal antibody, it
does not cross-react with other Src family kinases. Western blotting of
the anti-Fyn mAb immunoprecipitates with Jak2 gave results that were
virtually identical to those seen with the polyclonal antibody (Fig.
1C). Finally, reciprocal immunoprecipitation experiments
(immunoprecipitating VSMC with anti-Jak2 pAb and blotting with anti-Fyn
mAbs) produced results that were similar to those obtained using the
inverse protocol (Fig. 1D). Collectively, the data show that
in VSMC, activation of the seven-transmembrane AT1 receptor
by angiotensin II causes a specific in vivo association of
Jak2 and Fyn.
An Active Jak2 Kinase Is Required for Jak2/Fyn Association--
We
wanted to determine whether the kinase activity of Jak2 and/or Fyn was
required for this angiotensin II-mediated event. To determine this,
quiescent VSMC were either pretreated with Me2SO (control),
pretreated with the Jak2 kinase inhibitor AG-490 (25), or pretreated
with the Src family kinase inhibitor PP1 (26). For this study, we used
100 µM of the Jak inhibitor AG-490, the lowest dose that
in our hands fully inhibits Jak2 in cultured cells (27). After
inhibitor treatment, cells were then left untreated or stimulated with
107 M angiotensin II for either 5 min (Fig.
2A) or 60 min (Fig.
2B). The resulting lysates were immunoprecipitated with
anti-Fyn pAb and blotted with anti-Jak2 pAb. Association of Jak2 and
Fyn was observed in the control cells and the PP1-treated cells.
However, angiotensin II-dependent association was not seen
after treatment with the Jak2 inhibitor AG-490. To rule out the
possibility that the PP1 was not biologically active, in parallel
experiments, we pretreated VSMC with PP1 and demonstrated that the dose
and time of PP1 preincubation used in Fig. 2, A and
B, could completely block the angiotensin
II-dependent tyrosine phosphorylation of p130cas, a
known substrate of c-Src (28) (Fig. 2C). Thus, the data in
Fig. 2 suggest that Jak2 kinase activity, but not Fyn kinase activity,
is required for the angiotensin II-dependent association of
Jak2 and Fyn.
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Fig. 2.
Inhibition of angiotensin
II-dependent association of Jak2 and Fyn by a Jak2 tyrosine
kinase inhibitor. Quiescent VSMC were treated with either 2%
Me2SO (v/v) for 16 h (control), 100 µM
AG-490 for 16 h, or 20 µM PP1 for 1 h. Cells
were then stimulated with 107 M angiotensin
II for either 5 min (A) or 60 min (B), and the
anti-Fyn pAb immunoprecipitates were blotted with anti-Jak2 pAb. The
same membranes were blotted with anti-Fyn mAbs to demonstrate
equivalent loading. The results were observed in three separate
experiments. C, quiescent VSMC were treated with
Me2SO or with 20 µM PP1 for 1 h and then
stimulated with 107 M angiotensin II as
indicated. The anti-p130cas mAb immunoprecipitates were blotted
with anti-Tyr(P) mAbs. The same membrane was blotted with
anti-p130cas mAb.
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The data in Fig. 2 made use of pharmacologic enzyme inhibitors. As a
complementary approach to investigate this question, we used a protocol
employing a dominant negative Jak2 construct. Specifically, we
transiently transfected COS-7 cells (a cell line with very little
endogenous Jak2) with either a wild type Jak2 expression vector (Jak2
WT) or the mutant Jak2 vector pBODJK2DVIII in which two point mutations
in the kinase domain results in a catalytically inactive protein (Jak2
DN). In this protocol, Jak2 is activated not by angiotensin II ligand
binding but by autophosphorylation of overexpressed Jak2 protein. The
advantage of this system is that it avoids the high endogenous levels
of Jak2 present in VSMC; the only difference between each lysate is the
catalytic activity of Jak2. We have previously used this system to
delineate other Jak2 tyrosine phosphorylation-dependent
cellular events (27). Using this system, COS-7 cells were transfected
to express empty vector, Jak2 WT, or Jak2 DN. The resulting lysates
were immunoprecipitated with anti-Jak2 pAb, separated by SDS-PAGE, and
transferred onto nitrocellulose. The membrane was first blotted with
anti-Jak2 pAb to confirm equal precipitation of both the wild type and
kinase-deficient proteins (Fig.
3A, top). Next, we
measured the level of tyrosine phosphorylation of Jak2 WT by blotting
with anti-Tyr(P) mAbs (Fig. 3A, middle). As
expected, the level of phosphotyrosine of the dominant negative Jak2
was much less than wild type. Finally, the same membrane was blotted
with anti-Fyn mAbs to evaluate Jak2/Fyn physical association (Fig.
3A, bottom). While the Jak2 WT bound substantial
amounts of Fyn, the equally abundant Jak2 DN bound Fyn at a level
approximating that of the empty vector controls. The level of Jak2/Fyn
association was quantitated by densitometry over several experiments
(Fig. 3B). Cells transfected with Jak2 WT had a
statistically significant increased Jak2/Fyn association when compared
with cells transfected with Jak2 DN. Thus, two separate experimental
approaches indicate that a catalytically active Jak2 is necessary for
the efficient association of Jak2 and Fyn in vivo.
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Fig. 3.
A catalytically active Jak2 is required for
efficient association with Fyn. A, COS-7 cells were
transfected with an empty vector control, a wild type Jak2 (Jak2 WT),
or a dominant negative Jak2 (Jak2 DN). Jak2 activation (tyrosine
autophosphorylation) was attained via oligomerization of the highly
expressed Jak2 protein rather than by ligand-dependent
activation. The resulting lysates were immunoprecipitated with
anti-Jak2 pAb and subsequently blotted as indicated. These results were
observed in three separate experiments. B, the anti-Fyn
mAb Western blots from A (bottom) were scanned
and plotted graphically as increased Jak2/Fyn association as a function
of Jak2 status. All conditions are normalized to empty vector control.
Fold increases are expressed as the mean ± S.E.;
n = 3. *, p < 0.0025 (Student's
t test distribution).
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The N Terminus of Jak2 Is Required for Binding Fyn in Vivo--
We
wanted to characterize the region of Jak2 that is required to bind Fyn.
Previous work in other cell systems has shown that the N terminus of
Jak2 (JH7 and JH6 domains) mediates specific protein-protein
interactions between Jak2 and other signaling molecules (29, 30). We
therefore hypothesized that the N terminus of Jak2 is required for
binding Fyn. To test this hypothesis in vivo, BSC-40 cells
(a monkey kidney epithelial cell line) were transiently transfected
with a construct encoding either Jak2 WT or Jak2 lacking the
amino-terminal 240 amino acids (Jak2 ATD). Previously published work
has shown that modified Jak2 proteins lacking the amino-terminal
portion of the enzyme, such as Jak2 ATD, are catalytically active (19,
31). Expression of the Jak2 proteins in BSC-40 cells was augmented by
infecting the cells with vaccinia virus 4 h after transfection, a
protocol that results in high level expression of the transfected
constructs and facilitates the rapid mapping of the region of Jak2 that
binds Fyn. We assessed the relative levels of protein expression by
immunoprecipitating cell lysates with anti-Jak2 pAb and found roughly
equivalent expression of both the Jak2 WT and Jak2 ATD constructs (Fig.
4A, top). Next, the
relative levels of tyrosine phosphorylation were evaluated by reprobing
with anti-Tyr(P) mAbs (Fig. 4A, middle). As
expected, the Jak2 WT protein was heavily phosphorylated on tyrosine
due to oligomerization and autophosphorylation of the highly expressed protein. Tyrosine phosphorylation of the Jak2 ATD protein was detectable with longer exposures than that shown in Fig. 4A,
middle, but the Jak2 ATD mutant, lacking 16 tyrosine
residues on the amino terminus, was phosphorylated on tyrosine much
less than the wild type protein. Finally, association with Fyn was
measured by blotting with anti-Fyn mAbs (Fig. 4A,
bottom). Only the wild type Jak2 protein, containing the
amino-terminal portion of the molecule, efficiently bound Fyn.
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Fig. 4.
Deletion of the N-terminal portion of Jak2
markedly reduces association with Fyn in vivo.
BSC-40 cells were transfected with an empty vector control, Jak2 WT, or
Jak2 ATD and then infected with vaccinia clone vTF7-3 for enhanced
protein expression. Jak2 activation (tyrosine autophosphorylation) was
attained via oligomerization of the highly expressed Jak2 protein.
A, the resulting lysates were immunoprecipitated with
anti-Jak2 pAb and subsequently blotted as indicated. Shown is one of
four independent experiments. B, lysates were
immunoprecipitated with anti-Fyn pAb and then blotted as indicated.
Shown is one of three similar experiments.
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To control for the order of antibody addition, we conducted reciprocal
immunoprecipitation studies by first immunoprecipitating with anti-Fyn
pAb and then Western blotting with anti-Jak2 pAb (Fig. 4B,
top). These data were identical to those in Fig.
4A and show that the Jak2 ATD mutant, which lacks the
amino-terminal 240 amino acids, is far less efficient in associating
with Fyn when compared with wild type Jak2. In contrast to these
results, expression of a Jak2 molecule lacking the pseudokinase domain (amino acids 523-746) bound Fyn in a manner that was no different than
wild type Jak2 (data not shown). Thus, these data implicate the
amino-terminal portion of Jak2 as critical to its ability to physically
bind Fyn in vivo.
Jak2 Binds the SH2 Domain of Fyn--
We wanted to determine which
region of Fyn binds Jak2. This was evaluated using two separate
approaches. First, Jak2 was transcribed and translated in
vitro using a rabbit reticulocyte lysate. Translation was
performed in the presence of [35S]methionine. The Jak2
protein was then added to binding buffer containing GST/Fyn fusion
proteins composed of the SH2, SH3, or SH2 plus SH3 domains of Fyn.
Unmodified GST protein served as the control. The GST fusion proteins
were then isolated and washed, and the association of
35S-labeled Jak2 was detected by SDS-PAGE followed by
autoradiography (Fig. 5A). GST
alone bound virtually no Jak2. In contrast, GST/SH2 bound significantly
higher amounts of Jak2 as compared with control. The GST/SH3 construct
bound Jak2 at levels that were near or slightly above GST controls.
Finally, GST/SH2 + SH3 bound significant amounts of Jak2 in a manner
similar to the GST/SH2 construct. After each in vitro
synthesis of 35S-labeled Jak2, an aliquot of the reaction
was separated by SDS-PAGE to confirm translation of the protein. The
corresponding starting material for these data is also shown for
comparison (Fig. 5A).
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Fig. 5.
The SH2 domain of Fyn binds Jak2 in a
tyrosine phosphorylation-dependent manner.
A, 35S-labeled Jak2 was incubated with the
indicated GST/Fyn fusion proteins and captured by centrifugation. After
separation by SDS-PAGE, the gel was dried and exposed to film in order
to detect bound 35S-labeled Jak2. Also shown is a sample of
the 35S-labeled Jak2 starting material. This result was
observed in four independent experiments. B, COS-7 cells
were transfected to express no Jak2, a wild type Jak2, or a dominant
negative Jak2. The resulting lysates were incubated with GST/Fyn fusion
proteins as indicated. Bound proteins were separated by SDS-PAGE and
transferred onto a nitrocellulose membrane. The membrane was blotted
with an anti-Jak2 pAb to measure Jak2/Fyn association. The
figure represents one of three separate experiments.
C, partially purified Jak2 (~45 nM) was
injected over GST/Fyn (GST/SH2 + SH3) and GST surfaces to generate
binding and background sensograms, respectively. Corrected binding
sensograms were derived by subtracting background from binding
sensograms. Five corrected sensograms were generated (two are shown).
The five corrected binding sensograms were fit to a single equation
using BIAevaluation 3.0 (global analysis). The smooth
solid lines depict the fit to corrected response
versus time data. The kinetic fit parameters were
ka = 4.20 × 105 ± 9.95 × 103 M1 s1 and
kd = 9.91 × 104 ± 5.15 × 105 s1. The S.D. value for the relative
residual plot was 0.15, and  2 = 0.303.
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This same experimental question was investigated using a second
protocol. Here, Jak2 WT or Jak2 DN was transiently overexpressed in
COS-7 cells, and Jak2 was activated via oligomerization and autophosphorylation of the highly expressed protein. Cell lysates were
then prepared, and Jak2/Fyn association was assessed by pull down
experiments using GST/Fyn fusion proteins composed of the SH2, SH3, or
SH2 plus SH3 domains of Fyn. The fusion proteins were collected by
centrifugation, separated by SDS-PAGE, and transferred onto
nitrocellulose. The membrane was then blotted with anti-Jak2 pAb (Fig.
5B). GST did not bind Jak2. GST/SH2 bound the Jak2 WT and,
to a substantially lesser degree, the Jak2 DN. GST/SH3 bound small
amounts of both Jak2 WT and Jak2 DN equally. The GST/SH2 + SH3
construct bound Jak2 WT and Jak2 DN with a pattern similar to the
GST/SH2 protein. Thus, the two experimental approaches presented
in Fig. 5, A and B, suggest an important role of
the Fyn SH2 domain in its association with Jak2. Given the affinity of
SH2 domains for phosphotyrosine, it is not surprising that this domain
associates more efficiently with the catalytically active Jak2 WT as
compared with Jak2 DN.
Dissociation Equilibrium Constant of Jak2/Fyn
Association--
Having demonstrated an interaction between Jak2 and
Fyn, we wanted to quantitate this interaction. This was accomplished by surface plasmon resonance analysis using a BiacoreTM 2000 instrument. This instrument permits a quantitative and sensitive analysis of protein-protein interaction. The Biacore machine provides four separate flow cells (surfaces), which may be used independently or
in series. In the current study, an equivalent amount of anti-GST monoclonal antibody was covalently immobilized in flow cell 1 (fc1) and
flow cell 2 (fc2). The anti-GST surfaces were used to capture GST/Fyn
(SH2 plus SH3) on fc1 and GST on fc2. We chose to use the SH2 plus SH3
fusion protein because it more closely approximated full-length Fyn.
Partially purified Jak2 (~45 nM) was then passed over the
GST/Fyn (fc1) and GST (fc2) surfaces in series, thereby generating a
binding sensogram and a background sensogram with a single injection.
The anti-GST surfaces were restored by treatment with low pH and SDS
(see "Experimental Procedures"). Thus, each binding cycle consisted
of three steps: 1) fusion protein capture; 2) Jak2 binding interaction
(association followed by disassociation); and 3) regeneration. A total
of five such cycles were conducted. For data analysis, background
sensograms (fc2) were subtracted from binding sensograms (fc1),
yielding five corrected binding sensograms (two are shown in Fig. 5C).
Global analysis, fitting all five corrected sensograms to a single
equation, was performed. This analysis gave an association rate
constant (ka) of 4.20 × 105 ± 9.95 × 103 M1
s1 and a dissociation rate constant
(kd) of 9.91 × 104 ± 5.15 × 105 s1, resulting in a dissociation
equilibrium constant (Kd) of 2.36 nM. It
must be noted that the kinetic parameters reported here were derived
using an estimated value (~45 nM) for Jak2 concentration. Hence, these constants are estimates of the kinetic values and are
probably within 3-fold of the true values, assuming a 3-fold error in
the approximated Jak2 concentration. Thus, our data not only
demonstrate an interaction between Jak2 and Fyn; they also demonstrate
an interaction of high affinity.
Transphosphorylation of the N-terminal Region of Jak2 in
Vitro--
The data presented to this point indicate that Fyn binds
tyrosine-phosphorylated Jak2 far better than unphosphorylated Jak2 and
that the amino-terminal portion of Jak2 is critical for the efficient
physical association with Fyn. We next wanted to investigate the
obvious hypothesis that Jak2 activation leads to the Jak2-mediated tyrosine phosphorylation of its amino terminus. If Jak2
autophosphorylation is able to generate an N-terminal phosphotyrosine
capable of binding the SH2 domain of Fyn, we predicted that incubation
of a GST/Jak2 fusion protein (encoding Jak2 amino acids 1-294) with an
active Jak2 molecule would also result in transphosphorylation of the fusion protein. To test this hypothesis, we transfected COS-7 cells to
produce a cell lysate with either no Jak2 (empty vector), catalytically
active wild type Jak2 (Jak2 WT), or kinase-deficient Jak2 (Jak2 DN).
The GST/Jak2 fusion protein was added to each lysate and incubated.
After collection by centrifugation and separation by SDS-PAGE, the
nitrocellulose membrane was blotted with anti-Tyr(P) mAbs. We found
that the GST/Jak2 fusion protein was transphosphorylated by wild type
Jak2 but not by the dominant negative Jak2 (Fig. 6, top). To demonstrate that
equivalent amounts of GST/Jak2 were loaded into each lane, the membrane
was blotted with anti-GST mAb. Although, in this experiment, there was
poor capture of GST/Jak2 from the lysate expressing no Jak2 (Fig. 6,
bottom, lane 1), there was no
significant difference in the amount of GST/Jak2 between wild type Jak2
and the dominant negative Jak2 (Fig. 6, bottom, lanes 2 and 3). In experiments where
GST alone was added to the lysates, absolutely no increased tyrosine
phosphorylation was observed (data not shown). Thus, the
transphosphorylation of the GST/Jak2 fusion protein by wild type Jak2
is specific for the sequence containing the N-terminal 294 amino acids
of Jak2 and not GST.
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Fig. 6.
Transphosphorylation of the N-terminal region
of Jak2 in vitro. COS-7 cells were transfected to
express no Jak2, wild type Jak2, or dominant negative Jak2. Jak2
activation (tyrosine autophosphorylation) was attained via
oligomerization of the highly expressed Jak2 protein. The resulting
lysates were incubated with a GST/Jak2 fusion protein (amino acids
1-294) to allow for transphosphorylation of the fusion protein.
Analysis of the tyrosine phosphorylation of GST/Jak2 was measured by
immunoblotting with anti-Tyr(P) mAbs. The same membrane was blotted
with anti-GST mAb to confirm equal loading. Similar results were seen
in three separate experiments.
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Tyrosine-phosphorylated Jak2 Is Required, but Is Not Sufficient,
for Fyn Activation--
The data from Figs. 2 and 3 demonstrated that
catalytically active Jak2 is required for association with Fyn. To
investigate whether an active Jak2, per se, can activate
Fyn, COS-7 cells were transfected to overexpress either no Jak2, wild
type Jak2, or kinase-deficient Jak2. Again, Jak2 was activated by
overexpression. The cell lysates were immunoprecipitated with anti-Fyn
pAb and, after extensive washing, the Fyn precipitates were resuspended in kinase buffer containing [-32P]ATP and the specific
Src family kinase substrate, GAP p62 (32-34). Any change in Fyn kinase
activity as a function of Jak2 activity would be detected by increased
32P incorporation into the substrate. After separation by
SDS-PAGE and transfer onto nitrocellulose, the radiolabeled proteins
were exposed to film. Analysis of the film demonstrated that there was
no increased 32P incorporation into GAP p62 (Fig.
7A, top). The
membrane was later blotted with anti-Fyn mAbs to demonstrate equivalent
precipitation of Fyn (Fig. 7A, bottom). The
inability of wild type Jak2 to activate Fyn was observed in three
separate experiments as well as in four equivalent experiments using
the Src kinase substrate enolase (data not shown). Thus, while an
active Jak2 is required for association with Fyn, it appears that an
active Jak2 molecule per se is not sufficient for Fyn
activation as measured in this system.
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Fig. 7.
Tyrosine-phosphorylated Jak2 is not
sufficient but is required for Fyn activation. A, COS-7
cells were transfected to express no Jak2, wild type Jak2, or dominant
negative Jak2. Jak2 activation (tyrosine autophosphorylation) was
attained via overexpression of Jak2 protein rather than by
ligand-dependent activation. The resulting lysates were
immunoprecipitated with anti-Fyn pAb. The immunoprecipitates were
thoroughly washed, and Fyn kinase activity was measured by the addition
of the Src family kinase substrate, GAP p62. The membrane was
subsequently blotted with anti-Fyn mAbs to demonstrate equal Fyn
precipitation. Similar results were seen in three separate experiments.
B, COS-7 cells were cotransfected with plasmids encoding the
AT1 receptor (2.0 µg) and either wild type Jak2 (2.0 µg) or dominant negative Jak2 (2.4 µg). The cells were then
stimulated with 107 M angiotensin II, and the
resulting lysates were immunoprecipitated with anti-Fyn pAb. Kinase
reactions were carried out as described above. Similar results were
observed in three separate experiments. C, COS-7 cells were
cotransfected with plasmids encoding the wild type AT1
receptor (2.0 µg) and wild type Jak2 (2.0 µg). At 48 h
post-transfection, the cells were treated for 1 h with
Me2SO (control) or 20 µM PP1. The cells were
then treated with 107 M angiotensin II as
indicated, and kinase reactions were performed on the resulting
anti-Fyn pAb immunoprecipitates. The membrane was subsequently blotted
with anti-Fyn mAbs to demonstrate equivalent precipitation of Fyn.
Similar results were seen in four separate experiments.
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Although an active Jak2 kinase was in of itself insufficient for Fyn
activation, we wanted to determine if an active Jak2 was required for
Fyn activation. To test this possibility, COS-7 cells were transfected
with plasmids encoding either the wild type AT1 receptor
and Jak2 WT or with the wild type AT1 receptor and Jak2 DN.
This system is different from that described for Fig. 7A,
because in the previous experiment, neither the AT1
receptor nor angiotensin II were used. Here, treatment of cells with
angiotensin II allows for activation of the AT1 receptor
and all of the downstream signaling events that contribute to the
activation of Fyn. What is different about the two arms of this
experiment is that cells are transiently transfected with either Jak2
WT or Jak2 DN. Two days after transfection, the cells were treated with
angiotensin II for the indicated times, and normalized lysates were
immunoprecipitated with anti-Fyn pAb. Fyn kinase activity was measured
by resuspending the precipitates in kinase buffer containing
[-32P]ATP and GAP p62. Cells transfected with Jak2 WT
showed an angiotensin II-dependent increased
phosphorylation of GAP p62, indicating that Fyn kinase activity was
increased as a function of angiotensin II treatment (Fig.
7B, top). However, in three separate experiments, cells transfected with Jak2 DN did not show increased 32P
incorporation into GAP p62. To demonstrate equal loading, the membrane
was subsequently blotted with anti-Fyn mAbs (Fig. 7B, bottom). GAP p62 is not thought to be a Jak2 substrate.
However, to eliminate the possibility that we were measuring the
catalytic activity of Jak2 and not Fyn, we performed an equivalent
protocol in the presence of the Src family kinase inhibitor PP1. Cells were transfected with the wild type AT1 receptor and Jak2
WT constructs. The cells were then treated with either
Me2SO or PP1 and subsequently stimulated with angiotensin
II. After immunoprecipitation with anti-Fyn pAb, kinase reactions were
performed. Treatment with Me2SO had no effect on the
32P incorporation into GAP p62, whereas PP1 blocked the
angiotensin II-dependent increase (Fig. 7C,
top). To demonstrate equal loading, the membrane was blotted
with anti-Fyn mAbs (Fig. 7C, bottom). The
combination of 1) using Fyn immunoprecipitates, 2) using a Src family
kinase substrate, and 3) observing that the phosphorylation of GAP p62
is blocked with PP1 strongly indicates that the kinase that is acting
on GAP p62 is Fyn and not Jak2. Collectively, these data indicate that
an active Jak2 molecule, capable of tyrosine autophosphorylation, is
required for the angiotensin II-dependent activation of Fyn.
Catalytically Active Jak2 Is Required for Angiotensin
II-dependent Fyn Kinase Activation in VSMC--
Thus far
our data indicate that in response to angiotensin II, Jak2 and Fyn
physically associate. The consequence of this association is that a
tyrosine-phosphorylated Jak2 serves as a docking site for Fyn
activation. We wanted to determine whether these findings are seen in
physiologically relevant VSMC. Therefore, quiescent VSMC were
pretreated with either Me2SO or AG-490 and then stimulated
with angiotensin II. The resulting lysates were immunoprecipitated with
anti-Fyn pAb, and kinase assays were performed using the substrate GAP
p62 (Fig. 8A). AG-490
completely blocked the angiotensin II-induced activation of Fyn,
indicating that Jak2 kinase activity is required for Fyn activation in
VSMC. Equal loading of Fyn was confirmed by blotting the same membrane
with anti-Fyn mAbs (Fig. 8A).
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Fig. 8.
Characterization of the VSMC/Neo and
VSMC/Jak2 DN cell lines. A, quiescent VSMC were treated
for 16 h with either Me2SO or 100 µM
AG-490. The cells were then treated with 107
M angiotensin II, and kinase reactions were performed on
the resulting anti-Fyn pAb immunoprecipitates using the substrate
GAP p62. The same membrane was blotted with anti-Fyn mAbs to
demonstrate equivalent Fyn precipitation. These results were observed
in four separate experiments. B, whole cell lysates were
prepared from the VSMC/Neo and VSMC/Jak2 DN cell lines, and 30 µg
from each was Western blotted with anti-Jak2 pAb to assess Jak2 protein
expression. C, quiescent VSMC/Neo and VSMC/Jak2 DN cells
were treated with 107 M angiotensin II, and
the resulting anti-Tyr(P) mAb immunoprecipitates were Western blotted
with anti-Jak2 pAb. Shown is one of two representative results.
D, quiescent VSMC/Jak2 DN cells were treated with
107 M angiotensin II, and the resulting
anti-Fyn pAb immunoprecipitates were Western blotted with anti-Tyr(P)
mAbs. Shown is one of two representative results. E,
quiescent VSMC/Neo and VSMC/Jak2 DN cells were treated with
107 M angiotensin II, and the resulting
anti-Jak2 pAb immunoprecipitates were Western blotted with anti-Fyn
mAbs. The same membrane was then blotted with anti-Jak2 pAb. Shown is
one of two representative results. F, quiescent VSMC/Neo and
VSMC/Jak2 DN cells were stimulated with 107 M
angiotensin II, and kinase reactions were performed on the resulting
anti-Fyn pAb immunoprecipitates. The same membrane was then blotted
with anti-Fyn mAbs. This result was observed in three separate
experiments.
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To investigate the role of Jak2 in Fyn activation using an approach
different from pharmacologic inhibition, we generated a stable VSMC
cell line overexpressing the Jak2 DN construct (VSMC/Jak2 DN). Control
cells were VSMC stably transfected with a vector containing only the
neomycin cassette (VSMC/Neo). Jak2 protein expression was studied by
Western blotting equal amounts of whole cell lysate from both cell
lines with anti-Jak2 pAb (Fig. 8B). As expected, the
VSMC/Jak2 DN cell line contained significantly more Jak2 protein than
the VSMC/Neo cell line. Because the Jak2 DN protein migrates at a
position similar to endogenous Jak2, we confirmed the expression of the
Jak2 DN mRNA transcript in VSMC/Jak2 DN cell lines by Northern blot
analysis (data not shown). To test the functional inhibition of the
Jak2 DN protein in VSMC, quiescent VSMC/Neo and VSMC/Jak2 DN cell lines
were stimulated with angiotensin II, and the resulting anti-Tyr(P) mAb
immunoprecipitates were Western blotted with anti-Jak2 pAb (Fig.
8C). We observed that the angiotensin
II-dependent tyrosine phosphorylation of Jak2, seen in the
VSMC/Neo cell line, was lacking in the VSMC/Jak2 DN cell line. Thus,
the Jak2 dominant negative protein blocks angiotensin II-mediated Jak2
activation in the VSMC/Jak2 DN cell line.
To test the hypothesis that a catalytically active Jak2 is required for
the co-association and activation of Fyn, we now repeated several of
our earlier studies using the VSMC/Jak2 DN cell line. Fig.
1A demonstrated in VSMC that, in response to angiotensin II,
Fyn bound several tyrosine-phosphorylated proteins. When this study was
repeated using VSMC/Jak2 DN cells, we observed that the phosphorylation
pattern of Fyn-associated proteins was significantly different (Fig.
8D). The bands in the 130-kDa range were virtually undetectable, and a prominent band of about 60-62 kDa was now observed. The IgG heavy chain was faintly visible at the 50-kDa position. When we repeated the specific Jak2/Fyn co-association assays,
we observed that angiotensin II elicited a ligand-dependent association of Jak2 and Fyn in the VSMC/Neo control cells but not in
the VSMC/Jak2 DN cells, again indicating that a catalytically active
Jak2 is required for this ligand-dependent association (Fig. 8E). To demonstrate equal loading, the same membrane
was blotted with anti-Jak2 pAb (Fig. 8E). To directly
measure angiotensin II-mediated Fyn kinase activity, we stimulated
VSMC/Neo and VSMC/Jak2 DN cells with angiotensin II and conducted
kinase assays on the resulting anti-Fyn pAb immunoprecipitates using
the substrate GAP p62 (Fig. 8F). Treatment of the VSMC/Neo
cells resulted in an angiotensin II-dependent activation of
Fyn. However, this was blocked by expression of the Jak2 DN construct,
again indicating that Jak2 activation is a prerequisite for Fyn
activation in VSMC. We confirmed equal precipitation by blotting the
membrane with anti-Fyn mAbs (Fig. 8F). Collectively, the
data in Fig. 8 strongly argue that in VSMC, a catalytically active Jak2
is required for the angiotensin II-dependent association
with and activation of Fyn kinase.
Functional Consequences of Jak2 Activation in Angiotensin II
Signaling--
We now wanted to identify a putative downstream
signaling substrate of Jak2 in order to determine whether Jak2 is in
fact important for angiotensin II signaling. A well established
downstream signaling molecule of Jak2 in cytokine signaling is Stat1
(1, 2). Recent study has also demonstrated that angiotensin II stimulation leads to Stat1 phosphorylation and activation (10, 15-17).
To determine whether Stat1 is a physiological substrate of Jak2 in
angiotensin II signaling, we stimulated quiescent VSMC/Neo and
VSMC/Jak2 DN cells with angiotensin II. The resulting lysates were
immunoprecipitated with anti-Tyr(P) mAb and blotted with anti-Stat1 mAb
(Fig. 9A). We observed an
angiotensin II-dependent tyrosine phosphorylation of Stat1
in the VSMC/Neo cell line but not in the VSMC/Jak2 DN cell line. To
rule out the possibility that the VSMC/Jak2 DN cell line was somehow
nonspecifically altered by the dominant negative Jak2 protein, we
tested whether these cells could mediate the tyrosine phosphorylation
of p130cas, a protein whose phosphorylation is dependent on
Ca2+/c-Src/Pyk2 but independent of Jak2 (28, 35, 36).
Analysis of p130cas tyrosine phosphorylation indicated that the
VSMC/Jak2 DN cells phosphorylated p130cas in a fashion
identical to the VSMC/Neo control cells (Fig. 9B).
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Fig. 9.
Functional consequence of Jak2 DN expression
in angiotensin II-mediated signaling. Quiescent VSMC/Neo and
VSMC/Jak2 DN cells were treated with 107 M
angiotensin II, and the resulting anti-Tyr(P) mAb immunoprecipitates
were Western blotted with either anti-Stat1 mAb (A) or
anti-p130cas mAb (B). Shown is one of three
representative results for each. C, VSMC/Neo ( ) and
VSMC/Jak2 DN ( ) cells were transiently transfected with a plasmid
encoding firefly luciferase under the control of the c-fos
promoter. The cells were subsequently treated with 107
M angiotensin II for the indicated times, and luciferase
activity was measured. Values are expressed as the mean ± S.E.;
n = 6 for each time point. *, p < 0.025; **, p < 0.0005 (Student's t test
distribution). Shown is one of two representative results.
D, Northern blot analysis of RNA from VSMC/Neo and VSMC/Jak2
DN cells treated with 107 M angiotensin II.
The blot was probed with the mouse c-fos cDNA, stripped,
and then probed with the human GAPDH cDNA. Shown is one of two
representative results.
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To determine whether the inhibition of Stat1 tyrosine phosphorylation
by Jak2 DN had a functional consequence in angiotensin II signaling, we
examined the angiotensin II-dependent activation of the
c-fos promoter. Angiotensin II induces expression of several early response genes including c-fos (37, 38). Therefore, we
transiently transfected both the VSMC/Neo and VSMC/Jak2 DN cell lines
with a luciferase reporter under the control of the c-fos
promoter (20). This plasmid contains two copies of the c-fos
5'-regulated enhancer element (357 to 276), a core thymidine kinase
TATA-containing promoter (200 to +70), and the firefly luciferase
cDNA. Each copy of the c-fos enhancer contains the Stat1-binding sis-inducible element, the serum response
element, and an AP-1 binding site. After transfection, both cell lines were made quiescent and then treated with angiotensin II. Fig. 9C shows the quantitation of luciferase activity as a
function of time after angiotensin II addition. Expression of the Jak2 DN construct significantly reduced the angiotensin
II-dependent induction of the c-fos promoter
when compared with the VSMC/Neo control cells, suggesting that the
angiotensin II-dependent tyrosine phosphorylation of Stat1
by Jak2 is important for maximal induction of the c-fos
promoter. To ascertain whether the Jak2 DN-expressing allele had a
similar effect on endogenous c-fos mRNA, Northern blot
analysis was performed. The VSMC/Neo and VSMC/Jak2 DN cell lines were
treated with angiotensin II for periods ranging from 0 to 60 min (Fig.
9D). Treatment with angiotensin II resulted in the
ligand-dependent accumulation of c-fos mRNA
in the VSMC/Neo control cells but not in the VSMC/Jak2 DN cells.
Prolonged exposures of the blot indicated some
ligand-dependent increase in c-fos mRNA in
the VSMC/Jak2 DN cells, but it was vastly less than that seen from
similarly treated VSMC/Neo cells (data not shown). To demonstrate that
the difference in c-fos mRNA levels was not a loading
artifact, the membrane was subsequently probed with GAPDH (Fig.
9D).
While these data come short of proving a physiologic role of Jak2 in
whole animals, they do suggest that in physiologically relevant
vascular smooth muscle cells, Jak2 activation is required for the
angiotensin II-dependent association with and activation of
Fyn kinase. One functional consequence of this appears to be that a
catalytically active Jak2 is critical for the angiotensin II-dependent tyrosine phosphorylation of Stat1 and maximal
induction of the c-fos promoter.
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DISCUSSION |
The studies in this report were done to better understand the
relationship between the Jak2 and Fyn tyrosine kinase signaling pathways. To this end, we showed that activation of the
seven-transmembrane AT1 receptor by angiotensin II results
in a rapid physical association of Jak2 and Fyn. This
ligand-dependent association requires an active Jak2
kinase, since association can be blocked either by pharmacological
means or by a kinase-deficient Jak2 molecule. We demonstrated that the
portion of Jak2 that mediates this interaction is contained in the
first 240 amino acids of the protein. While both the SH2 and SH3
domains of Fyn participate in Jak2/Fyn association, the data indicate
that the SH2 domain is the dominant interaction and binds Jak2 in a
phosphotyrosine-dependent manner. Surface plasmon resonance
analysis estimated the dissociation equilibrium constant
(Kd) for this interaction to be 2.36 nM,
indicating that the interaction is of high affinity. Finally, our
in vitro and in vivo analysis collectively
suggest that an active Jak2 molecule, which is capable of
autophosphorylation, is required for Fyn activation in response to
angiotensin II in both COS-7 cells and in VSMC.
Based on the observations described in previous work and the results
obtained from this study, we propose a model for Fyn activation by the
AT1 receptor (Fig. 10).
Because the AT1 receptor lacks intrinsic kinase activity,
activation of cellular tyrosine kinase signal transduction cascades
must occur in a manner that is different from the classical tyrosine
kinase growth factor receptors. Activation of the AT1
receptor by angiotensin II results in Jak2 autophosphorylation,
including phosphorylation of the amino-terminal tyrosines (10). A
tyrosine-phosphorylated Jak2 binds to the AT1 receptor at
amino acids 319-322 (YIPP) of the AT1 receptor carboxyl
terminus (11). Fyn then binds to the tyrosine-phosphorylated N terminus
of Jak2 via a high affinity, phosphotyrosine/SH2 linkage, resulting in
Fyn activation. Other factors, such as kinase binding to the
AT1 receptor, may play a role in this process, since
tyrosine phosphorylation of Jak2 alone is insufficient for Fyn
activation.
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Fig. 10.
Model of Jak2-dependent
activation of Fyn by the AT1 receptor. Top,
in the absence of ligand, Jak2 is unphosphorylated and not bound to the
AT1 receptor. Bottom, binding of angiotensin II
to the AT1 receptor results in Jak2 tyrosine
phosphorylation and its coincident binding to amino acids 319-322
(YIPP) of the AT1 receptor. The tyrosine-phosphorylated N
terminus of Jak2 acts as a scaffold and directly binds the SH2 domain
of Fyn, allowing for Fyn activation.
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|
The observation that seven-transmembrane receptors cause an in
vivo convergence of the Jak2 and Fyn tyrosine kinase pathways in a
manner that is similar to the cytokine superfamily of receptors is a
novel observation. However, the significance of this report is that we
have defined a role for Jak2 that, to date, has not been described.
Specifically, our data indicate that in addition to serving as a signal
transduction kinase, Jak2 also functions as a scaffold protein
providing phosphotyrosine residues that participate in activating other
signaling pathways. It appears that Jak2 itself is serving as a docking
site for Fyn activation. A recent report described the crystal
structure of pp60c-src (39). Resolution of
pp60c-src in the inactive state revealed a
conformation in which the tyrosine phosphorylated tail of Src binds its
SH2 domain. This conformation simultaneously blocks the kinase domain
and sequesters the SH2 and SH3 domains. When a phosphotyrosine with
higher affinity binds the SH2 domain, the result is a conformational
change in the protein that frees the kinase domain, the SH2 domain, and
the SH3 domain. Previous studies using Biacore have measured the
affinity of the Src SH2 domain for synthetic phosphotyrosine-containing
peptides and reported Kd values in the range of 600 nM (40). Our data demonstrate that Jak2 binds the SH2
domain of Fyn and that this is tyrosine
phosphorylation-dependent. The estimated Kd for this interaction is 2.36 nM.
Thus, assuming a 3-fold error in our Biacore data (see "Results"),
the affinity of a Jak2 phosphotyrosine/Fyn SH2 interaction is at least
1 order of magnitude higher than those reported for peptides. This
strong interaction could presumably displace the Fyn phosphotyrosine from its SH2 domain and therefore lead to Fyn activation. Our observation indicating a high affinity interaction between a Jak2 phosphotyrosine and the SH2 domain of Fyn resembles a report describing an interaction between Jak2 and the SH2-containing protein termed SH2-B
. Carter-Su and co-workers (41) report that the
carboxyl terminus of SH2-B, which contains the SH2
domain, specifically interacts with kinase-active, tyrosine-phosphorylated Jak2 but not the kinase-inactive,
unphosphorylated Jak2 in the yeast two-hybrid system. Recruitment of
SH2-B to signaling complexes in PC12 cells appears to
mediate nerve growth factor-induced neuronal differentiation (42).
Hand-in-hand with defining a new role for Jak2 in cellular signal
transduction is the observation that we have defined a pathway of Fyn
activation by a seven-transmembrane receptor. The Src family tyrosine
kinases are known to be activated by a broad range of seven-transmembrane receptor ligands including angiotensin II, platelet-activating factor, lysophosphatidic acid, bombesin, and thrombin (12, 43, 44). To date, the precise mechanism of this
activation remains unclear. In the case of angiotensin II, our data
suggests that the activation of Fyn is a Jak2-dependent event. Indeed, the data in this paper give some functional insight into
the role of Jak2 in angiotensin II signaling. Not only does a dominant
negative Jak2 block Fyn activation, but it also interrupts the
signaling by which angiotensin II stimulates the early response gene
c-fos. In summary, this work provides insight into the
fundamental mechanism of how the seven-transmembrane AT1
receptor initiates tyrosine phosphorylation signal transduction
cascades and helps define the cross-talk that is observed between the
Jak and Src tyrosine kinase pathways.
| |
ACKNOWLEDGEMENTS |
We thank Kim Hawks for outstanding technical
assistance and Shaun Benford for administrative assistance. The
plasmids pBOSwtJk2 and pBODJK2VIII were a generous gift from Dr.
D.M. Wojchowski. The p2FTL plasmid was kindly provided by Dr. W. S. Chen. The c-fos and GAPDH encoding plasmids were
graciously provided by Drs. Jessica Schwartz and Randolph Hennigar,
respectively. The recombinant vaccinia virus clone vTF7-3 was
graciously provided by Dr. Bernard Moss and was used in compliance with
the Materials Transfer Agreement.
| |
FOOTNOTES |
*
This work was supported by National Institutes of Health
(NIH) Grants DK39777, DK44280, DK45215, DK51445, and HL47035.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by NIH Grants T32-DK07298 and F32-HL09678.
¶
Supported by NIH Grant HL61710.
**
To whom correspondence should be addressed: Dept. of Pathology and
Laboratory Medicine, 1639 Pierce Dr., 7107 WMB, Emory University School
of Medicine, Atlanta, GA 30322. Tel.: 404-727-3134; Fax: 404-727-8540;
E-mail: kbernst@emory.edu.
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ABBREVIATIONS |
The abbreviations used are:
SH2 and SH3, Src
homology 2 and 3, respectively;
VSMC, vascular smooth muscle cells;
GST, glutathione S-transferase;
DN, dominant negative;
mAb, monoclonal antibody;
pAb, polyclonal antibody;
PAGE, polyacrylamide gel
electrophoresis;
WT, wild type;
Jak2 ATD, Jak2 lacking the
amino-terminal 240 amino acids;
fc1 and fc2, flow cell 1 and 2, respectively;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
IB, immunoblot;
IP, immunoprecipitation;
DMEM, Dulbecco's modified
Eagle's medium.
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REFERENCES |
| 1.
|
Briscoe, J.,
Kohlhuber, F.,
and Muller, M.
(1996)
Trends Cell Biol.
6,
336-340 |
TOP
ABSTRACT
INTRODUCTION
