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 at Birmingham,
Birmingham, Alabama 35294
Received for publication, September 28, 2000, and in revised form, January 2, 2001
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ABSTRACT |
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In response to angiotensin II, Jak2
autophosphorylates and binds the angiotensin II
AT1 receptor. By studying a variety of Jak2
deletion proteins, we now show that the Jak2 protein motif 231YRFRR is required for the co-association of this kinase
with the AT1 receptor. We also used a full-length Jak2
protein containing a 231FAAAA amino acid substitution.
Although this protein still autophosphorylated in response to
angiotensin II, it did not co-associate with the AT1
receptor. This uncoupling indicates that AT1/Jak2
co-association is not necessary for angiotensin II-induced Jak2
autophosphorylation and that Jak2 autophosphorylation per
se is insufficient for AT1 receptor co-association.
In response to angiotensin II, the Jak2-231FAAAA mutant
will tyrosine phosphorylate Stat1. However, in the absence of
AT1/Jak2 co-association, Stat1 did not translocate into the
cell nucleus and failed to mediate gene transcription. This notable
result indicates that Stat1 tyrosine phosphorylation alone is
insufficient for Stat1 nuclear translocation. In summary, we now show
that, although Jak2-mediated tyrosine phosphorylation of Stat1 is
independent of receptor co-association, Jak2-mediated recruitment of
Stat1 to the AT1 receptor is critical for Stat1 nuclear
translocation and subsequent gene transcription.
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 (1). In
addition to promoting the hydrolysis of heterotrimeric G proteins,
activation of the AT1 receptor by angiotensin II also
results in the activation of several non-receptor tyrosine kinases,
including Jak2 (2-5).
Jak2 is a member of the Janus family of non-receptor
tyrosine kinases that also includes Jak1, Jak3, and Tyk2. These
proteins are ~130 kDa in mass and contain seven conserved Jak
homology domains. Typically, Jak activation by a cytokine receptor
leads to STAT1 activation and
thus transmission of a signal from the extracellular surface of the
cell into the nucleus (6). Studies by our laboratory (2) and by Baker
and co-workers (7-9) have shown that, similar to cytokines, activation
of the AT1 receptor by angiotensin II promotes 1) STAT
tyrosine phosphorylation, 2) STAT nuclear translocation, 3) STAT DNA
binding activity, and 4) STAT-dependent transcriptional activation.
Recently, our laboratory examined proximal signaling events that
mediate Jak/STAT activation by the AT1 receptor.
Specifically, we demonstrated that the physical co-association of the
AT1 receptor with Jak2 is dependent on the AT1
receptor motif 319YIPP found within the carboxyl terminus
of the receptor protein (10). Subsequently, we found that Jak2 must be
catalytically active to associate with the AT1 receptor, as
inhibition of Jak2 kinase activity either by pharmacological means or
by a dominant-negative Jak2 protein greatly reduces
AT1/Jak2 co-association (11).
The studies in this report examined whether, in addition to a
functional kinase domain, there are any other requirements for Jak2
co-association with the AT1 receptor. Here we demonstrate that the Jak2 protein motif 231YRFRR, located in the
amino-terminal portion of the molecule, is also required for
co-association with the AT1 receptor. Conversion of the
Jak2 protein motif from 231YRFRR to 231FAAAA
functionally uncouples Jak2 autophosphorylation from AT1 receptor binding, demonstrating that Jak2 autophosphorylation occurs
independently of receptor co-association. When Jak2 fails to
co-associate with the AT1 receptor, a functional
consequence is that Stat1 fails to translocate into the nucleus and
mediate gene transcription.
Cell Culture--
COS-7 cells and BSC-40 cells were cultured and
growth-arrested exactly as described (12, 13). Cell culture reagents
were obtained from Life Technologies, Inc. All other reagents were purchased from Sigma.
Plasmid Constructs--
The HA-tagged AT1 cDNA
was kindly provided by Dr. R. J. Lefkowitz (14) and was cloned
into pcDNA3 at the HindIII/NotI restriction sites. Construction of the pRC-WT, pRC-ATD, pRC-PKD, pRC-CTD, and
pRC-AFL Jak2 expression vectors has been described (13, 15, 16). The
following Jak2 deletion constructs were generated by polymerase chain
reaction using Pfu DNA polymerase and cloned into pRC-AFL at
the NotI/AflII restriction sites. The top
strand oligonucleotides were as follows: pRC-68,
5'-CATGATAATGCGGCCGCAATGGTGGCTGCTTCTAAAGCTTG-3'; pRC-120,
5'-CATGATAATGCGGCCGCAATGCCTCATTGGTACTGTAGTGG-3'; pRC-168, 5'-CATGATAATGCGGCCGCAATGCCTGTGACTCATGAAACTCAG-3'; pRC-186,
5'-CATGATAATGCGGCCGCAATGAGAATAGCTAAGGAG-3'; pRC-211,
5'-CATGATAATGCGGCCGCAATGAAGTGCGTTCGAGCGAAG-3'; pRC-221, 5'-CATGATAATGCGGCCGCAATGCACATTTTAACCCGGAAG-3'; pRC-225,
5'-CATGATAATGCGGCCGCAATGCGGAAGCGAATCAGGTACAG-3'; pRC-230,
5'-CATGATAATGCGGCCGCAATGTACAGATTTCGCAGATTC-3'; and pRC-235, 5'-CATGATAATGCGGCCGCAATGTTCATTCAGCAATTCAGTC-3' (the
initiation methionine for each oligonucleotide is underlined). The
bottom strand oligonucleotide for all constructs was
5'-GATACTGTCTGAGCGCACAGTTTC-3' and utilized an internal
AflII site. The pRC-240 plasmid was made by cutting pRC-ATD
with AflII and closing with ligase. The pRC-FLAG-225, pRC-FLAG-235, and pRC-FLAG-240 constructs were made by placing the FLAG
peptide sequence (DYKDDDDK) between the initiation methionine and the
respective Jak2 sequence. Other than pRC-CTD, which lacks the Jak2
kinase domain, all deletion constructs were found to have readily
detectable levels of tyrosine phosphorylation as measured by
anti-phosphotyrosine Western blotting. The Jak2-Y231F and
Jak2-231FAAAA mutants were generated using the QuikChange
site-directed mutagenesis system (Stratagene). All constructs were
confirmed by DNA sequence analysis.
Transient Cell Transfection--
AT1/Jak2
co-association in BSC-40 cells was done using the vaccinia virus
transfection/infection protocol (17, 18). Cells were seeded in 100-mm
dishes and transfected at near-confluency with 10 µg of
pHA-AT1 and 10 µg of Jak2 expression vector in 20 µl of
Lipofectin. 4 h later, vaccinia virus clone vTF7-3 was added at a
multiplicity of infection of 1.0 and incubated for 1 h. The medium
was aspirated, and cells were incubated overnight in serum-containing medium and lysed 18-20 h after infection. COS-7 cells were transiently transfected exactly as described (10).
Immunoprecipitation--
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 radioimmune precipitation assay buffer containing protease
inhibitors (18). The samples were 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 for 2-16 h at 4 °C with 2 µg of antibody and
20 µl of Protein A/G Plus-agarose beads (Santa Cruz Biotechnology).
After centrifugation, protein complexes were washed three to five times
with radioimmune precipitation assay buffer and resuspended in sample
buffer. Bound proteins were boiled, separated by SDS-PAGE, and
transferred onto nitrocellulose membranes. The immunoprecipitating
anti-Jak2 pAb (HR758) and anti-HA mAb (12CA5) were from Santa Cruz
Biotechnology and Roche Molecular Biochemicals, respectively. The
anti-Tyr(P) mAb (PY20) and anti-Stat1 mAb (S21120) were from
Transduction Laboratories.
Western Blotting--
Proteins were detected using enhanced
chemiluminescence exactly as described (19). Blotting antibodies were
anti-Jak2 pAb (Upstate Biotechnology, Inc.), anti-Tyr(P) mAb (Upstate
Biotechnology, Inc.), anti-HA mAb (Roche Molecular Biochemicals),
anti-Stat1 mAb (Transduction Laboratories), anti-FLAG mAb (Sigma),
anti-Eps15 pAb (Santa Cruz Biotechnology), anti-phospho-Stat1 pAb
(Oncogene Research Products), and anti-SV40 large T antigen mAb (Santa
Cruz Biotechnology).
Preparation of Nuclear Extracts--
After treatment with
angiotensin II, nuclear extracts were prepared using the high salt
method of extraction (20-22). Nuclear proteins were then dialyzed for
2 h at 4 °C against a 500× volume of solution
containing 20 mM HEPES (pH 7.9 at 4 °C), 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM
dithiothreitol, and protease inhibitors. Nuclear proteins were
quantitated, and equal amounts were separated by SDS-PAGE. The samples
were Western-blotted as indicated.
Luciferase Assay--
100-mm dishes of 70% confluent
COS-7 cell lines were transfected with the indicated DNA in 20 µl of
Lipofectin for 5 h. The cells were then trypsinized and seeded
into 6-well plates at 4.5 × 105 cells/well and
allowed to attach overnight in serum-containing medium. The following
afternoon, the cells were washed and placed into serum-free Dulbecco's
modified Eagle's medium containing 0.5% (w/v) bovine serum albumin.
The following morning, 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). Luciferase
values are reported as relative light units.
The Jak2 Protein Motif 231YRFRR Is Required for
AT1Receptor Binding in Vivo--
To determine
regions of Jak2 that are required for co-association with the
AT1 receptor, we utilized an in vivo vaccinia
virus-based expression system that exploits two aspects of Jak2 protein
expression. First, we previously demonstrated that overexpression of
wild-type Jak2 in COS-7 cells results in AT1/Jak2
co-association independent of angiotensin II treatment (11). The
expressed Jak2 is activated not by angiotensin II, but rather by
oligomerization and autophosphorylation of the highly expressed
protein. Although the COS-7 expression system worked well for wild-type
Jak2, we (12, 13, 15, 16) and others (23) observed that some Jak2
deletion proteins are poorly expressed. To overcome this, BSC-40 cells
(a vaccinia virus permissive cell line) are transfected with an
expression vector containing the Jak2 cDNA under the control of the
T7 promoter. The cells are then infected with a vaccinia virus that
produces T7 polymerase. This system greatly enhances Jak2 protein
expression and has been used to examine various aspects of Jak2 signal
transduction (12, 13, 15, 16, 23). This system was used to overexpress various Jak2 deletion constructs along with an HA-tagged
AT1 receptor cDNA (pHA-AT1). As with the
COS-7 cell system, Jak2 is activated by
oligomerization/autophosphorylation of the highly expressed protein,
which in turn binds the AT1 receptor. In vivo
AT1/Jak2 co-association is measured by immunoprecipitating
with anti-HA mAb and blotting with anti-Jak2 pAb.
Prior to assessing co-immunoprecipitation of Jak2 with the
AT1 receptor, several controls were performed. First, we
demonstrated that overexpression of Jak2 required the presence of both
the transfected cDNA and infection of the recombinant vaccinia
virus, as omission of either one resulted in the loss of overexpression (data not shown). Second, we confirmed that the vaccinia-derived Jak2
protein was post-translationally modified by tyrosine phosphorylation and was catalytically active (data not shown).
To demonstrate specificity of the co-immunoprecipitation system, BSC-40
cells were transfected with plasmids encoding wild-type Jak2 (pRC-WT)
and the HA-tagged AT1 receptor under the control of the T7
promoter. The cells were then infected with vaccinia virus clone
vTF7-3, which produces T7 polymerase. Lysates were prepared 18-20 h
after infection. The lysates were then immunoprecipitated with anti-HA
mAb and blotted with anti-Jak2 pAb (Fig.
1A). AT1/Jak2 co-association was seen only when the Jak2 plasmid was present (lane 3 versus lanes 1 and 2).
Furthermore, omission of either the HA-tagged AT1 receptor
plasmid (lane 4) or the immunoprecipitating anti-HA mAb
(lane 5) resulted in specific loss of Jak2 co-association, indicating that neither was Jak2 immunoprecipitated nonspecifically by
anti-HA mAb nor was Jak2 binding the Protein A/G-agarose
nonspecifically.
Using this system, we then coexpressed the HA-tagged AT1
receptor with several Jak2 deletion constructs. A schematic
representation of these constructs is shown as Fig. 1B.
Initially, expression vectors encoding wild-type Jak2, an
amino-terminal deletion (ATD), an internal deletion (PKD), or a
C-terminal deletion (CTD) were utilized. As shown in Fig.
2A (upper panel),
both wild-type Jak2 and the pseudo-kinase deletion (PKD;
To further analyze the amino terminus of Jak2 as being important for
binding the AT1 receptor, we tested the hypothesis that the
initial 240 amino acids of Jak2 are required for efficient co-association with the AT1 receptor. For these
experiments, we used the AflII deletion construct (AFL;
To test this hypothesis, progressive amino-terminal deletions of the
AFL mutant were made at amino acids 68, 120, 168, and 240. Using the
same system, BSC-40 cells were cotransfected with pHA-AT1
and the indicated Jak2 deletion expression vectors and then infected
with vaccinia virus. AT1/Jak2 co-association was measured by immunoprecipitating the cell lysates with anti-HA mAb and
Western blotting with anti-Jak2 pAb (Fig. 2C, upper
panel). We observed that the AFL, 68, 120, and 168 deletion
proteins all bound the receptor. However, the 240 deletion did not. We
confirmed expression of all the Jak2 deletion proteins by
immunoprecipitating and blotting equal aliquots from these same samples
with anti-Jak2 pAb (Fig. 2C, lower panel). These
results indicate that the AT1 receptor-binding region is
located between amino acids 168 and 240.
Jak2 deletion molecules were then generated at amino acids 186, 211, 221, and 240. Another round of co-association was performed using the
same vaccinia virus-based in vivo assay. We found that Jak2
deletions 168, 186, 211, and 221 all co-associated with the HA-tagged
AT1 receptor, whereas the 240 mutant did not (Fig.
2D, upper panel). As before, we confirmed
expression of all Jak2 deletions by immunoprecipitating and Western
blotting equal aliquots from these same samples with anti-Jak2 pAb
(Fig. 2D, lower panel). These data indicate that
an important AT1-binding region of Jak2 is located between
amino acids 221 and 240. Deletion mutants were then made at amino acids
225, 230, and 235, and AT1 co-association was assessed as
described above (Fig. 2E, upper panel). This
revealed that deletion proteins 221, 225, and 230 co-associated with
the AT1 receptor, whereas Jak2 deletion proteins 235 and
240 did not. Expression of all Jak2 proteins was confirmed by
immunoprecipitating and Western blotting equal aliquots of these
samples with anti-Jak2 pAb (Fig. 2E, lower
panel).
These data suggest that one region of Jak2 that facilitates
co-immunoprecipitation with the AT1 receptor is located
between amino acids 231 and 235. One issue that concerned us, however, was the appearance of a lower band of ~68 kDa that was observed with
many of the Jak2 deletion constructs. The protein appeared to be
Jak2-related, as it was immunoreactive against anti-Jak2 pAb and was
never seen when cells were transfected with the empty vector control.
To better understand the nature of this protein, we generated three
additional Jak2 deletion constructs: pRC-FLAG-225, pRC-FLAG-235, and
pRC-FLAG-240. These molecules were modified by placing the FLAG peptide
sequence (DYKDDDDK) between the initiator methionine and the respective
Jak2 sequence. This allowed us to Western blot these proteins with
either anti-Jak2 pAb or anti-FLAG mAb. These studies demonstrated that
the lower bands were a truncated form of Jak2 that lacked the
amino-terminal portion of the molecule; loss of the amino-terminal FLAG
peptide sequence resulted in a shorter protein that was no longer
immunoreactive with the anti-FLAG antibody, but was reactive with the
anti-Jak2 antibody (data not shown). Consistent with our previous
results, coexpression of these three constructs with the HA-tagged
AT1 receptor demonstrated that pRC-FLAG-225 was able to
coprecipitate with the HA-tagged AT1 receptor, whereas
pRC-FLAG-235 and pRC-FLAG-240 were not (data not shown).
In summary, these data confirm our previously published work
(11) demonstrating that Jak2 must possess a functional kinase domain to
bind the AT1 receptor; deletion of the kinase domain abrogates AT1/Jak2 co-association. In addition, the data
demonstrate for the first time that Jak2 must possess amino acids
231-235, encoding the 231YRFRR protein motif, to
coprecipitate with the AT1 receptor in vivo;
constructs lacking 231YRFRR fail to bind the
AT1 receptor.
A Jak2 Molecule Containing a 231FAAAA Mutation in Place
of 231YRFRR Fails to Bind the AT1 Receptor, but
Autophosphorylates in Response to Angiotensin II--
The vaccinia
virus deletion analysis studies presented in Figs. 1 and 2 were
performed without angiotensin II treatment. To examine the role of the
Jak2 231YRFRR motif in ligand-mediated binding to the
AT1 receptor, we generated two additional Jak2 molecules.
The first was a full-length Jak2 in which the 231YRFRR
motif was converted to 231FAAAA
(Jak2-231FAAAA). The second was a full-length Jak2
containing a single Y231F point mutation (Jak2-Y231F). To evaluate the
Jak2-231FAAAA construct, COS-7 cells were transiently
cotransfected with pHA-AT1 and this plasmid. The cells were
then treated with angiotensin II, and ligand-dependent
AT1/Jak2 co-association was measured. We found that the
Jak2-231FAAAA mutant did not co-associate with the
AT1 receptor in response to angiotensin II (Fig.
3A, upper panel).
We confirmed equal expression of both wild-type and
Jak2-231FAAAA mutant proteins by immunoprecipitating and
Western blotting equal aliquots from these samples with anti-Jak2 pAb
(Fig. 3A, lower panel). In contrast to this
result, the Y231F mutant bound the AT1 receptor in a manner
that was virtually identical to wild-type Jak2, indicating that in the
context of the 231YRFRR motif, tyrosine 231 appears to
provide a structural contribution rather than a phosphotyrosine binding
residue (data not shown).
To determine whether the Jak2-231FAAAA mutant
autophosphorylates in response to angiotensin II, COS-7 cells were
transfected as described for Fig. 3A, but the lysates were
then immunoprecipitated with anti-Jak2 pAb. The samples were first
blotted with anti-Tyr(P) mAb to measure the angiotensin
II-dependent tyrosine phosphorylation of Jak2 (Fig.
3B, upper panel). These data clearly show that
both the wild-type and 231FAAAA mutant proteins were
tyrosine-phosphorylated in response to angiotensin II. They also show
that, although Jak2-231FAAAA was unable to bind the
receptor in response to ligand, it was certainly capable of tyrosine
autophosphorylation. We confirmed equal precipitation of Jak2 protein
by blotting these same samples with anti-Jak2 pAb (Fig. 3B,
lower panel). When COS-7 cells were transfected with an
empty vector control in place of Jak2-231FAAAA, we observed
virtually no tyrosine phosphorylation of Jak2 in response to
angiotensin II (data not shown). This is because non-transfected COS-7
cells have very little endogenous Jak2. Thus, the response seen in
cells transfected with Jak2-231FAAAA is not due to
endogenous (wild-type) Jak2, but is due to the transfected construct.
The experiments in Fig. 3 indicate that, although
Jak2-231FAAAA was capable of tyrosine autophosphorylation,
it was unable to bind the AT1 receptor in response to
angiotensin II.
A Jak2 Molecule Containing a 231FAAAA Mutation in Place
of 231YRFRR Mediates the Angiotensin
II-dependent Tyrosine Phosphorylation of Stat1--
We
then measured the angiotensin II-dependent tyrosine
phosphorylation of Stat1. This protein is a substrate of Jak2 and is tyrosine-phosphorylated in response to angiotensin II (2, 6). COS-7
cells were transfected with plasmids encoding the AT1
receptor and an empty vector control, wild-type Jak2, or
Jak2-231FAAAA. 2 days later, the cells were treated with
angiotensin II, and the resulting lysates were immunoprecipitated with
anti-Tyr(P) mAb and blotted with anti-Stat1 mAb (Fig.
4A). In the absence of
transfected Jak2 expression vector, no Stat1 phosphorylation was
observed in this system. In contrast, addition of wild-type Jak2
plasmid resulted in the angiotensin II-dependent tyrosine phosphorylation of Stat1. When the Jak2-231FAAAA mutant
protein was transfected into cells, we found that it phosphorylated
Stat1 in a manner that was identical to that of wild-type Jak2.
To better establish a time course for angiotensin II-mediated Stat1
tyrosine phosphorylation and to measure Stat1 tyrosine phosphorylation
by an alternate protocol, transfected COS-7 cells were treated with
angiotensin II for periods of 0-30 min. A reciprocal protocol was then
performed whereby the lysates were immunoprecipitated with anti-Stat1
mAb and Western-blotted with anti-phospho-Stat1 pAb (Fig.
4B, upper panel). This blotting antibody is
specific for phosphorylated Stat1 protein at tyrosine 701, a site of
phosphorylation that correlates with Stat1 activation (24, 25). These
data showed that Jak2-231FAAAA phosphorylated Stat1 over a
time course that was nearly identical to that of wild-type Jak2, with
peak Stat1 tyrosine phosphorylation occurring between 3 and 6 min after
angiotensin II treatment for both groups. Furthermore, the alternate
protocol produced a result that was similar to that in Fig.
4A. Collectively, these results indicate that
Jak2-231FAAAA is not only capable of autophosphorylation,
but is also able to phosphorylate a substrate in response to
angiotensin II.
A Jak2 Molecule Containing a 231FAAAA Mutation in Place
of 231YRFRR Fails to Mediate the Angiotensin
II-dependent Nuclear Translocation of Stat1--
Given
that Jak2-231FAAAA is able to mediate Stat1 tyrosine
phosphorylation, we next investigated whether it could mediate Stat1 nuclear translocation. COS-7 cells were transfected with plasmids encoding the AT1 receptor and either wild-type Jak2 or
Jak2-231FAAAA. After angiotensin II treatment, nuclear
proteins were isolated and then separated via SDS-PAGE. After transfer
onto nitrocellulose membranes, Stat1 nuclear translocation was measured
by Western blotting the samples with anti-Stat1 mAb (Fig.
5A). In response to
angiotensin II, Stat1 was found only in the nucleus of cells transfected with wild-type Jak2 plasmid, but not in that of cells transfected with Jak2-231FAAAA. To establish a better time
course of Stat1 nuclear translocation and to determine the efficiency
and specificity of the nuclear extraction procedure, COS-7 cells were
transfected exactly as described for Fig. 5A, but were then
treated with 10
Collectively, these data show that, although Jak2-231FAAAA
is capable of autophosphorylation and tyrosine phosphorylating Stat1, its failure to co-associate with the AT1 receptor results
in the failure of Stat1 to translocate into the nucleus. This result is
reminiscent of the growth hormone-mediated activation of Stat3; recruitment of Stat3 to the growth hormone receptor is critically dependent on Jak2 kinase (30).
A Jak2 Molecule Containing a 231FAAAA Mutation in Place
of 231YRFRR Fails to Mediate Stat1-dependent
Gene Transcription--
To assess the functional impact of the
Jak2-231FAAAA mutation, we chose to investigate angiotensin
II-dependent, Stat1-mediated gene transcription. For this
assay, COS-7 cells were transfected as described for Fig. 5, but we
also included a luciferase reporter plasmid containing a Stat1-binding,
sis-inducible element. This plasmid contains a tandem repeat
of a minimal DNA enhancer element, the thymidine kinase TATA-containing
promoter, and the firefly luciferase cDNA (31). Each copy of the
DNA enhancer contains a sis-inducible element, a serum
response element, and an AP-1-binding site. We have previously
demonstrated that this plasmid is a good indicator of Jak/STAT-mediated
gene transcription (12, 16). The transfected cells were then treated
with angiotensin II for varying times, and luciferase activity was
measured (Fig. 6). Transfection of the
Jak2-231FAAAA mutant resulted in substantially reduced
angiotensin II-mediated luciferase activity with respect to wild-type
Jak2 controls. The greatest difference in luciferase values between the
two groups occurred 24 h after angiotensin II addition
(p < 0.00001; n = 6 for each point).
These data indicate that the failure of Jak2-231FAAAA to
facilitate Stat1 nuclear translocation also impairs
Stat1-dependent transcriptional activation.
This work is significant for three fundamental reasons. First, we
have identified a minimal region of Jak2 (231YRFRR) that is
required for Jak2 co-association with a cell-surface receptor. Second,
the generation of the Jak2-231FAAAA mutant functionally
uncoupled ligand-mediated Jak2 autophosphorylation from AT1
receptor co-association. Third, the work suggests that when Jak2 fails
to bind the AT1 receptor, one functional consequence is
that Stat1 does not translocate into the nucleus and therefore fails to
mediate gene transcription.
In regard to 231YRFRR-mediated receptor co-association,
exactly how 231YRFRR mediates the binding of Jak2 with the
AT1 receptor is not fully understood. Our data suggest that
it is probably a structural motif rather than a simple phosphotyrosine
interaction in that the Jak2-Y231F mutant bound the AT1
receptor, whereas the Jak2-231FAAAA mutant did not.
Consistent with our observation is a report describing a naturally
occurring Jak3 mutation from a patient with autosomal severe combined
immunodeficiency; a single Y100C amino acid substitution prevents
co-association of Jak3 with the common With respect to the second point, when COS-7 cells are transfected with
Jak2-231FAAAA and then treated with angiotensin II,
Jak2-231FAAAA autophosphorylates, but does not co-associate
with the AT1 receptor. This observation suggests that
ligand-mediated Jak2 autophosphorylation has therefore been uncoupled
from AT1 receptor co-association. Since the
Jak2-231FAAAA mutant can be activated independently of
receptor co-association, one interpretation is that Jak2
autophosphorylation temporally precedes receptor co-association. The
current findings are important in that they strongly suggest that
AT1/Jak2 co-association is not necessary for angiotensin
II-induced Jak2 activation and that Jak2 activation, although
necessary, is not sufficient for its interaction with the
AT1 receptor (11).
Perhaps most significant is the observation that when Jak2 fails to
bind the AT1 receptor, Stat1 does not translocate into the
nucleus and therefore fails to mediate gene transcription. The data in
Fig. 4 demonstrate that Stat1 tyrosine phosphorylation alone is
insufficient for Stat1 nuclear translocation, and the data in Fig. 5
indicate that failure of Jak2 to bind the AT1 receptor correlates with markedly decreased Stat1 nuclear translocation.
Our finding that Stat1 tyrosine phosphorylation alone is insufficient
for Stat1 nuclear localization has precedent in other published
studies. Herrington et al. (33) reported that a
Stat5b protein with a small mutation in the DNA-binding region
was defective in nuclear location despite its ability to be
tyrosine-phosphorylated and to dimerize in response to ligand.
Similarly, Strehlow and Schindler (34) reported a modified
Stat1 protein that was phosphorylated on tyrosine and that
dimerized, but was unable to be transported to the nucleus. Although
these reports described a phenotype similar to what we observed with
Jak2-231FAAAA, in both studies, the phenotype was a direct
result of a STAT structural mutation. Nonetheless, these studies
support our observation by providing other examples of STAT proteins
that are phosphorylated on tyrosine and that dimerize, but are unable to transport to the nucleus.
We recently reported that, in response to angiotensin II, Jak2 not only
serves as a Stat1 kinase, but also acts as a molecular bridge in
recruiting Stat1 to the AT1 receptor (35). One
interpretation of the Jak2-231FAAAA data presented here is
that recruitment of Stat1 to the AT1 receptor by wild-type
Jak2 further modifies Stat1 in a manner that promotes its nuclear
translocation. The exact modification(s) that occur at the
AT1 receptor and whether this phenomenon is applicable to
other STAT family members is under intense investigation. The concept
that Jak2 acts as a molecular bridge in recruiting STAT proteins to a
cell-surface receptor is not without precedent. In the case of growth
hormone, Jak2 acts as a scaffold in recruiting Stat3 to the growth
hormone receptor (30). The present data are significant in that they
extend this observation by demonstrating that when Jak2 fails to act as
a scaffold in recruiting Stat1 to the AT1 receptor, one
consequence is that Stat1-mediated gene transcription is severely compromised.
In summary, this work provides novel insight into the mechanism of Jak2
activation and receptor co-association. Specifically, it defines a
region of Jak2 that is required for receptor co-association and
advances our understanding of the temporal sequences that occur when
the Jak/STAT signaling pathway is activated. Most importantly, this
work establishes a correlation between the loss of a Jak2 signaling
complex at the level of a cell-surface receptor with the loss of
transcriptional regulation within the nucleus.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Specificity of vaccinia virus-mediated
AT1/Jak2 co-association. A, BSC-40 cells
were transfected with the indicated plasmids and infected with vaccinia
virus clone vTF7-3. Lysates were prepared and immunoprecipitated with
anti-HA mAb (lanes 1-4) or control IgG (lane 5)
and then Western-blotted with anti-Jak2 pAb to assess
AT1/Jak2 co-association. Shown is one of three independent
experiments. B, shown is a summary of Jak2 deletion analysis
studies and co-association with the HA-tagged AT1 receptor.
The schematic represents full-length Jak2 and the seven respective Jak
homology domains (1-7). Also shown are the deletions
generated and a table summarizing whether each was able to co-associate
with the HA-tagged AT1 receptor in vivo.
Co-IP, co-immunoprecipitate.
523-746)
co-associated with the receptor. However, the amino-terminal deletion
(ATD;
1-240) and the C-terminal deletion (CTD;
1000-1129) did
not. We confirmed that all Jak2 proteins were expressed by
immunoprecipitating and Western blotting aliquots from these same
samples with anti-Jak2 pAb (Fig. 2A, lower
panel). We believe the inability of the CTD mutant to bind the
receptor is due to the loss of the kinase domain since previous work
demonstrated that inhibition of Jak2 kinase activity either by
pharmacological means or by rendering the kinase domain inactive by a
dominant-negative mutation prevents AT1/Jak2 co-association
(11).
View larger version (55K):
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Fig. 2.
AT1/Jak2 co-association requires
the Jak2 protein motif 231YRFRR. BSC-40 cells were
transfected with the indicated plasmids and infected with vaccinia
virus clone vTF7-3. Lysates were prepared, and half of each lysate was
immunoprecipitated (IP) with anti-HA mAb and then
Western-blotted (immunoblotted (IB)) with anti-Jak2 pAb to
assess AT1/Jak2 co-association. The remaining half was
immunoprecipitated and Western-blotted with anti-Jak2 pAb to assess
Jak2 protein expression. Shown is one of three (A-C) or
four (D and E) independent experiments for each
construct. Constructs lacking either the kinase domain or
231YRFRR failed to co-associate with the HA-tagged
AT1 receptor in vivo. WT, wild-type
Jak2.
251-473). As shown in Fig. 2B (upper panel),
the AFL mutant was able to co-associate with the HA-tagged
AT1 receptor in vivo. In Fig. 2B
(lower panel), we confirmed expression of the individually
transfected Jak2 proteins. Although these data indicate that amino
acids 251-473 are not required for binding the AT1
receptor, they lead to the hypothesis that amino acids 1-240 of Jak2
are specifically required for binding the AT1 receptor
in vivo.
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[in a new window]
Fig. 3.
Conversion of 231YRFRR to
231FAAAA inhibits the angiotensin II-dependent
co-association of Jak2 with the AT1 receptor, but not Jak2
tyrosine autophosphorylation. COS-7 cells were transfected with 10 µg of HA-tagged AT1 receptor and either 5 µg of
wild-type Jak2 (Jak2 WT) or 12.5 µg of
Jak2-231FAAAA. The cells were treated with
10 7 M angiotensin II (Ang
II) for the indicated times, and lysates were prepared.
A, lysates were immunoprecipitated (IP) with
anti-HA mAb and immunoblotted (IB) with anti-Jak2 pAb
to assess AT1/Jak2 co-association (upper panel).
Equal aliquots from these samples were immunoprecipitated and blotted
with anti-Jak2 pAb to assess Jak2 expression (lower panel).
B, lysates were immunoprecipitated with anti-Jak2 pAb and
then blotted with either anti-Tyr(P) mAb to assess Jak2 tyrosine
autophosphorylation (upper panel) or anti-Jak2 pAb to
measure Jak2 expression (lower panel). Shown is one of three
independent experiments for each.
View larger version (47K):
[in a new window]
Fig. 4.
Conversion of 231YRFRR to
231FAAAA does not inhibit the angiotensin
II-dependent tyrosine phosphorylation of Stat1. COS-7
cells were transfected with 10 µg of HA-tagged AT1
receptor and either 5 µg of wild-type Jak2 (Jak2 WT) or
12.5 µg of Jak2-231FAAAA. The cells were treated with
10 7 M angiotensin II (Ang
II) for the indicated times, and lysates were prepared to assess
Stat1 tyrosine phosphorylation. A, lysates were
immunoprecipitated with anti-Tyr(P) mAb and Western-blotted
(immunoblotted (IB)) with anti-Stat1 mAb. B,
lysates were immunoprecipitated with anti-Stat1 mAb and Western-blotted
either with anti-phospho-Stat1 pAb to assess Stat1 tyrosine
phosphorylation (upper panel) or with anti-Stat1 mAb to
assess equal precipitation of Stat1 protein (lower panel).
Shown is one of three independent experiments for each.
7 M angiotensin II
for 0, 10, 20, 30, 45, and 60 min. First, the samples were
Western-blotted with anti-Stat1 mAb to assess the time course of Stat1
nuclear translocation (Fig. 5B, upper panel). This result reconfirmed that Stat1 translocated into the nucleus of
cells that received wild-type Jak2 plasmid and angiotensin II, but not
into that of cells that received Jak2-231FAAAA, regardless
of angiotensin II treatment. Analysis of three independent experiments
indicated that the accumulation of Stat1 in the nucleus of cells
transfected with wild-type Jak2 peaked ~20-30 min after angiotensin
II addition, whereas cells that received Jak2-231FAAAA
failed to mediate ligand-dependent Stat1 nuclear
translocation. To determine the efficiency and specificity of the
nuclear extraction procedure, the membrane was stripped and
Western-blotted with an antibody directed against the SV40 large T
antigen (Fig. 5B, middle panel). The large T
protein is highly abundant in COS-7 cells and is found only in the
nucleus of these cells (26, 27). The results indicated that the samples
were of nuclear origin as determined by the amount of large T protein
found in each sample relative to a COS-7 whole cell lysate sample. To
demonstrate that the nuclear preparations were devoid of cytoplasmic
contamination, the membrane was blotted with an antibody directed
against the cytoplasmic protein marker Eps15 (Fig.
5B, bottom panel) (28, 29). The lack of
Eps15 in the nuclear extracts indicated that the Stat1 was of nuclear
origin and not from cytoplasmic contamination.
View larger version (67K):
[in a new window]
Fig. 5.
Conversion of 231YRFRR to
231FAAAA inhibits the angiotensin II-dependent
nuclear translocation of Stat1. COS-7 cells were transfected
exactly as described in the legend to Fig. 4. A, after
treatment with 10 7 M angiotensin
II (Ang II) for the indicated times, nuclear proteins were
prepared and electrophoresed via SDS-PAGE. The proteins were
transferred onto a nitrocellulose membrane and blotted with anti-Stat1
mAb to assess Stat1 nuclear translocation. B, COS-7 cells
were treated with 10
7 M
angiotensin II for longer time periods. Nuclear extracts were prepared
from each time point and then subjected to SDS-PAGE along with a whole
cell lysate (WCL) from non-transfected COS-7 cells. The
proteins were transferred onto a nitrocellulose membrane and blotted as
indicated. Shown is one of three independent experiments. Jak2
WT, wild-type Jak2.
View larger version (13K):
[in a new window]
Fig. 6.
Conversion of 231YRFRR to
231FAAAA inhibits angiotensin II-mediated,
Stat1-dependent transcriptional activation. COS-7
cells were transfected with 10 µg of HA-AT1, 10 µg of
luciferase reporter plasmid, and either 5 µg of wild-type Jak2 ( )
or 12.5 µg of Jak2-231FAAAA (
). After angiotensin II
treatment, luciferase activity was measured from detergent-soluble
extracts. Values are expressed as the mean ± S.D.
(n = 6 for each time point). *, p < 0.0001; **, p < 0.001; ***, p < 0.00001 (Student's t test). Shown is one of three
independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain of the interleukin-2
receptor (32). Similar to our results, the authors of this report
concluded that Tyr100 does not mediate a
phosphotyrosine-dependent interaction, but rather
contributes to a larger structural motif, as the Y100C mutation
disrupts receptor co-association, whereas a Y100F mutation does not.
The amino acid sequence immediately downstream of Tyr100 is
100YRLRF, and this bears some homology to the
231YRFRR motif described in this report.
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ACKNOWLEDGEMENTS |
---|
We are indebted to Kim Hawks for outstanding technical assistance and thank Kevin Disher and Hui Zhao for administrative assistance. The HA-tagged AT1 receptor cDNA was kindly provided by Dr. R. J. Lefkowitz (Duke University). The recombinant vaccinia virus clone vTF7-3 was graciously provided by Dr. Bernard Moss (National Institutes of Health).
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants DK39777, DK44280, DK45215, DK51445, HL47035, and HL61710.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 National Institutes of Health Grants T32-DK07298 and F32-HL09678. Present address: Dept. of Physiology, P. O. Box 100274, University of Florida College of Medicine, Gainesville, FL 32610.
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.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M008856200
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ABBREVIATIONS |
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The abbreviations used are: STAT, signal transducer and activator of transcription; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; pAb, polyclonal antibody; mAb, monoclonal antibody.
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REFERENCES |
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---|
1. | Sayeski, P. P., Ali, M. S., Semeniuk, D. J., Doan, T. N., and Bernstein, K. E. (1998) Regul. Pept. 78, 19-29[CrossRef][Medline] [Order article via Infotrieve] |
2. | Marrero, M. B., Schieffer, B., Paxton, W. G., Heerdt, L., Berk, B. C., Delafontaine, P., and Bernstein, K. E. (1995) Nature 375, 247-250[CrossRef][Medline] [Order article via Infotrieve] |
3. | Sadoshima, J., and Izumo, S. (1996) EMBO J. 15, 775-787[Abstract] |
4. |
Ishida, M.,
Marrero, M. B.,
Schieffer, B.,
Ishida, T.,
Bernstein, K. E.,
and Berk, B. C.
(1995)
Circ. Res.
77,
1053-1059 |
5. |
Li, X.,
and Earp, H. S.
(1997)
J. Biol. Chem.
272,
14341-14348 |
6. | Briscoe, J., Kohlhuber, F., and Muller, M. (1996) Trends Cell Biol. 6, 336-340[CrossRef] |
7. |
Bhat, G. J.,
Thekkumkara, T. J.,
Thomas, W. G.,
Conrad, K. M.,
and Baker, K. M.
(1994)
J. Biol. Chem.
269,
31443-31449 |
8. |
Bhat, G. J.,
Thekkumkara, T. J.,
Thomas, W. G.,
Conrad, K. M.,
and Baker, K. M.
(1995)
J. Biol. Chem.
270,
19059-19065 |
9. | McWhinney, C. D., Hunt, R. A., Conrad, K. M., Dostal, D. E., and Baker, K. M. (1997) J. Mol. Cell. Cardiol. 29, 2513-2524[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Ali, M. S.,
Sayeski, P. P.,
Dirksen, L. B.,
Hayzer, D. J.,
Marrero, M. B.,
and Bernstein, K. E.
(1997)
J. Biol. Chem.
272,
23382-23388 |
11. | Ali, M. S., Sayeski, P. P., Safavi, A., Lyles, M., and Bernstein, K. E. (1998) Biochem. Biophys. Res. Commun. 249, 672-677[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Sayeski, P. P.,
Ali, M. S.,
Safavi, A.,
Lyles, M.,
Kim, S. O.,
Frank, S. J.,
and Bernstein, K. E.
(1999)
J. Biol. Chem.
274,
33131-33142 |
13. |
Sayeski, P. P.,
Ali, M. S.,
Hawks, K.,
Frank, S. J.,
and Bernstein, K. E.
(1999)
Circ. Res.
84,
1332-1338 |
14. |
Oppermann, M.,
Freedman, N. J.,
Alexander, R. W.,
and Lefkowitz, R. J.
(1996)
J. Biol. Chem.
271,
13266-13272 |
15. |
Zhao, Y.,
Wagner, F.,
Frank, S. J.,
and Kraft, A. S.
(1995)
J. Biol. Chem.
270,
13814-13818 |
16. | Frank, S. J., Yi, W., Zhao, Y., Goldsmith, J. F., Gilliland, G., Jiang, J., Sakai, I., and Kraft, A. S. (1995) J. Biol. Chem. 270, 14766-14785 |
17. | Fuerst, T. R., and Moss, B. (1989) J. Mol. Biol. 206, 333-348[Medline] [Order article via Infotrieve] |
18. | Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1997) Current Protocols in Molecular Biology , pp. 20.5.1-20.5.9, John Wiley & Sons, Inc., New York |
19. | Sayeski, P. P., Ali, M. S., Harp, J. B., and Bernstein, K. E. (1998) Circ. Res. 81, 1279-1288 |
20. | Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489[Abstract] |
21. | Shin, T. H., and Kudlow, J. E. (1994) Mol. Endocrinol. 8, 704-712[Abstract] |
22. |
Sayeski, P. P.,
Wang, D.,
Su, K.,
Han, I. O.,
and Kudlow, J. E.
(1997)
Nucleic Acids Res.
25,
1458-1466 |
23. |
Sakai, I.,
and Kraft, A. S.
(1997)
J. Biol. Chem.
272,
12350-12358 |
24. | Schindler, C., and Darnell, J. E., Jr. (1995) Annu. Rev. Biochem. 64, 621-651[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Darnell, J. E., Jr.
(1997)
Science
277,
1630-1635 |
26. | Lane, D. P., and Crawford, L. V. (1979) Nature 278, 261-263[Medline] [Order article via Infotrieve] |
27. | DeCaprio, J. A., Ludlow, J. W., Figge, J., Shew, J. Y., Huang, C. M., Lee, W. H., Marsillo, E., Paucha, E., and Livingston, D. M. (1988) Cell 54, 275-283[Medline] [Order article via Infotrieve] |
28. | Fazioli, F., Minichiello, L., Matoskova, B., Wong, W. T., and Di Fiore, P. P. (1993) Mol. Cell. Biol. 13, 5814-5828[Abstract] |
29. |
Yu, C. L.,
Jin, Y. J.,
and Burakoff, S. J.
(2000)
J. Biol. Chem.
275,
599-604 |
30. | Yi, W., Kim, S. O., Jiang, J., Park, S. H., Kraft, A. S., Waxman, D. J., and Frank, S. J. (1996) Mol. Endocrinol. 10, 1425-1443[Abstract] |
31. | Chen, W. S., Lazar, C. S., Poenie, M., Tsien, R. Y., Gill, G. N., and Rosenfeld, M. G. (1987) Nature 328, 820-823[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Cacalano, N. A.,
Migone, T. S.,
Bazan, F.,
Hanson, E. P.,
Chen, M.,
Candotti, F.,
O'Shea, J. J.,
and Johnston, J. A.
(1999)
EMBO J.
18,
1549-1558 |
33. |
Herrington, J.,
Rui, L.,
Luo, G., Yu-,
Lee, L. Y.,
and Carter-Su, C.
(1999)
J. Biol. Chem.
274,
5138-5145 |
34. |
Strehlow, I.,
and Schindler, C.
(1998)
J. Biol. Chem.
273,
28049-28056 |
35. |
Ali, M. S.,
Sayeski, P. P.,
and Bernstein, K. E.
(2000)
J. Biol. Chem.
275,
15586-15593 |