Requirement of Ca2+ and PKC
for Janus Kinase 2 Activation by Angiotensin II: Involvement of PYK2
Gerald D. Frank,
Shuichi Saito,
Evangeline D. Motley,
Terukatsu Sasaki,
Motoi Ohba,
Toshio Kuroki,
Tadashi Inagami and
Satoru Eguchi
Department of Biochemistry (G.D.F., S.S., T.I., S.E.), Vanderbilt
University School of Medicine, Nashville, Tennessee 37232; Department
of Anatomy and Physiology (E.D.M.), Meharry Medical College, Nashville,
Tennessee 37208; Department of Biochemistry (T.S.), Sapporo Medical
University, Sapporo 060-8556, Japan; and Institute of Molecular
Oncology (M.O., T.K.), Showa University, Tokyo 142-8555, Japan
Address all correspondence and requests for reprints to: Satoru Eguchi, M.D., Ph.D., Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232. E-mail:
satoru.eguchi{at}vanderbilt.edu
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ABSTRACT
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In vascular smooth muscle cells, angiotensin II (AngII) stimulates
association of its G protein-coupled AngII type 1 (AT1)
receptor with Janus kinase 2 (JAK2), resulting in the activation of
signal transducer and activator of transcription proteins. Although the
association and activation of subsequent signal transducer and
activator of transcription proteins appear to prerequire JAK2
activation, the signaling mechanism by which the AT1
receptor activates JAK2 remains uncertain. Here, we have examined the
signaling mechanism required for JAK2 activation by AngII in vascular
smooth muscle cells. We found that AngII, through the
AT1 receptor, rapidly stimulated JAK2 phosphorylation at
Tyr1007/1008, the critical sites for the kinase activation.
By using selective agonists and inhibitors, we demonstrated that PLC
and its derived signaling molecules, phosphatidylinositol
triphosphate/Ca2+ and diacylglycerol/PKC, were essential
for AngII-induced JAK2 phosphorylation. The PKC isoform required for
JAK2 activation appears to be PKC
since a selective PKC
but not
PKC
/ß inhibitor and dominant-negative PKC
overexpression
inhibited JAK2 activation. We further examined a link between JAK2 and
a Ca2+/PKC-sensitive tyrosine kinase, PYK2. We found
that PYK2 activation by AngII requires PKC
, and that PYK2 associates
with JAK2 constitutively. Moreover, transfection of two distinct PYK2
dominant-negative mutants markedly inhibited AngII-induced JAK2
activation. From these data we conclude that AT1-derived
signaling molecules, specifically Ca2+ and PKC
,
participate in AngII-induced JAK2 activation through PYK2. These data
provide a new mechanistic insight by which the hormone AngII exerts its
cytokine-like actions in mediating vascular remodeling.
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INTRODUCTION
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ANGIOTENSIN II (AngII) is a multifunctional
peptide hormone that not only controls cardiovascular homeostasis but
also promotes growth of its target cells. In cultured vascular smooth
muscle cells (VSMCs), cardiac myocytes, cardiac fibroblasts, and renal
mesangial cells, AngII has been shown to promote hypertrophy and/or
hyperplasia (1, 2). Thus, it is widely believed that AngII
plays a major role in the pathophysiology of cardiovascular remodeling
linked to the development of hypertension, atherosclerosis, and
restenosis after angioplastic injury (3, 4, 5).
VSMCs predominantly express the AngII type 1
(AT1) receptor, a G protein-coupled receptor
(GPCR) that is responsible for transducing the growth-promoting signal
evoked by AngII. The AT1 receptor coupled to Gq
leads to Ca2+ mobilization and PKC activation as
a result of PLC hydrolysis-generated second messengers,
phosphatidylinositol triphosphate (IP3)
and diacylglycerol, respectively (6). These second
messengers generated through the AT1 receptor
likely contribute to activation of downstream tyrosine and
serine/threonine kinases (6, 7, 8).
Recently, we and others have identified several key tyrosine kinases
activated by the AT1 receptor in VSMCs. These
tyrosine kinases belong to the family of receptor (epidermal growth
factor receptor and platelet-derived growth factor receptor) (9, 10) and nonreceptor [c-Src, PYK2, and Janus kinase 2
(JAK2) (11, 12, 13, 14)] tyrosine kinases. The JAK family kinases
consist of JAK1, JAK2, JAK3, and TYK2. JAK activation is required for
the initiation of multiple signaling pathways, including the signal
transducers and activators of transcription (STAT) pathway in response
to cytokine receptors (15, 16). However, it was found that
AngII stimulates association of JAK2 with the AT1
receptor and activates JAK2 in VSMCs (14), cardiac
myocytes (17), and renal mesangial cells
(18), which may partly explain the cytokine-like actions
of AngII in mediating cardiovascular remodeling. In addition, JAK2 is
required for cell growth induced by AngII in VSMCs (19),
further supporting the important role of JAK2 in the signal
transduction of AngII.
Similar to the mechanism by which cytokine receptors activate JAK/STAT,
it has been suggested that the AT1 receptor
provides docking sites for JAKs, thus initiating activation of the
JAK/STAT pathway (14, 20). In fact, Ali et al.
(21) demonstrated that the carboxyl-terminal region
containing the YIPP motif of the AT1 receptor
physically binds to JAK2 after AngII stimulation, supporting the
hypothesis. However, the same group subsequently showed that JAK2 must
first be catalytically active and autophosphorylated before JAK2 forms
a complex with the AT1 receptor
(22). In addition to AngII, a variety of GPCR agonists
(thrombin, serotonin, TSH-stimulating hormone, and
-MSH) have
recently been shown to activate JAK/STAT pathways
(23, 24, 25), further indicating that recruitment of JAK2
through the YIPP region cannot be a mechanism by which a GPCR activates
JAK2. However, there has been no study demonstrating mechanistically
how a GPCR such as the AT1 receptor activates
JAK2, or whether second messengers of the AT1
receptor, such as Ca2+ and diacylglycerol, are
required for AngII-induced JAK2 activation.
In this study, we hypothesized that JAK2 activation through the
AT1 receptor requires these second messengers in
VSMCs. Here, we demonstrate several lines of evidence showing that
Ca2+, PKC
, and their target PYK2 are essential
for the G protein-coupled AT1 receptor-mediated
JAK2 activation in VSMCs.
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RESULTS
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JAK2 Phosphorylation at Tyr1007/1008 Through the
AT1 Receptor
Two adjacent tyrosine residues of JAK2 at position 1,007 and 1,008
are believed to be autophosphorylation sites, and the tyrosine
phosphorylation at 1,007 is essential for JAK2 kinase activity
(26). To assess JAK2 activation in response to AngII, the
phosphorylation of JAK2 at Tyr1,007/1,008 was
measured by immunoblotting with a phosphospecific antibody that
selectively recognizes Tyr1,007/1,008
dual-phosphorylated JAK2 (27). AngII stimulated JAK2
phosphorylation as early as 1 min and maximally at 3 min (Fig. 1A
). AngII at a concentration as low as
10 nM stimulated JAK2 phosphorylation, whereas 100
nM AngII maximally stimulated JAK2 phosphorylation (Fig. 1B
).

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Figure 1. AngII-Induced JAK2 Phosphorylation at
Tyr1,007/1,008 Through the AT1 Receptor
Cells were stimulated with AngII (100 nM) at the indicated
time periods (panel A) and concentrations (panel B). The cell lysates
were immunoblotted by phosphospecific JAK2 antibody and anti-JAK2
antibody. C, Cells were pretreated with or without the AT1
antagonist, CV11974 (1 µM), for 30 min and stimulated
with either AngII (100 nM) for 3 min, EGF (50 ng/ml) for
the indicated time periods, or thrombin (10 U/ml) for 3 min. The cell
lysates were immunoblotted by phosphospecific JAK2 antibody and
anti-JAK2 antibody, as indicated. Results are representative of at
least three separate experiments giving similar results.
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Given the evidence that the AT1 receptor
specifically associates with JAK2 (14), we investigated
whether the AT1 receptor selectively activates
JAK2 or whether GPCRs such as the thrombin receptor can also activate
it in VSMCs. As shown in Fig. 1C
, AngII-induced JAK2 phosphorylation
was completely inhibited by the AT1 receptor
antagonist CV11974. In addition, thrombin markedly induced JAK2
phosphorylation. Since we have shown that AngII rapidly transactivates
the epidermal growth factor (EGF) receptor (10), we
further tested whether the EGF receptor was involved in
AngII-induced JAK2 activation. As shown in Fig. 1C
, EGF had no
effect on JAK2 phosphorylation. Moreover, AngII-induced JAK2
phosphorylation was not inhibited by AG1478 (250 nM, 30 min
pretreatment, data not shown), an EGF receptor kinase inhibitor the
concentration of which markedly inhibits AngII- induced ERK
activation (10). These results suggest that the
AT1 receptor, and not the EGF receptor, is
involved in the activation of JAK2 by AngII.
Role of Ca2+ and PKC in AngII-Induced JAK2
Activation
In VSMCs, the AT1 receptor activates
phosphatidylinositol-specific PLC, leading to intracellular
Ca2+ mobilization and PKC activation
(6). Figure 2A
shows that
edelfosine, a selective phosphatidylinositol-dependent PLC inhibitor
(28), markedly inhibited AngII-induced JAK2
phosphorylation, indicating that PLC is required for AngII-induced JAK2
activation in VSMCs. To test whether PLC-linked second messengers
participate in AngII-induced JAK2 activation, we stimulated VSMCs with
a Ca2+ ionophore, A23187, or a PKC activator,
phorbol 12-myristate 13-acetate (PMA). Both A23187 and PMA markedly
enhanced JAK2 phosphorylation at 1030 min. However, when VSMCs were
treated with both A23187 and PMA, there was a shift in the
phosphorylation of JAK2 to an early time point of 3 min (Fig. 2B
).
Alternatively, VSMCs were stimulated with A23187, PMA, or AngII and
immunoprecipitated with antiphosphotyrosine antibody and immunoblotted
by anti-JAK2 antibody to detect activation of JAK2. As shown in Fig. 2C
, A23187, PMA, and AngII enhanced the amount of JAK2
coprecipitated with antiphosphotyrosine antibody.

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Figure 2. Effect of the PLC Inhibitor, Edelfosine, a
Ca2+ Ionophore, A23187, or a Phorbol Ester, PMA, on JAK2
Phosphorylation
A, VSMCs were pretreated with or without edelfosine (25
µM) for 30 min and stimulated with AngII (100
nM) for 3 min. The cell lysates were immunoblotted by
phosphospecific JAK2 antibody and anti-JAK2 antibody. B, VSMCs were
stimulated with or without either the Ca2+ ionophore,
A23187 (10 µM), the PKC activator, PMA (100
nM), or a combination of both at various time periods. The
cell lysates were immunoblotted by phosphospecific JAK2 antibody and
anti-JAK2 antibody. C, Cells were stimulated with or without A23187 (10
µM) and PMA (100 nM) for 10 min or AngII (100
nM) for 3 min. Cell lysates were immunoprecipitated with
antiphosphotyrosine antibody and immunoblotted with anti-JAK2 antibody.
Results are representative of at least three separate experiments
giving similar results.
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To confirm the involvement of Ca2+ and PKC in the
activation of JAK2, we stimulated cells with AngII or a combination of
A23187 and PMA and immunostained them with phosphospecific JAK2
antibody. Figure 3
shows that AngII and a
combination of A23187 and PMA markedly enhanced JAK2 phosphorylation
specifically at the cytoplasm as is indicated by the increased
fluorescence.

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Figure 3. Immunostaining of JAK2 Phosphorylation in VSMCs
Cells were stimulated with or without AngII (100 nM) for 3
min or the combination of Ca2+ ionophore, A23187 (10
µM), and the PKC activator, PMA (100 nM), for
10 min. Fixed cells were incubated with phosphospecific JAK2 antibody.
Each picture panel shows fluorescent immunostaining of phosphorylated
JAK2 visualized by Cy3-conjugated secondary antibody. Results are
representative of at least three separate experiments giving similar
results.
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To further examine the role of intracellular
Ca2+ elevation and PKC activation in the
activation of JAK2 by AngII, we treated VSMCs with specific inhibitors,
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid acetoxymethyl ester (BAPTA-AM),
[8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate,
HCl] (TMB-8), or GF10920X. As shown in Fig. 4A
, BAPTA-AM, an intracellular
Ca2+ chelator, markedly reduced both basal
and AngII-stimulated JAK2 phosphorylation, whereas there remained a
noticeable AngII response. TMB-8, an intracellular
Ca2+ antagonist that blocks the release of
Ca2+ from intracellular stores, markedly but not
completely inhibited AngII-induced JAK2 phosphorylation (Fig. 4A
).
GF109203X is an inhibitor of several PKC isoforms, and pretreatment of
this agent markedly inhibited AngII-induced JAK2 phosphorylation (Fig. 4B
). Taken together, these results suggest that both
Ca2+ and PKC activated by second messengers of
PLC are involved in the activation of JAK2 by AngII.

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Figure 4. Effect of Intracellular Ca2+ and PKC
Inhibitors on AngII-Induced JAK2 Activation
A, VSMCs were pretreated with either the intracellular Ca2+
chelator BAPTA-AM (10 µM), the intracellular
Ca2+ antagonist TMB-8 (1 µM), or their
vehicle DMS0 (0.1%) for 30 min, and stimulated with AngII (100
nM) for 3 min. The cell lysates were immunoblotted by
phosphospecific JAK2 antibody and anti-JAK2 antibody. B, Cells were
pretreated with a selective PKC inhibitor, GF109203X (2
µM), or its vehicle, DMS0 (0.1%), for 30 min and
stimulated with AngII (100 nM) for 3 min. The cell lysates
were immunoblotted by phosphospecific JAK2 antibody and anti-JAK2
antibody. Results are representative of at least three separate
experiments giving similar results.
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Involvement of PKC
in AngII-Induced JAK2 Activation
We next determined the isoform(s) of PKC involved in AngII-induced
JAK2 activation by using selective PKC subtype inhibitors. VSMCs were
pretreated with rottlerin or Go6976 and stimulated with AngII.
Rottlerin is a selective PKC
inhibitor (29), and Go6976
is a selective PKC
and -ß inhibitor (30). As shown in
Fig. 5A
, rottlerin markedly inhibited
AngII-induced JAK2 phosphorylation. However, Go6976 had no effect on
AngII-induced JAK2 phosphorylation. These PKC inhibitors have been
shown to be selective toward their targets at the concentrations used
in this study (29, 30). To confirm these pharmacological
data, which support the involvement of PKC
in AngII-induced JAK2
activation, we transfected VSMCs with adenovirus encoding
dominant-negative PKC
and stimulated with AngII. As shown in
Fig. 5B
, dominant-negative PKC
transfection inhibited AngII-induced
JAK2 phosphorylation in a concentration-dependent manner.
Dominant-negative PKC
transfection had no inhibitory effect on basal
JAK2 phosphorylation. We could detect expression of PKC
(Fig. 5C
)
but not PKCßI or -ßII in our VSMCs. Dominant-negative PKC
transfection also had no inhibitory effect on AngII-induced JAK2
phosphorylation (Fig. 5C
). In addition, AngII rapidly increased the
amount of PKC
in the membrane fraction of VSMCs that was markedly
inhibited by rottlerin, thus confirming PKC
activation by AngII and
its inhibition by rottlerin (Fig. 6A
).
Rottlerin also inhibited PKC
activity as assessed by its in
vitro autophosphorylation (Fig. 6B
). These results strongly
indicate that the PKC isoform required for JAK2 activation by AngII is
the PKC
isoform.

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Figure 5. Involvement of PKC in AngII-Induced JAK2
Activation
A, VSMCs were pretreated with a PKC isoform inhibitor, rottlerin (10
µM), a PKC and -ß isoform inhibitor, Go6976 (2
µM), or their vehicle dimethylsulfoxide (0.1%) for 30
min and stimulated with AngII (100 nM) for 3 min. The cell
lysates were immunoblotted by phosphospecific JAK2 antibody and
anti-JAK2 antibody. B, Cells were transfected with adenovirus
expressing dominant-negative PKC for 48 h and stimulated with
AngII (100 nM) for 3 min. The cell lysates were
immunoblotted by phosphospecific JAK2 antibody, anti-JAK2 antibody, and
anti-PKC antibody. C, Cells were transfected with adenovirus
expressing dominant-negative PKC [100 moi (multiplicity of
infection)] for 48 h and stimulated with AngII (100
nM) for 3 min. The cell lysates were immunoblotted by
phosphospecific JAK2 antibody, anti-JAK2 antibody, and anti-PKC
antibody. Results are representative of at least three separate
experiments giving similar results.
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Involvement of PYK2 in AngII-Induced Activation of JAK2
We and others have previously shown that PYK2 activation by
AngII requires Ca2+ and/or PKC in VSMCs
(13, 31). These data prompted us to investigate the
possible link between PYK2 and JAK2 in AngII-stimulated VSMCs.
Tyr402 is one of the major autophosphorylation
sites of PYK2 (32). PYK2 Tyr402
phosphorylation was detected by the antibody that has been shown to
selectively recognize Tyr402-phosphorylated PYK2
in our VSMCs (33, 34). The PKC
inhibitor,
rottlerin, as well as dominant-negative PKC
transfection,
markedly inhibited AngII-induced PYK2 phosphorylation at
Tyr402 (Fig. 7A
),
clearly indicating that in addition to Ca2+,
PKC
is involved in AngII-induced PYK2 activation. Also, JAK2 was
constitutively associated with PYK2 before and after AngII stimulation
as detected by coimmunoprecipitation (Fig. 7B
).

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Figure 7. Role of PKC in PYK2 Activation and Association
of PYK2 with JAK2
A, After pretreatment with rottlerin (10 µM) for 30 min
or transfection with adenovirus expressing dominant-negative PKC
[100 moi (multiplicity of infection)] for 48 h, cells were
stimulated with AngII (100 nM) for 3 min. The cell lysates
were immunoprecipitated with anti-PYK2 antibody and immunoblotted with
phospho Tyr402-PYK2 antibody and anti-PYK2 antibody. B,
Cells were stimulated with or without AngII (100 nM) for 3
min. The cell lysates were immunoprecipitated with anti-PYK2 antibody
and immunoblotted with anti-JAK2 and anti-PYK2 antibody. Results are
representative of at least three separate experiments giving similar
results.
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To test the contribution of PYK2 on AngII-induced JAK2 activation,
VSMCs were transfected with adenovirus vector encoding PYK2
kinase-inactive mutant, K457A, or its autophosphorylation site mutant,
Y402F, and then stimulated with AngII. As shown in Fig. 8A
, overexpression of PYK2
kinase-inactive mutant markedly inhibited AngII-induced JAK2
phosphorylation, whereas it had no effect on the basal phosphorylation.
The PYK2 autophosphorylation site mutant also markedly inhibited
AngII-induced JAK2 phosphorylation compared with the control cells
transfected with vector alone (Fig. 8B
). In contrast, pretreatment with
the JAK2 kinase inhibitor, AG490 (10 µmol/liter, 30 min), the
condition that has been shown to inhibit JAK2 function in cultured rat
VSMCs (35), had no inhibitory effect on AngII-induced PYK2
phosphorylation at Tyr402 (data not shown). Taken
together, these data suggest that AT1-derived
signaling molecules, specifically Ca2+ and
PKC
, participate in AngII-induced JAK2 activation through its
association partner, PYK2.

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Figure 8. Involvement of PYK2 in AngII-Induced Activation of
JAK2
A, After transfection with adenoviral vector encoding K457A-PYK2 at 10
moi (multiplicity of infection) for 48 h, cells were stimulated
with AngII (100 nM) for 3 min. B, After transfection with
adenoviral vector encoding Y402F-PYK2 for 48 h at 10 moi, cells
were stimulated with AngII (100 nM) for 3 min. The cell
lysates were immunoblotted by phosphospecific JAK2 antibody, anti-JAK2
antibody, and anti-PYK2 antibody. Results are representative of at
least three separate experiments giving similar results.
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DISCUSSION
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The major finding of this study is that intracellular
Ca2+ elevation and PKC
activation initiated by
PLC-derived second messengers are involved in AngII-induced
JAK2 activation in VSMCs. We further found that PYK2 is required for
Ca2+- and PKC
-mediated JAK2 activation as
illustrated in Fig. 9
. Thus, our data
provide a new mechanistic insight by which AT1, a
GPCR, signals to JAK2, the kinase that explains the cytokine-like
actions of AngII in mediating vascular remodeling.

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Figure 9. Hypothetical Pathway of G Protein-Coupled
AT1 Receptor-Mediated JAK2 Activation in VSMCs
According to this model, AT1-derived second messengers,
Ca2+ and diacylglycerol, signal to PYK2 and thereby
activate JAK2 in VSMCs.
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In addition to the AT1 receptor, several distinct
GPCRs have recently been shown to activate JAK2. However, no reports
are available that reveal the general mechanism by which GPCRs activate
JAK2. In the present study, we have shown that eldelfosine, a
phosphatidylinositol-specific PLC inhibitor, inhibited AngII-induced
JAK2 phosphorylation at Tyr1,007/1,008,
suggesting the involvement of the Gq/PLC pathway in the activation of
JAK2 by AngII. To support this idea, we have demonstrated for the first
time that the PLC-linked second messengers, Ca2+
and diacylglycerol, are critical for the AngII-induced JAK2 activation.
In line with our findings, thrombin stimulates tyrosine phosphorylation
of JAK2 in platelets via second messengers derived from PLC hydrolysis
(23), and AngII was recently shown to stimulate induction
of the sis-inducing factor and tyrosine phosphorylation of
STAT3 through a Gq/PLC/PKC-mediated pathway in cardiac myocytes
(36). Interestingly, in our VSMCs as well as platelets
(23), the combination of A23187 with PMA produced greater
effects than either stimulus alone. In VSMCs, BAPTA-AM only partially
inhibited AngII-induced JAK2 activation. Thus, collaborative action
of the PLC-derived second messengers (Ca2+ and
PKC) can be considered as the common mechanism of JAK2 activation
shared by GPCRs, including the AT1 receptor.
Contrary to our data, AngII has recently been shown to activate JAK2
through a mutated AT1 receptor overexpressed in
COS-7 cells that lack Gq coupling (37), indicating that
several distinct mechanisms for JAK2 activation are used in a cell
type-dependent manner.
The novel PKC isoform, PKC
, is expressed abundantly in VSMCs
(38). By using PKC isotype inhibitors (Go6976, rottlerin),
and dominant-negative PKC
and PKC
overexpression, we found that
PKC
is the essential isoform responsible for AngII-induced JAK2
activation. We also confirmed that AngII rapidly activates this isoform
in our VSMCs. Recent studies suggest that PKC
associates with
different nonreceptor tyrosine kinases such as Src and Abl as a result
of agonist-induced PKC
tyrosine phosphorylation and thereby
activates these tyrosine kinases (39, 40). Thus, whether a
similar mechanism operates AngII-induced JAK2 activation through PKC
remains to be studied.
The interesting question is how do Ca2+ and
PKC
converge to activate JAK2 by AngII? We have previously shown
that Ca2+-dependent EGF receptor transactivation
is required for AngII-mediated signaling in VSMCs (10). In
addition, EGF has been shown to activate JAK1 (26).
Therefore, we speculated that the EGF receptor transactivation by AngII
may participate in the activation of JAK2 in VSMCs. However, in the
present study, as well as in our recent publication (41),
we found that the EGF receptor is not involved in the AngII-induced
JAK2 activation. In this regard, we further investigated the role of
PYK2 because PYK2 activation by AngII also requires
Ca2+ and PKC in VSMCs (12, 13, 31).
Tyr402 is a putative autophosphorylation site of
PYK2, the phosphorylation of which is critical for downstream signaling
activation (32). Similar to JAK2, we found AngII-induced
PYK2 Tyr402 phosphorylation was inhibited by
rottlerin and dominant-negative PKC
overexpression. Recently, it has
been shown that PYK2 constitutively associates with JAK2 in embryonic
fibroblasts and mediates JAK-dependent signaling in response to
cytokines (42). In the present study, we have shown that
PYK2 is also associated with JAK2 constitutively. Although AG490, a
JAK2 kinase inhibitor, had no effect on AngII- induced PYK2
phosphorylation, both kinase-inactive and Tyr402
PYK2 mutants markedly inhibited AngII-induced JAK2
phosphorylation. Therefore, our findings strongly indicate that
PYK2 is a point of convergence for JAK2 activation through second
messengers derived from the AT1 receptor.
How might PYK2 activate JAK2? AngII has been shown to induce
association of JAK2 with Src family tyrosine kinases such as c-Src and
Fyn in VSMCs (35, 43, 44). The kinase activity of PYK2
autophosphorylates Tyr402 of PYK2 and thereby
recruits and activates Src family kinases (32). We and
others have shown that c-Src and Fyn are associated with PYK2 in
response to AngII (13, 31, 45). In addition, JAK2
activation by H2O2 requires
Fyn in fibroblasts (46). Therefore, we speculate that PYK2
may activate preassociated JAK2 through recruitment and activation of
Src family tyrosine kinases.
Recently, Schieffer et al. (47) showed that the
NADPH oxidase-generated O2- was
required for the AngII-activated JAK/STAT pathway in VSMCs.
Moreover, H2O2 has been
shown to activate the JAK-STAT pathway in cultured fibroblasts
(46, 48). Also,
H2O2 activates PKC
(49), which leads to the activation of a tyrosine kinase
such as c-Abl through their interactive phosphorylation events
(40). In this regard, we have shown that reactive oxygen
species are essential for AngII-induced tyrosine kinase activation such
as PYK2 in VSMCs (33, 50, 51). Therefore, in addition to
IP3 and diacylglycerol, reactive oxygen species may also act as second
messengers required for AngII-induced JAK2 activation through their
input on PKC
and PYK2.
JAK family kinases are critical for the normal cell-cell signaling
important for biological functions such as proliferation and
development (16). Deleting JAK1 and JAK2 by gene
disruption has been reported to be lethal perinatally and embryonically
(15). In VSMCs, the AngII-induced JAK/STAT pathway plays
an essential role in proliferation (19). Moreover, the
AT1 receptor-mediated JAK/STAT pathway was shown
to be involved in cardiac hypertrophy induced by pressure overload
(52), and neointima formation in balloon-injured rat
artery (53). These findings, together with our present
data, suggest a pathophysiological implication of JAK2 activation in
influencing cellular responses involved in the initiation and
development of cardiovascular diseases.
In conclusion, we have demonstrated that
AT1-derived signaling molecules,
Ca2+, PKC
, and PYK2 play critical roles in
AngII-induced JAK2 activation in VSMCs. The activation mechanism
presented here will provide new therapeutic targets the inhibition of
which attenuates cytokine-like actions of the AT1
receptor and thereby reduces cardiovascular remodeling.
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MATERIALS AND METHODS
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Reagents and Antibodies
AngII and thrombin were purchased from Sigma (St.
Louis, MO). PMA, A23187, AG1478, AG490, edelfosine, BAPTA-AM, TMB-8,
Go6976, rottlerin, and GF109203X were purchased from
Calbiochem (La Jolla, CA). The Takeda Pharmaceutical Co.
(Osaka, Japan) generously provided the AT1
antagonist, CV11974. Phosphospecific anti-JAK2 antibody and
phosphospecific anti-PYK2 antibody were purchased from BioSource International (Camarillo, CA), and anti-JAK2 antiserum and EGF
were purchased from Upstate Biotechnology, Inc. (Lake
Placid, NY). Anti-PYK2 antibody was purchased from Transduction Laboratories, Inc. (Lexington, KY). Antiphosphotyrosine
antibody, anti-PKC
antibody, anti-PKCßI antibody, anti-PKCßII
antibody, and anti-PKC
antibody were purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Human
recombinant PKC
was purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA).
Cell Culture
VSMCs were prepared from thoracic aorta of Sprague Dawley rats
by the explant method as previously described (54).
Subcultured cells from passages 312 were used in the experiments and
showed 99% positive immunostaining with smooth muscle
-actin
antibody (Sigma). For subsequent experiments, cells at
8090% confluency in culture wells were incubated with serum-free
medium for 3 d and stimulated with AngII or other agonists at 37
C.
Adenovirus Transfection
To make the recombinant adenovirus,
BstUI-EcoRI fragments of K457A and Y402F mutant rat
PYK2 cDNAs were blunt ended and inserted into the SwaI site
of the adenovirus cosmid cassette, pAxCAwt. Recombinant adenoviruses
were obtained by transfecting 293 cells with different cosmids together
with adenoviral DNA excised with EcoT221 (55).
The generation of PKC
and PKC
dominant-negative adenovirus is
described in detail elsewhere (56). VSMCs were infected
with adenovirus as previously described (57). Almost 100%
of VSMCs in culture were infected under our transfection condition
(57).
Immunoprecipitation
After stimulation, the cells were lysed with ice-cold
immunoprecipitation buffer (150 mM NaCl, 50 mM
HEPES, pH 7.5, 1% Triton X-100, 1 mM EDTA, 10
mM NaF, 10 mM sodium pyrophosphate, 2
mM sodium orthovanadate 10% (vol/vol) glycerol and 10
µg/ml of leupeptin, 10 µg/ml of aprotinin, and 1 mM
phenylmethylsulfonyl fluoride). The cell lysates were centrifuged, and
the supernatant was immunoprecipitated with the antibody and protein
A/G plus agarose at 4 C for 16 h as described previously
(10). The antibodies and their concentrations used in
immunoprecipitation are antiphosphotyrosine antibody (2 µg/ml),
anti-PYK2 antibody (2 µg/ml), and anti-JAK2 antiserum (3
µl/ml).
Immunoblot Analysis
Cell lysates or immune complex lysates were subjected to
SDS-PAGE gel electrophoresis and transferred to a nitrocellulose
membrane. The membrane was exposed to the primary antibodies overnight
at 4 C. Immunoreactive proteins were visualized by an enhanced
chemiluminescence detection kit (Amersham Pharmacia Biotech, Arlington Heights, IL) as described previously
(54). The antibodies and their concentrations used
in immunoblotting are phosphospecific anti-JAK2 antibody (50
ng/ml), phosphospecific anti-PYK2 antibody (50 ng/ml), anti-JAK2
antiserum (0.1 µl/ml), antiphosphotyrosine antibody (20 ng/ml),
anti-PYK2 antibody (25 ng/ml), anti-PKC
antibody (20 ng/ml), and
anti-PKC
antibody (20 ng/ml).
Immunohistochemistry
VSMCs grown on eight-well chamber slides were stimulated with
agonists for specified doses and durations. The stimulation was stopped
by a single wash in PBS and the cells were fixed in cold acetone for 5
min. The slides were allowed to dry for a few seconds and incubated for
30 min with the phosphospecific JAK2 antibody (2 µg/ml) in PBS with
0.2% BSA. After the slides were washed three times in PBS, they were
then incubated for 30 min with second antibody conjugated with Cy3
(Zymed Laboratories, Inc., South San Francisco, CA), a
fluorescent dye, and washed two times with PBS and once with water. The
slides were mounted in Vectashield (Vector Laboratories, Inc., Burlingame, CA), an antifade agent, and the immunostained
slides were viewed with a fluorescent microscope (Carl Zeiss, Thornwood, NY) essentially as previously described
(58).
PKC
Membrane Translocation
PKC
translocation, as determined by collection of the
membrane fraction, was essentially performed as described previously
(59). After stimulation with agonist, cells were lysed in
a buffer containing 20 mM Tris-HCl, pH 7.4, 5
mM EGTA, 0.1 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride, and 20 µM
leupeptin and then sonicated briefly. Afterward, the cell lysates were
centrifuged at 100,000 x g for 60 min at 4 C. The
pellet was solubilized in SDS-PAGE buffer containing 2-
mercaptoethanol. The sample was sonicated briefly and
immunoblotted with anti-PKC
antibody.
In Vitro Kinase Assay for PKC
Autophosphorylation of PKC
was detected as previously
described. In brief, human recombinant PKC
(30 µg/ml) was
incubated in kinase buffer containing 10 mM
MgCl2 and 2.5 µCi
[
-32P]ATP for 20 min at 30 C. Phosphorylated
PKC
was separated by SDS-PAGE and analyzed by autoradiography
(39, 40).
Reproducibility of Results
Unless stated otherwise, these results are representative of at
least three separate experiments yielding similar results.
 |
ACKNOWLEDGMENTS
|
---|
We thank Kunie Eguchi and Trinita Fitzgerald for their excellent
technical assistance.
 |
FOOTNOTES
|
---|
This work was supported in part by NIH Grants HL-58205 (to T.I.),
HL-03320 (to E.D.M.), and DK-20593 (to T.I.), a United Negro College
Fund/Merck Postdoctoral Science Research Fellowship (to G.D.F.), an
American Heart Association Scientist Development Grant (to S.E.), and
Vanderbilt University Diabetes Center Pilot and Feasibility
Proposal (to S.E.).
Abbreviations: AngII, Angiotensin II; AT1,
AngII type 1; BAPTA-AM,
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid acetoxymethyl ester; EGF, epidermal growth factor; GPCR, G
protein-coupled receptor; JAK, Janus kinase; PMA, phorbol 12-myristate
13-acetate; STAT, signal transducer and activator of transcription;
TMB-8,
[8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate,
HCl]; VSMC, vascular smooth muscle cells.
Received for publication July 10, 2001.
Accepted for publication October 5, 2001.
 |
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