Requirement of Ca2+ and PKC{delta} 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{delta} since a selective PKC{delta} but not PKC{alpha} inhibitor and dominant-negative PKC{delta} 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{delta}, 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{delta}, 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-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{delta}, and their target PYK2 are essential for the G protein-coupled AT1 receptor-mediated JAK2 activation in VSMCs.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo). AngII at a concentration as low as 10 nM stimulated JAK2 phosphorylation, whereas 100 nM AngII maximally stimulated JAK2 phosphorylation (Fig. 1BGo).



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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.

 
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. 1CGo, 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. 1CGo, 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 2AGo 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 10–30 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. 2BGo). 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. 2CGo, 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.

 
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 3Go 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.

 
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. 4AGo, 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. 4AGo). GF109203X is an inhibitor of several PKC isoforms, and pretreatment of this agent markedly inhibited AngII-induced JAK2 phosphorylation (Fig. 4BGo). 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.

 
Involvement of PKC{delta} 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{delta} inhibitor (29), and Go6976 is a selective PKC{alpha} and -ß inhibitor (30). As shown in Fig. 5AGo, 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{delta} in AngII-induced JAK2 activation, we transfected VSMCs with adenovirus encoding dominant-negative PKC{delta} and stimulated with AngII. As shown in Fig. 5BGo, dominant-negative PKC{delta} transfection inhibited AngII-induced JAK2 phosphorylation in a concentration-dependent manner. Dominant-negative PKC{delta} transfection had no inhibitory effect on basal JAK2 phosphorylation. We could detect expression of PKC{alpha} (Fig. 5CGo) but not PKCßI or -ßII in our VSMCs. Dominant-negative PKC{alpha} transfection also had no inhibitory effect on AngII-induced JAK2 phosphorylation (Fig. 5CGo). In addition, AngII rapidly increased the amount of PKC{delta} in the membrane fraction of VSMCs that was markedly inhibited by rottlerin, thus confirming PKC{delta} activation by AngII and its inhibition by rottlerin (Fig. 6AGo). Rottlerin also inhibited PKC{delta} activity as assessed by its in vitro autophosphorylation (Fig. 6BGo). These results strongly indicate that the PKC isoform required for JAK2 activation by AngII is the PKC{delta} isoform.



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Figure 5. Involvement of PKC{delta} in AngII-Induced JAK2 Activation

A, VSMCs were pretreated with a PKC{delta} isoform inhibitor, rottlerin (10 µM), a PKC{alpha} 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{delta} 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{delta} antibody. C, Cells were transfected with adenovirus expressing dominant-negative PKC{alpha} [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{alpha} antibody. Results are representative of at least three separate experiments giving similar results.

 


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Figure 6. Effects of Rottlerin on PKC{delta} Translocation and Autophosphorylation

A, VSMCs were pretreated with or without rottlerin (10 µM) for 30 min, and cells were stimulated with AngII for 3 min and the membrane was prepared. The samples were immunoblotted by anti-PKC{delta} antibody. B, Recombinant PKC{delta} autophosphorylation was detected with or without rottlerin (10 µM) incubation. Results are representative of at least three separate experiments giving similar results.

 
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{delta} inhibitor, rottlerin, as well as dominant-negative PKC{delta} transfection, markedly inhibited AngII-induced PYK2 phosphorylation at Tyr402 (Fig. 7AGo), clearly indicating that in addition to Ca2+, PKC{delta} is involved in AngII-induced PYK2 activation. Also, JAK2 was constitutively associated with PYK2 before and after AngII stimulation as detected by coimmunoprecipitation (Fig. 7BGo).



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Figure 7. Role of PKC{delta} 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{delta} [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.

 
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. 8AGo, 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. 8BGo). 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{delta}, 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The major finding of this study is that intracellular Ca2+ elevation and PKC{delta} 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{delta}-mediated JAK2 activation as illustrated in Fig. 9Go. 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.

 
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{delta}, is expressed abundantly in VSMCs (38). By using PKC isotype inhibitors (Go6976, rottlerin), and dominant-negative PKC{delta} and PKC{alpha} overexpression, we found that PKC{delta} 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{delta} associates with different nonreceptor tyrosine kinases such as Src and Abl as a result of agonist-induced PKC{delta} tyrosine phosphorylation and thereby activates these tyrosine kinases (39, 40). Thus, whether a similar mechanism operates AngII-induced JAK2 activation through PKC{delta} remains to be studied.

The interesting question is how do Ca2+ and PKC{delta} 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{delta} 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{delta} (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{delta} 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{delta}, 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha} antibody, anti-PKCßI antibody, anti-PKCßII antibody, and anti-PKC{delta} antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Human recombinant PKC{delta} 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 3–12 were used in the experiments and showed 99% positive immunostaining with smooth muscle {alpha}-actin antibody (Sigma). For subsequent experiments, cells at 80–90% 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{delta} and PKC{alpha} 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{delta} antibody (20 ng/ml), and anti-PKC{alpha} 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{delta} Membrane Translocation
PKC{delta} 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{delta} antibody.

In Vitro Kinase Assay for PKC{delta}
Autophosphorylation of PKC{delta} was detected as previously described. In brief, human recombinant PKC{delta} (30 µg/ml) was incubated in kinase buffer containing 10 mM MgCl2 and 2.5 µCi [{gamma}-32P]ATP for 20 min at 30 C. Phosphorylated PKC{delta} 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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 DISCUSSION
 MATERIALS AND METHODS
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