The Angiotensin II–Dependent Association of Jak2 and c-Src Requires the N-Terminus of Jak2 and the SH2 Domain of c-Src
Abstract—The binding of angiotensin II (Ang II) to AT1 is known to increase the kinase activity of several nonreceptor tyrosine kinases including Jak2 and c-Src. In the present study, we demonstrate that treatment of vascular smooth muscle cells with Ang II results in a rapid and transient association of Jak2 and c-Src. This association is dependent on a catalytically active Jak2 kinase, because it is blocked both by pharmacological means and by the inability of a catalytically inactive Jak2 to associate with c-Src. c-Src bound tyrosine phosphorylated Jak2 but was unable to bind an equal amount of unphosphorylated Jak2 protein, indicating that the SH2 domain of c-Src mediates this association. In vivo studies indicated that c-Src binds the N-terminus of Jak2 as expression of a Jak2 molecule lacking the initial 240 amino acids, including 16 tyrosines, and was unable to bind c-Src. Lastly, using transiently transfected COS-7 cells, we found that Ang II treatment induced an association between c-Src and wild-type Jak2 but not between c-Src and the Jak2 molecule that lacks the initial 240 amino acids. Thus, our data suggest that in addition to increasing the kinase activities Jak2 and c-Src, treatment of cells with Ang II results in the physical association of Jak2 and c-Src; an association that is mediated by the SH2 domain of c-Src and the N-terminus of Jak2.
Angiotensin II (Ang II) is the effector molecule of the renin-angiotensin system.1 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. Activation of AT1 by Ang II results in the activation of several nonreceptor tyrosine kinases. For example, treatment of vascular smooth muscle cells (VSMCs) with Ang II activates two known Jak family tyrosine kinases: Jak2 and Tyk2.2 Similarly, treatment of cells with Ang II activates two known Src family tyrosine kinases: c-Src and Fyn.3 4
The Jak family of nonreceptor tyrosine kinases includes Jak1, Jak2, Jak3, and Tyk2. Each protein is ≈130 kDa in mass and contains seven conserved Jak homology domains (JH1 through JH7). The Jak kinases were initially shown to induce gene transcription through the signal transducers and activators of transcription (STATs).5 More recently, however, the Jaks have also been shown to signal through classical signaling pathways such as the Shc/Grb2/Sos pathway and through the serine/threonine kinase Raf-1.6 7 Unlike almost all other protein tyrosine kinases, members of the Jak family bear no SH2 or SH3 domains. In contrast to the Jaks, members of the Src family of nonreceptor tyrosine kinases are ≈55 to 62 kDa in mass and do possess SH2 and SH3 domains. Although the expression of most Src family members is restricted to hematopoietic cells, c-Src and Fyn are expressed in most tissues including vascular smooth muscle. The Src family of tyrosine kinases were initially shown to signal through the Shc/Grb2/Sos signaling pathway but more recent studies indicate that they play a critical role in transcriptional regulation by phosphorylating the STAT proteins.8 9 10
Because the Jak and Src families of nonreceptor tyrosine kinases phosphorylate identical substrates, we hypothesized that treatment of cells with Ang II might result in a ligand-dependent association of Jak2 and c-Src. Such a physical association would allow these very different nonreceptor tyrosine kinases to phosphorylate the same substrate. In this study, we demonstrate that treatment of VSMCs with Ang II does in fact lead to a ligand-dependent association of Jak2 and c-Src, an association that is mediated by an amino terminus Jak2 phosphotyrosine and the SH2 domain of c-Src. These studies support the hypothesis of a ligand-dependent physical association of the Jak and Src nonreceptor tyrosine kinases and, for the first time, define the region of each molecule that mediates this specific physical interaction.
Materials and Methods
VSMCs were grown in DMEM+10% FBS at 37°C in a 5% CO2 humidified atmosphere and used between passages 8 to 22; 100-mm dishes at ≈75% confluence were growth-arrested by incubation in serum-free DMEM for 48 hours before use. COS-7 cells were cultured in the same medium. BSC-40 cells were grown in DMEM+10% newborn calf serum. Cell culture reagents were obtained from Gibco/BRL. Inhibitors were purchased from Calbiochem. Losartan was a generous gift from Dupont Merck. All other reagents were purchased from Sigma Chemical Co.
Plasmid and GST Fusion Protein Constructs
pBOSwtJk2 (wild-type Jak2 [Jak2 WT]) and pBODJK2ΔVIII (dominant-negative Jak2 [Jak2 DN]) were a generous gift from Dr Don M. Wojchowski (Pennsylvania State University) and were previously described.11 Construction of the pRC-Jak2-WT, pRC-Jak2-ATD, and pRC-Jak2-PKD plasmids has been described elsewhere.12 The pRC-Jak2-AFL vector was made by digesting pRC-Jak2-WT with AflII and closing with ligase. The vector expressing wild-type AT1 cDNA (pZeo/AT1) has been previously described.13 The c-Src fusion proteins GST/SH2, GST/SH3, and GST/SH2+SH3 were a generous gift from Dr Sarah J. Parsons (University of Virginia).
COS-7 cells were transiently transfected exactly as previously described.13 For BSC-40 cell transfection, cells were seeded in 100-mm dishes and transfected at near confluency with either 4 μg pRC-Jak2-WT, 16 μg pRC-Jak2-ATD, 16 μg pRC-Jak2-AFL, or 8 μg pRC-Jak2-PKD in the presence of 20 μL lipofectin. Cells were infected with vaccinia virus clone vTF7-3 as described below. The total amount of transfected plasmid was normalized using empty expression vector.
Vaccinia Virus–Mediated Jak2 Overexpression
Jak2 constructs were overexpressed using the vaccinia virus–mediated transfection/infection protocol.14 15 Briefly, 100-mm dishes of nearly confluent BSC-40 cells were transfected as described. After 4 hours, vTF7-3 was added at a multiplicity of infection of 1.0 and incubated for 1 hour. The medium was removed, and cells were incubated overnight in DMEM+10% newborn calf serum. At 18 to 20 hours after infection, lysates were prepared and samples were immunoprecipitated as described. Vaccinia clone vTF7-3 was generously provided by Dr Bernard Moss (National Institutes of Health).
GST Fusion Protein Expression and Purification
The GST/Src fusion proteins and GST controls were expressed and purified using Glutathione Sepharose 4B (Pharmacia Biotech) as previously described.13
Protein-Protein Complex Formation and GST Pull-Down Assays
To prepare lysates, cells were washed with two volumes of ice-cold PBS containing 1 mmol/L Na3VO4 and lysed in 1.0 mL ice-cold gentle lysis buffer (25 mmol/L Tris [pH 7.5], 10% glycerol, 1% NP-40, 140 mmol/L NaCl, and protease inhibitors). Lysates were gently sonicated and incubated on ice for 1 hour. Samples were spun at 12 000g for 5 minutes at 4°C, and supernatants were normalized using the Dc protein assay (Bio-Rad). Normalized lysates (≈400 μg/mL) were immunoprecipitated with 2 μg of antibody and 20 μL of a 50% slurry of Protein A/G Plus agarose beads (Santa Cruz Biotechnology) for 6 to 16 hours at 4°C. The immunoprecipitating anti-Src (N-16) and anti-Jak2 (HR758) polyclonal antibodies were purchased from Santa Cruz Biotechnology. Immune complexes were washed 3 times with wash buffer (25 mmol/L Tris [pH 7.5], 150 mmol/L NaCl, and 0.1% Triton X-100) and resuspended in SDS sample buffer. For GST/Src pull-down assays, COS-7 cell lysates were precleared with 5 μg of Sepharose-bound GST for 1 hour at 4°C. To each sample, 0.2 μg of Sepharose-bound GST or GST/Src fusion protein was added and incubated for 30 to 60 minutes at 4°C. Beads were washed 4 to 5 times with wash buffer and resuspended in sample buffer. All sample buffer–containing proteins were separated on SDS-PAGE (National Diagnostics) and transferred onto nitrocellulose membranes (Schleicher & Scheull).
Membranes were blotted as previously described.13 Proteins were visualized with enhanced chemiluminescence according to the manufacturer’s instructions (Amersham). Protein bands were quantitated using UN-SCAN-IT digitizing software (Silk Scientific). Western blotting polyclonal anti-Jak2 and monoclonal anti-phosphotyrosine (clone 4G10) were from Upstate Biotechnology. Monoclonal anti-pp60c-src (clone GD-11) was a generous gift from Dr Sarah J. Parsons (University of Virginia).
Ang II–Dependent Association of Jak2 and c-Src
Ang II, acting through the seven-transmembrane AT1, has been shown to activate Jak2 and c-Src kinase activities as measured by autophosphorylation or phosphorylation of synthetic substrates.2 3 To determine whether Ang II could induce a physical association between Jak2 and c-Src, quiescent VSMCs were treated with Ang II for 0 to 60 minutes. The cell lysates were then immunoprecipitated with anti-Src-pAb (Src N16), which does not cross-react with other Src tyrosine kinases, and the immunoprecipitates were Western-blotted with anti-Jak2-pAb (Figure 1A⇓). These data demonstrated an Ang II–dependent association between Jak2 and c-Src. We confirmed equal precipitations across all lanes by blotting the same membrane with anti-Src-mAb (Figure 1A⇓). The three experiments represented in Figure 1A⇓ were quantitated by densitometry and plotted as a function of fold increase with respect to unstimulated controls (Figure 1B⇓). We found that maximal association between Jak2 and c-Src was ≈10-fold more than unstimulated controls and occurred 5 to 40 minutes after Ang II treatment. To demonstrate antibody specificity, we repeated the experiments but immunoprecipitated the lysates with either the same anti-Src-pAb or with control rabbit immunoglobulin (Ig) G. The use of IgG resulted in a loss of specific Jak2 signal that correlated with the lack of precipitated c-Src protein (Figure 1C⇓). In experiments in which we reversed the order of antibody addition (immunoprecipitated VSMC lysates with anti-Jak2-pAb and Western-blotted with anti-Src-mAb), we observed a similar Ang II–dependent association between Jak2 and c-Src (data not shown). The Ang II–dependent association of Jak2 and c-Src appears specific for these two molecules, because no association was observed when anti-Src-pAb immunoprecipitates were Western-blotted with anti-Jak1-mAb (data not shown).
Finally, to determine whether AT1 was mediating the Ang II–dependent association of Jak2 and c-Src, we pretreated VSMCs with the AT1-specific inhibitor losartan. Cells were then treated with Ang II, and the resulting anti-Src-pAb immunoprecipitates were Western-blotted with anti-Jak2-pAb (Figure 1D⇑). Losartan completely blocked the Ang II–dependent association of Jak2 and c-Src. However, the loss of Jak2 signal was not due to decreased immunoprecipitation of c-Src, given that blotting the same membrane with anti-Src-mAb demonstrated roughly equal precipitation (Figure 1D⇑). Collectively, the data in Figure 1⇑ indicate that treatment of VSMCs with Ang II results in an AT1-specific, ligand-dependent association of Jak2 and c-Src.
A Catalytically Active Jak2 Kinase Is Required for Jak2/c-Src Association
To determine whether the kinase activity of Jak2 and/or c-Src was required for this association, quiescent VSMCs were pretreated with either DMSO (control), the Jak2 kinase inhibitor AG-490, or the c-Src specific kinase inhibitor PP1.16 17 The concentration and the duration of inhibitor pretreatment used in the present study were based on our published and unpublished work that defined the lowest doses that fully inhibited Jak2 and c-Src kinase activities, respectively.18 19 Cells were then left untreated or treated with Ang II for the indicated times. The resulting lysates were immunoprecipitated with anti-Src-pAb and Western-blotted with anti-Jak2-pAb to assess the Ang II–dependent association of Jak2 and c-Src (Figure 2A⇓). An Ang II–dependent association was seen in the control cells and the PP1-treated cells. However, ligand-dependent association between Jak2 and c-Src was blocked by AG-490. To verify that AG-490 did not inhibit the immunoprecipitation of c-Src, we blotted the same membrane with anti-Src-mAb and confirmed that all lanes were precipitated equally well (Figure 2A⇓). To further test the inhibitory effect of AG-490 and the role of Jak2 kinase in mediating the association between Jak2 and c-Src, we pretreated VSMCs with varying doses of AG-490 and then stimulated them with Ang II. The anti-Src-pAb immunoprecipitates were Western-blotted with anti-Jak2-pAb to assess the Ang II–dependent association of Jak2 and c-Src (Figure 2B⇓). AG-490 blocked the Ang II–dependent association of Jak2 and c-Src in a dose-dependent manner, with maximal inhibition occurring at 100 μmol/L. This is the same dose that fully inhibits the Ang II–induced Jak2 tyrosine phosphorylation in VSMCs (data not shown).
The data in Figure 2A⇑ and 2B⇑ suggested that the catalytic activity of Jak2, but not c-Src, is required for the Ang II–dependent association of Jak2 and c-Src. This hypothesis was tested with an alternative approach that made use of a Jak2 DN protein. The dominant-negative construct contains two point mutations in the kinase domain rendering the molecule catalytically inactive and thus incapable of autophosphorylation.11 In this experiment, COS-7 cells were transiently transfected to express either no Jak2 (empty vector control), Jak2 WT, or the dominant negative Jak2 protein (Jak2 DN). The large amount of transfected plasmid used in this experiment results in Jak2 autophosphorylation through oligomerization of the Jak2 molecules rather than by a ligand dependent mechanism.18 The resulting lysates were then immunoprecipitated with anti-Jak2-pAb, separated on SDS-PAGE, and transferred onto nitrocellulose. The membrane was first blotted with anti-Jak2-pAb to confirm equal precipitation of both the wild-type and dominant-negative proteins (Figure 2C⇑, top). We then examined the level of tyrosine phosphorylation of the Jak2 proteins by blotting the same membrane with anti-Tyr(P)-mAb (Figure 2C⇑, middle). As expected, Jak2 WT was heavily phosphorylated on tyrosine whereas Jak2 DN was not. Finally, the same membrane was blotted with anti-Src-mAb to evaluate Jak2/Src association (Figure 2C⇑, bottom). We repeatedly observed that c-Src specifically bound kinase-active, tyrosine-phosphorylated Jak2 WT but not the equally abundant kinase-inactive, unphosphorylated Jak2 DN protein. The level of Jak2/Src association was quantitated in several experiments (Figure 2D⇑). Cells transfected with Jak2 WT, but not Jak2 DN, were found to have significantly increased Jak2/Src association compared with empty vector controls. Collectively, the data in Figure 2⇑ indicate that a catalytically active Jak2 protein, capable of tyrosine autophosphorylation, is necessary for efficient association of Jak2 and c-Src in vivo.
The SH2 Domain of c-Src Binds Jak2 in a Tyrosine Phosphorylation–Dependent Manner
To determine which region(s) of c-Src binds Jak2, we performed Jak2 pull-down experiments using GST/Src fusion proteins composed of either the SH2, SH3, or SH2+SH3 domains of c-Src. That is, we again transfected COS-7 cells with either empty vector control, Jak2 WT, or Jak2 DN. The resulting lysates were normalized, divided equally, and incubated with either GST, GST/SH2, GST/SH3, or GST/SH2+SH3. The fusion proteins were collected by centrifugation, separated on SDS-PAGE, and transferred onto nitrocellulose. The membrane was then blotted with anti-Jak2-pAb to measure Jak2 binding (Figure 3A⇓). We observed that only the fusion proteins containing the SH2 domain of c-Src bound Jak2, and this binding was tyrosine phosphorylation–dependent. That is, the SH2 domain of c-Src bound only the kinase-active, tyrosine-phosphorylated Jak2 WT but did not bind the equally abundant kinase-inactive, unphosphorylated Jak2 DN protein. To eliminate the possibility that the Jak2 DN protein was not expressed, equivalent amounts of cell lysate from each of the three transfected conditions were immunoblotted with anti-Jak2-pAb (Figure 3B⇓). Both Jak2 proteins were expressed at similar levels. Thus, the data in Figure 3⇓ strongly suggest that the region of c-Src that binds Jak2 is the SH2 domain, and this binding is tyrosine phosphorylation–dependent.
The N-Terminus of Jak2 Is Required for Binding c-Src In Vivo
The data in Figure 2⇑ demonstrate that Jak2 must possess a functional kinase domain to efficiently bind c-Src. Interestingly, previous work in other cell systems has shown that the N-terminus of Jak2 (JH7 and JH6 domains) mediates specific protein-protein interactions between Jak2 and other signaling molecules.20 21 In addition, work using Jak1/Jak2 chimeras suggested that the N-terminus of Jak2 mediates specific protein-protein interactions whereas the kinase domain simply phosphorylates substrate that is bound by the N-terminus of Jak2.22 We therefore hypothesized that the N-terminus of Jak2 is required for binding c-Src. To test this, we made several Jak2 N-terminal deletant constructs (Figure 4A⇓). We then used the vaccinia virus/T7 bacteriophage expression system to generate high-level expression of all constructs. This allowed for the rapid mapping of the region of Jak2 that binds c-Src. Specifically, we transiently transfected BSC-40 cells with the indicated Jak2 cDNA and then subsequently infected cells with vaccinia virus clone vTF7-3 to significantly increase protein expression. At 18 to 20 hours after infection, cells were lysed and immunoprecipitated with anti-Src-pAb. After separation on SDS-PAGE and transfer onto nitrocellulose, the samples were blotted with anti-Jak2-pAb to assess Jak2/c-Src association. Cells transfected with empty vector alone (pRC) contained a single, nonspecific 94-kDa band, presumably from the vaccinia virus (Figure 4B⇓). We observed that Jak2 WT (pRC-WT), the AflII deletant (pRC-AFL), and the pseudokinase deletant (pRC-PKD) all coimmunoprecipitated with c-Src. In contrast, the amino terminal deletant (ATD) did not. We confirmed equal precipitation of c-Src by blotting the same membrane with anti-Src-mAb (Figure 4B⇓). To rule out the possibility that the ATD protein was being masked by the 94-kDa nonspecific band, we repeated the experiment but reversed the order of antibody addition. Specifically, BSC-40 cells were transfected/infected with the same constructs, but this time we immunoprecipitated with anti-Jak2-pAb and then Western-blotted with anti-Src-mAb. This protocol clearly showed that c-Src did not coimmunoprecipitate with the ATD deletant whereas it could associate with either Jak2 WT or the other two deletant constructs (Figure 4C⇓, top). To demonstrate that all Jak2 constructs were precipitated equally, we blotted the same samples with anti-Jak2-pAb (Figure 4C⇓, middle). In the experiment shown, both the AFL and ATD deletants were precipitated at levels below that of WT and PKD. However, both the AFL and ATD proteins were precipitated at similar levels; the AFL deletant associated with c-Src whereas the ATD protein did not. Finally, because Figure 3⇑ indicated that the SH2 domain of c-Src bound Jak2 in a tyrosine phosphorylation–dependent manner, we Western-blotted these Jak2 immunoprecipitates with anti-Tyr(P)-mAb to measure the tyrosine phosphorylation of each Jak2 protein (Figure 4C⇓, bottom). Other than the ATD construct, all Jak2 proteins contained some readily visible degree of tyrosine phosphorylation. With prolonged exposure, we could see some tyrosine phosphorylation of Jak2 ATD, but it was significantly less than the other deletant proteins (data not shown). The lack of tyrosine phosphorylation of the ATD construct is not due to a lack of catalytic activity of the molecule because expression of similar ATD Jak2 proteins has been shown to be catalytically active and capable of autophosphorylation.21 23 In addition, we have demonstrated that the ATD mutant mediates the Ang II–dependent tyrosine phosphorylation of STAT1 in a manner that is no different than Jak2 WT (data not shown). Collectively, the data in Figure 4⇓ indicate that the amino terminal 240 amino acids of Jak2 are required for binding c-Src in vivo. Furthermore, expression of the Jak2 molecule lacking the initial 240 amino acids (the c-Src binding region) is not heavily phosphorylated on tyrosine, supporting the idea that the interaction between Jak2 and c-Src is dependent on a Jak2 N-terminal phosphotyrosine and the SH2 domain of c-Src.
The N-Terminus of Jak2 Is Required to Bind c-Src in Response to Ang II
The data in Figure 4⇑ indicate that the N-terminus of Jak2 is required for efficient binding of c-Src in vivo. However, those experiments were done in the absence of Ang II. To determine whether the N-terminus of Jak2 is required for an Ang II–mediated association between Jak2 and c-Src, we transiently transfected COS-7 cells with cDNAs encoding either AT1 and Jak2 WT or AT1 and Jak2 ATD. In contrast to the COS-7 cell transfections done in Figures 2C⇑, 3A⇑, and 3B⇑, the amount of Jak2 plasmid used here (Figure 5⇓) was significantly less to avoid Jak2 autophosphorylation in the absence of ligand. At 48 hours after transfection, the quiescent COS-7 cells were stimulated with Ang II, and the resulting lysates were immunoprecipitated and blotted with anti-Jak2-pAb to assess Jak2 protein expression (Figure 5⇓, top). Both Jak2 WT and Jak2 ATD protein expression were observed as was the endogenous Jak2 protein found in COS-7 cells.12 20 The samples were then blotted with anti-Tyr(P)-mAb to measure the Ang II–induced tyrosine phosphorylation of both Jak2 WT and Jak2 ATD (Figure 5⇓, middle). In these experiments, the Jak2 WT protein was tyrosine-phosphorylated in response to Ang II whereas the ligand-dependent tyrosine phosphorylation of the Jak2 ATD protein was significantly less. Finally, the membrane was blotted with anti-Src-mAb to assess Jak2/c-Src association (Figure 5⇓, bottom). Cells that were transfected with Jak2 WT and treated with Ang II bound more c-Src than similarly treated cells transfected with Jak2 ATD. These results indicate that the N-terminus of Jak2 is required for efficient binding of c-Src in response to Ang II.
One hypothesis that is consistent with these findings is that in response to Ang II, a catalytically active Jak2 autophosphorylates tyrosines at the N-terminus of the molecule. This region then binds the SH2 domain of c-Src, allowing for ligand-dependent association of Jak2 and c-Src. The time course of Jak2/c-Src association appears to coincide with the duration of Ang II–mediated Jak2 tyrosine phosphorylation as previously described.2 13 As indicated earlier, one functional consequence of this ligand-dependent binding would be that both Jak2 and c-Src would be physically colocalized and could therefore act on the same substrate. Several molecules are known to be phosphorylated by both the Jak and Src families of tyrosine kinases. These include STAT1, STAT3, Raf-1, and Shc.6 7 8 9 Interestingly, it appears that the Jak and Src kinases may act synergistically, because they phosphorylate different tyrosine residues on a given substrate.7 With respect to Ang II, a recent report indicated that STAT1, Jak2, and Fyn form a ligand-dependent protein complex, in vitro.24 These data support the hypothesis of an Ang II–dependent physical colocalization of the Jak and Src family tyrosine kinases with a putative substrate. However, the specific protein domains that mediate this complex, the requirement for catalytically active Jak2 and the demonstration of an in vivo STAT1:Jak2:Fyn ternary complex, were not presented.
A natural question that might be posed from our data is whether Ang II mediates the formation of a ternary complex among Jak2:Src:AT1. We have previously demonstrated that Jak2 binds AT1 in response to Ang II.2 13 18 The present observation that Jak2 binds c-Src in response to Ang II lends itself to the possibility that some c-Src protein may also bind AT1 as well. This is currently under investigation.
Finally, these data are the first to demonstrate that in response to Ang II, Jak2 and c-Src form a specific protein-protein complex. We have defined the requirement of a catalytically active Jak2 for this association and the regions that mediate this binding. The data support the hypothesis that the N-terminus of Jak2 mediates specific protein-protein interaction and suggest that the tyrosine phosphorylation of the N-terminus of Jak2 is critical for this physical interaction consisting of a Jak2 phosphotyrosine and the SH2 domain of c-Src.
This work was supported by National Institutes of Health grants DK39777, DK44280, DK45215, DK51445, and HL47035. Dr Sayeski has been supported by grants T32-DK07298 and F32-HL09678. We wish to thank Shaun Benford for administrative assistance.
- Received February 1, 1999.
- Accepted April 16, 1999.
- © 1999 American Heart Association, Inc.
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