Role of Gβ-Subunit in Angiotensin II-Type 1 Receptor Signaling

Biochemical and Biophysical Research Communications 280, 756 –760 (2001) doi:10.1006/bbrc.2000.4222, available online at http://www.idealibrary.com on

Role of G␤-Subunit in Angiotensin II-Type 1 Receptor Signaling Maren Luchtefeld, Helmut Drexler, and Bernhard Schieffer 1 Abteilung Kardiologie und Angiologie, Medizinische Hochschule Hannover, Hannover, Germany

Received December 26, 2000

The G-protein-coupled angiotensin II-type 1 (AT1) receptor activates the mitogen-activated protein (MAP) kinase cascade and the Janus kinase 2/signal transducers and activators of transcription (JAK2/ STAT) cascade via tyrosine phosphorylation. Recent observations indicated that the G␤-subunit of heterotrimeric G-proteins interacts with tyrosine phosphorylated proteins. We investigated whether angiotensin II (ANG II) activates MAP-kinases and JAK/STAT cascades via the G␤-subunit. In rat aortic smooth muscle (RASM) cells we found phosphorylated proteins associated with the G␤-subunit SHC (Sequence Homology of Collagen) and JAK2. We demonstrate that JAK2 activity increased upon G␤-binding. The activity of pp60 c-src kinase also increased, but upon activation pp60 c-src dissociates from the G␤-complex. Immunoprecipitations revealed that SHC forms a complex with JAK2. Blockade of JAK2 with AG490 abolished this complex formation; therefore, JAK2 may be the kinase responsible for SHC phosphorylation. Thus, the G␤subunit may play a pivotal role in AT1-receptor signaling by connecting signaling cascades leading to cell growth and differentiation. © 2001 Academic Press Key Words: angiotensin II; JAK2; pp60c-src; SHC; G␤; AT1-receptor.

Cell growth, differentiation, and cell-to-cell communication are regulated by release and activation of intracellular signaling proteins in response to their distinct receptors. Upon stimulation by a ligand of cell surface receptors, signaling proteins become activated by a variety of mechanisms, including reversible phosphorylation (1). Traditionally these reversible phosphorylation events were described for growth factor This work was supported by grants from the Deutsche Forschungsgemeinschaft (Schie 386/3-1 and Dre 147/9-1). 1 To whom correspondence and reprint requests should be addressed at Abteilung Kardiologie und Angiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Stra␤e 1, 30625 Hannover, Germany. Fax: ⫹49-511-532-5412. E-mail: Schieffer.Bernhard@ MH-Hannover.de. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

receptors, such as platelet derived growth factor (PDGF) and fibroblast growth factor (FGF) (2, 3). These receptors contain extracellular ligand binding domains and a large cytoplasmic catalytic domain with intrinsic tyrosine kinase activity (4). Upon ligand binding, autophosphorylation of the intracellular domain occurs on defined tyrosine residues which activates downstream effector molecules. Characteristic proteins, such phospholipase C␥1 (PLC␥1), SHC (sequence homology of collagen) and growth factor receptor binding protein 2 (GRB2) contain one or more Src homology 2 (SH2) domains (4). However, recent observations suggested that seven transmembrane G-protein coupled receptors, although lacking intrinsic tyrosine kinase domains, activate signaling proteins via tyrosine phosphorylation (5). These events were described for vasoactive peptides e.g., ANG II, arginin– vasopressin, endothelin, and bradykinin (6, 7). However, the mechanisms involved in the receptormediated induction of protein tyrosine phosphorylation remained unknown. Intracellular signaling events stimulated by the G-protein coupled AT1-receptor are critically dependent on intracellular tyrosine phosphorylation events (4). Upon AT1-receptor stimulation, ANG II activates Ca 2⫹ from intracellular stores via PLC␥1-dependent generation of 1,4,5-inositol trisphosphate (IP 3) and diacylglycerol (DAG). PLC␥1 becomes tyrosine phosphorylated in vascular smooth muscle cells in response to AT1-receptor activation and associates with the AT1receptor (8). This activation appears to be downstream of pp60 c-src activation. Thus, one of the earliest signaling events of the AT1-receptors seems to be the activation of pp60 c-src (9). Moreover, ANG II was shown to be a growth promoting factor like PDGF and EGF. This growth stimulation involves the activation of p21 ras (10). Recent observations demonstrated that this activation is also dependent on pp60 c-src activation and thereby regulates the activity of p21 ras (9). Stimulation of the AT1-receptor further activates the cytokine-specific Janus kinase (JAK) and signal

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transducers and activators of transcription (STAT) pathway (11). The JAK/STAT cascade connects activated cell surface receptors to nuclear transcriptional changes, leading to cell growth and differentiation (12, 13). This pathway was first elucidated for IL-6-family cytokines, but recent observations indicated that activation of the AT1-receptor by ANG II induces the rapid tyrosine phosphorylation of JAK2-kinase (14) and subsequently the phosphorylation of its substrates STAT1, STAT2, and STAT3 factors. Although a variety of downstream signaling events were identified in the past, the mechanism by which the G-protein coupled AT1-receptor activates these signaling pathways still remains unclear. In this regard, recent observations indicated that the G␤-subunit of heterotrimeric G-proteins interacts with tyrosine phosphorylated proteins, such as p184 neu and EGF-receptor, which leads to the activation of downstream signaling molecules. Therefore, we investigated the role of the G␤-subunit in connecting the early ANG II-AT1receptor signaling events. Here we were able to show that ANG II-stimulation of the AT1-receptor resulted in the binding of upstream signaling proteins e.g., JAK2, SHC and pp60 c-src to the G␤-subunit of the heterotrimeric G-protein and that pp60 c-src, once activated by tyrosine phosphorylation, dissociates from the G␤protein, whereby JAK2 and SHC remains associated with the G␤-subunit. MATERIALS AND METHODS Reagents. Tween 20, acrylamide, sodium dodecyl sulfate (SDS), N,N⬘-methylene-bisacrylamide, N,N,N⬘,N⬘-tetramethylenediamine and nitrocellulose membranes were purchased from Bio-Rad Laboratories. Molecular weight standards, immunoprecipitin, protein A– and G–agarose, Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum, trypsin and all medium additives were obtained from Life Technologies, Inc. Monoclonal anti-phosphotyrosine (4G10), polyclonal JAK2, SHC, pp60 c-src, G␤-antibody were obtained from Santa Cruz Biotechnology, Inc. AG-490 was from Calbiochem. [ 32P]Orthophosphate, the enhanced chemiluminescence kit was from Amersham Corp. Angiotensin II, goat anti-mouse IgG, and all other chemicals were purchased from Sigma Chemical Corp. Cell culture. Rat aortic smooth muscle (RASM) cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 10 mg/ml streptomycin, and 100 U/ml penicillin. Cells were grown to 75– 85% confluence and growth arrested in serum-free DMEM for 48 h prior to us (14). Immunoprecipitation and Western blotting. RASM cells were stimulated with ANG II for times indicated. Immunoprecipitation and Western blotting were performed as described previously (9, 8, 11). To immunoprecipitate proteins we used the following antibodies: anti-phosphotyrosine (PY20 clone, 10 ␮g/ml lysate), anti-G␤ (1 ␮g/ ml), anti-JAK2 (1 ␮g/ml). The recovered immunoprecipitates were separated by SDS–PAGE, transferred to nitrocellulose membrane and blotted with anti-JAK2, anti-SHC, anti-pp60 c-src, or phosphotyrosine antibodies. Proteins were visualized by enhanced chemiluminescence. In vitro kinase assay. JAK2 and pp60 c-src tyrosine kinases activity were measured by autophosphorylation as reported previously (11). In brief, following ANG II stimulation, JAK2 or pp60 c-src was immu-

FIG. 1. Time course of ANG II-stimulated G␤-associated proteins. RASM cells were labeled with [ 32P]-orthophosphate and stimulated with ANG II. At indicated times, cells were harvested and G␤-subunit was immunoprecipitated. G␤-associated proteins were separated by SDS–PAGE, transferred to membranes analyzed by using specific antisera and exposed to autoradiography. The molecular mass standards are indicated on the right of each figure. Arrows indicate the proteins of interest. In the lower panel, identical experiments were performed in the presence of pertussis toxin (10 ng/ml, PTX).

noprecipitated and washed in kinase buffer. The pellet was resuspended in kinase buffer and allowed to autophosphorylate in vitro in presence of 15 ␮mol/L ATP. Samples were separated by SDS–PAGE, transferred to nitrocellulose membrane, and probed with phosphotyrosine antisera. The activities of JAK2 or pp60 c-src were visualized by enhanced chemiluminescence.

RESULTS Stimulation of the AT1-receptor in RASM cells with ANG II resulted in the rapid induction of protein phosphorylation (4, 5, 16). RASM cells were labeled with [ 32P]orthophosphate and stimulated with ANG II. The lysates were immunoprecipitated with G␤-antisera (Fig. 1, upper panel). Western blot analysis revealed the phosphorylation of proteins at 72 and 130 kDa size as early as 30 s after ANG II-stimulation. Preincubation with pertussis toxin (PTX), a Gi-protein inhibitor, had no effect on protein phosphorylation (Fig. 1, lower panel). Proteins were subsequently identified by specific antibodies as JAK2 and SHC. Immunoprecipitations with G␤-antibodies followed by Western Blot analysis with JAK2-antisera revealed that JAK2 transiently binds to the G␤-subunit upon ANG IIstimulation. In vitro kinase assays with JAK2 immunoprecipitations revealed that JAK2 tyrosine kinase activity increases in parallel to its G␤-binding (Fig. 2, lower panel). In contrast, pp60 c-src kinase seems to be constitutively bound to the G␤-subunit and dissociates from the receptor upon ANG II. Subsequently, time course of p60 c-src kinase activity demonstrated that

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FIG. 2. Time course of ANG II-induced of G␤-JAK2 complex formation and activation. RASM cells were stimulated with ANG II for the indicated times. Cells were harvested and G␤-subunit was immunoprecipitated. Western blot analysis was performed with JAK2 antisera and visualized by enhanced chemiluminescence. The molecular mass standards are indicated on the right of each figure. In the lower panel, in vitro kinase assay was performed from JAK2immunoprecipitations, indicating the synergistic JAK2 activation in parallel to the G␤-JAK2 complex formation.

pp60 c-src kinase activity increased following dissociation of the G␤-subunit (Fig. 3, lower panel). Moreover, SHC a connector protein to the ras-raf-MAP kinase cascade, forms a complex with the G␤-subunit upon ANG IIstimulation (Fig. 4, left panel). Since the AT1-receptor activates different kinds of heterotrimeric G-proteins, like Gq or Gi, we tested, which G-protein was responsible for these complex formations. PTX is a known inhibitor of Gi-proteins, therefore the sensitivity of these complex formations to PTX was tested. Preincubation with PTX had no influence on G␤-JAK2-SHC– complex formation (Fig. 4, right panel). Subsequently, JAK2 complex formation with SHC was investigated in JAK2 immunoprecipitations. Results revealed that SHC forms a complex with JAK2 (Fig. 5). Preincubation with PTX had no influ-

FIG. 3. Time course of ANG II-induced of G␤-pp60 c-src complex formation and activation. RASM cells were stimulated with ANG II for the indicated times. Cells were harvested and the G␤-subunit was immunoprecipitated. Western blot analysis was performed with pp60 c-src antisera and visualized by enhanced chemiluminescence. Molecular mass standards are indicated on the right of each figure. In the lower panel, in vitro kinase assay was performed from pp60 c-src-immunoprecipitations, indicating the synergistic activation of pp60 c-src in parallel to the G␤– pp60 c-src complex formation.

FIG. 4. Time course of ANG II-induced G␤-SHC complex formation. RASM cells were stimulated with ANG II for the indicated times. Cells were harvested and G␤-subunit was immunoprecipitated. Western blot analysis was performed with SHC antisera and visualized by enhanced chemiluminescence (left panel). In the right panel, SHC phosphorylation was tested after preincubation with pertussis toxin (PTX), a G␣-subunit inhibitor. The molecular mass standards are indicated on the right of each figure. Results demonstrate that PTX had no influence on G␤-SHC complex formation.

ence on the complex formation. Blockade of the tyrosine kinase activity by genistein abolished complex formation of JAK2 and SHC, indicating that complex formation of JAK2-SHC depends on JAK2 kinase activity. In fact, experiments using the JAK2 inhibitor AG490 (17) revealed, that complex formation of JAK2SHC can be abolished by JAK2 blockade (Fig. 5). Thus, early phosphorylation events of the AT1-receptor are mediated by the interaction of the G␤-subunit with the tyrosine kinases JAK2 and pp60 c-src. Activation of the connector protein SHC seems to be dependent on JAK2 kinase. DISCUSSION The present study demonstrated, that the G␤subunit associates with JAK2, SHC and pp60 c-src. Fur-

FIG. 5. Time course of ANG II-induced JAK2-SHC complex formation. RASM cells were stimulated with ANG II for the indicated times. Complex formation of JAK2-SHC was analyzed by immunoprecipitation with JAK2 specific antibody. Pretreatment of RASM cells with DMSO 10% alone, a carrier for the specific inhibitors, or with PTX, a specific inhibitor for Gi-proteins did not influence complex formation of JAK2 and SHC. In contrary to this findings genistein, an unspecific tyrosine kinase inhibitor, or AG490 a JAK2 specific kinase inhibitor abolished complex formation. Molecular mass standards are indicated on the right. Results demonstrate, that PTX or DMSO had no influence on JAK-SHC complex formation, whereas AG490 and Genistein abolished complex formation of JAK2 and SHC.

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thermore, JAK2 and SHC become tyrosine phosphorylated upon ANG II-AT1-receptor stimulation within minutes. Phosphorylation of JAK2 and SHC occurs while binding to the G␤-subunit. This process seems to be independent of Gi-proteins, since PTX influenced neither protein phosphorylation of SHC, nor protein phosphorylation of JAK2. JAK2-G␤-complex formation is associated with an increasing JAK2 kinase activity. In contrast, pp60 c-src seems to be constitutively bound to the G␤-subunit, while activation of pp60 c-src by ANG II resulted in a dissociation from the G␤-subunit. JAK2–SHC complex formation seems to be dependent on JAK2 kinase activity since the JAK2-kinase inhibitor (AG 490) abolished JAK2-SHC complex formation. These findings are consistent with the notation, that JAK2 induced SHC phosphorylation depends on G␤-subunit, thereby connecting formerly unconnectable pathways and leading to the activation of the p21 ras-Raf-MAP-Kinase cascade. However, JAK2 is unable to bind SHC directly, since JAK2 lacks SH3 domains, necessitated for SHC binding. Therefore, it is tempting to speculate that a connector protein is needed for JAK2 induced SHC activation. In contrast to JAK2, pp60 c-src contains a SH3 domain and therefore may act as the kinase which phosphorylates SHC. Sayeski and co-workers (18) demonstrated, that a SRC-kinase rapidly associates with JAK2 upon ANG II stimulation depending on the phosphorylation state of JAK2. In addition, Sadoshima and co-workers demonstrated that Fyn, another member of the SRC kinase family, activates SHC by tyrosine phosphorylation (7). Here, we demonstrate that JAK2 binds to G␤-subunit upon ANG II stimulation and that its binding to the receptor induces kinase activity. We also show, that pp60 c-src seems to be constitutively associated with G␤-subunit and upon activation this tyrosine kinase dissociate from G␤. This complex formation is associated with an increasing kinase activity. Therefore, we suggest a model in which pp60 c-src— upon activation— binds to phosphorylated JAK2 and both kinases stimulate synergistically their downstream targets. SHC a connector protein of the RasRaf-Sos-pathway was shown to be activated by c-SRC in COS7 cells (19). We suggest that SHC could bind to pp60 c-src, where it will be activated. Previous studies have been demonstrated that, upon AT1-receptor activation, JAK2 and pp60 c-src will be activated. The findings of the presents study are consistent with the notion that the G␤-subunit may be the missing link between a classic G-protein coupled receptor and signaling proteins activated by tyrosine phosphorylation. We present a potential link in early signaling events between the JAK/STAT-pathway and the MAP-kinase cascade after ANG II-stimulation in RASM cells,

whereby JAK2 seems to be the kinase connecting these formerly unconnectable pathways. Consistent with this hypothesis are our recent observations that the pharmacological JAK2 inhibitor AG490 inhibits ANG II-induced RASM cells proliferation and DNA synthesis by abolishing Raf-1 kinase activity (14). Several investigators have demonstrated, that a PHdomain is required for binding to the G␤-subunit (20, 21). Since JAK2 does not contain a PH-domain, additional adapter proteins seem to be required for the binding of JAK2 to the G␤-subunit. The identification of this potential adapter protein needs further investigations. ACKNOWLEDGMENT The authors thank Nicole Brauer for her technical assistance.

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