Regulation of calcium-sensitive tyrosine kinase Pyk2 by angiotensin II in endothelial cells. Roles of Yes tyrosine kinase and tyrosine phosphatase SHP-2.

Calcium-sensitive tyrosine kinase Pyk2 has been implicated in the regulation of ion channels, cellular adhesion, and mitogenic and hypertrophic reactions. In this study, we have investigated the regulation of Pyk2 by angiotensin II (Ang II) in pulmonary vein endothelial cells. We found that the Ang II-induced tyrosine phosphorylation of Pyk2, which requires the activity of Src family kinase, was specifically regulated by the Src family kinase member, Yes kinase. Moreover, we identified for the first time the constitutive association of Pyk2 with an Src homology 2 (SH2) domain-containing tyrosine phosphatase SHP-2. SHP-2 interacts with Pyk2 through a region other than its SH2 domains. Pyk2 can be dephosphorylated in vitro in SHP-2 immunoprecipitates and in intact cells expressing an NH(2) terminus-truncated form of SHP-2, which lacks the two SH2 domains but has an enhanced phosphatase activity. Ang II activates the endogenous SHP-2. Finally, the SHP-2-mediated dephosphorylation of Pyk2 correlates with the negative effect of SHP-2 on the Ang II-induced activation of extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase. Thus, the balance of Pyk2 tyrosine phosphorylation in response to Ang II is controlled by Yes kinase and by a tyrosine phosphatase SHP-2 in endothelial cells.

Protein tyrosine phosphorylation and dephosphorylation are fundamental cellular signaling mechanisms that control cell growth and differentiation (1). Angiotensin II (Ang II) 1 type 1 receptor (AT 1 ) belongs to the superfamily of heterotrimeric G protein-coupled receptors (GPCR). The AT 1 receptor activates phospholipase C ␤ through G q to generate inositol trisphosphate and diacylglycerol, which in turn releases calcium from intracellular stores and activates protein kinase C, respectively (2). In recent years, the AT 1 receptor has been recognized to acti-vate some key protein-tyrosine kinases and initiate cascades of phosphotyrosine phosphorylation, which in turn activates cellular migration, adhesion, and mitogenic and hypertrophic reactions (3)(4)(5)(6). These tyrosine kinases include Src family kinases, calcium-sensitive tyrosine kinase Pyk2, epidermal growth factor (EGF) receptor, focal adhesion kinase (FAK), JAK kinases, and Tyk2. Emerging data suggest that Pyk2 and Src family kinases are critical in the early events of AT 1 receptor signaling (5)(6)(7).
Pyk2 (also called RAFTK for related adhesion focal tyrosine kinase, CAK␤ for cell adhesion kinase ␤, CADTK, and FAK2) is related to FAK and is activated by tyrosine phosphorylation in response to various agonists for GPCRs such as Ang II that increase intracellular calcium concentration (8 -10), stress stimuli (11), and membrane depolarization (8). Pyk2 has been implicated in the regulation of ion channels (8), cell adhesion and motility (12,13), extracellular signal-regulated kinase (ERK) (7,8,14,15), c-Jun NH 2 -terminal kinase (JNK) (9,11,(15)(16)(17)(18), and p70 S6 kinase (19). It has been shown that autophosphorylation of Pyk2 on Tyr-402 leads to binding of the SH2 domain of Src and subsequent Src activation and Pyk2 tyrosine phosphorylation in response to GPCRs in PC12 cells (14). Overexpression of Csk, a protein-tyrosine kinase that negatively regulates Src family kinase (20), or of a dominant negative mutant of Src, inhibited the Pyk2 tyrosine phosphorylation (14,15). Recent studies showed that the activated Src bound to Pyk2 may directly phosphorylate Pyk2 at Tyr-579, Tyr-580, and Tyr-881. In turn, this promotes Grb2 binding to Pyk2 and enhances Pyk2 kinase activity (15,21). Co-expression experiments indicate that Pyk2 can be phosphorylated by Src family kinase members Src, Fyn, and Yes, but not by Lck (21). In T cells, Pyk2 was found specifically associated with Fyn and was phosphorylated by Fyn during stimulation of T cell antigen receptor (22). It is likely that different members of Src family kinase may bind to Pyk2 and phosphorylate Pyk2 in response to different stimuli in different cell types. On the other hand, since tyrosine phosphorylation of Pyk2 is transient in response to various stimuli, it seems reasonable to expect involvement of a phosphatase in the tight control of this signaling event. However, this issue has not been addressed as far as we know.
We have recently reported the Fyn kinase-directed activation of the SH2 domain-containing protein-tyrosine phosphatase SHP-2 by Gi-coupled receptors in Madin-Darby Canine Kidney (MDCK) cells (23). Among the Src family members (Src, Fyn, Lck, Yes, Lyn) present in MDCK cells, Fyn was the only one specifically associated with SHP-2 through an intermediary molecule which can be phosphorylated by a Src family kinase. This suggests that Pyk2 may likely be the intermediary molecule. SHP-2 (previously known as SH-PTP2, PTP1D, Syp, PTP2C, and SHPTP3), a protein-tyrosine phosphatase with two Src homology 2 (SH2) domains at the NH 2 terminus, is expressed ubiquitously and is suggested to play an important role in the signal transduction of growth factor and insulin receptors (24). A targeted deletion of 65 amino acids in the NH 2 -terminal SH2 domain of SHP-2 leads to an embryonic lethality at midgestation in homozygous mutant mice (25). SHP-2 functions as either a positive or negative mitogenic regulator, depending on the specific receptor pathways stimulated (25)(26)(27)(28)(29)(30). However, the mechanisms and the active sites of SHP-2 are still not clear.
Since the endothelium is now being recognized as a target of renin-angiotensin system and endothelial dysfunction plays important roles in the pathogenesis of atherosclerosis and other cardiovascular diseases, elucidation of the signal mechanisms of the endothelial AT 1 receptor is essential for understanding the physiological and pathological effects of Ang II in endothelial cells. In the present study, we have investigated the regulation of Ang II-induced activation of Pyk2, a key tyrosine kinase in the early events of AT 1 receptor signaling, using pulmonary vein endothelial cells. We found that the Ang II-induced tyrosine phosphorylation of Pyk2 was regulated by Yes tyrosine kinase and by a tyrosine phosphatase SHP-2 in the endothelial cells. Furthermore, we have obtained evidence for the constitutive association of SHP-2 with Pyk2 in an SH2 domain-independent mechanism, which has not been reported as far as we are aware. Pyk2 can be dephosphorylated by SHP-2 in vitro and in intact cells. SHP-2-mediated dephosphorylation of Pyk2 correlates with the negative effect of SHP-2 on the Ang II-induced ERK and JNK activation in the endothelial cells.

EXPERIMENTAL PROCEDURES
Materials-Ang II and [Sar 1 ,Ile 8 ]Ang II were obtained from Peninsula Laboratories. The AT 1 receptor antagonist losartan was a generous gift of DuPont Merck Pharmaceutical Co., and the AT 2 receptor antagonist PD123319 was purchased from Research Biochemical, Inc.. Protein A-Sepharose was purchased from Amersham Pharmacia Biotech. Polyvinylidene difluoride membranes were obtained from Millipore. Monoclonal antibody against phosphospecific JNK, and polyclonal antibodies against SHP-2 (C-18), Src (N-16), Fyn (FYN3), Yes, Lck, Lyn, and JNK2 were obtained from Santa Cruz Biotechnology. Monoclonal anti-Pyk2, anti-phosphotyrosine (PY20), and anti-SHP-2 antibodies were obtained from Transduction Laboratories. FuGENE 6 transfection reagent was obtained from Roche Molecular Biochemicals. Polyclonal antibody to phosphospecific ERK, alkaline phosphatase-conjugated secondary antibodies, and reagents for chemiluminescence detection were purchased from New England Biolabs. All other reagents were from Sigma.
Cell Culture and Transfection-Rat pulmonary vein endothelial cells (PVEC) were kindly provided by Michael E. Mendelsohn (New England Medical Center, Boston, MA). PVECs were cultured in RPMI 1640 containing 10% fetal calf serum and grown overnight in a CO 2 incubator at 37°C to 70% confluence. PVECs were transfected with a catalytically inactive mutant of Src (kindly provided by S. A. Courtneidge, Sugen Inc.) or a catalytically inactive mutant of Fyn (23) using FuGENE 6 according to the manufacturer's protocol. The recently developed Fu-GENE 6 reagent has been shown to produce high levels of transfection with minimal cytotoxicity for many eukaryotic cells.
Stable Expression of NH 2 Terminus-truncated (⌬SH2SHP-2) and C Terminus-truncated (SH2(NϩC)) Forms of SHP-2-The NH 2 terminustruncated form of SHP-2, designated ⌬SH2SHP-2 (residues 192-593), which contains the COOH-terminal phosphatase domain but lacks the two SH2 domains at NH 2 terminus, was generated by cleaving a fulllength SHP-2 cDNA at a convenient BgIII site and re-ligating into pRC/CMV vector with an appropriate linker. The COOH terminustruncated form of SHP-2, designated SH2(NϩC), which contains both NH 2 -and COOH-terminal SH2 domains (residues 1-215) but lacks the COOH-terminal phosphatase domain, was generated by limited digestion of SHP-2 cDNA with XbaI and ScaI to give a fragment (residues 1-215) and subcloned into the pRc/CMV vector with an appropriate linker. Transfection was performed according to the standard calcium phosphate co-precipitation technique. To obtain stable cell lines, cells were selected in medium containing 0.5 mg/ml G418 sulfate, and single cell-derived colonies with high expression levels were obtained after 2-3 weeks.
Subcellular Fractionation-Cell cytosolic and membrane fractions were prepared as described previously (31). Membranes were suspended in Nonidet P-40 lysis buffer (25 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 10 mM NaF, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, 10 g/ml each leupeptin and aprotinin). The suspension was centrifuged at 100,000 ϫ g for 60 min, and the resulting supernatant was referred to as the solubilized membrane. All procedures were performed at 4°C.
Immunoprecipitation and Immunoblotting-Cells were washed twice with ice-cold phosphate-buffered saline containing 1 mM Na 3 VO 4 and then lysed on ice in Nonidet P-40 lysis buffer. The extract was clarified by centrifugation and incubated sequentially (4 h for each incubation at 4°C) with antibodies as indicated and protein A-Sepharose. The immunoprecipitates were collected and washed four times with the lysis buffer. For immunoblotting, whole cell lysates or immnunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. The membrane was probed with various primary antibodies as indicated and detected using the ECL system with alkaline phosphatase-conjugated secondary antibodies according to the manufacturer's protocol.
In Vitro Dephosphorylation Assay-SHP-2 immune complexes prepared from 50 M pervanadate-treated cells were washed three times with vanadate-free Nonidet P-40 lysis buffer and twice with dephosphorylation assay buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM dithiothreitol) and then incubated in the latter buffer for indicated time periods at 37°C. Reactions were terminated by washing the immune complexes with ice-cold Nonidet P-40 lysis buffer containing 2 mM Na 3 VO 4 . 1 Receptor in Rat Pulmonary Vein Endothelial Cells-Radioligand binding assay was performed as described previously (32). The binding of 125 I-[Sar 1 ,Ile 8 ]Ang II, a peptidic AT 1 receptor antagonist, to PVEC was dose-dependently inhibited by Ang II, [Sar 1 ,Ile 8 ]Ang II, and the AT 1 receptor-selective antagonist losartan with the potency order [Sar 1 ,Ile 8 ]Ang II Ͼ Ang II Ͼ losartan. In contrast, PD123319, an AT 2 receptor-selective antagonist, had no effect on the binding of 125 I-[Sar 1 ,Ile 8 ]Ang II. Scatchard analysis revealed that the dissociation constant (K d ) of AT 1 receptor in PVEC for 125 I-[Sar 1 ,Ile 8 ]Ang II is 1.0 nM, and the binding site number (B max ) is 190 fmol/mg of protein. This K d value is in good agreement with that previously reported for the endothelial cells derived from different origins (33).

Pharmacological Properties of AT
Calcium and Src Family Kinase-dependent Activation of Pyk2 by Ang II in PVEC-Pyk2 tyrosine phosphorylation is associated with an increase in its kinase activity (8 -10, 15, 21). To determine the effect of Ang II on Pyk2 tyrosine phosphorylation, lysates from control-or Ang II-stimulated cells were immunoprecipitated with anti-phosphotyrosine antibody (PY20) and subjected to immunoblotting with a Pyk2 antibody. As shown in Fig.  1, A and B, Ang II even at physiological concentration (10 pM) markedly increased the tyrosine phosphorylation of Pyk2. The stimulatory effect occurred very fast (Ͻ5 s), reached the maximal level at 30 s, and then declined. In agreement with previous reports (9, 10), the Ang II-induced tyrosine phosphorylation of Pyk2 was abolished by an intracellular calcium chelator BAPTA but not by the extracellular calcium chelator EGTA (Fig. 1C). Pretreatment of the cells with PP1 (34), a selective Src family kinase inhibitor, markedly inhibited the Ang II-induced tyrosine phosphorylation of Pyk2 (Fig. 1D). PP1 interacts specifically with Src family kinase and is a competitive inhibitor of ATP. PP1 selectively inhibits members of Src family kinase as compared with other tyrosine kinases such as ZAP-70, JAK2, and the EGF receptor (34). These results indicate that the Ang II-induced tyrosine phosphorylation of Pyk2 is dependent on intracellular calcium mobilization and requires the activity of Src family kinase in PVEC.

Specific Interaction of Yes Kinase with Pyk2 in Response to
Ang II in PVEC-Autophosphorylation of Pyk2 on Tyr-402 leads to binding of the SH2 domain of Src family kinases such as Src in PC12 cells and Fyn in T cells and their subsequent activation (14,22). The activated Src family kinase in turn phosphorylates Pyk2, generating docking sites for binding of additional signal proteins such as Grb2/Sos (14). We determined the association of Src family kinase with Pyk2 in response to Ang II in PVEC. As shown in Fig. 2A, among the Src family members (Src, Fyn, Lck, Yes) present in PVEC, Yes kinase was the only one specifically associated with Pyk2 in response to Ang II. We also found that Ang II did not induce the association of Lyn with Pyk2 in PVEC (data not shown). Fig. 2B shows the time course of Ang II-induced association of Yes with Pyk2. The association of Yes with Pyk2 by Ang II peaked at 30 s and then declined gradually. Losartan, a selective AT 1 receptor antagonist, blocked the Ang II-induced association of Yes with Pyk2. In contrast, an AT 2 receptor antagonist PD123319 had no effect (Fig. 2B). Src-and Fyn-independent Tyrosine Phosphorylation of Pyk2 by Ang II in PVEC-Ang II-induced tyrosine phosphorylation of Pyk2 was inhibited by the selective Src family kinase inhibitor PP1, and Yes kinase was the only one specifically associated with Pyk2 in response to Ang II. This suggests that Yes may contribute to the Ang II-induced tyrosine phosphorylation of Pyk2 in PVEC. To confirm this hypothesis, we transfected PVEC with a catalytically inactive mutant of Src (SrcK Ϫ ) or Fyn (FynK Ϫ ) (23) and determined the Ang II-induced tyrosine phosphorylation of Pyk2. As shown in Fig. 3, overexpression of SrcK Ϫ or FynK Ϫ did not affect the Pyk2 tyrosine phosphorylation by Ang II. These data support the specific role of Yes kinase in the regulation of Pyk2 by Ang II in PVEC.
Constitutive Association of SHP-2 with Pyk2 in PVEC-Ang II causes a transient tyrosine phosphorylation of Pyk2 in PVEC, suggesting involvement of a phosphatase in the regulation of Pyk2. We have recently reported a specific association of the Src family kinase member Fyn with SHP-2 upon stimulation of GPCR in MDCK cells. Fyn associates with SHP-2 through an intermediary protein that can be tyrosine-phosphorylated by a Src family kinase (23). To determine whether SHP-2 can interact with Pyk2 and to identity SHP-2-associated proteins in endothelial cells, cell cytosolic fraction and solubilized membrane from growing PVEC were prepared, immunoprecipitated with a polyclonal SHP-2 antibody, and subjected to immunoblot analysis with a monoclonal antibody to phosphotyrosine (PY20) as shown in Fig. 4A. A tyrosyl phosphoprotein band (116 kDa) and a 70-kDa band, which represents the phosphorylated SHP-2, were detected in SHP-2 immune complexes from the cytosolic fraction. The same blot was stripped and reprobed with a monoclonal anti-Pyk2 antibody. Pyk2 was detected in the SHP-2 immunoprecipitates (Fig. 4A). In an alternative experiment, SHP-2 is detected in the Pyk2 immunoprecipitates from the cytosolic fraction (data not shown). In the membrane fraction, three major phosphotyrosyl proteins, with apparent molecular masses of approximately 175, 116, and 97 kDa, were found associated with SHP-2. The 116-kDa protein was again identified as Pyk2. In addition, we identified the 97-kDa protein as the recently cloned SHP-2 substrate SIRP/SHPS-1 (35) by a specific antibody to this protein (data not shown). The association of SHP-2 with Pyk2 was also observed in bovine pulmonary artery endothelial and rat vascular smooth muscle cells (VSMC) as shown in Fig. 4B. We were unable to detect the association of SHP-2 with the Pyk2related kinase FAK, or STAT2, or the p120 Src substrate in all the tested cells (data not shown). Such a modality of association of SHP-2 with Pyk2 has not been reported to date as far as we know. We next analyzed the mechanism of the interaction between SHP-2 and Pyk2. Pervanadate has been shown to enhance protein tyrosine phosphorylation (36). As shown in Fig. 4C, pretreatment of PVEC with 50 M pervanadate for 20 min markedly increased the tyrosine phosphorylation of Pyk2 and SHP-2; however, it did not affect the SHP-2 association with Pyk2. Fig. 4D (left panel) shows that Ang II caused a rapid and robust tyrosine phosphorylation of Pyk2 with a maximum at 30 s. However, amounts of Pyk2 bound to SHP-2 immunoprecipitates from quiescent and Ang II-treated cells were similar. Moreover, EGF and serum, which have been reported to activate and promote the tyrosine phosphorylation of SHP-2 (37), did not alter the association of SHP-2 with Pyk2 in PVEC (Fig. 4D, right panel). Pyk2, like FAK, lacks SH2 and SH3 domains; in contrast, SHP-2 contains two SH2 domains at the NH 2 terminus (8,24). Therefore, our data indicate that the association of SHP-2 with Pyk2 is not mediated by the SH2 domains of SHP-2 because their interaction is not affected by tyrosine phosphorylation, and their association is constitutive.
Dephosphorylation of Pyk2 by SHP-2 in Vitro and in Intact Cells-To determine whether Pyk2 is a potential substrate of SHP-2, we performed experiments, in vitro and in intact cells, to address this question. First, PVECs were pretreated with pervanadate to enhance protein tyrosine phosphorylation, and the cell cytosolic fraction was incubated with SHP-2 antibodies. SHP-2 immune complexes containing Pyk2 (116 kDa) were then incubated in the dephosphorylation assay buffer for the indicated time periods. Incubation of the SHP-2 immunoprecipitates resulted in a significant dephosphorylation of the 116-kDa protein (Pyk2), a 55-kDa protein, and a 170-kDa protein within 5 min, reaching a higher dephosphorylation level at 20 min (Fig. 5). These dephosphorylation events are likely due to the presence of endogenous SHP-2 in the immune complexes. Thus, SHP-2 dephosphorylates Pyk2 in vitro.
Next, we determined whether Pyk2 is a substrate of SHP-2 in intact cells. Since the association of SHP-2 with Pyk2 is mediated by a region other than the SHP-2 SH2 domains, and since truncation of the SHP-2 SH2 domains enhances the phosphatase activity, compared with the wild-type full-length protein (38, 39), we transfected PVEC with a NH 2 terminus-truncated form of SHP-2, designated ⌬SH2SHP-2 (residues 192-593), which contains the COOH-terminal phosphatase domain but lacks the two SH2 domains at NH 2 terminus, and prepared cell clones stably expressing this mutant (Fig. 6, A and B). Expression of the ⌬SH2SHP-2 markedly inhibited (clone 34) or virtually abolished (clone 64) the Ang II-induced tyrosine phosphorylation of Pyk2 (Fig. 6C). The inhibitory effect of the SHP-2 mutant ⌬SH2SHP-2 on the Ang II-induced Pyk2 tyrosine phosphorylation was dependent on the expression levels of the ⌬SH2SHP-2 (Fig. 6, B and C). Similar results were observed in the other two cell clones expressing the ⌬SH2SHP-2 (data not shown). These results indicate that SHP-2 interacts with Pyk2 through a region other than its SH2 domains and dephosphorylates Pyk2. The catalytic activity of SHP-2 is regulated by its two SH2 domains. Binding of the SH2 domains with a specific tyrosine-phosphorylated partner activates the SHP-2 enzyme (40). Thus, overexpression of the SH2 domains will interrupt the binding of endogenous SHP-2 SH2 domains with its specific binding partner(s) and inhibit the activation of endogenous SHP-2 in response to agonists. As shown in Fig. 6 (D-F), expression of a COOH terminus-truncated form of SHP-2, designated SH2(NϩC), which contains both of the SH2 domains in the NH 2 -terminal half of the SHP-2 (residues 1-215) but lacks the COOH-terminal phosphatase domain, significantly increased the basal and Ang II-induced Pyk2 tyrosine phosphorylation in PVEC. This result further supports our conclusion that Pyk2 is a potential substrate of SHP-2.
Expression of ⌬SH2SHP-2 Suppresses Ang II-induced ERK and JNK Activation in PVEC-Pyk2 tyrosine phosphorylation is associated with an increase in its kinase activity and is involved in the JNK and ERK activation evoked by various stimuli including Ang II that increase intracellular calcium concentration (7-10, 14 -18). The ERK or JNK kinase is activated upon phosphorylation of a Thr and a Tyr residue in a TEY or TPY motif, respectively (41,42). Specific antibodies against phosphorylated ERK or JNK detects the activated form of ERK or JNK kinase by immunoblotting. Using these phosphospecific antibodies, we performed immunoblot analysis to determine the effects of the ⌬SH2SHP-2 mutant on Ang IIinduced ERK and JNK activation in PVEC. As shown in Fig.  7A, JNK2 was activated by Ang II. The effect of Ang II was virtually abolished by the expression of ⌬SH2SHP-2. Fig. 7B shows that Ang II-induced rapid ERK activation was also markedly inhibited by ⌬SH2SHP-2. Densitometric quantification revealed that phosphorylations of ERK1 and ERK2 were inhibited 75% and 55%, respectively, by ⌬SH2SHP-2. The Ang II-induced ERK and JNK activation are mediated through the AT 1 receptor in PVEC. Similar inhibitory effects of ⌬SH2SHP-2 on the ERK and JNK activation were observed in two other PVEC clones tested (data not shown). These results indicate that SHP-2 plays a negative role in the activation of ERK and JNK by Ang II in PVEC. The inhibition of Ang II-induced ERK/JNK activation by ⌬SH2SHP-2 correlates with ⌬SH2SHP-2-mediated dephosphorylation of Pyk2 in PVEC.
Activation of Endogenous SHP-2 by Ang II in PVEC-We have recently reported that SHP-2 but not SHP-1 was specifically activated by G i protein-coupled receptors in MDCK cells (23). SHP-2 tyrosine phosphorylation is correlated with an increase in its phosphatase activity. Tyrosine-phosphorylated SHP-2 directly binds to the adaptor protein Grb2 through the SH2 domain of Grb2 (23). In addition, SHP-2 could also associate with Grb2 through its binding protein such as Pyk2 (8).
Our overexpression experiments indicate that SHP-2 can dephosphorylate Pyk2 when SHP-2 is activated. To determine whether endogenous SHP-2 can be specifically activated by Ang II under physiological condition, we measured the SHP-2 tyrosine phosphorylation and its association with Grb2 in response to Ang II. Fig. 8 shows that Ang II rapidly stimulates the SHP-2 tyrosine phosphorylation and promotes the association of SHP-2 but not SHP-1 with Grb2, with maximal effects at 3 min. Losartan, a specific antagonist of AT 1 receptor blocked the Ang II-induced association of SHP-2 with Grb2. In contrast, an AT 2 receptor antagonist PD123319 had no effect (data not shown). These data indicate that Ang II specifically activates SHP-2 through the AT 1 receptor in PVEC. DISCUSSION In the present study, we have investigated the regulation of Ang II-induced activation of Pyk2, a key tyrosine kinase in the early events of AT 1 receptor signaling, using pulmonary vein endothelial cells. We found that the Ang II-induced tyrosine phosphorylation of Pyk2 was regulated by Yes tyrosine kinase and by a tyrosine phosphatase SHP-2. We found the constitutive association of SHP-2 with Pyk2, a finding which has not been documented as far as we know. SHP-2 interacts with Pyk2 through a region other than its SH2 domains and dephosphorylates Pyk2 when SHP-2 is activated. The SHP-2-mediated dephosphorylation of Pyk2 correlates with the negative effect of the SHP-2 on the Ang II-induced ERK and JNK activation in endothelial cells.
Ang II, a major effector peptide of the renin-angiotensin system, is believed to play a critical role in the pathogenesis of cardiovascular remodeling associated with hypertension, heart failure, and atherosclerosis (43). The AT 1 receptor activates phospholipase C ␤ through G q to generate inositol trisphosphate and diacylglycerol, which in turn releases calcium from intracellular stores and activates protein kinase C, respectively (2). It has been shown that the Ang II-induced tyrosine phosphorylation of Pyk2 in vascular smooth muscle and GN4 liver epithelial cells is dependent on intracellular calcium mobilization (9,10). Consistent with these studies, we found that the Ang II-induced tyrosine phosphorylation of Pyk2 in endothelial cells was virtually abolished by an intracellular calcium chelator BAPTA but not by the extracellular calcium chelator EGTA.
We also found that PP1 (34), a selective inhibitor of Src family kinase, markedly inhibited the Ang II-induced tyrosine phosphorylation of Pyk2. This indicates that the activity of Src family kinase is required for the tyrosine phosphorylation of Pyk2 by Ang II in endothelial cells. Co-expression experiments in 293T cells have shown that Pyk2 can be tyrosine phosphorylated by Src family members Src, Fyn, and Yes but not by Lck (21). Src family kinase may directly phosphorylate Pyk2 within the carboxyl terminus (Tyr-881) and within the catalytic domain (Tyr-579 and Tyr-580), which promotes Grb2 binding to Pyk2 and enhances Pyk2 kinase activity, respectively (15,21). The non-Src family kinases Syk and ZAP-70 could not directly phosphorylate Pyk2 (21). However, not all members of Src family kinase can be activated by the AT 1 receptor. Specific activation of Src in VSMC and Fyn in cardiac myocytes by Ang II has been reported (44,45). Src family kinase can be activated by three major mechanisms. One way is dephosphorylation of Tyr 527 at the COOH tail by a tyrosine phosphatase. A second way is binding of the Src SH2 domain to a tyrosine phosphorylated protein thus opening the COOH tail. A third way is binding of the Src SH3 domain to a SH3-binding protein. The full activation of a Src family kinase requires these three major ways, even though the order of dephosphorylation and binding events may differ (46). Mutagenesis studies have demonstrated that autophosphorylation of Tyr-402 of Pyk2 leads to binding of the SH2 domain of Src and results in Src activation by GPCRs in PC12 cells (14). The activated Src bound to Pyk2 will phosphorylate Pyk2 and Pyk2-associated proteins, thus amplifying signals from Pyk2 to downstream effectors (8,14,15). In endothelial cells, we found that, among the Src family members (Src, Fyn, Yes, Lck, and Lyn), Yes kinase was the only one specifically associated with Pyk2 in response to Ang II. Overexpression of a catalytically inactive mutant of Src and Fyn did not affect the Ang II-induced tyrosine phosphorylation of Pyk2 in PVEC. Therefore, our data indicate that Yes kinase is the major Src family member that contributes to the Pyk2 tyrosine phosphorylation in response to Ang II in endothelial cells. The association of Src with Pyk2 in VSMC in response to Ang II and in PC12 cells in response to lysophosphatidic acid and bradykinin has been reported (10,14). However, in T cells, Pyk2 was found specifically associated with Fyn and was phosphorylated by Fyn during stimulation of T cell antigen receptor (22).
The factors determining the specific association of Src family members with Pyk2 in response to various stimuli are not known. The expression levels of the Src family members seem not to be critical since comparable amounts of Src, Fyn, Yes, Lck, and Lyn were detected in PVEC. Members of Src family kinase share a common structure. The major structural difference among them is located in the NH 2 terminus, which contains sites for myristoylation and palmitoylation (not for Src and Blk) as well as a basic residues in the SH4 region (only for Src and Blk). Src family kinase members can locate to membrane and cytoskeleton through their NH 2 terminus. However, their final localization is dependent on cell types. For example, in fibroblasts, Src is found in perinuclear membranes. In T cells, Lck is on the plasma membrane and pericentriolar vesicles, but Fyn is closely associated with the centrosome and microtubule bundles (46). Interestingly, Pyk2 protein staining was found along actin microfilament-like structures that extended into focal adhesions in VSMC (47). In addition, pretreatment of the VSMC with cytochalasin D blocked the Ang IIinduced Pyk2 tyrosine phosphorylation (47). In the present study of PVEC, Pyk2 was detected in membranes as well as in cytosol. Taken together, these data suggest that differences in the localization of members of Src family kinase in different cell types may determine the specific association of members of the Src family with Pyk2 in response to various stimuli.
Our studies have revealed a site of action of SHP-2 in AT 1 receptor signaling. We identified the constitutive association of SHP-2 with Pyk2, a new finding that has not been reported so far as we know. The association of SHP-2 with Pyk2 is not mediated by the SH2 domains of SHP-2 since their interaction is not affected by protein tyrosine phosphorylation of either Pyk2 or SHP-2. The constitutive association of SHP-2 with JAK kinases has recently been reported in certain lymphoma cells (48). Cotransfection experiments in Cos-1 cells have revealed that a linker region of SHP-2 (residues 232-272), which contains a sequence (GFWEEFE) unique to SH2 domain-containing tyrosine phosphatases (SHP-1 and SHP-2), is required for the SHP-2 association with JAKs (49). Whether this unique sequence is involved in the SHP-2 interaction with Pyk2 remains to be determined. Moreover, we found that Pyk2 is a potential substrate of SHP-2. First, Pyk2 can be dephosphorylated in vitro in SHP-2 immunoprecipitates. Second, overexpression of a SHP-2 mutant ⌬SH2SHP-2 (residues 192-593), which contains the COOH-terminal phosphatase domain but lacks the two SH2 domains at NH 2 terminus and displays an enhanced phosphatase activity (38,39), markedly inhibited the Ang II-induced tyrosine phosphorylation of Pyk2. Third, expression of SHP-2 SH2 domains (residues 1-215), which is predicted to inhibit the activation of endogenous SHP-2 (40), significantly increased basal and Ang II-induced Pyk2 tyrosine phosphorylation in PVEC. Finally, we showed that endogenous SHP-2 was activated by Ang II in PVEC. The maximal activation of SHP-2 (3-10 min after Ang II treatment) seen in Fig. 8 correlates with the profound decrease in the Ang II-induced tyrosine phosphorylation of Pyk2. The specific activation of SHP-2 but not SHP-1 by G i -coupled lysophosphatidic acid and ␣ 2A -adrenergic receptors in MDCK cells (23) and by AT 1 receptor in VSMC (50) has been reported. We have recently reported the Fyn kinase-directed activation SHP-2 by G i -coupled receptors in MDCK cells (23). Among the Src family members present in MDCK cells, Fyn was the only one specifically associated with SHP-2 through an intermediary molecule. In PVEC, we found that Yes kinase is the only member of the Src family associated with Pyk2 in response to Ang II. The formation of the Yes/Pyk2/SHP-2 complex activates Yes kinase and allows Yes to phosphorylate Pyk2, and thus amplifying the signals from Pyk2 to downstream ERK and JNK activation as one model described recently (14,15). On the other hand, activated Yes bound to Pyk2 may directly phosphorylate the Pyk2 adjacent protein SHP-2 within the complex and activate SHP-2 as described in a model we have presented in MDCK cells (23). The activated SHP-2 could dephosphorylate Pyk2 and thus turn off the signals to the downstream ERK and JNK pathways. Therefore, it is not surprising that the SHP-2-mediated dephosphorylation of Pyk2 correlates with the negative effect of the SHP-2 on the Ang II-induced ERK and JNK activation in PVEC.
In addition to the regulation of ERK and JNK activation, Pyk2 has also been implicated in the regulation of cell adhesion and motility (12,13). Several focal adhesion associated-proteins such as paxillin, p130 Cas , and its homologue HEF1 (human enhancer of filamentation 1) have recently been shown associated constitutively with Pyk2 (12,13,51). It has been reported that Ang II and other stimuli cause tyrosine phosphorylation of these focal adhesion associated-proteins (52)(53)(54). Given the constitutive association of SHP-2 with Pyk2, SHP-2 may regulate the tyrosine phosphorylation of paxillin, p130 Cas , and HEF1 within the complexes, thus modulating the cell motility. Yu et al. (55) recently reported that SHP-2 plays an important role in cell spreading, migration, and focal adhesion. Additional cellular proteins, such as Graf (the GTPase-activating protein for Rho), Nirs (amino-terminal domain-interacting receptors), and Pap proteins, were also shown to be constitutively associated with the Pyk2/SHP-2 complex and participate in broad range of signaling pathways in cells (56 -59). Whether they are directly or indirectly regulated by SHP-2 remains to be investigated.
The balance of protein phosphorylation and dephosphorylation is controlled tightly in living cells (1). Constitutive association of calcium-calmodulin-dependent protein serine-threonine kinase IV (CaMKIV) and protein serine-threonine phosphatase 2A has been shown to play a role in T antigen signaling (60). Protein serine-threonine phosphatase 2A dephosphorylates CaMKIV and functions as a negative regulator of CaMKIV signaling. The stable complex of tyrosine kinase JAK2 and tyrosine phosphatase SHP-1 has also been shown to play a role in erythropoietin signaling (61). SHP-1 dephosphorylates JAK2 and results in termination of JAK2-mediated signaling. In the present study, we added an another example, i.e. the stable complex formation of SHP-2 with Pyk2. SHP-2 may dephosphorylate Pyk2 and terminate the signaling from Pyk2 or Yes/Pyk2 complex, thus playing a negative role in the regulation of AT 1 receptor signaling.