Fyn Kinase-directed Activation of SH2 Domain-containing Protein-tyrosine Phosphatase SHP-2 by Gi Protein-coupled Receptors in Madin-Darby Canine Kidney Cells*

Hua TangDagger , Zhizhuang Joe Zhao§, Xin-Yun Huang, Erwin J. Landonparallel , and Tadashi InagamiDagger **

From the Departments of Dagger  Biochemistry and parallel  Pharmacology and the § Division of Hematology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and the  Department of Physiology, Cornell University Medical College, New York, New York 10021

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SHP-2, an SH2 domain-containing protein-tyrosine phosphatase, plays an important role in receptor tyrosine kinase-regulated cell proliferation and differentiation. Little is known about the activation mechanisms and the participation of SHP-2 in the activity of G protein-coupled receptors lacking intrinsic tyrosine kinase activity. We show that the activity of SHP-2 (but not SHP-1) is specifically stimulated by the selective alpha 2A-adrenergic receptor agonist UK14304 and by lysophosphatidic acid (LPA) in Madin-Darby canine kidney (MDCK) cells. UK14304 and LPA promote the tyrosine phosphorylation of SHP-2 and its association with Grb2. The agonist-induced direct interaction of Grb2 with SHP-2 is mediated by the SH2 domain of Grb2 and the tyrosine phosphorylation of SHP-2. Rapid activation of Src family kinase by UK14304 preceded the SHP-2 activation. Among the Src family members (Src, Fyn, Lck, Yes, and Lyn) present in MDCK cells, Fyn was the only one specifically associated with SHP-2, and the physical interaction between them, which requires the Src family kinase activity, was increased in response to the agonists. Pertussis toxin, PP1 (a selective Src family kinase inhibitor), or overexpression of a catalytically inactive mutant of Fyn blocked the UK14304- or LPA-stimulated activity of SHP-2, SHP-2 tyrosine phosphorylation, and SHP-2 association with Grb2. Therefore, we have demonstrated for the first time that the activation of SHP-2 by these Gi protein-coupled receptors requires Fyn kinase and that there is a specific physical interaction of Fyn kinase with SHP-2 in MDCK cells.

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Protein tyrosine phosphorylation and dephosphorylation are fundamental cellular signaling mechanisms that control cell growth and differentiation (1). In mammalian cells, the SH2 domain-containing tyrosine phosphatases, which include SHP-1 and SHP-2, share a general structure: two SH2 domains at the N terminus followed by a catalytic domain and a C-terminal region containing two putative sites of tyrosine phosphorylation. SHP-2 (previously known as SH-PTP2, PTP1D, Syp, PTP2C, and SHPTP3) is expressed ubiquitously and is suggested to play a positive role in the regulation of proximal events downstream of receptor protein-tyrosine kinases (2, 3). Genetic investigation of SHP-2 indicates that this phosphatase is crucial for gastrulation during mammalian development, as mice homozygous for the mutant allele die in utero at mid-gestation (4-6). In contrast, SHP-1 (previously known as SH-PTP1, PTP1C, HCP, and SHP) is expressed predominantly in hematopoietic cells, where it seems to function primarily as a negative regulator of multiple cytokine and growth factor signaling pathways (2, 3).

It has been postulated that SHP-2 associates with activated EGF1 and PDGF receptors via its SH2 domains and becomes rapidly tyrosine-phosphorylated upon ligand stimulation, which may alter the catalytic activity or regulate its interaction with other SH2 domain-containing proteins/regulators (7, 8). In the case of PDGF stimulation, tyrosine phosphorylation of SHP-2, which occurs within its C terminus, creates a binding site for the Grb2·Sos complex, thereby leading to activation of the Ras/mitogen-activated protein (MAP) kinase cascade (9, 10). Previous studies have indicated that enzymatic activity or the SH2 domains of SHP-2 are required for MAP kinase activation evoked by EGF, insulin, insulin-like growth factor-1, and fibroblast growth factor (11-14). However, the mechanisms and the active sites of SHP-2 in the regulation of MAP kinase activation are not clear.

Little is known about the participation and regulation of SHP-2 in the activity of G protein-coupled receptors that lack intrinsic tyrosine kinase activity. It was reported recently that alpha -thrombin induced tyrosine phosphorylation of SHP-2 in fibroblasts (15). A catalytically inactive mutant of SHP-2 strongly inhibited the stimulatory effects of alpha -thrombin on c-fos transcription and DNA synthesis. This inhibition could be reversed by cotransfection of SHP-2, but not SHP-1 (15). However, the mechanism by which the G protein-coupled receptor regulates SHP-2 is unknown. In this study, we found that SHP-2 (but not SHP-1) was specifically activated by UK14304, an agonist of the alpha 2A-adrenergic receptor, and by lysophosphatidic acid (LPA) in Madin-Darby canine kidney (MDCK) cells. We have demonstrated for the first time that the activation of SHP-2 by these Gi protein-coupled receptors is directly mediated by Fyn kinase and that there is a specific physical interaction of Fyn with SHP-2 in MDCK cells.

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Materials-- Protein A- and glutathione-Sepharose and [gamma -32P]ATP were purchased from Amersham Pharmacia Biotech. The Src family kinase assay kit and monoclonal anti-phosphotyrosine antibody (4G10) were purchased from Upstate Biotechnology, Inc. Raytide, pp60c-Src tyrosine kinase, and PP1 were purchased from Calbiochem. UK14304 was purchased from Research Biochemical Inc. Polyvinylidene difluoride (PVDF) membranes were obtained from Millipore. Polyclonal antibodies against SHP-2 (C-18), SHP-1, Src (N-16 and SRC-2), Fyn (FYN3), Yes, Lck, and Lyn; monoclonal anti-glutathione S-transferase (GST) antibody; and GST fusion proteins containing the SH2 or SH3 domains of SHP-2, Fyn, and Grb2 were obtained from Santa Cruz Biotechnology. Monoclonal anti-SHP-1, anti-SHP-2, anti-EGF receptor, anti-Grb2, anti-phosphotyrosine (PY20), and anti-Fyn antibodies were obtained from Transduction Laboratories. Alkaline phosphatase-conjugated secondary antibody and reagents for chemiluminescence detection were purchased from New England Biolabs Inc. All other reagents were from Sigma.

Cell Culture and Expression of a Catalytically Inactive Mutant of Fyn-- MDCK-Tag3 cells stably expressing a hemagglutinin epitope-tagged version of the porcine alpha 2A-adrenergic receptor (alpha 2A-AR) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (16). A catalytically inactive mutant cDNA of mouse Fyn (Lys296-Met296) was subcloned into a pApuro vector (17, 18). At 50% confluence, MDCK-Tag3 cells were transfected with the catalytically inactive mutant of Fyn plasmid DNA using a standard calcium phosphate precipitation/glycerol shock procedure. After selection in puromycin (1 µg/ml), single cell-derived colonies were isolated, amplified, and screened for the expression of mutated Fyn by immunoblotting. Three clones with high expression of the mutated Fyn were obtained and propagated in the complete medium under a selection pressure of 0.5 µg/ml puromycin.

Immunoprecipitation and Immunoblotting-- MDCK-Tag3 cells were normally serum-starved in Dulbecco's modified Eagle's medium for ~24 h before agonist stimulation. Stimulated and unstimulated cells were washed twice with ice-cold phosphate-buffered saline containing 1 mM Na3VO4 and then lysed on ice in Nonidet P-40 lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each leupeptin and aprotinin). The extract was clarified by centrifugation at full speed in a microcentrifuge. The clarified lysates were 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 Nonidet P-40 lysis buffer. For immunoblotting, whole cell lysates or immunoprecipitates were separated by SDS-PAGE and transferred to a PVDF membrane. The membranes were 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.

GST Fusion Protein Binding Assay-- Whole cell lysates were incubated with GST fusion proteins bound to glutathione-Sepharose for 12 h at 4 °C with rotation. Beads were pelleted and washed four times with ice-cold Nonidet P-40 lysis buffer. Bound proteins were released by boiling in SDS-PAGE sample buffer for 4 min.

Far-Western Blotting-- Protein blots on PVDF membranes were first subjected to 6 M guanidine hydrochloride denaturation at 4 °C in a buffer containing 20 mM Hepes, pH 7.6, 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 1 mM dithiothreitol, and 0.05% Nonidet P-40 and then to renaturation at 4 °C by five dilution steps (10 min each) to a final concentration of 0.185 M guanidine. The PVDF membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline/Tween, incubated with GST fusion proteins in blocking buffer overnight, probed with monoclonal anti-GST antibody, and then developed using the appropriate secondary antibodies and the ECL system.

Immune Complex Tyrosine Phosphatase and Kinase Assays-- Cell lysates were prepared in lysis buffer (25 mM Tris-HCl, pH 7.5, 10 mM 2-mercaptoethanol, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml each leupeptin and aprotinin, 5 mM benzamidine, and 1% Nonidet P-40). The cell lysates were precleared with protein A-Sepharose and control IgG from normal mouse or rabbit serum, and SHP-2 was immunoprecipitated with monoclonal or polyclonal anti-SHP-2 antibodies. Immune complexes were washed four times with lysis buffer and once with phosphatase reaction buffer (25 mM Hepes, pH 7.4, 5 mM EDTA, and 10 mM dithiothreitol). The in vitro phosphatase activity was measured as described previously (19). Briefly, the synthetic peptide Raytide was labeled at its tyrosine residue using [gamma -32P]ATP and pp60c-Src tyrosine kinase. The washed immune complexes were mixed with 32P-labeled Tyr-Raytide in 30 µl of phosphatase reaction buffer and incubated at 30 °C for 5 min. The reaction was terminated by the addition of acidic charcoal mixture (0.9 M HCl, 90 mM sodium pyrophosphate, 2 mM NaH2PO4, and 4% (w/v) Norit A). After centrifugation in a microcentrifuge, the amount of radioactivity present in the supernatant was determined by scintillation counting. The phosphatase activity was evaluated by the extent of Tyr-Raytide dephosphorylation in vitro. For measuring the activities of Src family kinases, cell lysates prepared in Nonidet P-40 lysis buffer were precleared with protein A-Sepharose and control IgG from normal rabbit serum, and the lysates were immunoprecipitated with a polyclonal antibody (SRC-2) that recognizes the C-terminal sequence of the Src family members Src, Fyn, and Yes. Immunoprecipitates were washed four times with Nonidet P-40 lysis buffer and once with phosphate-buffered saline and resuspended in 15 µl of a buffer solution containing 40 mM Hepes, pH 7.0, 2% (v/v) glycerol, and 0.02% Nonidet P-40. Kinase reactions were initiated by the addition of 100 µM ATP, 25 mM MgCl2, 5 mM MnCl, 50 µM Na3VO4, and 10 µCi of [gamma -32P]ATP in the presence of 300 µM Src family kinase-specific peptide (KVEKIGEGTYGVVKK) in a total volume of 35 µl. After incubation at 30 °C for 10 min, peptide phosphorylation was stopped by the addition of 15 µl of 50% acetic acid, and the reaction mixture was then applied onto P-81 phosphocellulose filter paper. Papers were washed four times with 0.75% phosphoric acid and washed once with acetone, dried, and then counted in a scintillation counter.

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Specific Activation of SHP-2 (but Not SHP-1) by the alpha 2A-AR and LPA Receptor-- To determine whether SHP-2 or SHP-1 can be activated by the Gi protein-coupled alpha 2A-AR or LPA receptor, we measured the enzymatic activities of the tyrosine phosphatases following the stimulation of MDCK-Tag3 cells with the receptor's agonist. MDCK-Tag3 cells express the endogenous LPA receptor and porcine alpha 2A-AR (16). Lysates from control or agonist-treated cells were immunoprecipitated with monoclonal anti-SHP-2 or anti-SHP-1 antibody and subjected to immune complex tyrosine phosphatase assay. The phosphatase activity was measured using 32P-labeled Tyr-Raytide as a substrate. As shown in Fig. 1, both UK14304 (an agonist of the alpha 2A-AR) and LPA markedly stimulated the enzymatic activity of SHP-2 with similar kinetics. The stimulatory effects occurred fast (<1 min), reached a maximal level (4-5-fold increase) at 3 min, and declined after 10 min. Similar results were observed when the cell lysates were immunoprecipitated with polyclonal anti-SHP-2 antibody. The UK14304-stimulated phosphatase activity of SHP-2 was blocked by the selective alpha 2A-AR antagonist rauwolscine (data not shown). In contrast, we did not detect any change of phosphatase activity in SHP-1 immunoprecipitates by stimulation of the cells with UK14304 and LPA under these conditions (Fig. 1), although the cells express about equivalent amounts of SHP-1 and SHP-2.


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Fig. 1.   Specific activation of SHP-2 (but not SHP-1) by the alpha 2A-AR and LPA receptor. MDCK-Tag3 cells were stimulated with either 500 nM UK14304 or 10 µM LPA for the indicated time periods. Cell lysates were immunoprecipitated with monoclonal anti-SHP-2 or anti-SHP-1 antibodies, and the immunoprecipitates were then subjected to in vitro immune complex phosphatase assay using 32P-labeled Tyr-Raytide as a substrate. The phosphatase activity was evaluated by the extent of 32Pi release from 32P-labeled Tyr-Raytide. Data are the average of duplicate values and are representative of three independent experiments.

Since the catalytic activity of SHP-2 can be stimulated by tyrosine phosphorylation of the phosphatase (8, 20), we determined the tyrosine phosphorylation of SHP-2. MDCK-Tag3 cells were stimulated with UK14304 or LPA for 3 min, and the SHP-2 immunoprecipitates were subjected to immunoblot analysis with a mixture of monoclonal antibodies to phosphotyrosine (5:1 (v/v) PY20/4G10). As shown in Fig. 2A, both agonists stimulated the tyrosine phosphorylation of SHP-2, which migrates at 70 kDa. UK14034 and LPA also promoted the co-immunoprecipitation of SHP-2 with three tyrosine-phosphorylated proteins with apparent molecular masses of ~175, 125, and 115 kDa, respectively (data not shown). To verify the tyrosine phosphorylation of SHP-2 and to determine the dynamic effect of UK14304, we performed an alternative experiment. Cell lysates were immunoprecipitated with antibody PY20 and analyzed by immunoblotting with a polyclonal anti-SHP-2 antibody. Unexpectedly, two SHP-2 protein bands (~68 and 70 kDa) were detected by the anti-SHP-2 antibody (Fig. 2B). The upper 70-kDa protein band represents the tyrosyl-phosphorylated form of SHP-2 since tyrosine-phosphorylated SHP-2 migrates at 70 kDa as shown in Fig. 2A. It has been shown that phosphorylation of SHP-2 increases its apparent molecular size to ~70 kDa in response to growth factors and cytokines (8, 20-22). The phosphorylated form of SHP-2 (70 kDa) was not detected in the whole cell lysates. We estimate that <1% of SHP-2 protein was phosphorylated by UK14304 in MDCK-Tag3 cells. The lower 68-kDa protein band represents the non-tyrosine-phosphorylated form of SHP-2, and this may be complexed via the interaction of its SH2 domains with phosphotyrosine proteins including the tyrosine-phosphorylated SHP-2. The intensity of the phosphorylated and non-phosphorylated SHP-2 (70- and 68-kDa bands) was increased in a time-dependent manner, with a maximum at 3 min following the stimulation with UK14304. The stimulatory effects of UK14304 on SHP-2 tyrosine phosphorylation and SHP-2 association with tyrosine-phosphorylated proteins were abolished by PP1, a selective Src family kinase inhibitor (Fig. 2B). PP1 interacts specifically with Src family kinase and is a competitive inhibitor of ATP. PP1 selectively inhibits Lck, Fyn, and Hck as compared with other tyrosine kinases such as ZAP-70, JAK2, and the EGF receptor (23). In addition, the adaptor protein Grb2, which mediates signals to Ras through the nucleotide exchange factor Sos, was found co-immunoprecipitated with SHP-2 in response to the UK14304 treatment. The kinetics of the Grb2 association with SHP-2 was similar to that of the SHP-2 tyrosine phosphorylation evoked by UK14304. The agonist-induced SHP-2 association with Grb2 was also abolished by the Src family kinase inhibitor PP1 (Fig. 2C). In contrast, we could not detect the tyrosine phosphorylation of SHP-1 and its association with Grb2 in SHP-1 immunoprecipitates either before or after UK14304 or LPA treatment (data not shown). Therefore, although SHP-1 shares a high degree of homology with SHP-2, only SHP-2 was specifically activated by the alpha 2A-AR and LPA receptor in MDCK-Tag3 cells.


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Fig. 2.   Tyrosine phosphorylation of SHP-2 and its association with Grb2. A, MDCK-Tag3 cells were either left unstimulated (-) or stimulated with 500 nM UK14304 (UK) or 10 µM LPA for 3 min. Lysates were prepared and immunoprecipitated (IP) with polyclonal anti-SHP-2 antibody. Immune complexes were subjected to immunoblotting (IB) with a mixture of monoclonal antibodies to phosphotyrosine (5:1 (v/v) PY20/4G10) (upper panel). The same blot was stripped and reprobed with monoclonal anti-SHP-2 antibody (lower panel). B, MDCK-Tag3 cells were pretreated with or without 10 µM PP1 for 30 min as indicated and then were either left unstimulated (-) or stimulated with 500 nM UK14304 for the indicated time periods. Cell lysates were immunoprecipitated with control IgG from normal mouse serum or monoclonal anti-phosphotyrosine antibody PY20. The immunoprecipitates and whole cell lysates from UK14304-treated (3 min) MDCK-Tag3 cells were subjected to immunoblotting with a polyclonal antibody to SHP-2. C, MDCK-Tag3 cells were pretreated with or without 10 µM PP1 for 30 min as indicated. The cells were then unstimulated (-) or stimulated with 500 nM UK14304 for the indicated time periods. Lysates were immunoprecipitated with polyclonal anti-SHP-2 antibody. Immune complexes were subjected to immunoblotting with monoclonal anti-Grb2 antibody (upper panel). The same blot was stripped and reprobed with monoclonal anti-SHP-2 antibody (lower panel).

Grb2 Binds Directly via Its SH2 Domain to SHP-2-- The SH2 domain of Grb2 is predicted to bind to the consensus sequence pYXNX (24), of which there are two in SHP-2, namely at tyrosine 304 (Y304INA) and at tyrosine 542 (Y542TNI). To analyze the interaction between SHP-2 and Grb2, we first performed an in vitro binding assay as shown in Fig. 3A. The glutathione-agarose-bound GST fusion proteins containing the SH2 or SH3 domain of Grb2 were incubated with lysates of UK14304-stimulated MDCK-Tag3 cells and washed extensively, and the beads were resolved by SDS-PAGE. Immunoblot analysis with a monoclonal anti-SHP-2 antibody showed that SHP-2 coprecipitated only with a GST fusion protein containing the SH2 domain of Grb2 (Fig. 3A). The next set of in vitro binding assays and far-Western analyses was carried out to determine whether Grb2 interacts directly with SHP-2 and to resolve the issue of whether SHP-2 must be tyrosine-phosphorylated to affect this association. MDCK-Tag3 cells were stimulated with LPA and UK14304 to promote the tyrosine phosphorylation of SHP-2, and the SHP-2 immunoprecipitates were incubated with a GST fusion protein containing the SH2 domain of Grb2. As shown in Fig. 3B, LPA and UK14304 markedly increased the association of SHP-2 with the GST-Grb2 SH2 fusion protein. However, the results using GST fusion protein may also imply that Grb2 binds indirectly to SHP-2 through an association with proteins bound to SHP-2. We therefore used far-Western blots to resolve this issue. SHP-2 immunoprecipitates were transferred to PVDF membranes, denatured in 6 M guanidine HCl, and then gradually renatured. The membranes were subsequently incubated with the GST fusion protein containing the SH2 domain of Grb2. Proteins on the blot that interacted directly with the GST fusion protein were visualized using anti-GST antibodies. As shown in Fig. 3C, the GST-Grb2 SH2 fusion protein could bind directly to a 70-kDa protein band migrating with the tyrosine-phosphorylated form of SHP-2, and the binding was markedly increased upon UK14304 treatment. These results demonstrate that Grb2 binds directly via its SH2 domain to SHP-2 following stimulation with UK14304 and LPA.


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Fig. 3.   Grb2 interacts directly via its SH2 domain with SHP-2. A, MDCK-Tag3 cells were stimulated with 500 nM UK14304 for 3 min, and the cell lysates were incubated with GST or the GST fusion protein containing the SH2 (GST-SH2) or SH3 (GST-SH3) domain of Grb2 overnight at 4 °C. Beads were pelleted, washed, resolved by SDS-PAGE, and immunoblotted with monoclonal anti-SHP-2 antibody. B, MDCK-Tag3 cells were either left unstimulated (-) or stimulated with 500 nM UK14304 (UK) or 10 µM LPA for 3 min. Lysates were immunoprecipitated (IP) with control IgG from normal rabbit serum or a polyclonal antibody to SHP-2. The immunoprecipitates were washed and incubated with the GST-Grb2 SH2 fusion protein (Grb2 SH2). Beads were pelleted, washed, resolved by SDS-PAGE, and immunoblotted (IB) with monoclonal anti-GST antibody (upper panel). The same blot was stripped and reprobed with a monoclonal antibody to SHP-2 (panel b). C, cells were left unstimulated (-) or stimulated with 500 nM UK14304 for 3 min, and the lysates were immunoprecipitated with polyclonal anti-SHP-2 antibody. The SHP-2 immunoprecipitates were transferred to a PVDF membrane, denatured in 6 M guanidine HCl, and then gradually renatured. The membranes were subsequently incubated with the GST fusion protein containing the SH2 domain of Grb2. Proteins on the blot that interacted directly with the GST fusion protein were visualized using anti-GST antibody (upper panel). The same blot was stripped and reprobed with monoclonal anti-SHP-2 antibody (lower panel).

Activation of SHP-2 Is Sensitive to Pertussis Toxin and Requires Src Family Tyrosine Kinase-- Pretreatment of the cells with the selective Src family kinase inhibitor PP1 abolished the UK14304-induced tyrosine phosphorylation of SHP-2 and its association with tyrosine-phosphorylated proteins and Grb2 as shown in Fig. 2 (B and C). This suggests an involvement of a Src family kinase in the activation of SHP-2. Pertussis toxin-sensitive activation of Src family kinase by UK14304 and LPA has been reported (25, 26). To measure the UK14304-stimulated activity of Src family kinase, cell lysates were immunoprecipitated with polyclonal antibody SRC-2, which recognizes the common C-terminal sequence of the family members Src, Fyn, and Yes. The immunoprecipitable kinase activity was determined by its ability to phosphorylate a Src kinase-specific substrate peptide (KVEKIGEGTYGVVKK) (27). We found that the Src family kinase activity was increased as early as 30 s after the addition of UK14304, peaked at 2 min (2.3-fold; 56,270 ± 6200 cpm with UK14304 versus 24,465 ± 2800 cpm without UK14304), and returned to basal levels by 5 min. We next examined the effects of pertussis toxin and the selective Src family kinase inhibitor PP1 on the activation of SHP-2 by UK14304 and LPA. As shown in Fig. 4, UK14304 and LPA markedly increased the activity of SHP-2, and the increase in activity was completely suppressed by pertussis toxin and PP1 pretreatment of the cells. PP1 also markedly inhibited the basal activity of SHP-2. These results indicate that UK14304- and LPA-induced activation of SHP-2 in MDCK-Tag3 cells is mediated by a pertussis toxin-sensitive G protein and may require the activity of Src family kinase.


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Fig. 4.   Pertussis toxin and PP1 block the agonist-stimulated activity of SHP-2. MDCK-Tag3 cells were pretreated with 625 ng/ml pertussis toxin (PTX) overnight or with 10 µM PP1, a specific Src family kinase inhibitor, for 30 min as indicated, and the cells were then stimulated with 500 nM UK14304 or 10 µM LPA for 3 min. Lysates were immunoprecipitated with monoclonal anti-SHP-2 antibody, and the immunoprecipitates were then subjected to in vitro immune complex phosphatase assay using 32P-labeled Tyr-Raytide as a substrate. The phosphatase activity was evaluated by the extent of 32Pi release from 32P-labeled Tyr-Raytide. Data are the average of duplicate values and are representative of three independent experiments. PP1 also suppressed the basal activity of SHP-2.

Physical Interaction of SHP-2 with Fyn Kinase-- Since the UK14304- or LPA-stimulated activation of SHP-2 was inhibited by the selective Src family kinase inhibitor PP1, and the activation of Src family kinase was prior to that of SHP-2 in MDCK-Tag3 cells, we addressed the question as to whether SHP-2 could interact directly or indirectly with one of the members of the Src kinase family. Cell lysates were immunoprecipitated with specific polyclonal antibodies against Src, Fyn, Lck, Yes, and Lyn, which are present in MDCK-Tag3 cells (data not shown), and the immunoprecipitates were then immunoblotted with a monoclonal anti-SHP-2 antibody. As shown in Fig. 5A, among the members of the Src family, only Fyn was specifically co-immunoprecipitated with SHP-2. Two SHP-2 protein bands of ~68 and 70 kDa, representing non-tyrosine-phosphorylated and tyrosine-phosphorylated SHP-2, respectively, were detected in the Fyn immune complex. Comparing the amount of SHP-2 present in total cell lysates versus the amount coprecipitated with Fyn, we found that <1% of non-phosphorylated SHP-2 (68 kDa) was recovered in the Fyn immunoprecipitates and that the phosphorylated SHP-2 (70 kDa) associated with Fyn accounted for only <0.5% of total SHP-2. The association of both forms of SHP-2 with Fyn was increased following 3 min of stimulation with UK14304 (Fig. 5A) and LPA (data not shown). The UK14304-promoted association of SHP-2 with Fyn was blocked by pretreatment of the cells with the Src family kinase inhibitor PP1. The PP1 treatment also blocked the basal interaction of SHP-2 with Fyn (Fig. 5A). Blotting of the same membrane with specific antibodies to each member of the Src kinase family revealed that a comparable amount of Fyn (Fig. 5A, lower panel) and Src, Yes, Lck, and Lyn (data not shown) was immunoprecipitated. These results indicate that the specific interaction between SHP-2 and Fyn requires the activity of Src family kinase. To characterize the interaction between SHP-2 and Fyn, we performed an in vitro binding assay using GST or the GST fusion protein containing the SH2 or SH3 domain of Fyn. As shown in Fig. 5B, two SHP-2 protein bands (68 and 70 kDa) coprecipitated with the GST-Fyn SH2 fusion protein, and the coprecipitation was increased upon UK14304 treatment, whereas the GST fusion protein containing the SH3 domain of Fyn coprecipitated only the lower 68-kDa band of SHP-2 (the non-phosphorylated form). The association of the non-phosphorylated form of SHP-2 with the GST-Fyn SH3 fusion protein was also increased by UK14304.


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Fig. 5.   Physical interaction of SHP-2 with Fyn kinase. A, MDCK-Tag3 cells were pretreated with or without 10 µM PP1 for 30 min and then either left unstimulated (-) or stimulated (+) with 500 nM UK14304 (UK). Lysates were immunoprecipitated (IP) with a polyclonal antibody to Src, Fyn, Yes, Lck, or Lyn as indicated. The immunoprecipitates were subjected to immunoblotting (IB) with monoclonal anti-SHP-2 antibody. B, MDCK-Tag3 cells were either left unstimulated (-) or stimulated (+) with 500 nM UK14304 for 3 min. Lysates were prepared and incubated with GST or the GST fusion protein containing the SH2 (GST-SH2) or SH3 (GST-SH3) domain of Fyn. Beads were pelleted and washed, and the released proteins were subjected to immunoblotting with a monoclonal antibody to SHP-2 (left panel). The left panel was overexposed, and the agonist-promoted association of SHP-2 with the Fyn SH3 domain is shown in the right panel.

Fyn Kinase-directed Activation of SHP-2-- The physical and specific interaction between SHP-2 and Fyn suggests that the activation of SHP-2 evoked by UK14304 and LPA may be directly mediated by Fyn kinase in MDCK-Tag3 cells. To examine the role of Fyn kinase in the activation of SHP-2, we transfected MDCK-Tag3 cells with a catalytically inactive mutant cDNA of mouse Fyn (Lys296-Met296) and prepared cell clones stably overexpressing this inactive Fyn mutant, Fyn(K-) (Fig. 6A). Immune complex kinase assay reconfirmed that the exogenously expressed Fyn(K-) mutant was totally inactive. Overexpression of the Fyn(K-) mutant did not affect the expression of other members of the Src kinase family in MDCK-Tag3 cells (data not shown). As shown in Fig. 6 (B and C), overexpression of the Fyn(K-) mutant markedly inhibited the UK14304-induced tyrosine phosphorylation of SHP-2, SHP-2 association with Grb2, and the increase in SHP-2 enzymatic activity evoked by UK14304 and LPA. These results demonstrate that Fyn kinase plays a direct role in mediating the activation of SHP-2 by these Gi protein-coupled receptors in MDCK-Tag3 cells. Similar results were observed in the other two cell clones expressing the Fyn(K-) mutant (data not shown).


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Fig. 6.   Fyn kinase-directed activation of SHP-2. A, lysates from vector- or Fyn(K-) mutant-transfected MDCK-Tag3 cells were subjected to immunoblotting with monoclonal anti-Fyn antibody. B, MDCK-Tag3 cells stably expressing vector or the Fyn(K-) mutant were either left unstimulated (-) or stimulated with 500 nM UK14304 (UK) for 3 min. Lysates were immunoprecipitated (IP) with polyclonal anti-SHP-2 antibody, and the immunoprecipitates were subjected to immunoblotting (IB) with a mixture of monoclonal anti-phosphotyrosine antibodies (5:1 (v/v) PY20/4G10) (upper panel) or monoclonal anti-Grb2 antibody (center panel). The same blot was stripped and reprobed with monoclonal SHP-2 antibody (lower panel). C, MDCK-Tag3 cells stably expressing vector or the Fyn(K-) mutant were stimulated with 500 nM UK14304 or 10 µM LPA for 3 min. Lysates were immunoprecipitated with monoclonal anti-SHP-2 antibody, and the immunoprecipitates were then subjected to in vitro immune complex phosphatase assay using 32P-labeled Tyr-Raytide as a substrate. The phosphatase activity was evaluated by the extent of 32Pi release from 32P-labeled Tyr-Raytide. Data are presented as -fold increase over unstimulated controls and represent the means ± S.E. of three separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we found that SHP-2 (but not SHP-1) was specifically activated following the stimulation of the alpha 2A-AR and LPA receptor, which lack intrinsic tyrosine kinase activity in MDCK-Tag3 cells. We demonstrated for the first time that the activation of SHP-2 by these Gi protein-coupled receptors was mediated by Fyn kinase specifically associated with SHP-2.

It has been shown that the catalytic activity of SHP-2 is critical for its ability to modulate certain cellular responses to agonist. The phosphatase activity of SHP-2 is required for alpha -thrombin-induced early gene transcription and DNA synthesis, fibroblast growth factor-induced Xenopus development, EGF-induced cell cycle progression, and, in some cell types, PDGF-induced mitogenesis (12, 15, 28). In addition, the catalytic activity of SHP-2 is also required for the MAP kinase activation in response to EGF, insulin, insulin-like growth factor-1, and fibroblast growth factor (11-14). Our findings that SHP-2 (but not SHP-1) is specifically activated and tyrosine-phosphorylated by UK14304 (an agonist of the alpha 2A-AR) and by LPA indicate that SHP-2 is involved in signaling of the Gi protein-coupled receptors. SHP-2 is tyrosine-phosphorylated in response to EGF, PDGF, and an agonist (alpha -thrombin) of a G protein-coupled receptor as well as ligands for cytokine receptors that activate Janus tyrosine kinases JAK1 and JAK2, such as interleukin-2 and -3 and the granulocyte-macrophage colony-stimulating factor (7, 8, 15, 20, 29). Recently, Ali et al. (30) reported that angiotensin II stimulated the activity of SHP-2 and SHP-2 tyrosine phosphorylation through the Gq protein-coupled receptor AT1. The activity of SHP-2 has been reported to increase upon its tyrosine phosphorylation (8), whereas others found no difference in activity upon tyrosine phosphorylation of SHP-2 (7). Our data show that the tyrosine phosphorylation of SHP-2 is associated with its increased phosphatase activity in response to UK14304 and LPA in MDCK-Tag3 cells.

Tyrosine phosphorylation of SHP-1 by the Src family member Lck has been reported in T lymphocytes (31). SHP-2 is constitutively tyrosine-phosphorylated in cells transformed by v-Src (7), suggesting a potential involvement of Src family kinase in the activation of SHP-2. In MDCK-Tag3 cells, Src family kinase was activated rapidly in response to UK14304 and peaked at 2 min, which preceded the maximal activation of SHP-2. PP1 (23), a newly developed selective inhibitor of Src family kinase with a preferential effect on Lck, Fyn, and Hck, blocked the UK14304- and LPA-induced activation of SHP-2, SHP-2 tyrosine phosphorylation, and SHP-2 association with Grb2 and tyrosine-phosphorylated proteins in MDCK-Tag3 cells. Similar inhibitory effects on the agonist-stimulated activation of SHP-2 were obtained by overexpressing a catalytically inactive mutant of Fyn in MDCK-Tag3 cells. Thus, the activation of SHP-2 by the Gi protein-coupled receptors is mediated by Fyn kinase in MDCK-Tag3 cells.

Among the Src family members (Src, Fyn, Lck, Yes, and Lyn) present in MDCK-Tag3 cells, only Fyn was physically and specifically associated with SHP-2, and the interaction between them was increased in response to UK14304 and LPA. The interaction between Fyn and SHP-2 is dependent on the Src family kinase activity since the selective inhibitor of Src family kinase (PP1) blocked the basal and UK14304-induced association of SHP-2 with Fyn. As evidenced by the in vitro pull-down experiments, the GST-Fyn SH2 domain fusion protein can associate with both the phosphorylated form (70 kDa) and the non-phosphorylated form (68 kDa) of SHP-2, and the association is increased upon UK14304 stimulation. SHP-2 contains several potential tyrosine phosphorylation sites, such as Y304INA, Y542TNI, and Y580ENV. Y542TNI and Y580ENV are potential binding sites for the Src and Fyn SH2 domains (32). It has been shown that tyrosine 542 is the major in vivo site of tyrosine phosphorylation on SHP-2 (20, 33). Fyn could bind directly via its SH2 domain to tyrosine-phosphorylated SHP-2. Fyn could also possibly bind directly via its SH2 domain to an intermediary molecule that can be tyrosine-phosphorylated by a Src family kinase and that then interacts with the SH2 domains of SHP-2. Indeed, we observed that UK14034 and LPA promoted the co-immunoprecipitation of SHP-2 with three tyrosine-phosphorylated proteins with apparent molecular masses of ~175, 125, and 115 kDa, respectively, in MDCK-Tag3 cells. The 115-kDa protein band was identified as SHPS-1 (SHP substrate-1) by a specific antibody (data not shown). SHPS-1 is a transmembrane glycoprotein that undergoes tyrosine phosphorylation and binds directly to the SH2 domains of SHP-2 in response to various agonists (34). Takeda et al. (35) recently reported that LPA-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2 are mediated by a Src family kinase in Chinese hamster ovary cells. In addition, using far-Western blotting, we found that the SH2 domains of SHP-2 cannot bind directly to Fyn (data not shown).

The adaptor protein Grb2 functions to couple signals to Ras by recruiting the nucleotide exchange factor Sos to the plasma membrane (36). It has been reported that PDGF promotes interaction of the SHP-2 SH2 domains with the tyrosine-phosphorylated PDGF receptor, SHP-2 tyrosine phosphorylation, and concomitant binding of the SHP-2 phosphotyrosine (Tyr542) to the Grb2 SH2 domain, thereby leading to activation of the Ras/MAP kinase pathway (9, 10). SHP-2 is also tyrosine-phosphorylated and binds to the SH2 domain of Grb2 in response to interleukin-3 and the granulocyte-macrophage colony-stimulating factor (20). We found that UK14304 and LPA induced association of Grb2 with SHP-2 in MDCK-Tag3 cells. Consistent with the previous reports, the association is mediated directly by the SH2 domain of Grb2 and the tyrosine phosphorylation of SHP-2 as evidenced by in vitro pull-down and far-Western blot experiments. Stimulation of the cells with UK14304 and LPA may result in the formation of SHP-2/Grb2·Sos complexes, which in turn may lead to Ras/MAP kinase activation. However, recent studies have indicated a more complicated role for SHP-2 than serving as a binding protein for Grb2 (34, 37). SHP-2 may dephosphorylate its physiological substrates such as the SIRP family of transmembrane proteins including SHPS-1 to exert its positive role in the cell signaling pathway. Recruitment of Grb2·Sos complexes to the plasma membrane requires tyrosine phosphorylation of Grb2-binding sites on membrane-associated scaffolds. Tyrosine phosphorylation of several proteins (such as the adaptor protein Shc, focal adhesion kinase, the EGF receptor, and p185neu) that contain potential Grb2-binding sites has been reported following stimulation of Gi protein-coupled receptors (38, 39). The data presented herein raise the possibility that Grb2 may also function to target SHP-2 to the membrane, where SHP-2 can bind to and dephosphorylate its physiological substrates associated with the membrane.

Src family kinase has been demonstrated to play a key role in signal pathways mediated by Gi protein-coupled receptors (38, 39). We found that SHP-2 (but not SHP-1) was selectively activated upon the stimulation of the alpha 2A-AR and LPA receptor in MDCK-Tag3 cells and that the activation of SHP-2 by the Gi protein-coupled receptors was directly mediated by Fyn kinase through its specific physical interaction with SHP-2. Thus, we have demonstrated that SHP-2 is a specific target regulated by Fyn in the activity of the Gi protein-coupled receptors in MDCK cells.

    ACKNOWLEDGEMENT

We thank J. R. Keefer for the MDCK-Tag3 cells.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants HL-58205 (to T. I.) and HL-57393 (to Z. J. Z.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: 663 Light Hall, Dept. of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: 615-322-4347; Fax: 615-322-3201.

    ABBREVIATIONS

The abbreviations used are: EGF, epidermal growth factor; PDGF, platelet-derived growth factor; MAP, mitogen-activated protein; LPA, lysophosphatidic acid; MDCK, Madin-Darby canine kidney; PVDF, polyvinylidene difluoride; GST, glutathione S-transferase; alpha 2A-AR, alpha 2A-adrenergic receptor; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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