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Blocking the Function of Tyrosine Phosphatase SHP-2 by Targeting Its Src Homology 2 Domains

2003; Elsevier BV; Volume: 278; Issue: 44 Linguagem: Inglês

10.1074/jbc.m306136200

ISSN

1083-351X

Autores

Runxiang Zhao, Xueqi Fu, Lirong Teng, Qingshan Li, Zhizhuang Joe Zhao,

Tópico(s)

Glycosylation and Glycoproteins Research

Resumo

SHP-2 is an Src homology 2 (SH2) domain-containing tyrosine phosphatase with crucial functions in cell signaling and major pathological implications. It stays inactive in the cytosol and is activated by binding through its SH2 domains to tyrosine-phosphorylated receptors on the cell surface. One such cell surface protein is PZR, which contains two tyrosine-based inhibition motifs responsible for binding of SHP-2. We have generated a glutathione S-transferase fusion protein carrying the tandem tyrosine-based inhibition motifs of PZR, and the protein was tyrosine-phosphorylated by co-expressing c-Src in Escherichia coli cells. The purified phosphoprotein displays a strong binding to SHP-2 and causes its activation in vitro. However, when introduced into NIH 3T3 cells by using a protein delivery reagent, it effectively inhibited the activation of ERK1/2 induced by growth factors and serum but not by phorbol ester, in reminiscence of the effects caused by expression of dominant negative SHP-2 mutants and deletion of functional SHP-2. The data suggest that the exogenously introduced PZR protein specifically binds SHP-2, blocks its translocation, and renders it functionally incompetent. This is further supported by the fact that the phosphorylated PZR protein had no inhibitory effects on fibroblasts derived from mice expressing only a mutant SHP-2 protein lacking most of the N-terminal SH2 domain. This study thus provides a novel and highly specific method to interrupt the function of SHP-2 in cells. SHP-2 is an Src homology 2 (SH2) domain-containing tyrosine phosphatase with crucial functions in cell signaling and major pathological implications. It stays inactive in the cytosol and is activated by binding through its SH2 domains to tyrosine-phosphorylated receptors on the cell surface. One such cell surface protein is PZR, which contains two tyrosine-based inhibition motifs responsible for binding of SHP-2. We have generated a glutathione S-transferase fusion protein carrying the tandem tyrosine-based inhibition motifs of PZR, and the protein was tyrosine-phosphorylated by co-expressing c-Src in Escherichia coli cells. The purified phosphoprotein displays a strong binding to SHP-2 and causes its activation in vitro. However, when introduced into NIH 3T3 cells by using a protein delivery reagent, it effectively inhibited the activation of ERK1/2 induced by growth factors and serum but not by phorbol ester, in reminiscence of the effects caused by expression of dominant negative SHP-2 mutants and deletion of functional SHP-2. The data suggest that the exogenously introduced PZR protein specifically binds SHP-2, blocks its translocation, and renders it functionally incompetent. This is further supported by the fact that the phosphorylated PZR protein had no inhibitory effects on fibroblasts derived from mice expressing only a mutant SHP-2 protein lacking most of the N-terminal SH2 domain. This study thus provides a novel and highly specific method to interrupt the function of SHP-2 in cells. SHP-2 is a widely distributed intracellular tyrosine phosphatase that contains two SH2 1The abbreviations used are: SH2, Src homology domain 2; PTP, protein-tyrosine phosphatase; ITIM, immunoreceptor tyrosine-based inhibitory motif; GST, glutathione S-transferase; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; PDGF, platelet-derived growth factor; PMA, phorbol 12-myristate 13-acetate. domains (1Streuli M. Curr. Opin. Cell Biol. 1996; 183: 182-188Crossref Scopus (165) Google Scholar, 2Feng G.S. Exp. Cell Res. 1999; 253: 47-54Crossref PubMed Scopus (252) Google Scholar, 3Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (464) Google Scholar). It has a crucial role in cell signaling. Earlier studies by overexpressing the catalytically inactive cysteine to serine mutant of SHP-2 in cell lines demonstrated that the enzyme plays a positive role in activation of ERK1/2 induced by growth factors (1Streuli M. Curr. Opin. Cell Biol. 1996; 183: 182-188Crossref Scopus (165) Google Scholar, 2Feng G.S. Exp. Cell Res. 1999; 253: 47-54Crossref PubMed Scopus (252) Google Scholar, 3Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (464) Google Scholar). In Xenopus, a dominant negative mutant of SHP-2 blocks fibroblast growth factor- and activin-mediated induction of mesoderm (4Tang T.L. Freeman Jr., R.M. O'Reilly A.M. Neel B.G. Sokol S.Y. Cell. 1995; 80: 473-483Abstract Full Text PDF PubMed Scopus (309) Google Scholar). In mice, disruption of the Shp-2 gene caused death of mouse embryos at mid-gestation (5Saxton T.M. Henkemeyer M. Gasca S. Shen R. Rossi D.J. Shalaby F. Feng G.S. Pawson T. EMBO J. 1997; 16: 2352-2364Crossref PubMed Scopus (406) Google Scholar). Further studies with cells derived from SHP-2-deficient mice demonstrated impairment in erythropoiesis and cell migration (6Qu C.K. Shi Z.Q. Shen R. Tsai F.Y. Orkin S.H. Feng G.S. Mol. Cell. Biol. 1997; 17: 5499-5507Crossref PubMed Scopus (150) Google Scholar, 7Yu D.H. Qu C.K. Henegariu O. Lu X. Feng G.S. J. Biol. Chem. 1998; 273: 21125-21131Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 8Qu C. Nguyen S. Chen J. Feng G.S. Blood. 2001; 97: 911-914Crossref PubMed Scopus (102) Google Scholar). SHP-2 also has major pathological implications. Mutation of Shp-2 causes Noonan syndrome (9Tartaglia M. Mehler E.L. Goldberg R. Zampino G. Brunner H.G. Kremer H. van der Burgt I. Crosby A.H. Ion A. Jeffery S. Kalidas K. Patton M.A. Kucherlapati R.S. Gelb B.D. Nat. Genet. 2001; 29: 465-468Crossref PubMed Scopus (1341) Google Scholar), an autosomal dominant disorder characterized by dysmorphic facial features, proportionate short stature, and heart disease. Excessive SHP-2 activity caused by mutation in the N-terminal SH2 domain is considered to be responsible for the pathogenesis. Furthermore, SHP-2 is an intracellular target of the CagA protein in Helicobacter pylori (10Higashi H. Tsutsumi R. Muto S. Sugiyama T. Azuma T. Asaka M. Hatakeyama M. Science. 2002; 295: 683-686Crossref PubMed Scopus (855) Google Scholar). H. pylori are associated with severe gastritis and gastric cancers, and CagA is introduced from the attached H. pylori into host cells and undergoes tyrosine phosphorylation and thereby activates SHP-2. Considering the important role of SHP-2 in cell signaling and its pathological implications, inhibition of the enzyme has great research and therapeutic values. However, a specific inhibitor of SHP-2 is still lacking. The catalytic domains of all classic protein-tyrosine phosphatases (PTPs) share extremely high similarity in ternary structures. In fact, the catalytic domains of SHP-2 and its closest homologue SHP-1 share 60% sequence identity, but the ternary structures of the two catalytic domains are almost superimposible (11Yang J. Liang X. Niu T. Meng W. Zhao Z. Zhou G.W. J. Biol. Chem. 1998; 273: 28199-28207Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 12Hof P. Pluskey S. Dhe-Paganon S. Eck M.J. Shoelson S.E. Cell. 1998; 92: 441-450Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar, 13Yang J. Liu L. He D. Song X. Liang X. Zhao Z.J. Zhou G.W. J. Biol. Chem. 2003; 278: 6516-6520Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). This makes it difficult to find highly selective inhibitors targeting the catalytic domains. On the other hand, PTPs are known to have highly specific functions, and this specificity is at least partly conferred by the non-catalytic domains or segments beyond the catalytic domains (3Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (464) Google Scholar). SHP-1 and SHP-2 both contain SH2 domains that play important regulatory and targeting roles. In vitro, purified SHP-1 and SHP-2 display very low specific activity (14Zhao Z. Bouchard P. Diltz C.D. Shen S.H. Fischer E.H. J. Biol. Chem. 1993; 268: 2816-2820Abstract Full Text PDF PubMed Google Scholar, 15Zhao Z. Larocque R. Ho W.T. Fischer E.H. Shen S.H. J. Biol. Chem. 1994; 269: 8780-8785Abstract Full Text PDF PubMed Google Scholar). This low level of activity is because of an internal suppression because the enzymes are significantly activated by removal of either C-terminal segments or SH2 domains (14Zhao Z. Bouchard P. Diltz C.D. Shen S.H. Fischer E.H. J. Biol. Chem. 1993; 268: 2816-2820Abstract Full Text PDF PubMed Google Scholar, 15Zhao Z. Larocque R. Ho W.T. Fischer E.H. Shen S.H. J. Biol. Chem. 1994; 269: 8780-8785Abstract Full Text PDF PubMed Google Scholar), by interaction with anionic phospholipids (15Zhao Z. Larocque R. Ho W.T. Fischer E.H. Shen S.H. J. Biol. Chem. 1994; 269: 8780-8785Abstract Full Text PDF PubMed Google Scholar, 16Zhao Z. Shen S.H. Fischer E.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4251-4255Crossref PubMed Scopus (109) Google Scholar, 17Tomic S. Greiser U. Lammers R. Kharitonenkov A. Imyanitov E. Ullrich A. Bohmer F.D. J. Biol. Chem. 1995; 270: 21277-21284Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), and by binding of SH2 domains to specific phosphopeptides (18Lechleider R.J. Sugimoto S. Bennett A.M. Kashishian A.S. Cooper J.A. Shoelson S.E. Walsh C.T. Neel B.G. J. Biol. Chem. 1993; 268: 21478-21481Abstract Full Text PDF PubMed Google Scholar, 19Sugimoto S. Wandless T.J. Shoelson S.E. Neel B.G. Walsh C.T. J. Biol. Chem. 1994; 269: 13614-13622Abstract Full Text PDF PubMed Google Scholar). Furthermore, x-ray crystal structures of SHP-1 and SHP-2 reveal blocking of the catalytic center by the N-terminal SH2 domains (12Hof P. Pluskey S. Dhe-Paganon S. Eck M.J. Shoelson S.E. Cell. 1998; 92: 441-450Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar, 13Yang J. Liu L. He D. Song X. Liang X. Zhao Z.J. Zhou G.W. J. Biol. Chem. 2003; 278: 6516-6520Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In vivo, SHP-1 and SHP-2 reside mainly in the cytosol where they are presumably inactive. It is generally believed that SHP-1 and SHP-2 are activated by binding to tyrosine-phosphorylated receptors and other proteins concomitant with translocation of the enzymes to the plasma membrane (1Streuli M. Curr. Opin. Cell Biol. 1996; 183: 182-188Crossref Scopus (165) Google Scholar, 2Feng G.S. Exp. Cell Res. 1999; 253: 47-54Crossref PubMed Scopus (252) Google Scholar, 3Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (464) Google Scholar). Therefore, activation of SHP-1 and SHP-2 is controlled by membrane or membrane-associated proteins that recruit them. We reasoned that physiological functions of SHP-1 and SHP-2 could be efficiently blocked by exogenously introduced tyrosine-phosphorylated peptides or proteins that bind the enzymes and keep them in the cytosol or other cellular compartments where the enzymes are not functional. SHP-1 and SHP-2 have been known to be recruited to many receptors. Recently, we have isolated a cell surface glycoprotein designated PZR that specifically recruits SHP-2 through its immunoreceptor tyrosine-based inhibition motifs (ITIMs) (20Zhao Z. Tan T. Wright J.H. Diltz C.D. Shen S.-H. Krebs E.G. Fischer E.H. J. Biol. Chem. 1995; 270: 11765-11769Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 21Zhao Z.J. Zhao R. J. Biol. Chem. 1998; 273: 29367-29372Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 22Zhao R. Zhao Z.J. J. Biol. Chem. 2000; 275: 5453-5459Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). PZR are efficiently phosphorylated by the Src family tyrosine kinases in vitro and in vivo (23Zhao R. Guerrah A. Tang H. Zhao Z.J. J. Biol. Chem. 2002; 277: 7882-7888Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We have prepared a glutathione S-transferase (GST) fusion protein carrying tyrosine-phosphorylated ITIMs of PZR from Escherichia coli cells coexpressing active c-Src. In this study, we introduced the GST fusion protein into cells by using the BioPorter reagent. This effectively inhibited growth factor- and serum-induced activation of ERK1/2 by blocking SHP-2 function. Our study thus provides a novel and highly specific method to interrupt the function of SHP-2 in intact cells. Materials—NIH 3T3 cells and fibroblasts cells from wild type (SHP-2+/+) and functional SHP-2-deficient (Shp-2Δ46–110) mice were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 units/ml each of penicillin and streptomycin. Human embryonic kidney 293 cells overexpressing SHP-2 were maintained in the same medium plus 0.25 mg/ml G418 sulfate (20Zhao Z. Tan T. Wright J.H. Diltz C.D. Shen S.-H. Krebs E.G. Fischer E.H. J. Biol. Chem. 1995; 270: 11765-11769Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Polyclonal anti-SHP-2, monoclonal anti-SHP-2, polyclonal anti-pERK(Thr202/Tyr204), monclonal anti-GST, and monoclonal anti-phosphotyrosine 4G10 were purchased from Santa Cruz Biotechnology, Signal Transduction Laboratories, Cell Signaling Technology, Sigma, and Upstate Biotechnology Inc., respectively. SHP-1, SHP-2, and ΔSH2-SHP-2 were purified as previously described (14Zhao Z. Bouchard P. Diltz C.D. Shen S.H. Fischer E.H. J. Biol. Chem. 1993; 268: 2816-2820Abstract Full Text PDF PubMed Google Scholar, 15Zhao Z. Larocque R. Ho W.T. Fischer E.H. Shen S.H. J. Biol. Chem. 1994; 269: 8780-8785Abstract Full Text PDF PubMed Google Scholar). Expression of GST Fusion Proteins Carrying the Intracellular Domain of PZR— Fig. 1A shows schematic structures of PZR and two recombinant fusion proteins. GST-ΔPZR represents a GST fusion protein carrying the intracellular domain of PZR corresponding to amino acid residues 192–269 of the molecule. The recombinant protein was expressed in E. coli cells and purified by using glutathione-Sepharose beads. GST-pΔPZR is a tyrosine-phosphorylated form of GST-ΔPZR. The phosphorylation occurred only at the ITIMs of PZR and was achieved by co-expressing c-Src carried by a pET9a vector in E. coli cells as previously described (23Zhao R. Guerrah A. Tang H. Zhao Z.J. J. Biol. Chem. 2002; 277: 7882-7888Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The protein was also purified from glutathione-Sepharose columns. SHP-2 Binding and Phosphatase Activity Assays—GST-pΔPZR, GST-PZR, and GST-pΔPZR immobilized on glutathione-Sepharose beads were incubated with extracts of 293 cells overexpressing SHP-2. The cell extracts were made in Buffer A containing 25 mm β-glycerol-phosphate (pH 7.3), 0.1 m NaCl, 1% Triton X-100, 5 mm EDTA, 0.5 mm Na3VO4, 10 mm β-mercaptoethanol, and a protease inhibitor mixture (Roche Diagnostics). After washing three times with Buffer A, proteins bound to the beads were analyzed by SDS gels followed by immunoblotting or Coomassie Blue staining. To investigate the specific activation of SHP-2 by GST-pΔPZR, PTP activities of SHP-1, SHP-2, and ΔSH2-SHP-2 were measured with 2 mm p-nitrophenyl phosphate as substrate in the presence of GST-ΔPZR or GST-pΔPZR. The assays were carried out at room temperature, and the assay system contained 25 mm HEPES-NaOH (pH 7.0), 1.0 mm EDTA, 0.5 mg/ml bovine serum albumin, and 1.0 mm dithiothreitol. BioPorter-mediated Transfer of Proteins into Cells—We employed the BioPorter reagent (Gene Therapy Systems) to transfer GST-ΔPZR and GST-pΔPZR into NIH 3T3 cells following the protocol provided by the manufacturer. Briefly, 5 μl of BioPorter reagent was air-dried under a tissue culture hood for 2–3 h and then resuspended in 50 μl of phosphate-buffered saline containing 3–6 μg of GST-ΔPZR or GST-pΔPZR. After 5 min incubation, the suspensions were mixed with 250 μl of plain Dulbecco's modified Eagle's medium and were then added to NIH 3T3 cells (∼80–90% confluency) cultured in 12-well cell culture plates. Prior to the addition of the protein-BioPorter complex, the cells were washed twice with phosphate-buffered saline. After addition of the complex, cells were further incubated at 37 °C in 5% CO2 culture incubator for 4–6 h before further treatment. Cell Stimulation, Extraction, and Western Blotting Analyses—After 3–6 h of incubation with GST-ΔPZR and GST-pΔPZR carried by the BioPorter reagent, cells were treated with fetal bovine serum (FBS), epidermal growth factor, platelet-derived growth factor (PDGF), insulin, and phorbol 12-myristate 13-acetate (PMA) for various periods of time, and the stimulation was stopped by washing with ice-cold phosphate-buffered saline. The cells were then extracted in the Buffer A. Cell lysates were cleared by centrifugation, and clear cell extracts were then separated by 10% SDS gels for Western blot analyses with specified antibodies. Detection was made by enhanced chemiluminescence, and quantification of the gel bands was performed by using a gel scanner. GST-pΔPZR Specifically Binds and Activates SHP-2 in Vitro—In our previous study (23Zhao R. Guerrah A. Tang H. Zhao Z.J. J. Biol. Chem. 2002; 277: 7882-7888Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), we demonstrated that Src family protein-tyrosine kinases are at least partly responsible for tyrosine phosphorylation of PZR in cells, and we further showed that co-expression of GST-ΔPZR with c-Src in E. coli cells causes strong phosphorylation of GST-ΔPZR. The phosphorylation occurs on the tyrosyl residues of PZR embedded in ITIMs because mutation of the residues diminished the phosphorylation (23Zhao R. Guerrah A. Tang H. Zhao Z.J. J. Biol. Chem. 2002; 277: 7882-7888Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). By using glutathione-Sepharose columns, we were able to purify a large amount of tyrosine-phosphorylated GST-pΔPZR from E. coli cell extracts. To analyze the ability of GST-pΔPZR to interact with SHP-2, we incubated glutathione-Sepharose beads carrying GST-pΔPZR with cell extracts obtained from 293 cells overexpressing SHP-2 (20Zhao Z. Tan T. Wright J.H. Diltz C.D. Shen S.-H. Krebs E.G. Fischer E.H. J. Biol. Chem. 1995; 270: 11765-11769Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). As shown in Fig. 1B, Western blotting analysis with anti-SHP-2 antibodies revealed a strong binding of SHP-2 with GST-pΔPZR but minimal binding with non-phosphorylated GST-ΔPZR. GST-pΔPZR-associated SHP-2 was also clearly detected by Coomassie Blue staining. In a sense, GST-pΔPZR provides a very effective method to enrich SHP-2 from cell extracts. Considering the fact that the GST-pΔPZR protein sample may not be 100% phosphorylated, the data suggest a near stoichiometric association of GST-pΔPZR with SHP-2. When similar experiments were conducted with cell extracts from 293 cells overexpressing SHP-1, a much smaller level of SHP-1 was detected by Western blotting, and no binding was seen in Coomassie Blue staining (data not shown). This suggests a much preferable interaction of GST-pΔPZR with SHP-2 over SHP-1. These data agree with our previous results obtained with immunoprecipitation (20Zhao Z. Tan T. Wright J.H. Diltz C.D. Shen S.-H. Krebs E.G. Fischer E.H. J. Biol. Chem. 1995; 270: 11765-11769Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 21Zhao Z.J. Zhao R. J. Biol. Chem. 1998; 273: 29367-29372Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 22Zhao R. Zhao Z.J. J. Biol. Chem. 2000; 275: 5453-5459Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The specific interaction of GST-pΔPZR with SHP-2 is also demonstrated by specific activation of SHP-2 but not SHP-1 or ΔSH2-SHP-2 by GST-pΔPZR. As shown in Fig. 2, the presence of GST-pΔPZR in phosphatase assay reactions resulted in near 20-fold activation of SHP-2, but non-phosphorylated GST-ΔPZR had no stimulatory effect. In contrast, GST-pΔPZR only caused marginal activation of SHP-1 and had no effects on ΔSH2-SHP-2, an SH2 domain-truncated form of SHP-2. These results further verify that PZR specifically interacts with the SH2 domains of SHP-2. Because GST-pΔPZR activate SHP-2 by binding to its SH2 domains, SHP-2 activation can be considered as a measure of the interaction between SH2 domains and GST-pΔPZR. The BioPorter Protein Delivery Reagent Efficiently Transfers GST-pΔPZR into Cultured Cells and Initiates its Interaction with SHP-2—To deliver GST-pΔPZR into live cells, we employed the BioPorter protein delivery reagent developed by Gene Therapy Systems, Inc. The BioPorter protein delivery reagent is a cationic lipid mixture that when complexed with proteins and peptides allows for direct intracellular delivery. The complex formed is non-covalent and it therefore does not interfere with the biological activity of the protein. This technique is rapid and relatively uncomplicated. With the BioPorter reagent, we were able to effectively deliver GST-ΔPZR and GST-pΔPZR into NIH 3T3 cells. As shown in Fig. 3, both tyrosine-phosphorylated GST-pΔPZR and non-phosphorylated GST-ΔPZR were recovered in the cell extracts when the proteins complexed with the BioPorter reagent were incubated with the cells. In contrast, no incorporation of GST-pΔPZR into the cells was observed in the absence of the BioPorter reagent. Furthermore, transferred GST-pΔPZR was able effectively interact with SHP-2 as indicated by the "pull down" assays with glutathione-Sepharose beads. Therefore, the BioPorter reagent provides an effective way to deliver functional GST-pΔPZR into 3T3 cells to target SHP-2. In fact, Western blotting analyses of cell extracts and culture medium revealed that 15–20% of the GST fusion proteins entered the cells, and immunofluorescent cell staining showed nearly 100% cells were uploaded with the exogenously introduced fusion proteins in the entire cytoplasma (data not shown). Note that the efficient transfer mediated by the BioPorter reagent was obtained in serum-free medium, which is also used as the serum starvation step for stimulation of cells. GST-pΔPZR Efficiently Blocks Activation of ERK1/2 Induced by Serum and Growth Factors—SHP-2 has been defined as a positive signal transducer in growth factor signaling. To find out whether introduction GST-pΔPZR into cells block normal functions of SHP-2, we investigated activation of ERK1/2. NIH 3T3 cells were incubated with mixtures of the BioPorter reagent and GST-ΔPZR or GST-pΔPZR in serum-free medium. After 3 and 6 h of incubation, the cells were treated with 15% FBS. Western blotting analyses of the cell extracts are shown Fig. 4. A 3-h incubation was sufficient to transfer the GST fusion proteins into the cells. A longer incubation period (6 h) did not significantly change the transfer efficiency or the stability of transferred proteins, but a higher concentration of GST-ΔPZR and GST-pΔPZR clearly favored the transfer. We employed a phospho-specific anti-ERK1/2 antibody to determine the activation of ERK1/2. The antibody detects ERK1 and ERK2 (p42 and p44 MAP kinase) only when they phosphorylated at the threonyl and tyrosyl residues that are required for activation of the enzymes. Upon stimulation with FBS, the control cells and the cells treated with non-phosphorylated GST-ΔPZR showed a marked increase in the phosphorylation of ERK1/2 over the basal level. In contrast, the cells treated with GST-pΔPZR at both lower and higher concentrations exhibited essentially no response. Mobility shifts of ERK1/2 detected by a regular anti-ERK1/2 antibody further verified the data. As shown in the bottom panel of Fig. 4, a clear up-shift of the ERK2 band was seen with control and GST-ΔPZR cells but not with the cell treated with GST-pΔPZR. The mobility shift is also an indication of ERK1/2 phosphorylation. Therefore, the data indicate that introduction of GST-pΔPZR into cells significantly inhibited FBS-induced activation of ERK1/2. We further analyzed the effects of GST-pΔPZR on activation of ERK1/2 induced by growth factors and the phorbol ester PMA. As shown in Fig. 5 (top panel), whereas GST-pΔPZR significantly reduced the activation of ERK1/2 induced by PDGF, epidermal growth factor, and insulin, it had no effect on that induced by PMA. This is reminiscent of the data obtained with cells overexpressing of the dominant negative mutant form of SHP-2 and cells derived from Shp-2Δ46–110 mice (20Zhao Z. Tan T. Wright J.H. Diltz C.D. Shen S.-H. Krebs E.G. Fischer E.H. J. Biol. Chem. 1995; 270: 11765-11769Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 24Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (350) Google Scholar, 25Shi Z.Q. Lu W. Feng G.S. J. Biol. Chem. 1998; 273: 4904-4908Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). A quantitative representation of the effects of GST-pΔPZR on the activation of ERK2 is shown in Fig. 6. The delivery of GST-pΔPZR into NIH 3T3 cells reduced FBS- and growth factor-induced ERK2 phosphorylation by 70–90% but had no effect on that induced by PMA. Taken together, the data indicate that introduction of GST-pΔPZR specifically inhibited growth factor- and serum-induced activation of ERK1/2 in a way similar to the expression of dominant negative SHP-2 or deletion of functional SHP-2.Fig. 5Different effects of GST-pΔPZR on activation of ERK1/2 induced by growth factors and phorbol ester PMA. NIH 3T3 cells were incubated with 4 μg of GST-ΔPZR or GST-pΔPZR premixed with the BioPorter reagent for 4 h at 37 °C. Control experiments were performed without addition of GST-ΔPZR or GST-pΔPZR. The cells were stimulated with 15% FBS for 15 min, 10 ng/ml PDGF for 5 min, 0.1 nm PMA for 15 min, 40 ng/ml epidermal growth factor (EGF) for 5 min, or 0.5 μm insulin for 5 min. The cells were then extracted in Buffer A, and samples containing equal amounts of total proteins (20 μg) were subjected to Western blotting analyses with the indicated antibodies. The bottom panel shows a section of the polyvinylidene difluoride membrane stained with Coomassie Blue to demonstrate equal protein loadings. A tyrosine-phosphorylated protein band at ∼90 kDa is indicated by arrows. The position of autophosphorylated PDGF receptor is also shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Quantitative representation of the effects of GST-pΔPZR on activation of ERK2 induced by growth factors and phorbol ester PMA. Experiments were performed as described in the legend to Fig. 5. The intensity of pERK2 bands was quantified by gel scanning and normalized against total proteins in cell extracts. Data represent relative intensity (mean ± S.D., n ≥ 3). The level of pERK2 at the basal level in control cells was defined as 1. EGF, epidermal growth factor.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Delivery of GST-pΔPZR into NIH 3T3 Cells Causes Hyper-phosphorylation of a 90-kDa Protein—By specifically targeting SHP-2, GST-pΔPZR may affect tyrosine phosphorylation of specific cellular proteins. We performed Western blotting analyses of cell extracts with an anti-phosphotyrosine antibody to find out whether this is indeed the case. When the whole cell extracts of NIH 3T3 cells were separated on SDS gels, the PDGF receptor running at ∼180 kDa was shown as a major tyrosine-phosphorylated band, and its tyrosine phosphorylation was markedly enhanced upon stimulation of cells with PDGF (Fig. 5). However, introduction of neither GST-ΔPZR nor GST-pΔPZR in the cells had significant effects on its tyrosine phosphorylation at the basal level or under PGDF stimulation. Immunoprecipitation of the PDGF receptor followed by anti-phosphotyrosine immunoblotting analyses further verified the results with the PDGF receptor, and similar data were obtained with the epidermal growth factor receptor and the insulin receptor (data not shown). This suggests that GST-pΔPZR does not affect growth factor signaling at the receptor level. Interestingly, treatment of cells with GST-pΔPZR but not with GST-ΔPZR resulted in appearance of a broad tyrosine-phosphorylated protein band around 90 kDa (see Fig. 5). Tyrosine phosphorylation of this protein was not altered in response to stimulation with growth factors. This protein likely corresponds to the tyrosine-phosphorylated protein of similar molecular size observed in fibroblast cells derived from the Shp-2Δ46–110 mice (26Shi Z.Q. Yu D.H. Park M. Marshall M. Feng G.S. Mol. Cell Biol. 2000; 20: 1526-1536Crossref PubMed Scopus (191) Google Scholar). This also suggests that GST-pΔPZR effectively blocks growth factor-induced activation of ERK1/2 by blocking the function of SHP-2. GST-pΔPZR Has No Inhibitory Effects on Activation of ERK1/2 in Functional SHP-2-deficient Fibroblast Cells—To prove further that the exogenously introduced GST-pΔPZR block the function of SHP-2, we employed functional SHP-2-deficient fibroblast cells derived from SHP-2Δ46–110 mice. The mice were generated by targeted deletion of exon 3 of the Shp-2 gene that results in expression of an N-terminal SH2 domain-truncated enzyme (5Saxton T.M. Henkemeyer M. Gasca S. Shen R. Rossi D.J. Shalaby F. Feng G.S. Pawson T. EMBO J. 1997; 16: 2352-2364Crossref PubMed Scopus (406) Google Scholar). Fibroblast cells derived from wild type mice were used as control. As obtained with NIH 3T3 cells, the BioPorter reagent effectively delivered GST-pΔPZR into both SHP-2+/+ and SHP-2Δ46–110 cells. However, while wild type SHP-2 was found to be associated with GST-pΔPZR, N-terminal SH2 domain-truncated SHP-2Δ46–110 was not, suggesting that the remaining C-terminal SH2 domain in SHP-2Δ46–110 is not sufficient to mediate efficient interactions with ITIMs of PZR (Fig. 7A). This is further supported by the fact that pervanadate failed to induce associations of SHP-2Δ46–110 with endogenous PZR in contrast to the robust associations seen with native SHP-2 (Fig. 7B). We further treated the GST-pΔPZR-loaded cells with FBS and PDGF. As shown in Fig. 7C, SHP-2+/+ cells displayed strong activation of ERK1/2, but the activation was significantly inhibited by treatment of cells with GST-pΔPZR. In contrast, SHP-2Δ46–110 cells exhibited a much weaker response to FBS and PDGF, but GST-pΔPZR had no inhibitory effects. These data provide strong evidence that GST-pΔPZR inhibits serum- and growth factor-induced activation of ERK1/2 by blocking the normal function of SHP-2. In this study, we have demonstrated that a GST fusion protein carrying tyrosine-phosphorylated ITIMs of PZR effectively inhibited serum- and growth factor-induced activation of ERK1/2. We believe that the inhibitory effects of GST-pΔPZR are mediated by blocking the normal function of tyrosine phosphatase SHP-2 in the cells. This is supported by the following facts. First, the inhibitory role is dependent on tyrosine phosphorylation of PZR, and tyrosine-phosphorylated ITIMs of PZR specifically interact with the SH2 domain of SHP-2 in vitro. Second, the inhibitory effects are specific for growth factor but not for phorbol ester PMA. This agrees with the expression of the dominant negative mutant of SHP-2 and targeted disruption of SHP-2 in mice (24Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (350) Google Scholar, 25Shi Z.Q. Lu W. Feng G.S. J. Biol. Chem. 1998; 273: 4904-4908Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 26Shi Z.Q. Yu D.H. Park M. Marshall M. Feng G.S. Mol. Cell Biol. 2000; 20: 1526-1536Crossref PubMed Scopus (191) Google Scholar, 27Xiao S. Rose D.W. Sasaoka T. Maegawa H. Burke Jr., T.R. Roller P.P. Shoelson S.E. Olefsky J.M. J. Biol. Chem. 1994; 269: 21244-21248Abstract Full Text PDF PubMed Google Scholar, 28Zhao R. Zhao Z.J. Biochem. 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We have turned a powerful in vitro activator of SHP-2 into an effective blocker of the enzyme in vivo. This is because of the structural and regulatory nature of the enzyme. The model shown in Fig. 8 provides a schematic explanation. The activity of SHP-2 is tightly regulated. During the resting state of cells, SHP-2 stays mostly in the cytosol where it remains inactive because of its internal suppressive structure. Upon stimulation, specific tyrosine-phosphorylated anchor proteins recruit SHP-2 through the SH2 domains and results in translocation and activation of the enzyme. Translocation is the key in regulation of the enzyme. In the cytosol or other cellular compartments where SHP-2 substrates do not exist, SHP-2 should be non-functional even if it is in an enzymatically active state. Therefore, a peptide or protein that binds the SH2 domains of SHP-2 and thereby blocks its translocation to desired locations should specifically inhibit the function of SHP-2. This notion is fully supported by our current experimental data. This view also explains the loss of SHP-2 function in SHP-2Δ46–110 mice in which exon 3 of the Shp-2 gene was deleted. Targeted deletion of exon 3 gave rise to an N-terminal SH2 domain-truncated enzyme (5Saxton T.M. Henkemeyer M. Gasca S. Shen R. Rossi D.J. Shalaby F. Feng G.S. Pawson T. EMBO J. 1997; 16: 2352-2364Crossref PubMed Scopus (406) Google Scholar). The truncated enzyme is expressed in mouse cells and is presumably more active. However, it is apparently nonfunctional in vivo because of its inability to be recruited to desired locations (5Saxton T.M. Henkemeyer M. Gasca S. Shen R. Rossi D.J. Shalaby F. Feng G.S. Pawson T. EMBO J. 1997; 16: 2352-2364Crossref PubMed Scopus (406) Google Scholar, 6Qu C.K. Shi Z.Q. Shen R. Tsai F.Y. Orkin S.H. Feng G.S. Mol. Cell. Biol. 1997; 17: 5499-5507Crossref PubMed Scopus (150) Google Scholar, 7Yu D.H. Qu C.K. Henegariu O. Lu X. Feng G.S. J. Biol. Chem. 1998; 273: 21125-21131Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 8Qu C. Nguyen S. Chen J. Feng G.S. Blood. 2001; 97: 911-914Crossref PubMed Scopus (102) Google Scholar). A recent study with a total knockout of SHP-2 showed similar embryonic lethality and thereby excluded any possible gain-of-function of the truncated SHP-2 (discussed in Ref. 3Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (464) Google Scholar). Both introduction of GST-pΔPZR in this study and the deletion of SHP-2 exon 3 in the earlier studies resulted in an enzymatically active but non-transferable enzyme, and the consequence is loss of SHP-2 function. It should be pointed out that because the affinity of SHP-2 to substrate is largely conferred by target domains and its catalytic domain possesses substrate specificity, this catalytically active but non-transferable enzyme per se should not cause nonspecific effects. On the other hand, if an exogenously introduced protein is localized to the plasma membrane and has the ability to recruit SHP-2, it should functionally activate SHP-2. This is probably the case with the CagA protein of H. pylori. CagA is localized to the plasma membrane of host cells and is constitutively phosphorylated on tyrosyl residues. Tyrosine-phosphorylated CagA recruits SHP-2 through SH2 domain binding to the plasma membrane and cause its functional activation. This is believed to be responsible for gastric tumors caused by bacteria. Recent studies have also linked mutations of Shp-2 with ∼50% of Noonan syndrome (9Tartaglia M. Mehler E.L. Goldberg R. Zampino G. Brunner H.G. Kremer H. van der Burgt I. Crosby A.H. Ion A. Jeffery S. Kalidas K. Patton M.A. Kucherlapati R.S. Gelb B.D. Nat. Genet. 2001; 29: 465-468Crossref PubMed Scopus (1341) Google Scholar, 33Tartaglia M. Kalidas K. Shaw A. Song X. Musat D.L. van der Burgt I. Brunner H.G. Bertola D.R. Crosby A. Ion A. Kucherlapati R.S. Jeffery S. Patton M.A. Gelb B.D. Am. J. Hum. Genet. 2002; 70: 1555-1563Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar, 34Maheshwari M. Belmont J. Fernbach S. Ho T. Molinari L. Yakub I. Yu F. Combes A. Towbin J. Craigen W.J. Gibbs R. Hum. Mutat. 2002; 20: 298-304Crossref PubMed Scopus (71) Google Scholar). Many of these mutations involve residues that participate in the interaction of the N-SH2 and PTP domains that cause autoinhibition of PTP activity. It is generally thought that activation of SHP-2 is responsible for the pathogenesis. Based on current data, the activation of SHP-2 per se may not be important. Instead, the enhanced ability of SHP-2 to bind tyrosine-phosphorylated peptide motifs and consequent translocation may be crucial. This explains the pathogenic effectiveness of some mutations of SHP-2 in Noonan syndrome that are not related to the N-SH2 and PTP domain interaction responsible for autoinhibition of the enzyme (33Tartaglia M. Kalidas K. Shaw A. Song X. Musat D.L. van der Burgt I. Brunner H.G. Bertola D.R. Crosby A. Ion A. Kucherlapati R.S. Jeffery S. Patton M.A. Gelb B.D. Am. J. Hum. Genet. 2002; 70: 1555-1563Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar, 34Maheshwari M. Belmont J. Fernbach S. Ho T. Molinari L. Yakub I. Yu F. Combes A. Towbin J. Craigen W.J. Gibbs R. Hum. Mutat. 2002; 20: 298-304Crossref PubMed Scopus (71) Google Scholar). It is apparent that inhibition of SHP-1 and SHP-2 function has important research and therapeutic values. Because the specific functions of SHP-1 and SHP-2 are defined by both catalytic and SH2 domains, inhibitors can target the SH2 domains as well as the catalytic domains. Determination of the crystal structures revealed very high structural similarity between SHP-1 and SHP-2, especially in the catalytic and N-SH2 domains (12Hof P. Pluskey S. Dhe-Paganon S. Eck M.J. Shoelson S.E. Cell. 1998; 92: 441-450Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar, 13Yang J. Liu L. He D. Song X. Liang X. Zhao Z.J. Zhou G.W. J. Biol. Chem. 2003; 278: 6516-6520Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Both SHP-1 and SHP-2 use the N-SH2 domain to keep the enzymes in the inactive conformation. However, the C-SH2 domain in the SHP-1 structure is differently orientated from that in SHP-2, and its secondary structure elements are relatively more openly organized (13Yang J. Liu L. He D. Song X. Liang X. Zhao Z.J. Zhou G.W. J. Biol. Chem. 2003; 278: 6516-6520Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Therefore, the C-SH2 domains may play a major role in determining the specificity of SHP-1 and SHP-2, and therefore, targeting the C-SH2 domain may be more likely to produce specific inhibitors. Main methodology of drug development in industry has centered on the screening of vast libraries of molecules for biological activity upon which variants are produced to maximize beneficial medical characteristics. Recent developments in the understanding of the biochemistry of intracellular processes and protein-protein interaction have given rise to an alternative method by using peptides and proteins targeted at specific protein domains. New methods of noninvasive delivery of functional peptides and proteins to cells have made this feasible (35Dunican D.J. Doherty P. Biopolymers. 2001; 60: 45-60Crossref PubMed Scopus (62) Google Scholar, 36Hawiger J. Curr. Opin. Chem. Biol. 1999; 3: 89-94Crossref PubMed Scopus (133) Google Scholar, 37Rojas M. Donahue J.P. Tan Z. Lin Y.Z. Nat. Biotechnol. 1998; 16: 370-375Crossref PubMed Scopus (142) Google Scholar). In this study, we employed the BioPorter reagent to deliver GST-ΔPZR into NIH 3T3 cells. This method worked very well for NIH 3T3 cells when the transfer is conducted in serum-free conditions. We believe other delivery methods including attachment of cell-permeable peptide sequences can also be employed. We employed a tyrosine-phosphorylated GST fusion protein in our current study. In principle, synthetic diphosphotyrosyl peptides derived from the ITIM sequences should be equally effective. In addition, the strategy for functional inhibition of SHP-2 can be applied to SHP-1. SHP-1 and SHP-2 share over 50% sequence identity but their functions are often opposite. Our earlier studies demonstrated that SHP-1 preferentially binds LAIR-1, and the specificity is conferred by the interactions between the dual ITIMs and the dual SH2 domains (38Xu M. Zhao R. Zhao Z.J. J. Biol. Chem. 2000; 275: 17440-17446Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Therefore, tyrosine-phosphorylated ITIMs of LAIR-1 should serve as a selective blocker of SHP-1. We are grateful to Dr. Gen-Sheng Feng (Burnham Institute, La Jolla, CA) for providing us with SHP-2+/+ and SHP-2Δ46–110 mouse fibroblast cells.

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