Selective Inhibition of Growth Factor-stimulated Mitogenesis by a Cell-permeable Grb2-binding Peptide

Abstract

The activation of the mitogen-activated protein kinase (MAPK) cascade by a variety of growth factors and other agents is central to a mitogenic response. In the case of polypeptide growth factors such as the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), the steps leading to activation of MAPK require the function of the adaptor protein Grb2 (growth factor receptor binding protein 2), which can bind either directly or indirectly via its Src homology 2 domain to activated receptor tyrosine kinases. A cell-permeable mimetic of the EGF receptor Grb2 binding site has been investigated for its ability to inhibit biological responses stimulated by a variety of growth factors. Pretreatment of cells with this peptide results in the accumulation of the peptide in cells and its association with Grb2. This is associated with a complete inhibition of the mitogenic response stimulated by EGF and PDGF. In contrast, the peptide has no effect on the mitogenic response stimulated by fibroblast growth factor. The peptide could also inhibit the phosphorylation of MAPK stimulated with EGF and PDGF in the absence of an effect on the fibroblast growth factor response. These data demonstrate that cell-permeable mimetics of Src homology 2 binding sites can selectively inhibit growth factor-stimulated mitogenesis, and also directly demonstrate specificity in the coupling of activated receptor tyrosine kinases to the MAPK cascade. The activation of the mitogen-activated protein kinase (MAPK) cascade by a variety of growth factors and other agents is central to a mitogenic response. In the case of polypeptide growth factors such as the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), the steps leading to activation of MAPK require the function of the adaptor protein Grb2 (growth factor receptor binding protein 2), which can bind either directly or indirectly via its Src homology 2 domain to activated receptor tyrosine kinases. A cell-permeable mimetic of the EGF receptor Grb2 binding site has been investigated for its ability to inhibit biological responses stimulated by a variety of growth factors. Pretreatment of cells with this peptide results in the accumulation of the peptide in cells and its association with Grb2. This is associated with a complete inhibition of the mitogenic response stimulated by EGF and PDGF. In contrast, the peptide has no effect on the mitogenic response stimulated by fibroblast growth factor. The peptide could also inhibit the phosphorylation of MAPK stimulated with EGF and PDGF in the absence of an effect on the fibroblast growth factor response. These data demonstrate that cell-permeable mimetics of Src homology 2 binding sites can selectively inhibit growth factor-stimulated mitogenesis, and also directly demonstrate specificity in the coupling of activated receptor tyrosine kinases to the MAPK cascade. Growth factors such as EGF, 1The abbreviations used are: EGF, epidermal growth factor; Ahx, aminohexanoic acid; BrdUrd, 5-bromo-2-deoxyuridine/5-fluoro-2-deoxyuridine; CHAPS, 3-[(3-cholamidopropyl)dimethylammionio]-1-propanesulfonate; DMEM, Dulbecco's modified Eagle's medium; Me2SO, dimethyl sulfoxide; FCS, fetal calf serum; FGF, fibroblast growth factor; FITC, fluorescein isothiocyanate; Grb2, growth factor receptor-binding protein 2; GS, goat serum; GST, glutathione S-transferase; HRP, horseradish peroxidase; MAPK, mitogen-activated protein kinase; PDGF, platelet-derived growth factor; PLCγ, phospholipase C gamma; Shc, Src homology and collagen protein; SH2, Src homology 2; Fmoc,N-(9-fluorenyl)methoxycarbonyl; FGFR, FGF receptor; EGFR, EGF receptor; bFGF, basic FGF; PBS, phosphate-buffered saline; AMEM, amphibian modified Eagle's medium.1The abbreviations used are: EGF, epidermal growth factor; Ahx, aminohexanoic acid; BrdUrd, 5-bromo-2-deoxyuridine/5-fluoro-2-deoxyuridine; CHAPS, 3-[(3-cholamidopropyl)dimethylammionio]-1-propanesulfonate; DMEM, Dulbecco's modified Eagle's medium; Me2SO, dimethyl sulfoxide; FCS, fetal calf serum; FGF, fibroblast growth factor; FITC, fluorescein isothiocyanate; Grb2, growth factor receptor-binding protein 2; GS, goat serum; GST, glutathione S-transferase; HRP, horseradish peroxidase; MAPK, mitogen-activated protein kinase; PDGF, platelet-derived growth factor; PLCγ, phospholipase C gamma; Shc, Src homology and collagen protein; SH2, Src homology 2; Fmoc,N-(9-fluorenyl)methoxycarbonyl; FGFR, FGF receptor; EGFR, EGF receptor; bFGF, basic FGF; PBS, phosphate-buffered saline; AMEM, amphibian modified Eagle's medium. PDGF, and FGF stimulate cell proliferation by binding to and activating membrane-spanning receptors that have cytoplasmic tyrosine kinase domains (1Schlessinger J. Ullrich A. Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1291) Google Scholar, 2Fantl W.J. Johnson D.E. Williams L.T. Annu. Rev. Biochem. 1993; 62: 453-481Crossref PubMed Scopus (929) Google Scholar). Ligand binding induces receptor dimerization, and this is associated with autophosphorylation on tyrosine residues both within and outside the catalytic domain in the receptor dimer (e.g. see Refs. 3Lemmon M.A. Schlessinger J. Trends Biochem. Sci. 1994; 19: 459-463Abstract Full Text PDF PubMed Scopus (432) Google Scholar and4Mohammadi M. Dikic I. Sorokin A. Burgess W.H. Jaye M. Schlessinger J. Mol. Cell. Biol. 1996; 16: 977-989Crossref PubMed Scopus (341) Google Scholar). Whereas the former are required for catalytic activity, the latter can serve as high affinity binding sites for effector molecules or adaptor molecules that recruit effectors to the receptor. Grb2 is a key adaptor molecule that couples some receptor tyrosine kinases to the Ras/mitogen-activated protein kinase (MAPK) cascade, and this is central to the mitogenic response stimulated by many growth factors (5Downward J. FEBS Lett. 1994; 338: 113-117Crossref PubMed Scopus (162) Google Scholar). Grb2 binds to defined motifs, containing phosphorylated tyrosine (designated Y in the single-letter amino acid code), via its SH2 domain, and these motifs are found in a wide range of molecules including the EGF and PDGF receptors and the Shc (Src homology and collagen protein) adaptor (6Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2379) Google Scholar, 7Okutani T. Okabayashi Y. Kido Y. Sugimoto Y. Sakaguchi K. Matuoka K. Takenawa T. Kasuga M. J. Biol. Chem. 1994; 269: 31310-31314Abstract Full Text PDF PubMed Google Scholar, 8Batzer A.G. Rotin D. Urena J.M. Skolknik E.Y. Schlessinger J. Mol. Cell. Biol. 1994; 14: 5192-5201Crossref PubMed Google Scholar). In this context, when phosphorylated, Tyr1068 in the EGF receptor acts as a docking site for the SH2 domain of Grb2 and synthetic phosphopeptides corresponding to this site inhibit Grb2 binding to the activated EGF receptor in vitro (8Batzer A.G. Rotin D. Urena J.M. Skolknik E.Y. Schlessinger J. Mol. Cell. Biol. 1994; 14: 5192-5201Crossref PubMed Google Scholar). These peptides can also inhibit Grb2 function in intact cells, e.g. a 9-amino acid phosphopeptide corresponding to this site can inhibit the ability of EGF to activate Ras in permeabilized cells (9Buday L. Downward J. Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (931) Google Scholar). Moreover, when made in tandem with a peptide that allows for translocation across the cell membrane, this phosphopeptide retains its ability to bind to Grb2 and can inhibit Grb2 binding to the activated EGF receptor in live cells (10Rojas M. Yao S. Lin Y. J. Biol. Chem. 1996; 271: 27456-27461Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). This was associated with a competitive inhibition of EGF stimulation of the MAPK cascade, with the peptide being able to partially (∼60%) inhibit the effects of EGF at 10 ng/ml but being ineffective against EGF at 50 ng/ml. In both systems phosphorylation of Tyr1068 was required for inhibitory activity. We have recently shown that a cell-permeable peptide mimetic of the PLCγ binding site on the FGF receptor (the FGFR766Y-P peptide) can inhibit phosphatidylinositol hydrolysis stimulated by FGF in intact cells in the absence of any effect on the same response stimulated by PDGF or neurotrophin-3 (11Hall H. Williams E.J. Moore S.E. Walsh F.S. Prochiantz A. Doherty P. Curr. Biol. 1996; 6: 580-587Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). This level of specificity was unexpected and might be related to lower levels of FGF receptors as compared with the other receptors and/or differences in the affinity of PLCγ binding to the various receptors. Moreover, we were able to show that this peptide could inhibit the neurite outgrowth response stimulated by FGF, but had no effect on neurite outgrowth stimulated by agents that act downstream from PLCγ activation in the FGF receptor signal transduction cascade, or other growth factors such as nerve growth factor (11Hall H. Williams E.J. Moore S.E. Walsh F.S. Prochiantz A. Doherty P. Curr. Biol. 1996; 6: 580-587Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 12Saffell J.L. Williams E.J. Mason I.J. Walsh F.S. Doherty P. Neuron. 1997; 18: 231-242Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Again, phosphorylation of Tyr766was required for inhibitory activity. Thus, cell-permeable phosphopeptides that mimic receptor binding sites for specific SH2 domain-containing proteins have the potential to be developed as tools that will help us understand how growth factor receptors couple to signaling cascades and biological responses. In the present study we report on the effects of cell-permeable mimetics of the EGF receptor Grb2 binding site and FGF receptor PLCγ binding site for their ability to inhibit mitogenic responses stimulated by EGF, PDGF, and FGF. All three responses were inhibited by PD 098059, which inhibits MAPK kinase (13Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3249) Google Scholar), and this is consistent with activation of the MAPK cascade being required for these responses. We find that the EGF receptor Grb2 binding site mimetic was able to bind to Grb2 in cells and inhibit the mitogenic response stimulated by EGF and PDGF, but had no effect on the response stimulated by FGF. This peptide could also inhibit the phosphorylation of MAPK stimulated by EGF and PDGF, but not FGF. In contrast, the distinct phosphopeptide corresponding to the FGF receptor PLCγ binding site had no effect on the mitogenic response stimulated by EGF, PDGF, or FGF. These data demonstrate for the first time that cell-permeable peptide mimetics of SH2 domain binding sites can selectivity inhibit activation of the MAPK cascade and mitogenic responses stimulated by polypeptide growth factors. Peptides were synthesized on a 431A Applied Biosystem peptide synthesizer usingp-hydroxymethylphenoxymethyl polystyrene resin and standard Fmoc chemistry. The EGFR Grb2-binding peptide sequence was as follows, Biot-Ahx-Arg-Arg-Trp-Arg-Arg-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg-Trp-Arg-Arg-Pro-Val-Pro-GluTyr(P)-Ile-Asn-Gln-Ser, where the underlined sequence corresponds to the 9-amino acid sequence containing phosphotyrosine 1068 of the EGF receptor that has been established to bind to the SH2 domain of Grb2 (the full peptide is called the EGFR1068Y-P peptide throughout the study). A phosphorylated Fmoc tyrosine (Novabiochem) was used in this sequence. The rest of the peptide sequence corresponds to a functional analogue of a sequence derived from the third helix of the Antennapedia protein, which has been established to act as a vector that can carry other peptides across the cell membrane into the cytosol (for full details of internalization properties of Antennapedia-based peptides, see Refs. 11Hall H. Williams E.J. Moore S.E. Walsh F.S. Prochiantz A. Doherty P. Curr. Biol. 1996; 6: 580-587Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholarand 14Prochiantz A. Curr. Opin. Neurobiol. 1996; 6: 629-634Crossref PubMed Scopus (195) Google Scholar). This peptide also contained an aminohexanoic acid (Ahx) spacer (Novabiochem) and a biotin at the N terminus. Synthesis of the cell-permeable Antennapedia-FGFR PLCγ-binding peptide (called the FGFR766Y-P peptide throughout the study) was as described previously with the exception that this peptide also contained an N-terminal biotin (11Hall H. Williams E.J. Moore S.E. Walsh F.S. Prochiantz A. Doherty P. Curr. Biol. 1996; 6: 580-587Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). After synthesis and deprotection, the peptides were desalted on a Sephadex G-10 column and lyophilized. The peptides were analyzed for purity by analytical high pressure liquid chromatography, and in each case a single peak was observed. Peptides were made as stock solutions at 25 mg/ml in dimethyl sulfoxide (Me2SO) and stored at −20 °C before use. All surface plasmon resonance experiments were carried out using a BIAcore X™ biosensor (Pharmacia Ltd., Biacore, the of which has been described L. H. J. S. H. M. Google Scholar). cells of were using the coupling with in 10 to the resonance of were corresponding to a of peptides were via to cell (the other as a by at in at a of and at 25 of were carried out to any peptide and also to proteins were both cells at in at a of After a of any to the surface was with a of the signal to binding of the proteins to each peptide was by the by of from cell in called resonance The proteins used in the present study the full sequence and SH2 domain of Grb2, the SH2 domain of PLCγ, and the SH2 domain of the of were a of cells were in in of with fetal calf with and as described previously P. J. PubMed Scopus Google Scholar). were at 25 °C in a of cells D. 270: PubMed Scopus Google were in Dulbecco's modified Eagle's with and at °C in were by either of previously with or of were by in for was with containing or with basic FGF EGF or PDGF from in the or absence of the EGFR1068Y-P peptide or the MAPK kinase PD In some experiments were with the EGFR1068Y-P peptide for 2 before being three and the with growth factors in the absence of peptide and After were for with mg/ml using the proliferation to were for with and for as described previously F. I. G. 1994; PubMed Scopus Google Scholar). cells were for 10 with by with for 10 with phosphate-buffered containing goat the and were and fluorescein in were with mg/ml and with cells were to on and for 2 was with or with either the PD or the EGFR1068Y-P peptide and growth factors for and The were with before being and in of at 50 at the time were for and for before being on and were for at with in in and for at °C with a MAPK from in were three with with in for at and three with before with of cells were established on of an The were and with containing 10 EGFR1068Y-P After 2 the were three with and and permeabilized with acid by for at −20 protein binding sites were for with in The were for with The were three with before being for by cells were to on in with and for and with or peptide for at The cell were with at and 2 at to peptide before being in containing CHAPS, and complete from to were by and the peptides from the with at °C for 2 were three with containing was to both the and and were for on and to were for in in and for with in were three in in before being with in for to in were with We have synthesized a peptide of a functional analogue of the acid sequence derived from the third helix of the Antennapedia protein that acts as an internalization vector for other peptide 14Prochiantz A. Curr. Opin. Neurobiol. 1996; 6: 629-634Crossref PubMed Scopus (195) Google Scholar for a of the internalization properties of peptides and 11Hall H. Williams E.J. Moore S.E. Walsh F.S. Prochiantz A. Doherty P. Curr. Biol. 1996; 6: 580-587Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar for an in tandem with the Grb2 docking site on the activated EGF receptor see for of the binding properties of this peptide using BIAcore The peptide a protein containing the full Grb2 molecule and also containing the single SH2 The peptide not bind a protein or a protein containing the N-terminal SH2 domain, and a protein containing the N-terminal SH2 domain of PLCγ In contrast, a peptide mimetic of the FGF receptor PLCγ binding site (the FGFR766Y-P peptide) PLCγ, and binding of Grb2 and In a range of phosphopeptides that not the sequence for PLCγ or Grb2 binding molecule not the peptides show the specificity in of SH2 domain Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2379) Google Scholar for the of the internalization sequence. The of the EGFR1068Y-P peptide into cells was by were with the peptide at 10 for before being and permeabilized the cell the of the peptide within the results were with the other phosphopeptides (e.g. see 11Hall H. Williams E.J. Moore S.E. Walsh F.S. Prochiantz A. Doherty P. Curr. Biol. 1996; 6: 580-587Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). the EGFR1068Y-P peptide could with Grb2 cells, of were with the peptide at 50 for before being to The cells were and the peptide with Grb2 with the EGFR1068Y-P peptide and the peptide of the Grb2 from the cell including the FGFR766Y-P to Grb2 or from the cell and data not EGF, PDGF, and FGF stimulate proliferation of as by was established that a response was by and ng/ml EGF, PDGF, and FGF, and that these responses could be inhibited by the MAPK kinase PD the lower of growth factors the peak activation of MAPK was at and this was inhibited by with PD at the of growth factors activation of the MAPK cascade was of with the growth factors for EGF and FGF in and EGF in of PD 2 the of the growth factors the MAPK to levels and also inhibited the mitogenic response not that this activation of the MAPK cascade is required for the full mitogenic MAPK kinase PD inhibits MAPK phosphorylation stimulated by EGF, PDGF, and FGF. In the shown in the cells were and in and or with PD and the were to EGF, PDGF, or FGF at ng/ml as for before being for MAPK as by with an MAPK that the phosphorylated of the for In the shown in the cells were and in and or containing ng/ml FGF and or 10 ng/ml EGF and After 2 PD was to the as and After 2 the were and for the of experiments with an that the MAPK that the of the the level of MAPK protein not EGFR1068Y-P peptide inhibits the phosphorylation of MAPK stimulated by EGF and PDGF but not by FGF. In the shown in the cells were and with either and or containing EGF at 10 ng/ml for and and or This was in both in the and and absence and of the EGFR1068Y-P peptide at 10 The were and for In the shown in cells were and in or with of the EGFR1068Y-P peptide and cells were with ng/ml FGF and EGF and or PDGF After an the were and for The effect of the EGFR1068Y-P peptide on the mitogenic of EGF, PDGF, and FGF on cells was also from three experiments that the of 10 peptide the inhibited the mitogenic response stimulated by EGF and PDGF, but had no effect on the response to FGF The peptide also had no effect on the or of cells not that its effects on are the of a specific and this is in with the of the peptide to the FGF response. The show that the peptide will in cells a and in this we find that a of cells with the peptide is to inhibit the mitogenic responses stimulated by EGF and PDGF Pretreatment of with 50 peptide was also associated with a full inhibition of the mitogenic response stimulated by EGF and PDGF in the absence of any effect on the response stimulated by FGF not This also that the peptide has a in cells, and this might the of the peptide from Grb2, which be to from and also that the peptide is in both and The Grb2-binding peptide was for its ability to inhibit the activation of MAPK stimulated by the of EGF 10 peptide inhibited the activation of the MAPK cascade, and this was at time The competitive of the inhibition was by that peptide could inhibit the activation of MAPK stimulated by ng/ml EGF at see the same the peptide also inhibited the activation of MAPK by PDGF, but had no effect of the response stimulated by FGF The specificity of of the EGFR1068Y-P peptide was by that the FGFR766Y-P which inhibits accumulation and growth responses (11Hall H. Williams E.J. Moore S.E. Walsh F.S. Prochiantz A. Doherty P. Curr. Biol. 1996; 6: 580-587Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), had no effect on the FGF, EGF, or PDGF mitogenic response In we have three other cell-permeable phosphopeptides that not bind to the SH2 domain of Grb2 as by and found that also have no effect on the mitogenic response stimulated by EGF and PDGF not the inhibitory of the EGFR1068Y-P is specific and can be for by its ability to bind to the SH2 domain of Grb2 and inhibit activation of the MAPK cascade. This is by the ability of peptide mimetics of the Grb2 binding site on the EGF receptor to out binding of Grb2 to the activated vitro (8Batzer A.G. Rotin D. Urena J.M. Skolknik E.Y. Schlessinger J. Mol. Cell. Biol. 1994; 14: 5192-5201Crossref PubMed Google Scholar), as as in live cells (10Rojas M. Yao S. Lin Y. J. Biol. Chem. 1996; 271: 27456-27461Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). we have that peptide will bind to Grb2 in intact cells, and that not with PLCγ, or Shc not we the that the peptide with other is from the results that its effects are specific and that are by the ability to bind to the SH2 domain of FGFR766Y-P peptide not inhibit mitogenic responses by EGF, PDGF, or cells were before in or with EGF PDGF or FGF in the or absence of 10 FGFR766Y-P The results show the of cells in from three in a cells were before in or with EGF PDGF or FGF in the or absence of 10 FGFR766Y-P The results show the of cells in from three The that the peptide can inhibit both EGF and PDGF responses is not as the SH2 domain of Grb2 is required for both the and of this adaptor with the EGF receptor and an association via with the activated PDGF receptor Schlessinger J. B. 1994; 9: Google Scholar, Sugimoto S. Walsh Neel B.G. Sci. S. A. 1994; PubMed Scopus Google Scholar). The that the of the peptide to inhibit the FGF response the of a mitogenic is for a of FGF activated the MAPK cascade, and both this and the mitogenic response were inhibited by the PD MAPK kinase The that the selectivity of the Grb2-binding peptide differences in the coupling of the receptors and the FGF receptor to the MAPK cascade, is a and is that a protein might couple the FGF receptor to the via Grb2 P. S. L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, P. L. Biochem. 1996; PubMed Scopus Google Scholar, H. M. 1996; Google Scholar, J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), and in this a FGF receptor called has recently been tyrosine can bind Grb2 and the FGF receptor to MAPK activation H. T. J. D. I. Schlessinger J. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Shc can also act as an adaptor molecule that can Grb2 to the activated FGF receptor P. S. L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, H. T. J. D. I. Schlessinger J. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, the of the EGFR1068Y-P peptide to inhibit the FGF response might be for by its to inhibit Grb2 binding to and/or In has been previously that the EGFR1068Y-P peptide is ineffective at the association of Grb2 with Shc (10Rojas M. Yao S. Lin Y. J. Biol. Chem. 1996; 271: 27456-27461Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). A of peptides that not bind to the Grb2 SH2 domain had no effect on the mitogenic response stimulated by EGF and PDGF. The of these is a cell-permeable peptide mimetic of the PLCγ binding site on the FGF receptor that inhibits hydrolysis and growth responses stimulated by FGF (11Hall H. Williams E.J. Moore S.E. Walsh F.S. Prochiantz A. Doherty P. Curr. Biol. 1996; 6: 580-587Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). is that this peptide had no effect on the mitogenic response stimulated by FGF, and this is in with a of that has that of can the FGF receptor from activating PLCγ, has no effect on the mitogenic response stimulated by this factor M. Dikic I. Sorokin A. Burgess W.H. Jaye M. Schlessinger J. Mol. Cell. Biol. 1996; 16: 977-989Crossref PubMed Scopus (341) Google Scholar). In we have shown that cell-permeable peptides that have binding for SH2 domain-containing molecules can be used to inhibit the mitogenic of polypeptide growth factors. This (11Hall H. Williams E.J. Moore S.E. Walsh F.S. Prochiantz A. Doherty P. Curr. Biol. 1996; 6: 580-587Abstract Full Text Full Text PDF PubMed Scopus (115) Google that the ability of cell-permeable phosphopeptides to with high affinity and specificity to the SH2 domains of and effectors can be to tools for the of signaling in The functional analogue of the internalization sequence derived from the third helix of the Antennapedia protein was developed by Prochiantz and and their and we for this sequence with