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Cell Growth & Differentiation Vol. 11, 293-303, June 2000
© 2000 American Association for Cancer Research

Specific Inhibition of Growth Factor-stimulated Extracellular Signal-regulated Kinase 1 and 2 Activation in Intact Cells by Electroporation of a Growth Factor Receptor-binding Protein 2-Src Homology 2 Binding Peptide1

Leda Raptis2, Heather L. Brownell3, Adina M. Vultur, Gregory M. Ross, Eric Tremblay and Bruce E. Elliott

Departments of Microbiology and Immunology [L. R., H. L. B., A. M. V., B. E. E.], Pathology [L. R., E. T., B. E. E.], and Medicine [G. M. R.], Queen’s University, Kingston, Ontario, K7L 3N6 Canada


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Activation of the Ras pathway is central to mitogenesis by a variety of growth factors such as the epidermal growth factor, platelet-derived growth factor, or hepatocyte growth factor. Ras activation requires the function of adaptors such as growth factor receptor-binding protein 2, which can bind either directly or indirectly through Src homology 2 domains to the activated receptor. To examine the role of the Src homology 2 domain of growth factor receptor-binding protein 2 in the mitogenic response triggered by these growth factors, we introduced a peptide (PVPE-phosphono-methylphenylalanine-INQS) that can selectively bind this domain into mouse, rat, or human cells growing on conductive indium-tin oxide-coated glass by in situ electroporation. Cells were subsequently stimulated with growth factors and assessed for activation of a downstream target, extracellular signal-regulated kinase (ERK) 1/2, by probing with antibodies specific for its activated form. Electrodes and slides were configured to provide nonelectroporated control cells side by side with the electroporated ones, both growing on the same type of indium-tin oxide-coated glass surface. The data demonstrate that the peptide can cause a dramatic inhibition of epidermal growth factor or platelet-derived growth factor-mediated ERK1/2 activation and DNA synthesis in vivo, compared with its control phenylalanine-containing counterpart. In contrast, the peptide had a very limited effect on hepatocyte growth factor-triggered ERK1/2 activation and DNA synthesis. These results demonstrate the potential of the in situ electroporation approach described here in the study of the coupling of activated receptor tyrosine kinases to the ERK1/2 cascade.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Growth factors such as the EGF,4 PDGF, and HGF stimulate cell proliferation by binding to and activating membrane receptors with cytoplasmic tyrosine kinase domains. Ligand binding induces receptor dimerization, which is associated with autophosphorylation of the receptor at distinct tyrosine residues (reviewed in Refs. 1 and 2 ). These constitute docking sites for a number of effector molecules including c-Src, PLC-{gamma}, the p85 subunit of PI3k, Syp, Nck, Crk (3) , signal transducers and activators of transcription 3 (4) , the multiadaptor Gab1 (5 , 6) , and Grb2, some of which are recruited to specific receptors through binding modules termed SH2 domains.

In vitro binding and receptor mutagenesis studies have established Grb2 as an adaptor molecule that is able to couple a number of receptor tyrosine kinases to the Ras/ERK1/2 cascade, an event that is central to the mitogenic response stimulated by many growth factors (7 , 8) . Grb2 binds to the receptors for PDGF and EGF directly at a number of sites and indirectly through other adaptors such as Shc and Syp (9, 10, 11, 12) . The HGF receptor, Met, on the other hand, has a sequence in the cytoplasmic domain containing two contiguous SH2 binding sites (Y1349 VHVNATY1356VNV), which function as multifunctional docking sites for the majority of SH2-containing cytoplasmic effectors either directly or through the Gab1 adaptor (5 , 6) . Grb2, however, selectively binds to one site (Y1356 VNV) because of the presence of an asparagine residue in the +2 position (13 , 14) . Using a mutational approach, it was further shown that different HGF-induced effects are regulated by these separate Met binding sites for cytoplasmic transducers (15) and that complementation in trans between these two binding sites is required for the invasive and metastatic phenotype. In addition to Grb2, the Sos/Ras/Raf/ERK pathway has also been shown to be activated through other adaptors such as Nck and Crk in different systems (3 , 16 , 17) . Moreover, recent reports indicate that growth factors can act cooperatively with integrins in activation of ERK2 (18 , 19) and that integrin-mediated ERK activation can occur via a Ras-independent pathway (20) .

Previous results indicated that a synthetic phosphopeptide corresponding to the Grb2 binding site of the EGFR (flanking EGFR Y1068) specifically inhibits Grb2 attachment to the activated receptor in vitro and Ras activation by EGF in streptolysin O-permeabilized cells (21) . Furthermore, when made in tandem with peptides that allow for translocation across the cell membrane, this peptide could inhibit EGF-mediated mitogenesis and ERK activation in newt A1 myoblasts when stimulated with 1 ng/ml EGF as opposed to the less effective 10 ng/ml EGF (7 , 8) . To determine the functional consequences of disrupting the association of Grb2 per se with different receptors or adaptors in vivo in mammalian cells, we delivered large quantities of this peptide into intact cells using electroporation in situ, a technique developed recently in our laboratory.

Cells are grown on an ITO-coated glass slide and loaded with the peptide through an electrical pulse, which opens transient pores on the cell membrane (22) . Cells can be subsequently lysed for large-scale experiments, or their morphology and biochemical properties can be examined using immunocytochemistry techniques. The latter offers the advantage that cell stress, as revealed by changes in morphology, can be examined simultaneously. In addition, the demonstration of the effects of peptide introduction can be especially powerful when a slide configuration providing nonelectroporated cells growing side by side with electroporated ones on the same type of surface is used. This can be achieved by plating the cells on a partly conductive glass slide, and it permits precise assessment of small background changes in morphology or ERK activation levels (23) . Previous results indicated that in situ electroporation does not affect cellular metabolism in any detectable way, presumably because the pores reseal rapidly, so that the cell interior is restored to its original state (23 , 24) . Moreover, the instant introduction of the molecules into essentially 100% of the cells makes this technique especially suitable for kinetic studies of effector activation. The results indicate that the Grb2-SH2 blocking peptide can cause a dramatic inhibition of both EGF- and PDGF-mediated ERK activation in mouse NIH-3T3 cells at growth factor concentrations permitting full receptor stimulation. In contrast, the same peptide had only a limited effect or no effect on ERK activation triggered by HGF or the PKC stimulator TPA, respectively. These findings demonstrate that the in situ electroporation approach described here can achieve a precise inhibition of growth factor-stimulated mitogenic effects and can thereby detect differential specificity in the coupling of activated receptor tyrosine kinases to the mitogen-activated protein kinase cascade.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Inhibition of Grb2 Binding to PDGFR, EGFR, or HGFR through Electroporation of a Grb2-SH2 Blocking Peptide.
EGF and PDGF can stimulate DNA synthesis in NIH-3T3 fibroblasts, as measured by [3H]thymidine incorporation. After growth arrest in spent medium or total serum deprivation, maximal response was achieved at 100 ng/ml EGF and 40 ng/ml PDGF (23) . HGF, on the other hand, stimulated DNA synthesis of A549 cells or NIH-3T3 cells expressing methu (NIH-3T3-methu cells), with maximal effect at 10–40 ng/ml (see "Materials and Methods"). To examine the role of the SH2 domain of Grb2 in signal transduction, we took advantage of a sequence derived from the well-characterized Grb2 docking site on the EGFR (PVPE-Pmp-INQS; see "Materials and Methods"). To determine whether, under the conditions used, this peptide can complex with Grb2 inside the cell and inhibit Grb2 binding to the EGFR or PDGFR, the peptide was electroporated into mouse NIH-3T3 fibroblasts that express both receptor types. Cells were plated on conductive slides of 32 x 10 mm (Fig. 1B)Citation and growth-arrested by serum starvation. The Grb2-SH2 blocking peptide or the control peptide containing phenylalanine at the position of phosphotyrosine were added to the cells and introduced by electroporation (see "Materials and Methods"). After a 5-min incubation at 37°C, cells were treated with PDGF or EGF for 5 min. After extensive washing to remove unincorporated peptide, cell lysates were immunoprecipitated with an anti-Grb2 antibody. Precipitated proteins were resolved on SDS-PAGE and transferred to a nitrocellulose membrane that was probed with an antiphosphotyrosine antibody (see "Materials and Methods"). To assess the effect of the Grb2-SH2 blocking peptide on HGF signaling, the same approach was undertaken with HGF in the human lung carcinoma A549 line, which expresses methu. As shown in Fig. 2Citation , electroporation of the Grb2-SH2 binding peptide caused a dramatic inhibition of Grb2 binding to HGF receptor or PDGFR (Lanes 2 and 5), compared with its phenylalanine-containing counterpart (Lanes 3 and 6). Similar results were obtained with EGFR (data not shown).



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  		Fig. 1. Electroporation electrode and slide assembly. Cells are grown on glass slides coated with conductive and transparent ITO within a "window" cut into a Teflon frame as shown. The peptide solution is added to the cells and introduced by an electrical pulse delivered through the electrode set, which is placed directly on the frame. Two configurations (A and B) were used. A, partly conductive slide assembly, with electroporated (a) and nonelectroporated (c) cells growing on the same type of ITO-coated surface. b, area where the conductive coating has been stripped, exposing the nonconductive glass underneath. Cells growing in areas b and c are not electroporated. B, fully conductive slide assembly with a window of 32 x 10 mm (reprinted from Ref. 23 with permission).

 


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  		Fig. 2. Electroporation of a synthetic Grb2-SH2 blocking peptide or compound 8 inhibits Grb2 binding to growth factor receptors. The Grb2-SH2 binding peptide containing Pmp at the position of phosphotyrosine (Lanes 2 and 5), a control phenylalanine-containing peptide (Lanes 3 and 6), or compound 8 (Lane 7) was electroporated into mouse NIH-3T3 fibroblasts (Lanes 1–3), human lung carcinoma A549 cells (Lanes 4–6), or NIH-3T3-methu cells (Lanes 7–9). Cells were subsequently stimulated with PDGF (Lanes 2 and 3), HGF (Lanes 5 and 6), or EGF (Lanes 8 and 9), as indicated. Lanes 1, 4, and 8, unstimulated controls. Detergent cell extracts were precipitated with a Grb2-specific antibody, and proteins were resolved by PAGE and transferred to a nitrocellulose membrane probed with antibodies to phosphotyrosine (Lanes 1–3), Met (Lanes 4–6), or EGFR (Lanes 7–9). Horizontal bar, the position of the Mr 116,000 marker.

 
Previous results indicated that two compounds, [(dimethylamino)methyl]acrylo-para-[(benzoylsulfonyl)-oxy]phenone and [(dimethylamino)methyl]acrylo-para-[(hydroxy-benzoylsulfonyl)-oxy]phenone [compounds 7 and 8, respectively (25) ], selectively inhibit phosphorylation of the EGFR (23 , 25) . To further explore the mechanism of the Grb2-receptor interaction, we examined whether binding of Grb2 to EGFR depends on receptor phosphorylation in NIH-3T3-methu cells. To this effect, compounds 7 and 8 were introduced by in situ electroporation, and after a 5-min incubation at 37°C, cells were treated with EGF for 5 min. Detergent cell lysates were immunoprecipitated with the anti-Grb2 antibody, and precipitated proteins were resolved on SDS-PAGE and transferred to a nitrocellulose membrane that was probed with an anti-EGFR antibody (see "Materials and Methods"). As shown in Fig. 2Citation (Lanes 7–9), inhibition of EGFR phosphorylation by compound 8 caused a dramatic inhibition of EGFR binding to Grb2. Similar results were obtained with compound 7 (data not shown). These data indicate that EGFR phosphorylation may be necessary for Grb2 binding to EGFR in this system.

Inhibition of ERK Activation by EGF or PDGF but not HGF through Electroporation of the Grb2-SH2 Blocking Peptide.
Previous results indicated that the introduction of a Grb2-SH2 binding peptide using a homeobox-derived leader sequence caused a partial inhibition of the EGF- or PDGF-mediated ERK activation in newt A1 myoblasts (8) . To definitively demonstrate the role of the SH2 domain of Grb2 in EGF- or PDGF-mediated signaling, higher amounts of the Grb2-SH2 blocking peptide were introduced into NIH-3T3 cells by in situ electroporation. Cells were plated on conductive slides with a cell growth area of 32 x 10 mm (Fig. 1B)Citation , growth-arrested by serum starvation, and electroporated with the Grb2-SH2 blocking peptide or the control peptide containing phenylalanine at the position of phosphotyrosine. After incubation at 37°C for 5 min to allow the peptide to bind to its intracellular target, cells were stimulated with EGF (100 ng/ml) or PDGF (40 ng/ml) for 5 min, lysed, and probed for activated ERK by Western blotting (see "Materials and Methods"). As shown in Fig. 3ACitation , EGF-mediated activation of ERK was dramatically reduced by the Grb2-SH2 binding peptide electroporated at 10 mg/ml (Lane 4). At the same time, the phenylalanine-containing control peptide had no detectable effect, even at concentrations of 20 mg/ml, at all voltages tested (Fig. 3A, Lane 3Citation ). Similar results were obtained with PDGF (data not shown), indicating that the Grb2-SH2 domain is an essential component of ERK activation by these growth factors in NIH-3T3 cells.



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  		Fig. 3. Effect of the Grb2-SH2 blocking peptide on growth factor-mediated ERK activation in vivo. A, NIH-3T3 cells. The Grb2-SH2 blocking peptide was electroporated into NIH-3T3 cells growing on fully conductive slides (Fig. 1Citation B) and growth-arrested by serum starvation. After a 5-min incubation in DMEM, cells were stimulated with 100 ng/ml EGF (Lanes 2–4) for 5 min. Proteins in detergent cell lysates were resolved by PAGE and analyzed by Western blotting using the antibody against the dually phosphorylated active ERK enzymes (see "Materials and Methods"). Lane 1, control unstimulated cells. Lane 2, control nonelectroporated EGF-treated cells. Lane 3, cells electroporated with the control phenylalanine-containing peptide and EGF-stimulated. Lane 4, cells electroporated with the Grb2-SH2 binding peptide and EGF-stimulated. B, methu-expressing NIH-3T3 cells. The Grb2-SH2 binding peptide containing Pmp at the position of phosphotyrosine (Lane 4), a control phenylalanine-containing peptide (Lane 3), or the EGFR phosphorylation inhibitor, compound 8 (Lanes 10 and 12), was electroporated into NIH-3T3-methu cells. Cultures were subsequently stimulated with HGF (Lanes 2–5, 7, and 8), EGF (Lanes 10 and 11), or PDGF (Lanes 12 and 13), as indicated. Lanes 1, 6, and 9, unstimulated controls. Lane 5, cells treated with 100 µM of the MEK inhibitor PD98059 for 2 h and stimulated with HGF. Lane 7, cells treated with 40 µM of the PI3k inhibitor LY294002 for 2 h and stimulated with HGF. After treatment, cell extracts were probed for activated ERK as described above. Arrows point to the position of the p42 (ERK2) and p44 (ERK1) forms of the activated ERK kinases.

 
Contrary to the above-mentioned results, the peptide had only a marginal effect on HGF-mediated ERK activation in A549 cells (Fig. 3BCitation , compare Lanes 3 and 4), indicating that the Grb2-SH2 domain is dispensable for ERK activation in this system. To examine the importance of MEK in ERK activation by HGF, cells were treated with the MEK inhibitor PD98059. As shown in Fig. 3B, Lane 5Citation , this drug dramatically inhibited ERK activation by HGF, indicating that this pathway may be involved in HGF signaling, but in a manner independent of the Grb2-SH2 domain. On the other hand, the PI3k inhibitor LY294002 had no effect on ERK activation (Fig. 3B, Lane 7Citation ), indicating that PI3k does not play a significant role in ERK activation by HGF in this system (26) .

To further examine the importance of EGFR phosphorylation subsequent to ligand binding upon ERK activation, we electroporated compounds 7 and 8 into NIH-3T3 methu cells. After a 5-min incubation, cells were stimulated with EGF, lysed, and probed for activated ERK by Western blotting as described above. As shown in Fig. 3BCitation , compound 8 electroporation dramatically inhibited ERK activation by EGF in these cells (compare Lane 10 with Lane 11). Similar results were obtained with compound 7 (data not shown). On the other hand, as demonstrated previously (23) , compound 8 did not significantly inhibit PDGF-triggered ERK activation in this system (Fig. 3B, compare Lane 12 and Lane 13Citation ). The above-mentioned data, taken together, indicate that Grb2, through binding to the phosphorylated EGFR with its SH2 domain, may be an essential mediator of the EGF signal to ERK in this system.

Specificity of the Inhibitory Activity of the Grb2-SH2 Blocking Peptide.
Previous results revealed a strong antiproliferative activity of a number of pharmacological inhibitors of the ERK pathway, such as the [(alkylamino)methyl]-acrylophenone blockers of the EGFR, at substantially lower concentrations than the levels required for inhibition of the tyrosine kinase activity of the EGFR, pointing to the possibility of additional sites of action in vivo for this class of compounds (25) . Therefore, to better demonstrate the specificity of action of the Grb2-SH2 binding peptide and to examine the distribution of signal inhibition across the cell layer, the peptide was introduced into NIH-3T3 cells plated on partly conductive slides (Fig. 1A)Citation and growth-arrested in spent medium. After growth factor stimulation as described above for 1, 5, 10, or 20 min, ERK activation was assessed by immunocytochemistry (see "Materials and Methods"). As shown in Fig. 4Citation , electroporation of the Grb2-SH2 blocking peptide totally inhibited EGF-induced ERK activation (Fig. 4A, a)Citation , whereas the control phenylalanine-containing peptide had no effect (Fig. 4C, a)Citation . This inhibition was uniform across the cell layer, in agreement with previous results showing that in situ electroporation can introduce the material into essentially 100% of the treated cells (24) . The use of lower concentrations of the Grb2-SH2 blocking peptide resulted in progressively lower but uniform levels of inhibition. It is especially noteworthy that this inhibition extends into the adjacent nonelectroporated cells growing on the nonconductive part of the slide (Fig. 4, area bCitation ), probably due to movement of the Mr 1123 peptide through gap junctions (27) . This finding constitutes compelling evidence that the observed inhibition must be due to the peptide rather than an artifact of electroporation. At the same time, as shown by phase-contrast microscopy (Fig. 4B)Citation , there was no alteration in the morphology of the electroporated cells under these conditions, suggesting that the observed effect is a result of a specific inhibition rather than toxic action. EGF stimulation for up to 20 min after peptide electroporation did not result in lower levels of ERK signal inhibition, indicating that the binding of the peptide to Grb2 is stable during this period of time. As expected from the results described in Figs. 2Citation and 3Citation , the phenylalanine-containing control peptide (Fig. 4, C and D)Citation or an unrelated peptide (CVVLSKRAT; see "Materials and Methods") had no effect on ERK activation. Similar results were obtained with rat F111 cells (data not shown). In sharp contrast, although the peptide inhibited ERK activation by EGF (Fig. 5B)Citation or PDGF in the methu-expressing NIH-3T3 cells, it only marginally inhibited HGF-mediated ERK activation (compare Fig. 4E, area aCitation with Fig. 4, area cCitation and Fig. 5Citation ). Similarly, electroporation of the Grb2-SH2 binding peptide into the human lung carcinoma cell line A549 had only a slight effect on the HGF-induced ERK activation (Fig. 4G)Citation . For both met-expressing cell lines, the Grb2-SH2 binding peptide could not inhibit ERK activation by HGF, even on the addition of lower amounts of HGF (1–10 ng/ml) or stimulation for shorter periods of time (1–10 min instead of 20 min), which resulted in lower activation levels. The above-mentioned results, taken together, indicate that the Grb2-SH2 domain may be dispensable for ERK activation in HGF-mediated signaling. All of the above-mentioned experiments were repeated with cells growth-arrested by total serum starvation, with essentially identical results.



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  		Fig. 4. Specificity of the inhibitory activity of the Grb2-SH2 blocking peptide. The Grb2-SH2 blocking peptide (A, B, E, F, G, and H) or the control phenylalanine-containing peptide (C and D) was introduced by in situ electroporation into NIH-3T3 (A–D), methu-expressing NIH-3T3 (E and F) or A549 (G and H) cells growing on partly conductive slides (Fig. 1Citation A) and growth-arrested in spent medium. Five min after electroporation, cells were stimulated with EGF for 5 min (A–D) or HGF for 20 min (E–H), fixed, probed for activated ERK (see "Materials and Methods"), and photographed under bright-field (A, C, E, and G) illumination. B, D, F, and H, same frames as in A, C, E, and G, respectively, phase-contrast illumination. Magnification: A and B, x240; C–F, x40. The arrow points to the transition line between the stripped (b) and electroporated (a) areas, whereas the arrowhead points to the line between the control ITO-coated (c) and etched (b) areas (Fig. 1Citation A). Cells growing on the left side (a) were electroporated, whereas cells on the stripped zone (b) or right side (c) of the slide did not receive any pulse. Note that the Grb2-SH2 blocking peptide dramatically reduced the EGF signal (A, a), whereas the degree of ERK activation is the same on both sides of the slide (a or c) for cells electroporated with the control phenylalanine-containing peptide (C). In A, the inhibition of the signal extends into ~3–4 rows of adjacent cells in the nonelectroporated area (b), probably due to movement of the peptide through gap junctions (27) . On the other hand, the Grb2-SH2 blocking peptide caused only a slight reduction of the HGF signal (E and G, area a). At the same time, there is no detectable effect on cell morphology as shown by phase-contrast microscopy (B, D, F, and H).

 


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  		Fig. 5. Quantitation of ERK activation by densitometry of immunocytochemically stained cells. NIH-3T3 (A), methu-expressing NIH-3T3 (B and C), or A549 (D) cells were plated on partly conductive slides (Fig. 1Citation A) and electroporated with the control phenylalanine-containing peptide or the Grb2-SH2 binding peptide, as indicated. After stimulation with the indicated growth factors (100 ng/ml TPA for 2, 10, or 30 min or inactive TPA analogue 4{alpha}-phorbol-12,13-didecanoate for 10 min), cells were probed for activated ERK as described in the Fig. 4Citation legend. Densitometry was subsequently carried out using an MCID M5 image analysis program. Values represent staining intensity compared with the nonelectroporated side, with the average intensity of EGF-stimulated NIH-3T3 cells taken as 100%. Results are representative of at least three experiments. Error within each group is less than 10%. phe, control peptide containing phenylalanine at the position of the EGFR-Tyr1068. Pmp, Grb2-SH2 blocking peptide containing Pmp at the position of the EGFR-Tyr1068 4{alpha}PDD, 4{alpha}-phorbol-12, 13-didecanoate.

 
Previous results indicated that PKC stimulation with the phorbol ester TPA leads to ERK activation (28) . To determine the degree of ERK activation by TPA in NIH-3T3-methu cells, these cells were stimulated with 100 ng/ml TPA for 2, 10, or 30 min [conditions previously demonstrated to activate PKC to different levels in mouse or rat fibroblasts (29, 30, 31) ] or with its inactive analogue, 4{alpha}-phorbol-12,13-didecanoate, and ERK activation measured by immunocytochemistry as described above. As shown in Fig. 5CCitation , under optimal conditions, TPA activated ERK to approximately 50% of the level of activation by EGF in this system. To examine whether this activation is mediated through Grb2-SH2 domain binding to EGFR, the Grb2-SH2 blocking peptide or its phenylalanine-containing counterpart was electroporated into NIH-3T3-methu cells and, after TPA treatment for 2, 10, or 30 min, cells were fixed, and ERK activation was measured by immunocytochemistry as described above. As shown in Fig. 5CCitation , electroporation of the Grb2-SH2 blocking peptide at 20 mg/ml failed to inhibit ERK activation by TPA. These results reinforce previous observations indicating that ERK activation by PKC is Ras independent (28) and further confirm the specificity of signal inhibition by the peptide in this system.

Inhibition of PDGF- and EGF- but not HGF-induced DNA Synthesis by Electroporation of the Grb2-SH2 Blocking Peptide.
Because ERK phosphorylation has been shown to be an important step in growth factor-triggered DNA synthesis, the effect of inhibition of the Ras/Raf/ERK cascade through electroporation of the Grb2-SH2 binding peptide on DNA synthesis was studied. After growth arrest by serum starvation, the Grb2-SH2 binding peptide or its phenylalanine-containing counterpart was electroporated into NIH-3T3 cells at different concentrations (1–10 mg/ml), and cells were subsequently stimulated to divide by the addition of EGF or PDGF. As shown in Fig. 6ACitation , electroporation of the Grb2-SH2 binding peptide at 10 mg/ml caused a dramatic inhibition in [3H]thymidine incorporation. There was no incorporation above background at 12 h after stimulation with EGF, whereas at lower peptide concentrations, the inhibition was less pronounced. At the same time, the control peptide had no effect, even at concentrations as high as 20 mg/ml. DNA synthesis and [3H]thymidine incorporation resumed after a 40-h delay (Fig. 6A)Citation , presumably after the blocking peptide had been metabolized, indicating the absence of overt toxicity. These data suggest that the Grb2-SH2 domain is required for the EGF-mediated mitogenic response in NIH-3T3 cells. Similar results were obtained with PDGF (data not shown).



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  		Fig. 6. The Grb2-SH2 blocking peptide inhibits EGF-induced but not HGF-induced DNA synthesis. The Grb2-SH2 blocking peptide or its control phenylalanine-containing counterpart was introduced at the indicated concentrations by in situ electroporation into NIH-3T3 (A) or A549 (B) cells growing on fully conductive slides (Fig. 1Citation B) and growth-arrested by serum starvation. Five min after electroporation, cells were stimulated with 100 ng/ml EGF (A), 40 ng/ml HGF (B), or 10% fetal bovine serum (FBS), respectively, as indicated. Twelve h later, cells were labeled with [3H]thymidine for 2 h, and TCA precipitable radioactivity was determined. In A, the two lanes at the far right represent cells labeled at 40–42 h after electroporation and EGF stimulation. phe, control peptide containing phenylalanine at the position of the EGFR-Tyr1068. Pmp, Grb2-SH2 binding peptide containing Pmp at the position of Tyr1068. The results represent the mean ± SE cpm from three experiments.

 
A similar approach was undertaken with HGF in human lung carcinoma A549 cells (Fig. 6B)Citation . After electroporation of the above-mentioned peptides, cells were stimulated to divide by the addition of HGF. Contrary to the EGF or PDGF responses, the Grb2-SH2 binding peptide had only a small effect on HGF-induced DNA synthesis, even at concentrations of 20 mg/ml, indicating that mitogenesis by HGF in this system is largely independent of the Grb2-SH2 domain.

Inhibition of DNA Synthesis in Response to EGF, PDGF, and HGF by the MEK1/2 Inhibitor PD98059.
The above-mentioned results indicate that the Grb2-SH2 domain may be required for EGF- or PDGF-mediated but not HGF-mediated ERK activation and mitogenesis. To examine the potential involvement of the Ras/Raf/MEK/ERK pathway in proliferation triggered by HGF, we examined the effect of the MEK1/2 inhibitor PD98059 (32 , 33) on the DNA synthesis response to this growth factor. A549 cells were treated with PD98059 two h after stimulation with 40 ng/ml HGF. Twelve h later, cells were labeled with [3H]thymidine, and TCA-precipitable radioactivity determined (see "Materials and Methods"). The effect of this inhibitor on the EGF or PDGF signal was also examined for a comparison; NIH-3T3 cells were treated with PD98059 two h after stimulation with 100 ng/ml EGF or 40 ng/ml PDGF and labeled as described above. As shown in Fig. 7Citation , drug treatment dramatically inhibited [3H]thymidine incorporation by EGF or HGF, even when the drug was added 2 h after growth factor addition. Similar results were obtained with PDGF (data not shown). These data suggest that a sustained activation of the ERK cascade is required for full mitogenic response to all three growth factors.



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  		Fig. 7. The PD98059 MEK1/2 inhibitor blocks DNA synthesis in response to EGF or HGF. A, NIH-3T3 cells grown in 24-well plates were growth-arrested by serum starvation and then stimulated with 100 ng/ml EGF. Two h later, PD98059 was added at 50, 100, or 200 µM, as indicated. Twelve h later, cells were labeled with [3H]thymidine for an additional 2 h, and acid precipitable radioactivity was determined. Results are expressed as the mean ± SE cpm of four wells. B, A549 cells grown in 24-well plates were growth-arrested by serum starvation and then stimulated with 40 ng/ml HGF. Where indicated, PD98059 (50 or 100 µM) was added. [3H]Thymidine labeling followed, and cells were processed as described above. Results represent the mean ± SE cpm from six wells.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
To finely pinpoint the interactions of different regions of a growth factor receptor molecule with its effectors, a number of investigators have ectopically expressed receptor mutants into cellular variants lacking these receptors. Using such an approach, it was shown that a number of adaptors or effectors, such as Grb2, Gab1, Nck, Crk, PLC-{gamma}, Syp, or c-Src, may be able to mediate activation of the Ras/Raf/ERK pathway in different systems (3 , 34) . However, although such an experimental layout can determine whether a specific site is necessary for a particular function, it cannot yield any information on the requirement for a specific domain on the signal-transducing protein. Knowledge of domain specificity can more effectively contribute to peptidomimetic drug design. Therefore, to complement these earlier results, cell-permeable peptides specifically blocking the point(s) of contact of the signaling proteins were introduced. Using such an approach, it was shown that the Grb2-SH2 domain is required for mitogenesis by EGF or PDGF in newt A1 cells or mouse C2 myoblasts (8) . Such peptides, however, could not entirely inhibit ERK activation after full receptor stimulation, possibly because of toxicity or insufficient permeability at high concentrations (35) . The data presented in this report conclusively demonstrate that large amounts of material can be introduced by in situ electroporation in a nontraumatic manner, so that a GRB2-SH2 blocking peptide can also block PDGF- and EGF-mediated ERK activation and mitogenesis in NIH-3T3 cells even after full receptor activation. ERK inactivation by the peptide in nonelectroporated cells caused by movement of the peptide through gap junctions from adjacent cells makes this demonstration especially powerful. The fact that a control peptide containing phenylalanine at the position of Pmp or an unrelated peptide had no effect on ERK activation indicates that the inhibitory activity of the Grb2-SH2 binding peptide can most probably be accounted for by its ability to bind this domain and inhibit activation of the ERK cascade, especially because this module was shown to be required for both the direct and indirect interactions of this adaptor with the EGFR or PDGFR (13) .

Although in vitro binding and molecular modeling studies showed that phosphotyrosine peptides containing asparagine at the +2 position can bind a number of SH2 domains in addition to Grb2-SH2, their affinity for these domains was lower, and the in vivo relevance of these interactions has not been determined (36) . Thus, although previous results (8) indicated that the sequence used in our study (PVPEpYINQS) does not interact with the PLC-{gamma} or p85 SH2 domains, we cannot exclude the possibility that this peptide might interact with other, perhaps unknown targets. Nevertheless, the dramatic inhibition of ERK activation produced by this peptide implicates this domain as the main avenue to ERK stimulation in this system. Hence, as shown before for newt A1 cells (8) , the other adaptors (e.g., CrkII) either do not contribute significantly to ERK activation in this system or also somehow require the Grb2-SH2 domain.

Contrary to EGF or PDGF, the inhibition of ERK activation by HGF through electroporation of the Grb2-SH2 binding peptide was very weak under conditions in which binding of endogenous Grb2 to activated Met is strongly inhibited. Because both EGF-mediated ERK activation and HGF-mediated ERK activation were studied in methu-expressing NIH-3T3 cells, the dramatic differences observed cannot be due to differences in cellular background. The possibility that the failure of this peptide to significantly inhibit the HGF response reflects the use of a totally different mitogenic pathway is unlikely because HGF did activate ERK, and the HGF-mediated mitogenic response was inhibited by the MEK inhibitor PD98059. The alternative possibility that the selectivity of the Grb2-SH2 binding peptide reflects differences in the coupling of these receptors to the ERK cascade is more likely. In fact, previous point mutagenesis studies with this receptor indicated that inhibition of Grb2 binding by changing its binding site on Met to Y1356VHV inhibited ERK activation to only a limited extent (15) . Therefore, it appears that in HGF-stimulated cells, other effectors independent of Grb2 binding can pass enough signal to cause ERK activation. It was recently shown that multisubstrate docking protein Gab1 binds Met at Y1356 through Grb2 and at Y1349 in a Grb2-independent manner (5 , 6) . It follows that inhibition of Grb2 binding through electroporation of the peptide would inhibit Gab1 binding to Y1356 but not to Y1349. Gab1, in turn, could still bind Met and activate ERK through a number of downstream effectors (6 , 37) . In fact, it was shown that Nck is tyrosine-phosphorylated after HGF stimulation, although its binding site(s) on Met is presently unknown (16 , 17) . Nck, in turn, could bind Sos and activate the Ras/Raf/ERK pathway in a Grb2-independent manner (38) . It is also possible that the binding of other Ras-activating adaptors might be intensified after inhibition of Grb2 binding.

Previous results indicated that some Shc proteins might form a constitutive complex with Grb2 in NIH-3T3 cells overexpressing the EGFR (7) . This observation might indicate that in HGF-stimulated A549 cells, ERK could be activated through Shc/Grb2 binding Met at Y1349 because such complexes would not be easily disrupted by the peptide. However, the demonstrated inhibition of Grb2 binding to the HGF receptor after the introduction of large amounts of peptide by electroporation argues against this possibility. Finally, Ras-independent mitogen-activated protein kinase activation has been demonstrated in a variety of systems, such as in integrin (20 , 39) or v-Src (40) signaling or in Xenopus oocyte maturation (41 , 42) , hence, it is conceivable that in the HGF system, ERK might be activated independently of Grb2 and Ras.

A number of pharmacological inhibitors of the ERK pathway have been designed, such as the [(alkylamino)methyl]acrylophenone blockers of the EGFR tyrosine kinase (25) . However, these compounds display a considerable amount of nonspecific effects. The present data demonstrate that the Grb2-SH2 blocking peptide, even after reaching intracellular concentrations that totally inhibit ERK activation by EGF, had no effect on TPA-induced ERK activation. These results indicate that the peptide itself and the electroporation procedure used do not cause detectable toxicity, thus revealing the higher specificity of this peptide for the PDGF- or EGF-induced signal, compared with the above-mentioned compounds. The absence of nonspecific toxicity is also demonstrated by the fact that the same peptide had only a minimal effect on HGF-induced mitogenesis.

The introduction of nonpermeant peptides to interrupt signaling pathways using the modification of in situ electroporation described here is a powerful approach for in vivo assessment of the relevance of in vitro interactions. The results presented clearly demonstrate that an essentially complete and specific inhibition of growth factor-dependent ERK1/2 activation can be achieved through peptide electroporation. Consequently, this approach could yield information on the precise extent of the contribution of a specific domain (hence, specific branches of a pathway) to a particular signal. The stepwise dissection of signaling cascades is essential for a better understanding of normal proliferative pathways that could lead to the development of drugs for the rational treatment of neoplasia.


   Materials and Methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Cell Lines and Culture Techniques
EGF and PDGF signaling was studied in mouse NIH-3T3 and rat F111 fibroblasts (43 , 44) . For the study of HGF signaling, a number of cell lines were tested for their ability to express high levels of activated ERK on HGF stimulation with low background. These included the mouse breast carcinoma cell lines SP1 and SP1-3M (45) ; the human breast epithelial and carcinoma cell lines MCF10A1 and MCF10A1T3B, respectively (46) ; the mink lung epithelial cell line Mv1Lu (American Type Culture Collection), and the human non-small cell lung carcinoma line A549 (American Type Culture Collection). A549 cells were found to be very suitable for the study of HGF-mediated ERK activation. In addition, these cells displayed good margins of voltage tolerance for the introduction of peptides (24) , similar to NIH-3T3 cells (see below). However, although A549 cells possess EGFRs, EGF activates the c-Jun-NH2-terminal kinase/stress-activated protein kinase kinase, rather than ERK (47 , 48) , hence ERK activation by EGF cannot be studied in this system. On the other hand, NIH-3T3 cells do not express HGF receptors. Therefore, to study EGF, PDGF, and HGF signaling in the same cell type, we used mouse NIH-3T3 fibroblasts that ectopically express human Met (Methu; a gift from Dr. G. F. Vande Woude), in addition to A549. Due to weak autocrine activation of the Methu receptor by the mouse HGF secreted by these cells, there is some background of ERK activity in the absence of added HGF in this system (49 , 50) .

Tissue culture medium (DMEM) was from ICN (Aurora, OH), and fetal calf and calf sera were from Life Technologies, Inc. (Burlington, Ontario, Canada). NIH-3T3 and F111 cells were grown in DMEM supplemented with 5% calf serum. A549 and methu-expressing NIH-3T3 cells were grown in DMEM supplemented with 10% FCS. All cells were cultured at 37°C in a humidified 5% CO2 incubator.

Peptides
The Grb2-SH2 binding peptide was based on the sequence flanking the Tyr1068 of the EGFR (PVPE-Pmp-INQS; Mr 1123). To enhance peptide stability, the phosphotyrosine analogue Pmp, which cannot be cleaved by phosphatases yet binds to SH2 domains with high affinity and specificity (51 , 52) , was incorporated at the position of phosophotyrosine. The Pmp monomer was custom synthesized by Color your Enzyme, Inc. (Kingston, Ontario, Canada). As controls, the same peptide containing phenylalanine at the position of Pmp and a random peptide of similar size and charge (CVVLSKRAT; Mr 975) were used. Peptides were synthesized by the Queen’s University Core Facility, using standard Fmoc chemistry.

Growth Arrest
To achieve growth arrest before growth factor stimulation, cells were incubated for 48 h in serum-free DMEM or in 50% spent medium in DMEM. The latter was prepared from the supernatant of cell cultures that had been grown in 10% calf serum (NIH-3T3 or F111 cells) or 10% FCS (A549 or NIH-3T3-methu), respectively, for 7 days after confluence and then dialyzed against and diluted (1:1) with fresh DMEM. Cells were growth-arrested by incubation for 48 h in spent medium prepared from the same cell lines.

In Situ Electroporation
The electroporation equipment was provided by Ask Science Products, Inc. (Kingston, Ontario, Canada). The procedures described in Ref. 24 were used, with some modifications. Briefly, cells were grown on conductive and transparent indium-tin oxide-coated glass slides and growth-arrested as described above. Unless otherwise indicated, the peptides were added to the cells at 10 or 20 mg/ml for the Grb2-SH2 blocking or control peptides, respectively, in calcium-free DMEM, and the electrode set was placed on the slide (Fig. 1)Citation . Compounds 7 and 8 (Ref. 25 ; gifts from Dr. N. Lydon; Kinetix Pharmaceuticals, Inc., Boston, MA) were dissolved in DMSO at 1 M (389 mg/ml, compound 7; 405 mg/ml, compound 8) and diluted down to 50 µg/ml in serum-free DMEM immediately before use. As shown previously, the optimal voltage and capacitance settings depended on the area being electroporated, and they were determined as described previously (24 , 53) to ensure efficient incorporation of the peptides without damage to the cells. Essentially 100% of the cells were permeated under the conditions used, as revealed by Lucifer yellow fluorescence, without any disturbance to cellular metabolism, as shown by immunostaining with antibodies against activated forms of the stress-activated kinases [c-Jun-NH2-terminal kinase/stress-activated protein kinase or p38hog (23) ]. Under these conditions, the concentration of fluorescein-coupled peptide achieved inside the NIH-3T3 cells without or with methu expression or inside the A549 human lung carcinoma line was approximately 3–5% of the concentration applied to the cell (24 , 54) . For Western blotting experiments, cells were grown on fully conductive slides with a growth area of 32 x 10 mm (Fig. 1B)Citation and electroporated using six pulses of 28–32 V from a 10 µF capacitor. For the examination of the effect of the peptides on cellular morphology or measurement of ERK activation by immunocytochemistry, the conductive coating was removed from part of the slide to provide nonelectroporated cells side by side with the electroporated ones to serve as controls (23 , 55) . In this configuration, the electroporated area was 4 x 4 mm (Fig. 1A)Citation , and electroporation consisted of six pulses at 30–34 V from a 0.1 µF capacitor. The exact voltage range was found to be approximately 2 V lower for the A549 cells.

Growth Factor Stimulation and Measurement of DNA Synthesis
For DNA synthesis measurements, cells were plated on fully conductive slides (Fig. 1B)Citation with a window of 4 x 7 mm at a density of approximately 50% of confluence, and, after growth arrest, the peptides were introduced by electroporation as described above (0.2 µF, 30 V, six pulses). After a 5-min incubation at 37°C, cells were stimulated to divide through the addition of DMEM supplemented with either EGF (100 ng/ml; human recombinant, Intergen), PDGF [40 ng/ml; Upstate Biotechnologies (UBI)], or HGF (40 ng/ml; recombinant human HGF; Genentech Inc.). Twelve h after growth factor addition, cells were labeled with 50 µCi/ml [3H]thymidine (Amersham) for 2 h, and TCA precipitable radioactivity was determined by scintillation counting. To examine the role of MEK, the inhibitor PD98059 (Calbiochem, San Diego, CA) was added to a final concentration of 100–200 µM to the growth medium of cells growing in 24-well plates from a 20 mM stock in DMSO 2 h after growth factor addition. Twelve h later, cells were labeled with 20 µCi/ml [3H]thymidine for 2 h, and TCA precipitable radioactivity was determined.

Grb2 Receptor Binding Experiments
After peptide electroporation and growth factor stimulation of the cells, proteins were extracted using 150 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.2 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride, 0.5% NP40, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1% Triton X-100. One mg of total cell extract was precipitated with an antibody against Grb2 (Transduction Laboratories, Lexington, KY). Proteins in the precipitate were resolved on a 12% polyacrylamide-SDS gel and transferred to Hybond C Extra Nitrocellulose (Amersham). The membrane was probed with antibodies to the extracellular domain of the HGF receptor (UBI catalogue number 05-238), phosphotyrosine (4G10; UBI catalogue number 05-321), or EGFR (UBI catalogue number 06-129), followed by horseradish peroxidase-coupled secondary antibodies and enhanced chemiluminescence reagents according to the manufacturer’s instructions (New England Nuclear).

Measurement of ERK Activation
Cells were growth-arrested, electroporated with peptides or compounds, and stimulated with the indicated growth factors for various times from 1 to 60 min. Alternatively, cells were treated with the PI3k inhibitor LY294002 (40 µM, 2 h) before growth factor stimulation. Under these conditions, PI3k activity, as measured by Western blotting using antibodies against the phosphorylated form of its downstream target, Akt, was completely inhibited. In a separate experiment, cells were treated with the MEK inhibitor PD98059 (100 µM, 2 h) before growth factor stimulation. ERK activity levels were then measured as described below.

Immunocytochemistry.
At different times after growth factor stimulation, cells were fixed with 8% paraformaldehyde, incubated in 1% peroxide in PBS, permeabilized with 100% methanol at -20°C for 10 min, and blocked with 1% BSA for 30 min. Cells were subsequently incubated with the affinity-purified antibodies raised in rabbits against the dually phosphorylated, activated form of ERK (Promega, Madison, WI; catalogue number V667). The antibodies were visualized through incubation with a biotinylated goat antirabbit secondary antibody followed by avidin-biotin-horseradish peroxidase complex and diaminobenzidine staining according to the manufacturer’s instructions (Vectastain kit; Vector Laboratories). Cells were photographed under bright-field or phase-contrast illumination using an inverted Olympus IX70 microscope. Semiquantitative densitometry of immunocytochemical staining was carried out using a MCID M5 image analysis software program (Imaging Research, Inc., St. Catharines, Ontario, Canada). Results are expressed as relative arbitrary density units compared with background (unstained) monolayers.

Western Blotting.
At different times after electroporation and growth factor stimulation, proteins were extracted using 50 mM HEPES (pH 7.4), 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM Na3VO4, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1% Triton X-100 (54) . Total cell extract (300 µg) was resolved on a 12% polyacrylamide-SDS gel and transferred to Hybond C Extra Nitrocellulose (Amersham). This membrane was probed with the antibodies against the activated form of ERK, followed by a horseradish peroxidase-coupled secondary antibody and enhanced chemiluminescence reagents as described above.

Using either assay, EGF was previously found to be a more effective ERK activator than PDGF in NIH-3T3 cells when EGF and PDGF were added at their optimal concentrations of 100 and 40 ng/ml, respectively (23) . This difference may be due to differences in the numbers of respective receptors or to other signaling molecules upstream of ERK. A time course of ERK activation on EGF or PDGF stimulation of NIH-3T3 cells grown on ITO-coated glass indicated a detectable increase in ERK activity levels as early as 1 min after stimulation with either growth factor, with the maximal response seen at 5 min. A similar time course was obtained using rat F111 cells (23) . On the other hand, the peak ERK activation by HGF in A549 cells or methu-expressing NIH-3T3 cells was observed at 20–30 min, using the optimal concentration of 40 ng/ml HGF.


   Acknowledgments
 
We thank Kevin Firth and P. Eng (Ask Science Products, Inc.) for the design and development of the in situ electroporation apparatus, Dr. Erik Schaefer (Quality Controlled Biochemicals, Boston, MA) for the generous gift of the antiactive ERK antibodies, Dr. G. F. Vande Woude (National Cancer Institute, Frederick, MD) for the methu-expressing NIH-3T3 cell line, Lloyd Kennedy (Queen’s University) for help with image analysis, and Dr. Nick Lydon (Kinetix Pharmaceuticals, Inc.) for compounds 7 and 8, and Marina Walcer (Queen’s University) for technical assistance.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the Cancer Research Society, Inc. and the Natural Sciences and Engineering Research Council of Canada (to L. R.) and a grant from the Canadian Breast Cancer Foundation (to B. E. E.). H. L. B. was supported by a Medical Research Council of Canada studentship and was the recipient of a Microbix Biosystems, Inc. travel award. A. M. V. is the recipient of a studentship from the Natural Sciences and Engineering Research Council of Canada. Back

2 To whom requests for reprints should be addressed, at Department of Microbiology and Immunology, Botterell Hall, Room 716, Queen’s University, Kingston, Ontario, K7L 3N6 Canada. Phone: (613) 533-2462; Fax: (613) 533-6796; E-mail: raptisl{at}post.queensu.ca Back

3 Present address: Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Back

4 The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular signal-regulated kinase; Pmp, phosphono-methylphenylalanine; HGF, hepatocyte growth factor; ITO, indium oxide doped with tin; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; Grb2, growth factor receptor-binding protein 2; SH2, Src homology 2; PLC, phospholipase C; MEK, mitogen-activated protein/ERK kinase; PI3k, phosphatidylinositol 3'-kinase. Back

Received for publication 1/ 4/00. Revision received 4/18/00. Accepted for publication 4/24/00.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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