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 cellpermeable 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, 1 PDGF, and FGF stimulate cell proliferation by binding to and activating membrane-spanning receptors that have cytoplasmic tyrosine kinase domains (1, 2). 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. 3 and 4). 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/mitogenactivated protein kinase (MAPK) cascade, and this is central to the mitogenic response stimulated by many growth factors (5). 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 (6 -8). In this context, when phosphorylated, Tyr 1068 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 (8). 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 (9). Moreover, when made in tandem with a peptide that allows for translocation ac...

PSM, a mediator of PDGF-BB-, IGF-I-, and insulin-stimulated mitogenesis

“…We con®rmed transfer with biotinylated peptides which entered the cell cytoplasm in a peptide dose-responsive fashion (Table 1). A tyrosine-phosphorylated, cell-permeable Grb2 binding peptide based on EGF receptor sequences was shown to speci®cally block the mitogenic response to PDGF and EGF but not to FGF, demonstrating that selective pathways are inhibited by this approach (Williams et al, 1997). Activation of the MAP kinase cascade was inhibited in response to up to 10 ng/ml but not to higher 50 ng/ml EGF concentrations, suggesting that the peptide does not exert an excessive inhibitory function (Williams et al, 1997).…”

“…A tyrosine-phosphorylated, cell-permeable Grb2 binding peptide based on EGF receptor sequences was shown to speci®cally block the mitogenic response to PDGF and EGF but not to FGF, demonstrating that selective pathways are inhibited by this approach (Williams et al, 1997). Activation of the MAP kinase cascade was inhibited in response to up to 10 ng/ml but not to higher 50 ng/ml EGF concentrations, suggesting that the peptide does not exert an excessive inhibitory function (Williams et al, 1997). With this approach the IGF-I-and PDGF-BB-stimulated mitogenic responses were essentially eliminated with increasing concentration of PSM Pro-rich fusion peptide in a dose-responsive fashion whereas the EGF response remained unaected ( Figure 5).…”

Identification of a p65 Peptide That Selectively Inhibits NF-κ B Activation Induced by Various Inflammatory Stimuli and Its Role in Down-regulation of NF-κB-mediated Gene Expression and Up-regulation of Apoptosis

“…These include Grb2-binding peptide (16), mitogen-activated protein kinase (17), STAT3 (18), NEMO-IKK-interacting peptide (19), and peptides carrying nuclear localization sequences (20 -23). Besides peptides, PTD have also been used to deliver larger full-length polypeptides, including IB␣ (24), cyclin-dependent kinase inhibitory protein p27 (25), antiapoptotic protein Bcl-x L (26), and proapoptotic proteins (27).…”

True

“…To date, several members of the heterogeneous family of cell permeable proteins have been described: the third helix of the antennapedia homeodomain (pAntp), the HIV-Tat protein, the K-FGF and the HSV-1 structural protein VP22. 5,[18][19][20][21][22][23][24] While in the case of pAntp, an amphipatic ␣-helix of 16 amino acids mediates the cell permeability, 1 and an unstructured cluster of nine basic amino acids is responsible for the cell permeability of the HIV-1 Tat protein, 2 in case of VP22 4 and of K-FGF 3 the underlying structural motif is unknown.…”