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 peptide.
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.
Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada. raptisl@post.queensu.ca
L
L
Raptis
H L
HL
Brownell
A M
AM
Vultur
G M
GM
Ross
E
E
Tremblay
B E
BE
Elliott
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015153:Blotting, Western; D002455:Cell Division; D002478:Cells, Cultured; D018274:Electroporation / Q000295:instrumentation* / Q000379:methods*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D051380:GRB2 Adaptor Protein; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology*; D011506:Proteins / Q000378:metabolism; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism; D051381:Rats; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D013997:Time Factors; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D004791:Enzyme Inhibitors; D005419:Flavonoids; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495949:Grb2 protein, mouse; C495950:Grb2 protein, rat; D010455:Peptides; D011506:Proteins; D066246:ErbB Receptors; D019859:Proto-Oncogene Proteins c-met; D017479:Receptors, Platelet-Derived Growth Factor; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one