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 gro wth factors such as the epidermal growth factor, platelet-derived growth fa ctor, or hepatocyte growth factor. Ras activation requires the function of adaptors such as growth factor receptor-binding protein 2, which can bind e ither directly or indirectly through Src homology 2 domains to the activate d receptor. To examine the role of the Src homology 2 domain of growth fact or 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 cell s growing on conductive indium-tin oxide-coated glass by in situ electropor ation. Cells were subsequently stimulated with growth factors and assessed for activation of a downstream target, extracellular signal-regulated kinas e (ERK) 1/2, by probing with antibodies specific for its activated form. El ectrodes and slides were configured to provide nonelectroporated control ce lls side by side with the electroporated ones, both growing on the same typ e of indium-tin oxide-coated glass surface. The data demonstrate that the p eptide can cause a dramatic inhibition of epidermal growth factor or platel et-derived growth factor-mediated ERK1/2 activation and DNA synthesis in vi vo, compared with its control phenylalanine-containing counterpart. In cont rast, the peptide had a very limited effect on hepatocyte growth factor-tri ggered ERK1/2 activation and DNA synthesis. These results demonstrate the p otential of the in situ electroporation approach described here in the stud y of the coupling of activated receptor tyrosine kinases to the ERK1/2 casc ade.