Phosphoproteomic analysis of signal transduction dynamics induced by a modified cell-penetrating peptide-protein...

Cell-penetrating peptides (CPPs) deliver cargo proteins into cells; however, most CPPs rely on nonspecific electrostatic interactions, limiting their cell-type specificity. The CPP Pas2r12 delivers cargo, such as enhanced green fluorescent protein (EGFP) and immunoglobulin G, into the cytosol, primarily through caveolae-dependent endocytosis. To elucidate the intracellular signaling underlying Pas2r12-mediated delivery, time-resolved phosphoproteomic analyses were performed following treatment with Pas2r12 or the Pas2r12-EGFP complex. Early phosphorylation of mitogen-activated protein kinase 1 (MAPK1) and ephrin B1 (EFNB1) was observed following stimulation with either Pas2r12 or the Pas2r12-EGFP complex, whereas F11R phosphorylation occurred only in response to Pas2r12. Notably, MAPK1 phosphorylation consistently increased at all timepoints after stimulation with either treatment. In addition, lysophosphatidic acid receptor 1 (LPAR1) phosphorylation was induced by both treatments at 30 min. EFNB1, F11R, and LPAR1 are cell surface ligands or receptors that are potential targets of Pas2r12 or the Pas2r12-EGFP complex. These results suggest that sustained MAPK1 phosphorylation may play a central role in cellular response to Pas2r12-based delivery. Future studies should aim at establishing Pas2r12 as an efficient and versatile protein delivery tool, with particular focus on exploiting specific cell-surface molecules to enhance targeting specificity.