title: Quantification of growth factor signaling and pathway cross talk by live-cell imaging.
abstract: Peptide growth factors stimulate cellular responses through activation of their transmembrane receptors. Multiple intracellular signaling cascades are engaged following growth factor-receptor binding, leading to short- and long-term biological effects. Each receptor-activated signaling pathway does not act in isolation but rather interacts at different levels with other pathways to shape signaling networks that are distinctive for each growth factor. To gain insights into the specifics of growth factor-regulated interactions among different signaling cascades, we developed a HeLa cell line stably expressing fluorescent live-cell imaging reporters that are readouts for two major growth factor-stimulated pathways, Ras-Raf-Mek-ERK and phosphatidylinositol (PI) 3-kinase-Akt. Incubation of cells with epidermal growth factor (EGF) resulted in rapid, robust, and sustained ERK signaling but shorter-term activation of Akt. In contrast, hepatocyte growth factor induced sustained Akt signaling but weak and short-lived ERK activity, and insulin-like growth factor-I stimulated strong long-term Akt responses but negligible ERK signaling. To address potential interactions between signaling pathways, we employed specific small-molecule inhibitors. In cells incubated with EGF or platelet-derived growth factor-AA, Raf activation and the subsequent stimulation of ERK reduced Akt signaling, whereas Mek inhibition, which blocked ERK activation, enhanced Akt and turned transient effects into sustained responses. Our results reveal that individual growth factors initiate signaling cascades that vary markedly in strength and duration and demonstrate in living cells the dramatic effects of cross talk from Raf and Mek to PI 3-kinase and Akt. Our data further indicate how specific growth factors can encode distinct cellular behaviors by promoting complex interactions among signaling pathways.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon; and.
authors.forename: Sean M
authors.initials: SM
authors.lastname: Gross
authors.affiliation: Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech Health University Health Sciences Center, El Paso, Texas peter.rotwein@ttuhsc.edu.
authors.forename: Peter
authors.identifier: http://orcid.org/0000-0002-9505-1817
authors.initials: P
authors.lastname: Rotwein
mesh_terms: D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D000069416:Intravital Microscopy / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D020239:Receptor Cross-Talk / Q000502:physiology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D036341:Intercellular Signaling Peptides and Proteins
keywords: Raf; cell signaling; extracellular signal-regulated kinase; growth factors; mitogen/extracellular signal-regulated kinase; protein kinase B; signal transduction