SPATIO-TEMPORAL IMAGING OF EGF-INDUCED ACTIVATION OF PROTEIN KINASE A BY FRET IN LIVING CELLS
利用FRET技术在活细胞内观察EGF对PKA作用的时空成像

The cAMP-dependent protein kinase-A (PKA) is an intracellular enzyme with serine- threonine kinase activity that plays a key role in cell growth and differentiation. Epidermal growth factor (EGF), a well-characterized polypeptide cell growth factor, has been demonstrated to induce PKA activation through the ligation of EGFR. To investigate the spatial and temporal dynamics of PKA activation by EGF, we visualized the PKA activation in living cells using a fluorescent indicator (AKAR) composed of two green fluorescent protein (GFP) variants, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), joined by a phosphoamino acid binding domain (14-3-3) and a consensus substrate for protein kinase-A (PKA). Phosphorylation of the consensus substrate by PKA activation resulted in the transfer of energy from excited CFP to YFP within the AKAR molecule. A human lung cancer cell line (ASTC-a-1) that stably expressed the AKAR indicator protein was established and characterized. After treatment with EGF, the activation dynamics of PKA was visualized in living cells by fluorescence microscopic imaging system. Time-lapse imaging analysis reveals the spatio-temporal distribution of EGF-activated PKA in specific sites within the living cells. These data provide direct and in vivo evidence for PKA activation by EGF and add a new dimension for PKA signaling pathway.