Improved Methodology

FRETting Over cAMP Flux and PKA Activation

Science's STKE  01 Feb 2000:
Vol. 2000, Issue 17, pp. tw6
DOI: 10.1126/stke.2000.17.tw6

The second messenger cAMP plays a critical role in several signaling pathways. However, the current methods used to measure intracellular cAMP levels and cAMP-dependent protein kinase (PKA) activation preclude the ability to measure these signaling components easily in intact cells. Zaccolo et al. have successfully engineered chimeric PKA subunits that express fluorescent proteins at their COOH-termini. Expression of the PKA catalytic subunit fused to green fluorescent protein often resulted in morphological changes. However, coexpression with the RII regulatory subunit of PKA fused to blue fluorescent protein reverted the cells to normal morphology in the absence of stimulation. This indicated that both subunit chimeric proteins were functional and that overexpression of both proteins was not detrimental to the cells. Cells coexpressing both chimeric subunits exhibited fluorescence resonance energy transfer (FRET) in response to the pharmacological adenylate cyclase activator forskolin. Zaccolo et al. also utilized the more physiologically relevant stimulus norepinephrine and demonstrated the ability of the chimeric proteins to detect fluctuations in cAMP levels. Thus, these new reagents may enable investigators to study cAMP diffusion and PKA activation in whole cells in real time.

Zaccolo, M., De Giorgi, F., Cho, C.Y., Feng, L., Knapp, T., Negulescu, P.A., Taylor, S.S., Tsien, R.Y., and Pozzan, T. (2000) A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat. Cell Biol. 2: 25-29. [Online Journal]