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Science 26 March 1999:
Vol. 283. no. 5410, pp. 2085 - 2089
DOI: 10.1126/science.283.5410.2085
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Reports

Imaging Protein Kinase Calpha Activation in Cells

Tony Ng, 1* Anthony Squire, 2* Gurdip Hansra, 1 Frederic Bornancin, 1dagger Corinne Prevostel, 1 Andrew Hanby, 3 William Harris, 3 Diana Barnes, 3 Sandra Schmidt, 2 Harry Mellor, 1 Philippe I. H. Bastiaens, 2ddagger Peter J. Parker 1ddagger

Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase Calpha (PKCalpha ) activation is identified and exploited. Activation of PKCalpha is detected through the binding of fluorescently tagged phosphorylation site-specific antibodies; the consequent FRET is measured through the donor fluorophore on PKCalpha by FLIM. This approach enabled the imaging of PKCalpha activation in live and fixed cultured cells and was also applied to pathological samples.

1 Protein Phosphorylation Laboratory and
2 Cell Biophysics Laboratory, Imperial Cancer Research Fund (ICRF), 44 Lincoln's Inn Fields, London, WC2A 3PX, UK.
3 Hedley Atkins Laboratory, Imperial Cancer Research Fund, Guy's Hospital, St. Thomas Street, London, SE1 9RT, UK.
*   These authors contributed equally to this work.

dagger    Present address: Synthelabo Recherche (LERS), 31 Avenue Paul, Valliant-Counturier, 92220 Bagneux, France.

ddagger    To whom correspondence should be addressed. E-mail: (P.I.H.B.) bastiaen{at}icrf.icnet.uk, and (P.J.P.) parkerp{at}icrf.icnet.uk

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