Imaging protein kinase Calpha activation in cells.
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.
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T
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Protein Phosphorylation Laboratory and Cell Biophysics Laboratory, Imperial Cancer Research Fund (ICRF), 44 Lincoln's Inn Fields, London, WC2A 3PX, UK.
Squire
A
A
Hansra
G
G
Bornancin
F
F
Prevostel
C
C
Hanby
A
A
Harris
W
W
Barnes
D
D
Schmidt
S
S
Mellor
H
H
Bastiaens
P I
PI
Parker
P J
PJ
D016475:3T3 Cells; D000818:Animals; D001943:Breast Neoplasms / Q000201:enzymology; D019556:COS Cells; D002384:Catalysis; D003593:Cytoplasm / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004735:Energy Transfer; D004789:Enzyme Activation; D005453:Fluorescence; D005456:Fluorescent Dyes; D006056:Golgi Apparatus / Q000201:enzymology; D049452:Green Fluorescent Proteins; D006801:Humans; D007106:Immune Sera; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism*; D008164:Luminescent Proteins; D051379:Mice; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D010769:Phosphothreonine / Q000276:immunology / Q000378:metabolism; D011493:Protein Kinase C / Q000276:immunology / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D005456:Fluorescent Dyes; D007106:Immune Sera; D007527:Isoenzymes; D008164:Luminescent Proteins; D010769:Phosphothreonine; D049452:Green Fluorescent Proteins; C496628:PRKCA protein, human; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate