Imaging Protein Kinase Cα Activation in Cells

Abstract

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.

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…In particular, we focus on those phosphorylation sites on PKCs that are inducible or regulated in cells (Figure 3). PKC : PKC undergoes phosphorylation at Thr-250 (numbering of amino acids from human sequences are used throughout this manuscript) located in the C2 domain in response to phorbol esters that mimics DAG signalling [95] and also in response to cells adhering to fibronectin [107] (Figure 3). Thr-250 is an autophosphorylation site and hence the phosphorylation status of this isoform at this site has been used as a marker of PKC catalytic activity [95].…”

High-precision FLIM–FRET in fixed and living cells reveals heterogeneity in a simple CFP–YFP fusion protein

“…proximity of an acceptor chromophore, ion concentration or temperature). FLIM has been successfully used to probe energy transfer in cells [9][10][11]. However, the multiple decay pathways available to an excited molecule due to variations in local environment or chemical structure (e.g.…”

Defective IgG2a/2b Class Switching in PKCα−/− Mice

“…To assess whether PKC␣ is activated by the established autophosphorylation on Thr 250 (16) in intact T cells, the anti-(phospho)Thr 250 antiserum reacted strongly with recombinant transfected PKC␣ in PDBu-stimulated Jurkat T cells but much less in unstimulated cells (Fig. 1D).…”