scholarly journals Analysis by Fluorescence Resonance Energy Transfer of the Interaction between Ligands and Protein Kinase Cδ in the Intact Cell

2004 ◽  
Vol 280 (9) ◽  
pp. 8164-8171 ◽  
Author(s):  
Derek C. Braun ◽  
Susan H. Garfield ◽  
Peter M. Blumberg
Keyword(s):  
Energy Transfer ◽  
Protein Kinase ◽  
Fluorescence Resonance Energy Transfer ◽  
Intact Cell ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Resonance ◽  
Protein Kinase Cδ

scholarly journals Phospholemman Phosphorylation Alters Its Fluorescence Resonance Energy Transfer with the Na/K-ATPase Pump

2006 ◽  
Vol 281 (43) ◽  
pp. 32765-32773 ◽  
Author(s):  
Julie Bossuyt ◽  
Sanda Despa ◽  
Jody L. Martin ◽  
Donald M. Bers
Keyword(s):  
Protein Kinase C ◽  
Energy Transfer ◽  
Protein Kinase ◽  
Fluorescence Resonance Energy Transfer ◽  
Cardiac Myocyte ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Hek293 Cells ◽  
Kinase C ◽  
Fluorescence Resonance

Phospholemman (PLM) or FXYD1 is a major cardiac myocyte phosphorylation target upon adrenergic stimulation. Prior immunoprecipitation and functional studies suggest that phospholemman associates with the Na/K-pump (NKA) and mediates adrenergic Na/K-pump regulation. Here, we tested whether the NKA-PLM interaction is close enough to allow fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent (CFP/YFP) fusion proteins of Na/K pump and phospholemman and whether phospholemman phosphorylation alters such FRET. Co-expressed NKA-CFP and PLM-YFP in HEK293 cells co-localized in the plasma membrane and exhibited robust FRET. Selective acceptor photobleach increased donor fluorescence (FCFP) by 21.5 ± 4.1% (n = 13), an effect nearly abolished when co-expressing excess phospholemman lacking YFP. Activation of protein kinase C or A progressively and reversibly decreased FRET assessed by either the fluorescence ratio (FYFP/FCFP) or the enhancement of donor fluorescence after acceptor bleach. After protein kinase C activation, forskolin did not further reduce FRET, but after forskolin pretreatment, protein kinase C could still reduce FRET. This agreed with phospholemman phosphorylation measurements: by protein kinase C at both Ser-63 and Ser-68, but by protein kinase A only at Ser-68. Expression of PLM-YFP and PLM-CFP resulted in even stronger FRET than for NKA-PLM (FCFP increased by 37 ± 1% upon YFP photobleach), and this FRET was enhanced by phospholemman phosphorylation, consistent with phospholemman multimerization. Co-expressed PLM-CFP and Na/Ca exchange-YFP were highly membrane co-localized, but FRET was undetectable. We conclude that phospholemman and Na/K-pump are in very close proximity (FRET occurs) and that phospholemman phosphorylation alters the interaction of Na/K-pump and phospholemman.

scholarly journals Stimulus-Specific Distinctions in Spatial and Temporal Dynamics of Stress-Activated Protein Kinase Kinase Kinases Revealed by a Fluorescence Resonance Energy Transfer Biosensor

2009 ◽  
Vol 29 (22) ◽  
pp. 6117-6127 ◽  
Author(s):  
Taichiro Tomida ◽  
Mutsuhiro Takekawa ◽  
Pauline O'Grady ◽  
Haruo Saito
Keyword(s):  
Energy Transfer ◽  
Protein Kinase ◽  
Fluorescence Resonance Energy Transfer ◽  
Cell Population ◽  
Temporal Dynamics ◽  
Single Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
A Cell ◽  
Fluorescence Resonance

ABSTRACT The stress-activated protein kinases (SAPKs), namely, p38 and JNK, are members of the mitogen-activated protein kinase family and are important determinants of cell fate when cells are exposed to environmental stresses such as UV and osmostress. SAPKs are activated by SAPK kinases (SAP2Ks), which are in turn activated by various SAP2K kinases (SAP3Ks). Because conventional methods, such as immunoblotting using phospho-specific antibodies, measure the average activity of SAP3Ks in a cell population, the intracellular dynamics of SAP3K activity are largely unknown. Here, we developed a reporter of SAP3K activity toward the MKK6 SAP2K, based on fluorescence resonance energy transfer, that can uncover the dynamic behavior of SAP3K activation in cells. Using this reporter, we demonstrated that SAP3K activation occurs either synchronously or asynchronously among a cell population and in different cellular compartments in single cells, depending on the type of stress applied. In particular, SAP3Ks are activated by epidermal growth factor and osmostress on the plasma membrane, by anisomycin and UV in the cytoplasm, and by etoposide in the nucleus. These observations revealed previously unknown heterogeneity in SAPK responses and supplied answers to the question of the cellular location in which various stresses induce stimulus-specific SAPK responses.

scholarly journals Fluorescence Resonance Energy Transfer in Quantum Dot-Protein Kinase Assemblies

2007 ◽  
Vol 2007 ◽  
pp. 1-5 ◽  
Author(s):  
Ibrahim Yildiz ◽  
Xinxin Gao ◽  
Thomas K. Harris ◽  
Françisco M. Raymo
Keyword(s):  
Energy Transfer ◽  
Protein Kinase ◽  
Fluorescence Resonance Energy Transfer ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Kinase Domain ◽  
Binding Domain ◽  
Dependent Protein ◽  
Fluorescence Resonance

In search of viable strategies to identify selective inhibitors of protein kinases, we have designed a binding assay to probe the interactions of human phosphoinositide-dependent protein kinase-1 (PDK1) with potential ligands. Our protocol is based on fluorescence resonance energy transfer (FRET) between semiconductor quantum dots (QDs) and organic dyes. Specifically, we have expressed and purified the catalytic kinase domain of PDK1 with an N-terminal histidine tag [His6-PDK1(ΔPH)]. We have conjugated this construct to CdSe-ZnS core-shell QDs coated with dihydrolipoic acid (DHLA) and tested the response of the resulting assembly to a molecular dyad incorporating an ATP ligand and a BODIPY chromophore. The supramolecular association of the BODIPY-ATP dyad with theHis6-PDK1(ΔPH)-QD assembly encourages the transfer of energy from the QDs to the BODIPY dyes upon excitation. The addition of ATP results in the displacement of BODIPY-ATP from the binding domain of theHis6-PDK1(ΔPH) conjugated to the nanoparticles. The competitive binding, however, does not prevent the energy transfer process. A control experiment with QDs, lacking theHis6-PDK1(ΔPH), indicates that the BODIPY-ATP dyad adsorbs nonspecifically on the surface of the nanoparticles, promoting the transfer of energy from the CdSe core to the adsorbed BODIPY dyes. Thus, the implementation of FRET-based assays to probe the binding domain of PDK1 with luminescent QDs requires the identification of energy acceptors unable to interact nonspecifically with the surface of the nanoparticles.

Imaging protein–protein interactions in cell motility using fluorescence resonance energy transfer (FRET)

2004 ◽  
Vol 32 (3) ◽  
pp. 431-433 ◽  
Author(s):  
M. Parsons ◽  
B. Vojnovic ◽  
S. Ameer-Beg
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Intact Cell ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Fluorescence Resonance

Protein–protein interactions and signal transduction pathways have traditionally been analysed using biochemical techniques or standard microscopy. Although invaluable in the delineation of protein hierarchy, these methods do not provide information on the true spatial and temporal nature of complex formation within the intact cell. Recent advances in microscopy have allowed the development of new methods to analyse protein–protein interactions at very high resolution in both fixed and live cells. The present paper provides a brief overview of using fluorescence resonance energy transfer to analyse directly molecular interactions and conformational changes in various proteins involved in the regulation of cell adhesion and motility.

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