Quantitation of the interaction of protein kinase C with diacylglycerol and phosphoinositides by time-resolved detection of resonance energy transfer

Biochemistry ◽  
1993 ◽  
Vol 32 (48) ◽  
pp. 13310-13317 ◽  
Author(s):  
E. H. W. Pap ◽  
P. I. H. Bastiaens ◽  
J. W. Borst ◽  
P. A. W. van den Berg ◽  
A. van Hoek ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Energy Transfer ◽  
Protein Kinase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Kinase C

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 Screening for Protein Kinase C Ligands Using Fluorescence Resonance Energy Transfer

2014 ◽  
Vol 62 (10) ◽  
pp. 1019-1025 ◽  
Author(s):  
Nami Ohashi ◽  
Wataru Nomura ◽  
Natsuki Minato ◽  
Hirokazu Tamamura
Keyword(s):  
Protein Kinase C ◽  
Energy Transfer ◽  
Protein Kinase ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Kinase C ◽  
Fluorescence Resonance

scholarly journals A cGMP-Dependent Protein Kinase Assay for High Throughput Screening Based on Time-Resolved Fluorescence Resonance Energy Transfer

2001 ◽  
Vol 6 (4) ◽  
pp. 255-264 ◽  
Author(s):  
Benjamin Bader ◽  
Elke Butt ◽  
Alois Palmetshofer ◽  
Ulrich Walter ◽  
Thomas Jarchau ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Protein Kinase ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
Cyclic Gmp ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dependent Protein Kinase ◽  
Time Resolved ◽  
Dependent Protein

Activation of cyclic GMP-dependent protein kinase (cGK) is an important event in the regulation of blood pressure and platelet function. Upstream signals are the generation of nitric oxide (NO) by NO syntheses and the subsequent rise in cyclic GMP levels mediated by NO-dependent guanylyl cyclases (GCs). The identification of new cGK activators by high throughput sreening (HTS) may lead to the development of a novel class of therapeutics for the treatment of cardiovascular diseases. Therefore, a homogeneous, nonradioactive assay for cGK activity was developed using a biotinylated peptide derived from vasodilator-stimulated phosphoprotein (VASP), a well-characterized natural cGK substrate. The phosphorylated peptide could be detected by a VASP-specific monoclonal phosphoserine antibody and a fluorescent detection system consisting of a europium-labeled secondary antibody and allophycocyanin (APC)-labeled streptavidin. Fluorescence resonance energy transfer (FRET) from europium to APC was detected in a time-resolved fashion (TR-FRET). Activation and inhibition constants for known substances determined by this new fluorescence-based assay correlated well with published results obtained by conventional radioactive cGK activity assays. The assay proved to be sensitive, robust, highly specific for cGK, and suitable for HTS in 96- and 384-well formats. This assay is applicable to purified enzymes as well as to complex samples such as human platelet extracts.

Fluorescence Resonance Energy Transfer-based Screening for Protein Kinase C Ligands Using 6-Methoxynaphthalene-labeled 1,2-Diacylglycerol-lactones

2021 ◽  
Author(s):  
Kohei Tsuji ◽  
Takahiro Ishii ◽  
T Kobayakawa ◽  
Nami Ohashi ◽  
Wataru Nomura ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Resonance Energy Transfer ◽  
Signal Transduction Pathway ◽  
Resonance Energy ◽  
Alzheimer Type Dementia ◽  
Kinase C ◽  
P Protein ◽  
Cellular Signal ◽  
Cellular Signal Transduction Pathway

Protein kinase C (PKC) is associated with a central cellular signal transduction pathway and disorders such as cancer and Alzheimer-type dementia and is therefore a target for treatment of these...

Time-resolved Forster resonance energy transfer assays for the binding of nucleotide and protein substrates to p38α protein kinase

2005 ◽  
Vol 343 (1) ◽  
pp. 76-83 ◽  
Author(s):  
Wen Xiao Zhang ◽  
Ruixiu Wang ◽  
Douglas Wisniewski ◽  
Alice I. Marcy ◽  
Philip LoGrasso ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Protein Kinase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Time Resolved ◽  
Protein Substrates ◽  
Förster Resonance

Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels in mitochondrial inner membrane

2007 ◽  
Vol 293 (1) ◽  
pp. H322-H332 ◽  
Author(s):  
Vivek Garg ◽  
Keli Hu
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Significant Implication ◽  
Sulfonylurea Receptor ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Kinase C ◽  
Pkc Activation

The ATP-sensitive K+ (KATP) channels in both sarcolemmal (sarcKATP) and mitochondrial inner membrane (mitoKATP) are the critical mediators in cellular protection of ischemic preconditioning (IPC). Whereas cardiac sarcKATP contains Kir6.2 and sulfonylurea receptor (SUR)2A, the molecular identity of mitoKATP remains elusive. In the present study, we tested the hypothesis that protein kinase C (PKC) may promote import of Kir6.2-containing KATP into mitochondria. Fluorescence imaging of isolated mitochondria from both rat adult cardiomyocytes and COS-7 cells expressing recombinant Kir6.2/SUR2A showed that Kir6.2-containing KATP channels were localized in mitochondria and this mitochondrial localization was significantly increased by PKC activation with phorbol 12-myristate 13-acetate (PMA). Fluorescence resonance energy transfer microscopy further revealed that a significant number of Kir6.2-containing KATP channels were localized in mitochondrial inner membrane after PKC activation. These results were supported by Western blotting showing that the Kir6.2 protein level in mitochondria from COS-7 cells transfected with Kir6.2/SUR2A was enhanced after PMA treatment and this increase was inhibited by the selective PKC inhibitor chelerythrine. Furthermore, functional analysis indicated that the number of functional KATP channels in mitochondria was significantly increased by PMA, as shown by KATP-dependent decrease in mitochondrial membrane potential in COS-7 cells transfected with Kir6.2/SUR2A but not empty vector. Importantly, PKC-mediated increase in mitochondrial Kir6.2-containing KATP channels was blocked by a selective PKCε inhibitor peptide in both COS-7 cells and cardiomyocytes. We conclude that the KATP channel pore-forming subunit Kir6.2 is indeed localized in mitochondria and that the Kir6.2 content in mitochondria is increased by activation of PKCε. PKC isoform-regulated mitochondrial import of KATP channels may have significant implication in cardioprotection of IPC.

scholarly journals Time-resolved luminescence resonance energy transfer imaging of protein-protein interactions in living cells

2010 ◽  
Vol 107 (31) ◽  
pp. 13582-13587 ◽  
Author(s):  
H. E. Rajapakse ◽  
N. Gahlaut ◽  
S. Mohandessi ◽  
D. Yu ◽  
J. R. Turner ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Protein Protein Interactions ◽  
Time Resolved

Time-Resolved Fluorescence Resonance Energy Transfer Shows that the Bacterial Multidrug ABC Half-Transporter BmrA Functions as a Homodimer†

Biochemistry ◽  
2005 ◽  
Vol 44 (11) ◽  
pp. 4312-4321 ◽  
Author(s):  
Olivier Dalmas ◽  
Marie-Ange Do Cao ◽  
Miguel R. Lugo ◽  
Frances J. Sharom ◽  
Attilio Di Pietro ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Fluorescence Resonance
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