scholarly journals Temperature-Dependent Expression of a CFP-YFP FRET Diacylglycerol Sensor Enables Multiple-Read Screening for Compounds That Affect C1 Domains

2019 ◽  
Vol 24 (6) ◽  
pp. 682-692 ◽  
Author(s):  
Xiuyi Alexander Yang ◽  
Adam Zweifach
Keyword(s):  
Phorbol Ester ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Temperature Dependent ◽  
Kinase C ◽  
C1 Domain ◽  
Plate Reader ◽  
Ratio Change ◽  
Robust Measurements ◽  
Fluorescence Plate Reader

Intramolecular CFP-YFP fluorescence resonance energy transfer (FRET) sensors expressed in cells are powerful research tools but have seen relatively little use in screening. We exploited the discovery that the expression of a CFP-YFP FRET diacylglycerol sensor (DAGR) increases over time when cells are incubated at room temperature to assess requirements for robust measurements using a Molecular Devices Spectramax i3x fluorescence plate reader. Expression levels resulting in YFP fluorescence >10-fold higher than untransfected cells and phorbol ester-stimulated FRET ratio changes of 60% or more were required to consistently give robust Z′ > 0.5. As a means of confirming that these conditions are suitable for screening, we developed a novel multiple-read protocol to assay the NCI’s Mechanistic Set III for agonists and antagonists of C1 domain activation. Sixteen compounds prevented C1 domain translocation. However, none blocked phorbol ester-stimulated protein kinase C (PKC) activity assessed using a phospho-specific antibody—six actually stimulated PKC activity. Cytometry, which produces higher Z′ for a given FRET ratio change, might have been a better approach for discovering antagonists, as it would have allowed lower phorbol ester concentrations to be used. We conclude that CFP-YFP FRET measured in a Spectramax i3x plate reader can be used for screening under the conditions we defined. Our strategy of varying expression level and FRET ratio could be useful to others for determining conditions needed for robust cell-based intramolecular CFP-YFP FRET measurements on their instrumentation.

The factor V C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis

2004 ◽  
Vol 91 (01) ◽  
pp. 16-27 ◽  
Author(s):  
Mary Quinn-Allen ◽  
Sandra Macedo-Ribeiro ◽  
Pablo Fuentes-Prior ◽  
Wolfram Bode ◽  
Mahasen Saleh ◽  
...  
Keyword(s):  
Molecular Model ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Aromatic Amino Acids ◽  
Phospholipid Vesicles ◽  
Factor V ◽  
Alanine Scanning Mutagenesis ◽  
Important Amino Acid ◽  
C1 Domain ◽  
Prothrombinase Activity

SummaryThe contribution of the factor Va C1 domain (fVa-C1) to assembly of the prothrombinase complex has not been previously investigated. The homologous fVa-C2 domain contains a binding site for phosphatidylserine (PS) that includes the indole moieties of Trp2063/Trp2064 at the apex of spike-1. In order to investigate the structure and function of fVa-C1 a molecular model was constructed based on the structure of fVa-C2. The aromatic and hydrophobic side chains of Tyr1956/Leu1957 in fVaC1 are located at the predicted apex of spike-3. Exposed charged and hydrophobic residues in fVa-C1 were changed to alanine in clusters of 1-3 mutations per construct. The resultant 20 mutants were expressed in COS cells and screened for binding to immobilized PS and prothrombinase activity on phospholipid vesicles containing either 25% or 5% PS. Two mutants, (Y1956,L1957)A, and (R2023,R2027)A showed both decreased binding to immobilized PS and a selective decrease in prothrombinase activity on membranes containing 5% PS. The interaction of purified (Y1956,L1957)A with phospholipid vesicles was studied using fluorescence resonance energy transfer and prothrombinase assays. The affinity of (Y1956,L1957)A binding to 25% PS membranes was reduced 12-fold compared to rHFVa. Prothrombin activation in the presence of (Y1956,L1957)A was markedly impaired on phospholipid vesicles containing 10% or less PS. We conclude that solvent exposed hydrophobic and aromatic amino acids in both fVa-C1 and fVa-C2 contribute to the interaction of factor V with PS membranes.

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.

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...

Origin of the temperature dependence of the rate of singlet energy transfer in a three-component truxene-bridged dyads

2014 ◽  
Vol 18 (01n02) ◽  
pp. 94-106 ◽  
Author(s):  
Adam Langlois ◽  
Hai-Jun Xu ◽  
Bertrand Brizet ◽  
Franck Denat ◽  
Jean-Michel Barbe ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Excited States ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Bond ◽  
Free Base ◽  
Temperature Dependent ◽  
Energy Transfers ◽  
Dual Mechanism ◽  
Limited Motion

We report a truxene-based dyad built upon one donor (tri-meso-phenylzinc(II)porphyrin) and two acceptors (octa-β-alkylporphyrin free base) in which the donor exhibits free rotation around a Ctruxene-Cmeso single bond at 298 K in fluid solution but not at 77 K in a glass matrix, whereas the acceptors have very limited motion as they are blocked by β-methyl groups. This case is interesting because all the structural and spectroscopic parameters affecting the rate for singlet energy transfer according to a Förster Resonance Energy Transfer are only weakly temperature dependent, leaving only the Dexter mechanism explaining the larger variation in rate of energy transfers with the temperature hence providing a circumstantial evidence for a dual mechanism (Föster and Dexter) in truxene-based dyads (or polyads) in the S1 excited states.

Temperature-dependent resonance energy transfer from CdSe–ZnS core–shell quantum dots to monolayer MoS2

Nano Research ◽  
2016 ◽  
Vol 9 (9) ◽  
pp. 2623-2631 ◽  
Author(s):  
Juan Li ◽  
Weina Zhang ◽  
Yao Zhang ◽  
Hongxiang Lei ◽  
Baojun Li
Keyword(s):  
Quantum Dots ◽  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Core Shell ◽  
Monolayer Mos2 ◽  
Temperature Dependent

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.

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