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...

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

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.

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

2133-P: Protein Kinase C Delta Mediates Cytokine-Induced Apoptosis in the Pancreatic Beta Cell

Diabetes ◽  
2019 ◽  
Vol 68 (Supplement 1) ◽  
pp. 2133-P
Author(s):  
NIKKI L. FARNSWORTH ◽  
ROBERT A. PISCOPIO ◽  
RICHARD K. BENNINGER
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Beta Cell ◽  
Pancreatic Beta Cell ◽  
Protein Kinase C Delta ◽  
Kinase C ◽  
P Protein ◽  
Induced Apoptosis

scholarly journals Targeted disruption of PKC from AKAP Signaling Complexes

2021 ◽  
Author(s):  
Ameya J. Limaye ◽  
George N. Bendzunas ◽  
Eileen Kennedy
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Signaling Pathways ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Targeted Disruption ◽  
Physiological Processes ◽  
Kinase C ◽  
P Protein

Protein Kinase C (PKC) is a member of the AGC subfamily of kinases and regulates a wide array of signaling pathways and physiological processes. Protein-protein interactions involving PKC and its...

p21ras and protein kinase C function in distinct and interdependent signaling pathways in C3H 10T1/2 fibroblasts

1993 ◽  
Vol 13 (3) ◽  
pp. 1471-1479
Author(s):  
A Krook ◽  
M J Rapoport ◽  
S Anderson ◽  
H Pross ◽  
Y C Zhou ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Signaling Pathways ◽  
Tyrosine Kinases ◽  
Signal Pathways ◽  
Mrna Levels ◽  
Ras Gene ◽  
Kinase C ◽  
Tetradecanoyl Phorbol Acetate ◽  
Cellular Signal

Both p21ras and protein kinase C (PKC) are believed to function downstream of plasma membrane-associated tyrosine kinases in cellular signal transduction pathways. However, it has remained controversial whether they function in the same pathway and, if so, what their relative position and functional relationship in such a pathway are. We investigated the possibilities that p21ras and PKC function either upstream or downstream of each other in a common linear pathway or that they function independently in colinear signal pathways. Either decreased expression of endogenous normal ras in fibroblasts transfected with an inducible antisense ras construct or overexpression of a mutant ras gene reduced the capacity of the phorbol ester tetradecanoyl phorbol acetate to trigger expression of the tetradecanoyl phorbol acetate-responsive and ras-dependent reporter gene osteopontin (OPN). PKC depletion decreased basal OPN mRNA levels, and the overexpression of ras restored OPN expression to the level of non-PKC-depleted cells. We propose a model in which ras and PKC function in distinct and interdependent signaling pathways.

scholarly journals Protein kinase D (PKD) activation in intact cells through a protein kinase C-dependent signal transduction pathway.

1996 ◽  
Vol 15 (22) ◽  
pp. 6220-6230 ◽  
Author(s):  
J. L. Zugaza ◽  
J. Sinnett-Smith ◽  
J. Van Lint ◽  
E. Rozengurt
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Signal Transduction Pathway ◽  
Protein Kinase D ◽  
Transduction Pathway ◽  
Intact Cells ◽  
Kinase C ◽  
Dependent Signal

Inhibition of voltage-gated K+ current in rat intrapulmonary arterial myocytes by endothelin-1

1998 ◽  
Vol 274 (5) ◽  
pp. L842-L853 ◽  
Author(s):  
Larissa A. Shimoda ◽  
J. T. Sylvester ◽  
James S. K. Sham
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Vascular Reactivity ◽  
Signal Transduction Pathway ◽  
Patch Clamp Technique ◽  
Arterial Smooth Muscle ◽  
Kinase C ◽  
Gf 109203X

Although endothelin (ET)-1 is an important regulator of pulmonary vascular tone, little is known about the mechanisms by which ET-1 causes contraction in this tissue. Using the whole cell patch-clamp technique in rat intrapulmonary arterial smooth muscle cells, we found that ET-1 and the voltage-dependent K+(KV)-channel antagonist 4-aminopyridine, but not the Ca2+-activated K+-channel antagonist charybdotoxin (ChTX), caused membrane depolarization. In the presence of 100 nM ChTX, ET-1 (10−10to 10−7 M) caused a concentration-dependent inhibition of K+ current (56.2 ± 3.8% at 10−7 M) and increased the rate of current inactivation. These effects of ET-1 on K+ current were markedly reduced by inhibitors of protein kinase C (staurosporine and GF 109203X) and phospholipase C (U-73122) or under Ca2+-free conditions and were mimicked by activators of protein kinase C (phorbol 12-myristate 13-actetate and 1,2-dioctanoyl- sn-glycerol). These data suggest that ET-1 modulated pulmonary vascular reactivity by depolarizing pulmonary arterial smooth muscle, due in part to the inhibition of KV current that occurred via activation of the phospholipase C-protein kinase C signal transduction pathway.

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