Novel, isotype-specific sensors for protein kinase A subunit interaction based on bioluminescence resonance energy transfer (BRET)

2006 ◽  
Vol 18 (10) ◽  
pp. 1616-1625 ◽  
Author(s):  
Anke Prinz ◽  
Mandy Diskar ◽  
Andrea Erlbruch ◽  
Friedrich W. Herberg
Keyword(s):  
Energy Transfer ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Subunit Interaction ◽  
A Subunit ◽  
Kinase A

scholarly journals A divergent protein kinase A in the human pathogen Leishmania is associated with developmental morphogenesis

2021 ◽  
Author(s):  
R. Fischer Weinberger ◽  
S. Bachmaier ◽  
R. Dandugudumula ◽  
I.Q. Phan ◽  
M. Almoznino ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Regulatory Subunit ◽  
Cell Cortex ◽  
Human Pathogen ◽  
Human Leishmaniasis ◽  
Stage Development ◽  
Kinase A

SummaryParasitic protozoa of the genus Leishmania cause human leishmaniasis. They cycle between the phagolysosome of mammalian macrophages, where they reside as round intracellular amastigotes, and the mid-gut of female sand flies, which are colonized by elongated extracellular promastigotes. Shifting promastigotes to a lysosome-like environment (pH 5.5 and 37°C, 5% CO2) initiates their development into amastigotes. Previous studies suggested a role for protein kinase A in this differentiation process. Here we describe a new, divergent, regulatory subunit of protein kinase A (PKAR3) present only in a limited group of kinetoplastids. In L. donovani, phosphorylation of PKAR3 is regulated by the differentiation signal and coincides with parasite morphogenesis during stage development. LdPKAR3 is bound to the subpellicular microtubules cell cortex via a formin FH2-like domain at the tip of a large and divergent N-terminal domain. Immunoprecipitation, fluorescence resonance energy transfer (FRET) and proteomics analyses show that PKAR3 selectively binds the C3 isoform of the PKA catalytic subunit in a holoenzyme complex, as supported by structural modeling. In promastigotes, PKAR3 recruits PKAC3 to the subpellicular microtubules at the cell’s central cortex. After exposure to the differentiation signal, PKAR3 relocates evenly to the entire cortex in concert with cell rounding. Deleting either the R3 or C3 subunit resulted in premature rounding of the promastigote population, indicating that PKA determines their normal elongated shape. Regulation of Leishmania developmental morphogenesis by interaction with the subpellicular microtubule corset is a novel function for an unusual PKA complex not present in the host cell.

scholarly journals Protein Kinase A-Dependent Step(s) in Hepatitis C Virus Entry and Infectivity

2008 ◽  
Vol 82 (17) ◽  
pp. 8797-8811 ◽  
Author(s):  
Michelle J. Farquhar ◽  
Helen J. Harris ◽  
Mandy Diskar ◽  
Sarah Jones ◽  
Christopher J. Mee ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Kinase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Viral Receptor ◽  
Kinase A

ABSTRACT Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced HCV entry into Huh-7.5 hepatoma cells. Bioluminescence resonance energy transfer methodology allowed us to investigate the PKA isoform specificity of the cAMP antagonists in Huh-7.5 cells, suggesting a role for PKA type II in HCV internalization. Since viral entry is dependent on the host cell expression of CD81, scavenger receptor BI, and claudin-1 (CLDN1), we studied the role of PKA in regulating viral receptor localization by confocal imaging and fluorescence resonance energy transfer (FRET) analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to an intracellular vesicular location(s) and disrupted FRET between CLDN1 and CD81, demonstrating the importance of CLDN1 expression at the plasma membrane for viral receptor activity. Inhibiting PKA activity in Huh-7.5 cells reduced the infectivity of extracellular virus without modulating the level of cell-free HCV RNA, suggesting that particle secretion was not affected but that specific infectivity was reduced. Viral particles released from H89-treated cells displayed the same range of buoyant densities as did those from control cells, suggesting that viral protein association with lipoproteins is not regulated by PKA. HCV infection of Huh-7.5 cells increased cAMP levels and phosphorylated PKA substrates, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.

scholarly journals Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomomi Kaku ◽  
Kazunori Sugiura ◽  
Tetsuyuki Entani ◽  
Kenji Osabe ◽  
Takeharu Nagai
Keyword(s):  
Energy Transfer ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Color Change ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Bacterial Luciferase ◽  
Bacterial Bioluminescence

AbstractUsing the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. However, applications of bacterial bioluminescence-based imaging have been limited because of its low brightness. Here, we engineered the bacterial luciferase (heterodimer of luxA and luxB) by fusion with Venus, a bright variant of yellow fluorescent protein, to induce bioluminescence resonance energy transfer (BRET). By using decanal as an externally added substrate, color change and ten-times enhancement of brightness was achieved in Escherichia coli when circularly permuted Venus was fused to the C-terminus of luxB. Expression of the Venus-fused luciferase in human embryonic kidney cell lines (HEK293T) or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhanced the autonomous bioluminescence. We believe the improved luciferase will forge the way towards the potential development of autobioluminescent reporter system allowing spatiotemporal imaging in live cells.

scholarly journals Greatly enhanced detection of a volatile ligand at femtomolar levels using bioluminescence resonance energy transfer (BRET)

2011 ◽  
Vol 29 (1) ◽  
pp. 119-124 ◽  
Author(s):  
Helen Dacres ◽  
Jian Wang ◽  
Virginia Leitch ◽  
Irene Horne ◽  
Alisha R. Anderson ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer
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