scholarly journals FRET and LRET Biosensors for Cell-based Imaging and Screening of Rac1 Activation

2021 ◽  
Author(s):  
Ha Pham ◽  
Mona Hoseini Soflaee ◽  
Andrei V Karginov ◽  
Lawrence Miller
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Hek293t Cell ◽  
Resonance Energy ◽  
Terbium Complex ◽  
A Cell ◽  
Fret Biosensors ◽  
Kinase A

Rac1 is a key regulator of several cell signaling pathways and dysregulated Rac1 activation has been implicated in cancer. Genetically encoded Forster resonance energy transfer (FRET) biosensors with enhanced dynamic range enabled live cell fluorescence imaging of Rac1 activity and a cell lysate-based assay of Rac1 inhibition in 96-well plates. We prepared HEK293T cell lines that stably expressed polypeptides with a general domain sequence (N- to C-terminus) of 1) FRET acceptor; 2) Rac/Cdc42 binding domain of human p21 protein kinase A (residues 68-150); 3) a linker domain; 4) FRET donor; and 5) full-length Rac1. Activated Rac1 binds to the protein kinase A domain, bringing donors and acceptors close together to increase FRET. We evaluated the effects on FRET signal dynamic range of alpha helical linkers comprised of alternating repeats of roughly four glutamate and four arginine or lysine residues. So-called ER/K linkers had limited effects on conventional FRET biosensors that incorporated the fluorescent protein (FP) pairs mCerulean/YPet, or mTFP1(cp227)/mVenus(cp229). Fluorometric measurements of cells that co-expresssed biosensors with positive (TIAM1) or negative (RhoGDI) Rac1 regulators revealed significant dynamic range enhancement in only one FP construct (mCerulean/YPet with 20 nm ER/K linker) relative to an analogous structure that incorporated an unstructured linker. We transfected this construct into a cell line that stably expressed a rapamycin-inducible c-Src analog (RapR-Src) and observed activation of Rac1 at protruding edges following rapamycin stimulation. In cells that expressed lanthanide-based FRET (LRET biosensors) that incorporated a luminescent terbium complex donor and GFP fluorescent acceptor, time-gated luminescence (TGL) measurements showed substantial gains in dynamic range that increased with linker length (up to 1200%). We robustly detected small molecule Rac1 inhibition following lysis of LRET biosensor-expressing cells grown directly in 96-well plates. The results herein highlight the potential of FRET and LRET biosensors with ER/K linkers for cell-based imaging and screening of protein activities.

scholarly journals Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter

2019 ◽  
Author(s):  
Allen K. Kim ◽  
Helen D. Wu ◽  
Takanari Inoue
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Electrostatic Interactions ◽  
Rational Design ◽  
Dynamic Range ◽  
Single Cell Analysis ◽  
Resonance Energy ◽  
Sequence Motif ◽  
Recognition Mechanism ◽  
Kinase A

AbstractProtein Kinase A (PKA) exists as a tetrameric holoenzyme which activates with increase of cAMP and plays an important role in many physiological processes including cardiac physiology, neuronal development, and adipocyte function. Although this kinase has been the subject of numerous biosensor designs, a single-fluorophore reporter that performs comparably to Förster resonance energy transfer (FRET) has not yet been reported. Here, we have used basic observations of electrostatic interactions in PKA substrate recognition mechanism and nucleus localization sequence motif to design a phosphorylation switch that shuttles between the cytosol and the nucleus, a strategy that should be generalizable to all basophilic kinases. The resulting reporter yielded comparable kinetics and dynamic range to the PKA FRET reporter, AKAR3EV. We also performed basic characterization and demonstrated its potential use in monitoring multiple signaling molecules inside cells using basic fluorescence microscopy. Due to the single-fluorophore nature of this reporter, we envision that this could find broad applications in studies involving single cell analysis of PKA activity.

scholarly journals Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A

2011 ◽  
Vol 300 (5) ◽  
pp. C989-C997 ◽  
Author(s):  
Pimthanya Wanichawan ◽  
William E. Louch ◽  
Kristin H. Hortemo ◽  
Bjørg Austbø ◽  
Per Kristian Lunde ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Mutational Analysis ◽  
Phosphorylation Site ◽  
Full Length ◽  
Kinase A

The cardiac Na+/Ca2+ exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ homeostasis and cardiac function. Several studies have indicated that NCX1 is phosphorylated by the cAMP-dependent protein kinase A (PKA) in vitro, which increases its activity. However, this finding is controversial and no phosphorylation site has so far been identified. Using bioinformatic analysis and peptide arrays, we screened NCX1 for putative PKA phosphorylation sites. Although several NCX1 synthetic peptides were phosphorylated by PKA in vitro, only one PKA site (threonine 731) was identified after mutational analysis. To further examine whether NCX1 protein could be PKA phosphorylated, wild-type and alanine-substituted NCX1-green fluorescent protein (GFP)-fusion proteins expressed in human embryonic kidney (HEK)293 cells were generated. No phosphorylation of full-length or calpain- or caspase-3 digested NCX1-GFP was observed with purified PKA-C and [γ-32P]ATP. Immunoblotting experiments with anti-PKA substrate and phosphothreonine-specific antibodies were further performed to investigate phosphorylation of endogenous NCX1. Phospho-NCX1 levels were also not increased after forskolin or isoproterenol treatment in vivo, in isolated neonatal cardiomyocytes, or in total heart homogenate. These data indicate that the novel in vitro PKA phosphorylation site is inaccessible in full-length as well as in calpain- or caspase-3 digested NCX1 protein, suggesting that NCX1 is not a direct target for PKA phosphorylation.

scholarly journals Label-Free Assay of Protein Kinase A Activity and Inhibition Using a Peptide-Based Electrochemical Sensor

Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 423
Author(s):  
Hyunju Cho ◽  
Chang-Seuk Lee ◽  
Tae Hyun Kim
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Self Assembly ◽  
Dynamic Range ◽  
Electrochemical Impedance ◽  
Protein Kinase Activity ◽  
Label Free ◽  
Transfer Resistance ◽  
Substrate Peptide ◽  
Kinase A

We propose a simple label-free electrochemical biosensor for monitoring protein kinase activity and inhibition using a peptide-modified electrode. The biosensor employs cys-kemptide (CLRRASLG) as a substrate peptide which was immobilized on the surface of a gold electrode via the self-assembly of the thiol terminals in cysteine (C) residues. The interaction between protein kinase A (PKA) and adenosine 5′-triphosphate (ATP) on the cys-kemptide immobilized electrode can cause the transfer of ATP terminal phosphates to the peptide substrates at serine (S) residues, which alters the surface charge of the electrode, thus enabling monitoring of the PKA activity via measuring the interfacial electron transfer resistance with electrochemical impedance spectroscopy. The proposed sensor showed reliable, sensitive, and selective detection of PKA activity with a wide dynamic range of 0.1–100 U/mL and a detection limit of 56 mU/mL. The sensor also exhibited high selectivity, rendering it possible to screen PKA inhibitors. Moreover, the sensor can be employed to evaluate the activity and inhibition of PKA in real samples.

scholarly journals Signaling of ghrelin at GHSR1b and OX1R receptor heterodimers

2019 ◽  
pp. mbc.E19-06-0326 ◽  
Author(s):  
Qiuling Wang ◽  
Weiwei Xiao ◽  
Yang Li ◽  
Zhenying Liu ◽  
Huafeng Li ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Growth Hormone Receptor ◽  
Signal Transduction Pathway ◽  
Resonance Energy ◽  
Signalling Pathway ◽  
Pathway Activity ◽  
Physiological Functions ◽  
Body Distribution ◽  
Kinase A

Growth hormone receptor 1b (GHSR1b) and orexin type 1 receptor (OX1R) have many similar characteristics in terms of their functions and body distribution and are involved in various physiological functions. In this study, we explored the possibility of GHSR1b and OX1R dimerization. Bioluminescence (BRET), fluorescence resonance energy transfer (FRET) and coimmunoprecipitation (Co-IP) were used to analyse the formation of GHSR1b and OX1R heteromers in cells. We also explored their signal transduction pathway mechanism. The results showed that ghrelin could stimulate GHSR1b/OX1R heterodimer cellsto increase Gαs protein activation and induce downstream signalling pathway activity. GHSR1b/OX1R heteromers triggered ghrelin-induced Gαs/protein kinase A signalling pathway activity. Thus, GHSR1b can form a heterodimer with OX1R, leading to increased protein kinase A activity. At the same time, stimulation with orexinA did not alter G protein-coupled receptor (GPCR) interactions with Gα protein subunits. Moreover, ghrelin induced a significant increase in cell proliferation. These results suggest that heterodimers of ghrelin and GHSR1b/OX1R promote the upregulation of a Gαs-cAMP-cAMP-responsive element signalling pathway. The nature of this signalling pathway may have significant implications in regulating physiological functions.

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.

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