Effects of Different Na+ Concentrations on cAMP-Dependent Protein Kinase Activity in Postmortem Meat.

cAMP-dependent protein kinase (PKA) activity regulates protein phosphorylation, with Na+ playing a crucial role in PKA activity. The aim of this study was to investigate the effects of different Na+ concentrations on PKA activity and protein phosphorylation level in postmortem muscle. The study consisted of two experiments: (1) NaCl of 0, 20, 100, 200 and 400 mM was added to a muscle homogenate incubation model to analyze the effect of Na+ concentration on PKA activity, and (2) the same concentrations were added to pure PKA in vitro incubation models at 4 °C to verify the effect of Na+ on PKA activity. The PKA activity of the muscle homogenate model increased with storage time in groups with different Na+ concentrations. High concentrations of Na+ inhibited sarcoplasmic protein phosphorylation. The PKA activity at 24 h of storage and the sarcoplasmic protein phosphorylation level at 12 h of storage in the group with 200 mM Na+ was lower than that of the other groups. After 1 h incubation, the PKA activity of samples in the 200 mM Na+ group was inhibited and lower than that in the other Na+ groups in the in vitro incubation model. These results suggest that the Na+ concentration at 200 mM could better inhibit PKA activity. This study provided valuable insights for enhancing curing efficiency and improving meat quality.

Zr4+-mediated hybrid chain reaction and its application for highly sensitive electrochemical detection of protein kinase A.

An electrochemical platform has been developed to detect protein kinase activity through the combined actions of Zr4+ mediated signal transition and hybridization chain reaction (HCR)-stimulated DNAzymes nanowires. First of all, protein kinase A (PKA) phosphorylates substrate peptides immobilized on gold electrode surface. Thereafter, the DNA1 containing 5'-phosphoryl ends is linked to the phosphorylated substrate peptide via the robust phosphate-Zr4+-phosphate linkages. By the introduction of molecular beacons (MBs), the DNA1 can open the hairpin structures of MBs through toehold mediated strand displacement (TMSDR), leading to an autonomous stem-opening process and subsequent assembly of G-quadruplex-containing DNA chains by HCR. After the addition of hemin, the formed HRP-mimicking DNAzymes can catalyze the hydroquinone-H2O2 system to generate amplified electrochemical signals. As expected, this method can achieve ultrahigh analytical performance with a low detection limit of 0.02U/mL and exhibit high cost-savings potential without the need for antibody, protease and labeling. Therefore, this method can serve as a new tool for the assay of protein kinase A and its inhibitor screening in the future.

Phosphorylation-Dependent SERS Readout for Activity Assay of Protein Kinase A in Cell Extracts.

Protein kinases are key regulators of cell function, the abnormal activity of which may induce several human diseases, including cancers. Therefore, it is of great significance to develop a sensitive and reliable method for assaying protein kinase activities in real biological samples. Here, we report the phosphorylation-dependent surface-enhanced Raman scattering (SERS) readout of spermine-functionalized silver nanoparticles (AgNPs) for protein kinase A (PKA) activity assay in cell extracts. In this assay, the presence of PKA would phosphorylate and alter the net charge states of Raman dye-labeled substrate peptides, and the resulting anionic products could absorb onto the AgNPs with cationic surface charge through electrostatic attraction. Meanwhile, the Raman signals of dyes labeled on peptides were strongly enhanced by the aggregated AgNPs with interparticle hot spots formed in assay buffer. The SERS readout was directly proportional to the PKA activity in a wide range of 0.0001-0.5 U·µL-1 with a detection limit as low as 0.00003 U·µL-1. Moreover, the proposed SERS-based assay for the PKA activity was successfully applied to monitoring the activity and inhibition of PKA in real biological samples, particularly in cell extracts, which would be beneficial for kinase-related disease diagnostics and inhibitor screening.

Link between steroidogenesis, the cell cycle, and PKA in adrenocortical tumor cells.

Adrenocortical tumors (ACTs) frequently cause steroid excess and present cell-cycle dysregulation. cAMP/PKA signaling is involved in steroid synthesis and play a role in cell-cycle regulation. We investigated, by cell synchronization in the different phases of the cell-cycle, the control of steroidogenesis and the contribution of PKA in adrenocortical cells (H295R and culture of primary pigmented nodular adrenocortical disease cells). Cells showed increased steroidogenesis and a maximal PKA activity at G2 phase, and a reduction at G1 phase. PRKACA overexpression, or cAMP stimulation, enhanced PKA activity and induced steroidogenesis in all synchronized groups but is not sufficient to drive cell-cycle progression. PRKAR1A inactivation enhanced PKA activity and induced STAR gene expression, only in cells in G1, and triggered cell-cycle progression in all groups. These findings provide evidence for a tight association between steroidogenesis and cell-cycle in ACTs. Moreover, PRKAR1A is essential for mediating the function of PKA activity on both steroidogenesis and cell-cycle progression in adrenocortical cells.

Construction of industrial baker's yeast with high level of cAMP.

Cyclic adenosine monophosphate (cAMP) plays an important role in modulating the activity of microbe cell. In this study, PKA (protein kinase A) activity was weakened through truncation of TPK2 promoter (-150 bp and -300 bp) and gene deletion of BCY1 (encodes the regulatory subunit of PKA), TPK1 and TPK3, generating strains BY9a-T2-150 and BY9a-T2-300, respectively. High-performance liquid chromatography analysis showed cAMP levels in BY9a-T2-150 and BY9a-T2-300 were increased by 5- and 18-fold, respectively, compared with that of parent strain, BY9a. The expression levels of TPK2 gene in two engineered strains were decreased by 95% and 97% compared with that of BY9a, respectively. The PKA activity reflected by heat resistance of engineered strains enhanced compared with parent strain BY9a. This study show a new method to increase the intracellular cAMP concentration in industrial yeast by fine-tuning of PKA activity, without influence in growth and fermentation properties. PRACTICAL APPLICATIONS: cAMP as the "second messenger," is essential for plant, animal, and microorganisms and human life. But its synthesis is still limited by expensive cost and time-consuming method. We constructed the industrial baker's yeast with high level of cAMP and desired to be used to produce functional food for relaxing smooth muscle, expanding blood vessels, improving liver function, and promoting nerve regeneration and as a food additive for treating hyperthyreosis and hepatopathy. The methods of two step homologous recombination and backcross operated in this study eliminate the exogenous gene in engineered strains, made it safety to be used in food production. Fine-tuning of PKA activity in engineered strains ensure produce high level of cAMP and exhibit normal growth performance in engineering strains. Therefore, this work is significant in functional foods product and has the potential to be used in practical application.

MRP4-mediated cAMP efflux is essential for mouse spermatozoa capacitation.

Mammalian spermatozoa must undergo biochemical and structural changes to acquire the capacity for fertilization, in a process known as capacitation. Activation of PKA enzymes is essential for capacitation, and thus cAMP levels are tightly regulated during this process. Previously, we demonstrated that during capacitation, bovine spermatozoa extrude cAMP through multidrug resistance-associated protein 4 (MRP4, also known as ABCC4), which regulates intracellular levels of the nucleotide and provides cAMP to the extracellular space. Here, we report the presence of functional MRP4 in murine spermatozoa, since its pharmacological inhibition with MK571 decreased levels of extracellular cAMP. This also produced a sudden increase in PKA activity, with decreased tyrosine phosphorylation at the end of capacitation. Blockade of MRP4 inhibited induction of acrosome reaction, hyperactivation and in vitro fertilization. Moreover, MRP4 inhibition generated an increase in Ca2+ levels mediated by PKA, and depletion of Ca2+ salts from the medium prevented the loss of motility and phosphotyrosine inhibition produced by MK571. These results were supported using spermatozoa from CatSper Ca2+ channel knockout mice. Taken together, these results suggest that cAMP efflux via MRP4 plays an essential role in mouse sperm capacitation.This article has an associated First Person interview with the first author of the paper.

Aptamer based electrochemical assay for protein kinase activity by coupling hybridization chain reaction.

The present work reported a simple, lable-free and sensitive electrochemical method for the detection of protein kinase A (PKA) activity. This method was based on the specific recognition of aptamer and the aptamer-induced hybridization chain reaction (HCR) amplification strategy. The aptasensor was constructed by immobilizing capture probe on a gold electrode via an Au-S bond. When adenosine triphosphate (ATP) aptamer was introduced, its one terminus hybridized with capture probe and the other hybridized with the complementary region of an auxiliary probe, which other region triggered HCR between two hairpin DNA (H1 and H2) to form a long DNA concatamer. At last a large number of electroactive methyle blue (MB) molecules were assembled on the dsDNA concatamer, which generated a significantly amplified electrochemical signal. In the presence of ATP, the HCR would not be performed because the aptamer specifically bond to ATP and the electrochemical response would decrease. However, when ATP and PKA coexisted, the electrochemical response would recovery because that ATP had been translated into ADP by PKA. So the activity of PKA could be effectively monitored according to the change of electrochemical signal. Based on the HCR amplification strategy, the aptasensor showed a wide linear range (4 - 4 ×105 U L-1) and a low detection limit (1.5 U L-1) for the detection of PKA. Furthermore, the method was applied to study the inhibitory effect of H-89 on PKA activity. The developed aptasensor was also used to the analysis of drug-induced PKA activity in cell lysates, indicating the potential application of the developed method in the fields of clinical diagnostics and discovery of new targeted drugs.

DNA-hosted copper nanoclusters/graphene oxide based fluorescent biosensor for protein kinase activity detection.

A novel fluorescent biosensor for protein kinase activity (PKA) detection was designed by applying double-strands DNA-hosted copper nanoclusters (dsDNA-CuNCs) and graphene oxide (GO). One DNA strand of the dsDNA consisted of two domains, one domain can hybridize with another complementary DNA strand to stabilize the fluorescent CuNCs and another domain was adenosine 5'-triphosphate (ATP) aptamer. ATP aptamer of the dsDNA-CuNCs would be spontaneously absorbed onto the GO surface through π-π stacking interactions. Thus GO can efficiently quench the fluorescence (FL) of dsDNA-CuNCs through fluorescence resonance energy transfer (FRET). In the present of ATP, ATP specifically combined with ATP aptamer to form ATP-ATP aptamer binding complexes, which had much less affinity to GO, resulting in the fluorescence recovery of the system. Nevertheless, in the presence of PKA, ATP could be translated into ADP and ADP could not combine with ATP aptamer resulting in the fluorescence quenching of dsDNA-CuNCs again. According to the change of the fluorescence signal, PKA activity could be successfully monitored in the range of 0.1-5.0 U mL-1 with a detection limit (LOD) of 0.039 U mL-1. Besides, the inhibitory effect of H-89 on PKA activity was studied. The sensor was performed for PKA activity detection in cell lysates with satisfactory results.

A label-free and sensitive fluorescent assay for one step detection of protein kinase activity and inhibition.

In this paper, a label-free, highly sensitive and simple assay for one step detection of protein kinase (PKA) activity and inhibition that avoids the fluorescent dye process has been established. The detection was based on the fluorescence (FL) quenching of peptide-Ag nanoclusters (Ag NCs) caused by antibody modified Au nanoparticles (anti-Au NPs) via fluorescence resonance energy transfer (FRET). With PKA and adenosine 5'-triphosphate (ATP) introduced, the substrate peptide of Ag NCs could react with PKA via targeted phosphorylation, and followed by the linking interactions between peptide-Ag NCs and anti-Au NPs. According to the fluorescence quenching of Ag NCs, the activity of protein kinase can be facilely monitored in the range of 0.1-2000 mU/µL with high sensitivity. The detection limit for PKA is 0.039 mU/µL. We further explored the inhibitory effect of H-89 for protein kinase activity. The developed method was also applied to the investigation of drug-induced PKA activation in HeLa cells, which provides a promising means for screening of kinase-related drugs and the clinical diagnosis of disease.

A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy.

The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes.

PKA tightly bound to human placental mitochondria participates in steroidogenesis and is not modified by cAMP.

INTRODUCTION: Protein phosphorylation plays an important role in the modulation of steroidogenesis and it depends on the activation of different signaling cascades. Previous data showed that PKA activity is related to steroidogenesis in mitochondria from syncytiotrophoblast of human placenta (HPM). PKA localization and contribution in progesterone synthesis and protein phosphorylation of HPM was assessed in this work. METHODS: Placental mitochondria and submitochondrial fractions were used. Catalytic and regulatory PKA subunits were identified by Western blot. PKA activity was determined by the incorporation of (32)P into proteins in the presence or absence of specific inhibitors. The effect of PKA activators and inhibitors on steroidogenesis and protein phosphorylation in HPM was tested by radioimmunoassay and autoradiography. RESULTS: The PKAα catalytic subunit was distributed in all the submitochondrial fractions whereas ßII regulatory subunit was the main isoform observed in both the outer and inner membranes of HPM. PKA located in the inner membrane showed the highest activity. Progesterone synthesis and mitochondrial protein phosphorylation are modified by inhibitors of PKA catalytic subunit but are neither sensitive to inhibitors of the regulatory subunit nor to activators of the holoenzyme. DISCUSSION: The lack of response in the presence of PKA activators and inhibitors of the regulatory subunit suggests that the activation of intramitochondrial PKA cannot be prevented or further activated. CONCLUSIONS: The phosphorylating activity of PKA inside HPM could be an important component of the steroidogenesis transduction cascade, probably exerting its effects by direct phosphorylation of its substrates or by modulating other kinases and phosphatases.