Calculation of centralities in protein kinase A

Topological analysis of amino acid networks is a common method that can help to understand the roles of individual residues. The most popular approach for network construction is to create a connection between residues if they interact. These interactions are usually weighted by absolute values of correlation coefficients or mutual information. Here we argue that connections in such networks have to reflect levels of cohesion within the protein instead of a simple fact of interaction between residues. If this is correct, an indiscriminate combination of correlation and anti-correlation, as well as the all-inclusive nature of the mutual information metrics, should be detrimental for the analysis. To test our hypothesis, we studied amino acid networks of the protein kinase A created by Local Spatial Pattern alignment, a method that can detect conserved patterns formed by Cα-Cβ vectors. Our results showed that, in comparison with the traditional methods, this approach is more efficient in detecting functionally important residues. Out of four studied centrality metrics, Closeness centrality was the least efficient measure of residue importance. Eigenvector centrality proved to be ineffective as the spectral gap values of the networks were very low due to the bilobal structure of the kinase. We recommend using joint graphs of Betweenness centrality and Degree centrality to visualize different aspects of amino acid roles.

Repercussion of cAMP and EPAC in Memory and Signaling

It is well recognized that cyclic adenosine monophosphate (cAMP) signaling within neurons plays a key role in the foundation of long-term memories. Memory storage is the process that demands the movement of signals, neural plasticity, and the molecules which can transfer the signals from the sensory neuron to the dorsal root ganglion (DRG) neurons and later into the temporal region of the brain. The discovery of cAMP in 1958 as the second messenger also had a role in memory formation and other neural aspects. Further, in 1998 the scientists found that cAMP does not just activate protein kinase A (PKA) but also exchange protein directly activated by cAMP (Epac) which has an active role to play in hyperalgesia, memory, and signaling. The cAMP has three targets, hyperpolarization-activated cyclic nucleotide modulated (HCN) channels, protein kinase A (PKA), and exchange protein activated by cAMP (Epac). Different research has exposed that both PKA and HCN channels are significant for long-term memory creation. Epac is a cAMP-dependent guanine nucleotide exchange factor for the small G proteins including Rap1. However, slight information is there about the role of Epac in this process. The effects of cAMP are predominantly imparted by activating protein kinase A (PKA) and the more newly discovered exchange proteins are directly activated by cAMP 1 and 2 (EPAC1 and EPAC2). This review provides an insight regarding the function and role of both of these secondary messengers in memory and nerve signaling.

Comparative Study of Active and Allosteric Interaction in Protein Kinases

Protein kinases are key regulators of cell function that constitute one of the largest and most functionally diverse gene families. By adding phosphate groups to substrate proteins, they direct the activity, localization and overall function of many proteins, and serve to orchestrate the activity of almost all cellular processes. The main protein kinases consist of protein kinase A (PKA), protein kinase B (PKB), and protein kinase C (PKC) and are distinguished from each other by the different intracellular second messengers involved in their regulation and by the selective substrates they use. They all have a binding site for Mg2+-ATP (phosphate donor) and for substrate protein as well as various regulatory sites. We formulated to compare the binding capacity of protein kinases at the active site to allosteric sites. By comparing the active site and allosteric site of the protein kinases – A, B and C, using molecular docking it was found that in most of the cases the binding energy is high when an inhibitor binds to an active site as compared to the allosteric site. This comparison gave us an understanding of the interaction and inhibition of compounds to protein kinases in order to inhibit the activity of protein kinase A, B and C. It was concluded that for inhibiting the protein kinase function such as cell division and proliferation, binding of inhibitor to the allosteric site will be more effective.

Protein Kinase A (PRKA) Activity Is Regulated by the Proteasome at the Onset of Human Sperm Capacitation

The proteasome increases its activity at the onset of sperm capacitation due to the action of the SACY/PRKACA pathway; this increase is required for capacitation to progress. PRKA activity also increases and remains high during capacitation. However, intracellular levels of cAMP decrease in this process. Our goal was to evaluate the role of the proteasome in regulating PRKA activity once capacitation has started. Viable human sperm were incubated in the presence and absence of epoxomicin or with 0.1% DMSO. The activity of PRKA; the phosphorylation pattern of PRKA substrates (pPRKAs); and the expression of PRKAR1, PRKAR2, and AKAP3 were evaluated by Western blot. The localization of pPRKAs, PRKAR1, PRKAR2, and AKAP3 was evaluated by immunofluorescence. Treatment with epoxomicin changed the localization and phosphorylation pattern and decreased the percentage of pPRKAs-positive sperm. PRKA activity significantly increased at 1 min of capacitation and remained high throughout the incubation. However, epoxomicin treatment significantly decreased PRKA activity after 30 min. In addition, PRKAR1 and AKAP3 were degraded by the proteasome but with a different temporal kinetic. Our results suggest that PRKAR1 is the target of PRKA regulation by the proteasome.

The 3-phosphoinositide–dependent protein kinase 1 is an essential upstream activator of protein kinase A in malaria parasites

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signalling is essential for the proliferation of Plasmodium falciparum malaria blood stage parasites. The mechanisms regulating the activity of the catalytic subunit PfPKAc, however, are only partially understood, and PfPKAc function has not been investigated in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown (cKD) mutant, we confirm the essential role for PfPKAc in erythrocyte invasion by merozoites and show that PfPKAc is involved in regulating gametocyte deformability. We furthermore demonstrate that overexpression of PfPKAc is lethal and kills parasites at the early phase of schizogony. Strikingly, whole genome sequencing (WGS) of parasite mutants selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the parasite orthologue of 3-phosphoinositide–dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we demonstrate that PfPDK1 is required to activate PfPKAc and that T189 in the PfPKAc activation loop is the crucial target residue in this process. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and imply that PfPDK1 acts as a crucial upstream regulator in this pathway and potential new drug target.

The effect of IBMX and prolactin on functional status of cryopreserved bull spermatozoa

Purpose: investigate the effect of IBMX (activator of protein phosphorylation) and prolactin (PRL) on the functional state of cryopreserved bovine spermatozoa using inhibitory analysis.Materials and methods. Frozen-thawed semen samples from 60 black-and-white bulls was used in the experiments. For capacitation, cells were incubated in Sp-TALP medium supplemented with 6 mg/ml bovine serum albumin and various compounds: an inductor of capacitation (IBMX at concentrations of 1 μM, 10 μM, 50 μM, 100 μM), hormone (PRL at concentrations of 1 ng, 10 ng, 50 ng, 100 ng) and inhibitors of protein kinases C (Ro 31-8220 at a concentration of 10 ng/ml) and protein kinase A (H-89 at a concentration of 10 μM). The incubation was carried out at 38°C in an atmosphere of 5% CO2, 98% humidity for 4 hours. The functional status of the cells was determined by the chlortetracycline test.Results. It was shown that IBMX at all experimental concentrations did not affect the post-ejaculatory maturation (capacitation and acrosome reaction) of spermatozoa, while all concentrations of PRL (1-100 ng/ml) promoted the acrosome reaction in capacitated cells. In the presence of a protein kinase A inhibitor, there was a decrease in number of capacitated and an increase in number of acrosome-reactive spermatozoa under the action of IBMX at a concentration of 100 μM and no changes under the action of a protein kinase C inhibitor. Also, in case of protein kinase C inhibition the PRL-related stimulation of the acrosome reaction was canceled, while the usage of H-89 did not affect the functional status of spermatozoa, mediated by PRL. Thus, the influence of IBMX and PRL on the processes of post-ejaculatory maturation in thawed bovine spermatozoa was studied using the inhibitory analysis.Conclusion. At the capacital stage, all studied IBMX concentrations did not affect the ratio of deconved cells with various functional status. Prode also contributed to the passage of the acrosomous reaction in the rolled spermatozoa after defrosting. Inhibition of protein kinase A when incubating cells with IBMX has mediated the processes of acrosomal exocytosis in ripped cells and did not affect this process under the action of the PRR, while the protein kinase inhibitor C changed the ratio of cells with various functional status in the direction of increasing the percentage of cells at the rate of occasion I did not participate in intracellular action provided IBMX on deconved cells.