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

Nanomaterials ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 575
Author(s):  
Renyong Liu ◽  
Chenggen Xie ◽  
Yehan Yan ◽  
Lin Hu ◽  
Suhua Wang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Biological Samples ◽  
Cell Function ◽  
Activity Assay ◽  
Cell Extracts ◽  
Disease Diagnostics ◽  
Wide Range ◽  
Surface Enhanced Raman ◽  
Kinase A

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.

Comparative Study of Active and Allosteric Interaction in Protein Kinases

2021 ◽  
Vol 8 (1) ◽  
pp. 23-31
Author(s):  
Jefrin Ahmed ◽  
Judith Mary Lamo ◽  
Baphilinia Jones Mylliemngap
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Protein Kinases ◽  
Active Site ◽  
Cell Function ◽  
Binding Capacity ◽  
Second Messengers ◽  
Gene Families ◽  
Allosteric Site ◽  
Kinase A

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.

scholarly journals Reciprocal Stimulation of Decay Between Serotonergic Facilitation and Depression of Synaptic Transmission

2008 ◽  
Vol 100 (2) ◽  
pp. 1113-1126 ◽  
Author(s):  
Sun Hee Cho Lee ◽  
Karen Taylor ◽  
Franklin B. Krasne
Keyword(s):  
Protein Kinase ◽  
Synaptic Transmission ◽  
Protein Kinase A ◽  
The Other ◽  
Command Neurons ◽  
Wide Range ◽  
History Of ◽  
Depressive States ◽  
Kinase A ◽  
Stimulation Of

Serotonin can produce multiple, contradictory modulatory effects on strength of synaptic transmission in both vertebrate and invertebrate nerve circuits. In crayfish, serotonin (5-HT) can both facilitate and depress transmission to lateral giant escape command neurons; however, which effect is manifest during application, as well as the sign and duration of effects that may continue long after 5-HT washout, may depend on history of application as well as on concentration. We report that protein kinase A (PKA) signaling is essential to the production of facilitation but depression is mediated by non-cAMP/PKA signaling pathways. However, we unexpectedly found that PKA activity is essential for the decay of depression when serotonin is washed out. This, and evidence from the effects of a variety of serotonin application regimens, suggest that facilitatory and depressive states coexist and compete and that the decay of each is dependent on stimulation by the other. A computational model that incorporates these assumptions can account for and rationalize the varied effects of a wide range of serotonin application regimens.

scholarly journals Integrative multi-omics profiling reveals cAMP-independent mechanisms regulating hyphal morphogenesis in Candida albicans

2021 ◽  
Vol 17 (8) ◽  
pp. e1009861
Author(s):  
Kyunghun Min ◽  
Thomas F. Jannace ◽  
Haoyu Si ◽  
Krishna R. Veeramah ◽  
John D. Haley ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Adenylyl Cyclase ◽  
Regulatory Subunit ◽  
Chromosome 2 ◽  
Casein Kinase 1 ◽  
Hyphal Morphogenesis ◽  
Wide Range ◽  
Kinase A

Microbial pathogens grow in a wide range of different morphologies that provide distinct advantages for virulence. In the fungal pathogen Candida albicans, adenylyl cyclase (Cyr1) is thought to be a master regulator of the switch to invasive hyphal morphogenesis and biofilm formation. However, faster growing cyr1Δ/Δ pseudorevertant (PR) mutants were identified that form hyphae in the absence of cAMP. Isolation of additional PR mutants revealed that their improved growth was due to loss of one copy of BCY1, the negative regulatory subunit of protein kinase A (PKA) from the left arm of chromosome 2. Furthermore, hyphal morphogenesis was improved in some of PR mutants by multigenic haploinsufficiency resulting from loss of large regions of the left arm of chromosome 2, including global transcriptional regulators. Interestingly, hyphal-associated genes were also induced in a manner that was independent of cAMP. This indicates that basal protein kinase A activity is an important prerequisite to induce hyphae, but activation of adenylyl cyclase is not needed. Instead, phosphoproteomic analysis indicated that the Cdc28 cyclin-dependent kinase and the casein kinase 1 family member Yck2 play key roles in promoting polarized growth. In addition, integrating transcriptomic and proteomic data reveals hyphal stimuli induce increased production of key transcription factors that contribute to polarized morphogenesis.

scholarly journals Noncanonical protein kinase A activation by oligomerization of regulatory subunits as revealed by inherited Carney complex mutations

2021 ◽  
Vol 118 (21) ◽  
pp. e2024716118
Author(s):  
Naeimeh Jafari ◽  
Jason Del Rio ◽  
Madoka Akimoto ◽  
Jung Ah Byun ◽  
Stephen Boulton ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Molecular Mechanisms ◽  
Carney Complex ◽  
Regulatory Subunit ◽  
Regulatory Subunits ◽  
Inhibitory Function ◽  
Wide Range ◽  
Liver Cancers ◽  
Kinase A

Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function.

Insulin sensitizes beta-agonist and forskolin-stimulated lipolysis to inhibition by 2',5'-dideoxyadenosine

1996 ◽  
Vol 270 (2) ◽  
pp. C562-C569 ◽  
Author(s):  
Y. Gokmen-Polar ◽  
E. C. Coronel ◽  
S. W. Bahouth ◽  
J. N. Fain
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Beta Agonist ◽  
Camp Accumulation ◽  
Fat Cell ◽  
Beta Agonists ◽  
Cell Extracts ◽  
Dependent Protein ◽  
Cellular Compartments ◽  
Kinase A

In isolated rat adipocytes incubated in the absence of insulin, 2',5'-dideoxyadenosine blocked the increase in total adenosine 3',5'-cyclic monophosphate (cAMP) accumulation due to beta 1- or beta 3-catecholamine agonists and forskolin without affecting their stimulation of lipolysis. The inhibition of cAMP accumulation by 2',5'-dideoxyadenosine was not reflected in the total cytosolic cAMP-dependent protein kinase A activity, suggesting that the inhibition of cAMP occurred in cellular compartments distinct from those involved in the regulation of bulk protein kinase A activity. However, there was a good correlation between effects of lipolytic agents on cytosolic protein kinase A activity in fat cell extracts and lipolysis. Furthermore, it was possible to see an inhibition of the increase due to beta-agonists in cAMP accumulation, protein kinase A activity, and lipolysis by 2',5'-dideoxyadenosine in the presence of insulin. These data suggest that the readily measurable accumulation of cAMP seen with catecholamines in the absence of insulin is in a compartment separate from that involved in protein kinase A activation.

Identification of a pathway by which glucose regulates β-catenin signalling via the cAMP/protein kinase A pathway in β-cell models

2013 ◽  
Vol 449 (3) ◽  
pp. 803-811 ◽  
Author(s):  
Emmanuelle Cognard ◽  
Coralie G. Dargaville ◽  
Deborah L. Hay ◽  
Peter R. Shepherd
Keyword(s):  
Insulin Secretion ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Cell Function ◽  
Regulation Of Gene Expression ◽  
Cell Models ◽  
Β Cells ◽  
Β Cell ◽  
Glucose Levels ◽  
Kinase A

Pancreatic β-cells are highly responsive to changes in glucose, but the mechanisms involved are only partially understood. There is increasing evidence that the β-catenin signalling pathway plays an important role in regulating β-cell function, but the mechanisms regulating β-catenin signalling in these cells is not well understood. In the present study we show that β-catenin levels and downstream signalling are regulated by changes in glucose levels in INS-1E and β-TC6-F7 β-cell models. We found a glucose-dependent increase in levels of β-catenin in the cytoplasm and nucleus of INS-1E cells. Expression of cyclin D1 also increased with glucose and required the presence of β-catenin. This was associated with an increase in phosphorylation of β-catenin on Ser552, which is known to stabilize the molecule and increase its transcriptional activity. In a search for possible signalling intermediates we found forskolin and cell-permeable cAMP analogues recapitulated the glucose effects, suggesting a role for cAMP and PKA (cAMP-dependent protein kinase/protein kinase A) downstream of glucose. Furthermore, glucose caused sustained increases in cAMP. Two different inhibitors of adenylate cyclase and PKA signalling blocked the effects of glucose, whereas siRNA (small interfering RNA) knockdown of PKA blocked the effects of glucose on β-catenin signalling. Finally, reducing β-catenin levels with either siRNA or pyrvinium impaired glucose- and KCl-stimulated insulin secretion. Taken together the results of the present study define a pathway by which changes in glucose levels can regulate β-catenin using a mechanism which involves cAMP production and the activation of PKA. This identifies a pathway that may be important in glucose-dependent regulation of gene expression and insulin secretion in β-cells.

Facile and Sensitive Method for Protein Kinase A Activity Assay Based on Fluorescent Off-On PolyU-peptide Assembly

2017 ◽  
Vol 89 (20) ◽  
pp. 10980-10984 ◽  
Author(s):  
Yanhui Xu ◽  
Wen Shi ◽  
Xinyuan He ◽  
Xiaofeng Wu ◽  
Xiaohua Li ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Activity Assay ◽  
Peptide Assembly ◽  
Sensitive Method ◽  
Kinase A

scholarly journals P06.04 Enhancing T cell function for cancer immunotherapy by microRNA mediated knockdown of PRKAR1A

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A43.1-A43
Author(s):  
A Poudel ◽  
SM Rad ◽  
G Tan ◽  
AD McLellan
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Cancer Immunotherapy ◽  
Cell Function ◽  
Luciferase Assay ◽  
T Cell Function ◽  
Car T ◽  
Kinase A

BackgroundProtein Kinase A (PKA) is a heterotetramer holoenzyme that consists of two regulatory and catalytic subunits. During T cell activation, one of the regulatory subunits (PRKAR1A) localizes to the immune synapse, inhibiting several central proteins in the T-cell signalling cascade and leading to T cell inactivation. Previously, the disruption of localisation of PKA type I R1α (PRKAR1A) to the immune synapse using disruptor peptides has been shown to improve chimeric antigen receptor (CAR) T cell function.1 2 However, the effect of PRKAR1A knockdown in T cells (including CAR T cells) has not been studied yet. In this study, we have utilized microRNAs (miR); miR96/183 or miR155 to knockdown PRKAR1A and explored the advantages of PRKAR1A knockdown on T cell activation and function.Materials and MethodsMicroRNAs (miR); miR96/183 or miR155 were cloned from human genomic DNA into a sleeping beauty system under a doxycycline inducible promoter (TCE). Overexpression of miRNA and target knockdown was assessed at both transcript level (by real time RT-PCR) and/or protein level (by western blot) respectively while target validation was done by luciferase assay. The fate of PRKAR1A knockdown on Jurkat T cells activated with anti-CD3 and anti-CD28 antibodies were determined by measuring IL-2 production (ELISA) and CD69 surface expression (flow cytometry). The effect of miR96/183 or miR155 overexpression in primary T cells expressing HER2-CAR were also compared.ResultsWe efficiently overexpressed both miRNAs and downregulated PRKAR1A expression in HEK293 cells at both mRNA and protein level. Luciferase assay confirmed miRNA mediated specific knockdown of PRKAR1A; mutated 3’UTR of PRKAR1A was used as negative control. Overexpression of miRNAs also downregulated PRKAR1A expression in Jurkat cells which resulted in enhanced activation (CD69 expression) and IL-2 production following anti-CD3/CD28 stimulation compared to untransfected controls (with normal PRKAR1A expression). Additionally, miRNA 96/183 and miRNA155 were found to target inhibitory proteins of TCR signalling such as CTLA4, Foxo3 and ptpn2 and resulted in superior T cell function. A third-generation lentiviral system has been optimised to express either miR96/183 or miR155 and HER2-CAR in the same vector and currently we are assessing the effect of PRKAR1A knockdown on primary CAR T cells.ConclusionsOverexpressing miRNA for knockdown of inhibitory proteins could be an efficient way of enhancing T cell function against solid tumours. Additionally, co-expressing CAR and miRNAs using lentiviral system would benefit such approaches for cancer immunotherapy.ReferencesNewick K, O’ Brien S, Sun J, Kappor V, Maceyko S, Lo A, Pure E, Moon E, Albelda SM. Augmentation of CAR T cell trafficking and antitumour efficacy by blocking protein kinase A (PKA) localization. Cancer Immunol Res 2016; 4(6): 541–551Hussain M, Shah Z, Abbas N, Javeed A, Mukhtar MM, Zhang J. Targeting tumour-associated immune suppression with selective protein kinase A type I (PKAI) inhibitors may enhance cancer immunotherapy. Medical Hypotheses 2016;86: 56–59Disclosure InformationA. Poudel: None. S.M. Rad: None. G. Tan: None. A.D. McLellan: None.

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