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 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.

A peptide array-based serological protein kinase A activity assay and its application in cancer diagnosis

The Analyst ◽  
2015 ◽  
Vol 140 (19) ◽  
pp. 6588-6594 ◽  
Author(s):  
Deok-Hoon Kong ◽  
Se-Hui Jung ◽  
Hye-Yoon Jeon ◽  
Woo-Jin Kim ◽  
Young-Myeong Kim ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Sensitivity And Specificity ◽  
Cancer Diagnosis ◽  
Crucial Role ◽  
Peptide Array ◽  
Biological Processes ◽  
Activity Assay ◽  
P Protein ◽  
Kinase A

Protein kinase A (PKA) plays a crucial role in several biological processes; however, there is no assay with sufficient sensitivity and specificity to determine serological PKA (sPKA) activity.

Regulation of spontaneous and induced resumption of meiosis in mouse oocytes by different intracellular pathways

Reproduction ◽  
2000 ◽  
pp. 377-383 ◽  
Author(s):  
L Leonardsen ◽  
A Wiersma ◽  
M Baltsen ◽  
AG Byskov ◽  
CY Andersen
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Mitogen Activated Protein Kinase ◽  
Meiotic Maturation ◽  
Mouse Oocytes ◽  
Mitogen Activated Protein ◽  
Dependent Signal ◽  
Kinase Pathway ◽  
Kinase A

The mitogen-activated protein kinase-dependent and the cAMP-protein kinase A-dependent signal transduction pathways were studied in cultured mouse oocytes during induced and spontaneous meiotic maturation. The role of the mitogen-activated protein kinase pathway was assessed using PD98059, which specifically inhibits mitogen-activated protein kinase 1 and 2 (that is, MEK1 and MEK2), which activates mitogen-activated protein kinase. The cAMP-dependent protein kinase was studied by treating oocytes with the protein kinase A inhibitor rp-cAMP. Inhibition of the mitogen-activated protein kinase pathway by PD98059 (25 micromol l(-1)) selectively inhibited the stimulatory effect on meiotic maturation by FSH and meiosis-activating sterol (that is, 4,4-dimethyl-5alpha-cholest-8,14, 24-triene-3beta-ol) in the presence of 4 mmol hypoxanthine l(-1), whereas spontaneous maturation in the absence of hypoxanthine was unaffected. This finding indicates that different signal transduction mechanisms are involved in induced and spontaneous maturation. The protein kinase A inhibitor rp-cAMP induced meiotic maturation in the presence of 4 mmol hypoxanthine l(-1), an effect that was additive to the maturation-promoting effect of FSH and meiosis-activating sterol, indicating that induced maturation also uses the cAMP-protein kinase A-dependent signal transduction pathway. In conclusion, induced and spontaneous maturation of mouse oocytes appear to use different signal transduction pathways.

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