Related Experiment Videos
Alterations of protein kinase C isozyme and substrate proteins in mouse brain after electroconvulsive seizures
1Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, ROC.
Abstract:
Protein kinase C (PKC) activity, Western blot analysis of PKC alpha, beta, gamma, epsilon and zeta with isozyme-specific antibodies, endogenous substrate protein phosphorylation, and Western blot analysis of neuromodulin, were studied in mouse brain after repeated electroconvulsive shock. The PKC isozymes and endogenous substrates in the crude cytosolic and membrane fractions were partially purified on DE-52 columns eluted with buffer containing 100 or 200 mM KCl. The kinase activity assayed by phosphorylation of exogenous histone was increased in the 200 mM KCl eluates of both the cytosol and membrane fractions from electroshocked mice. Further analysis by immunoblotting demonstrated that this increased activity was due to an increase in the PKC gamma isozyme. The level of the novel type isozymes, epsilon and zeta, was not altered in electroshocked mice. An in vitro phosphorylation study showed that the endogenous substrate, 17 kDa neurogranin, was mostly eluted by 100 mM KCl. In contrast, the 43 kDa neuromodulin only appeared in the 200 mM KCl eluate, according to autoradiography, SDS-PAGE and Western blot analysis; its level was found to be increased in the membrane fraction of electroshocked mice, as demonstrated by in vitro phosphorylation studies. Therefore, an increase in both PKC gamma and neuromodulin contributed to the increased phosphorylation of neuromodulin during electroshock seizure.
Insights
Electroconvulsive shock increases protein kinase C (PKC) gamma activity and neuromodulin levels in mouse brain. This leads to enhanced phosphorylation of neuromodulin, a key event during electroshock seizures.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Protein kinase C (PKC) signaling is crucial for neuronal function.
- Electroconvulsive shock (ECS) is a therapeutic intervention with complex molecular effects on the brain.
Purpose of the Study:
- To investigate the impact of repeated electroconvulsive shock (ECS) on PKC isozyme activity and substrate phosphorylation in the mouse brain.
- To identify specific PKC isozymes and their substrates affected by ECS.
Main Methods:
- Western blot analysis using isozyme-specific antibodies to quantify PKC alpha, beta, gamma, epsilon, and zeta.
- Partial purification of PKC isozymes and substrates using DE-52 column chromatography.
- In vitro kinase assays and phosphorylation studies of endogenous substrates like neuromodulin and neurogranin.
Main Results:
- ECS significantly increased kinase activity in both cytosolic and membrane fractions, primarily attributed to an upregulation of the PKC gamma isozyme.
- The level of neuromodulin, a 43 kDa substrate, was elevated in the membrane fraction of ECS-treated mice.
- Novel PKC isozymes (epsilon and zeta) and neurogranin were not significantly altered by ECS.
Conclusions:
- Increased PKC gamma activity and elevated neuromodulin levels contribute to the enhanced phosphorylation of neuromodulin following electroconvulsive shock.
- These molecular changes highlight specific signaling pathways modulated by ECS in the brain.