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Inhibition of protein phosphatase-1 is linked to phosphorylation of p53 and apoptosis
1Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA. xlong001@umaryland.edu
Abstract:
p53 is a multifunctional protein and its activity can be modulated by phosphorylation and dephosphorylation. In this study, we sought to examine the notion that serine/threonine phosphatases (PP-1 and PP-2A) are active modulators of the p53-dependent apoptotic pathway. Exposure of neonatal rat cardiomyocytes to the established apoptotic agents, bafilomycin A1 (BAF) or staurosporine (STAU) induced apoptosis and caused a decrease in PP-1 activity of 35%. This response was restricted to apoptotic stimuli as treatment with phenylephrine neither decreased PP-1 and PP-2A activity nor induced DNA fragmentation in cardiomyocytes. The level of phosphorylated p53 was increased as a result of BAF or STAU-treatment. We further examined the effect of PP-1 inhibition on cardiomyocytes by the use of the phosphatase inhibitor, okadaic acid, and an antisense strategy. Okadaic acid (100 nM) resulted in a decrease in PP-1 activity of 45%, enhanced phosphorylation of p53, and stimulated apoptosis. Furthermore, overexpression of the antisense PP-1 catalytic subunit transcript caused a 44% decrease in expression of PP-1, with no change in the levels of the PP-2A catalytic subunit, and also evoked DNA fragmentation. Our data support the view that decreased activity of PP-1 is an important signaling event in the apoptotic process.
Insights
Decreased protein phosphatase 1 (PP-1) activity is a key event in apoptosis. This study shows PP-1 inhibition enhances p53 phosphorylation and triggers programmed cell death in cardiomyocytes.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- p53 is a crucial protein regulating cell fate.
- Protein phosphorylation and dephosphorylation modulate p53 activity.
- Serine/threonine phosphatases (PP-1 and PP-2A) are potential regulators of p53.
Purpose of the Study:
- To investigate the role of PP-1 and PP-2A in the p53-dependent apoptotic pathway.
- To determine if PP-1 and PP-2A activity changes during apoptosis induction.
- To assess the impact of PP-1 inhibition on apoptosis.
Main Methods:
- Neonatal rat cardiomyocytes were treated with apoptotic agents (bafilomycin A1, staurosporine) or phenylephrine.
- Protein phosphatase activities (PP-1, PP-2A) were measured.
- p53 phosphorylation levels were assessed.
- Cardiomyocytes were treated with okadaic acid (PP-1 inhibitor) or an antisense strategy targeting PP-1.
Main Results:
- Apoptotic agents decreased PP-1 activity by 35% and increased p53 phosphorylation.
- Phenylephrine did not affect phosphatase activity or induce apoptosis.
- Okadaic acid and PP-1 antisense reduced PP-1 activity, enhanced p53 phosphorylation, and induced DNA fragmentation.
- PP-2A activity remained unchanged in response to apoptotic stimuli or PP-1 inhibition.
Conclusions:
- Decreased PP-1 activity is a significant signaling event in the apoptotic process.
- PP-1, but not PP-2A, plays a critical role in p53-mediated apoptosis in cardiomyocytes.
- Modulation of PP-1 activity represents a potential therapeutic target for controlling apoptosis.