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Regulation of protein phosphatase 2A activity by caspase-3 during apoptosis
M F Santoro1, R R Annand, M M Robertson
1Department of Biochemistry, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105, USA.
Abstract:
Although the available evidence suggests that whereas the caspase family plays a major role in apoptosis, they are not the sole stimulators of death. A random yeast two-hybrid screen of a lymphocyte cDNA library (using caspase-3 as the bait) found an interaction between caspase-3 and the regulatory subunit Aalpha of protein phosphatase 2A. This protein was found to be a substrate for caspase-3, but not caspase-1, and could compete effectively against either a protein or synthetic peptide substrate. In Jurkat cells induced to undergo apoptosis with anti-Fas antibody, protein phosphatase 2A (PP2A) activity increased 4.5-fold after 6 h. By 12 h, the regulatory Aalpha subunit could no longer be detected in cell lysates. There was no change in the amount of the catalytic subunit. The effects on PP2A could be prevented by the caspase family inhibitors acetyl-Asp-Glu-Val-Asp (DEVD) aldehyde or Ac-DEVD fluoromethyl ketone. The mitogen-activated protein (MAP) kinase pathway is regulated by PP2A. At 12 h after the addition of anti-Fas antibody, a decrease in the amount of the phosphorylated forms of MAP kinase was observed. Again, this loss of activated MAP kinase could be prevented by the addition of DEVD-cho or DEVD-fmk. These data are consistent with a pathway whereby induction of apoptosis activates caspase-3. This enzyme then cleaves the regulatory Aalpha subunit of PP2A, increasing its activity. These data show that the activated PP2A will then effect a change in the phosphorylation state of the cell. These data provide a link between the caspases and signal transduction pathways.
Insights
Caspase-3 activation during apoptosis cleaves the regulatory subunit of protein phosphatase 2A (PP2A), increasing its activity and impacting cell signaling pathways. This reveals a novel link between caspases and MAP kinase regulation.
Area of Science:
- Cellular Biology
- Biochemistry
- Molecular Biology
Background:
- Caspase proteases are key mediators of apoptosis.
- The role of caspases beyond direct cell death execution is an area of active investigation.
- Protein Phosphatase 2A (PP2A) is a crucial regulator of cellular signaling pathways.
Purpose of the Study:
- To investigate the interaction between caspase-3 and protein phosphatase 2A (PP2A).
- To elucidate the role of PP2A in apoptosis-induced signaling.
- To identify novel substrates and functions of caspase-3 in the apoptotic cascade.
Main Methods:
- Yeast two-hybrid screening to identify interacting proteins.
- In vitro enzymatic assays using protein and peptide substrates.
- Apoptosis induction in Jurkat cells using anti-Fas antibody.
- Western blot analysis to detect protein levels and phosphorylation.
- Inhibition studies using caspase family inhibitors (DEVD aldehyde, Ac-DEVD fluoromethyl ketone).
Main Results:
- Caspase-3 directly interacts with and cleaves the regulatory Aalpha subunit of PP2A.
- PP2A activity significantly increases during anti-Fas induced apoptosis.
- The Aalpha subunit of PP2A is degraded during apoptosis.
- Caspase inhibition prevents PP2A activation and subsequent dephosphorylation of MAP kinase.
- Activated PP2A influences the phosphorylation state of cellular components.
Conclusions:
- Caspase-3 activation during apoptosis leads to the cleavage and activation of PP2A.
- This caspase-mediated PP2A activation impacts downstream signaling pathways, including the MAP kinase pathway.
- These findings establish a novel link between caspases and cellular signal transduction in apoptosis.