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Cleavage and inactivation of antiapoptotic Akt/PKB by caspases during apoptosis
S Rokudai1, N Fujita, Y Hashimoto
1Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
Abstract:
The oncogene Akt/PKB/RAC-PK is a serine/threonine kinase that mediates survival signals and has protective effects against apoptosis induced by a variety of stimuli. The kinase activity of Akt has been demonstrated to be critical in transmitting survival signals. We found that Akt protein was down-regulated during apoptosis. The down-regulation was blocked by a caspase inhibitor, indicating that Akt was cleaved by caspases during apoptosis. The Akt protein incubation with active caspases in vitro revealed that it was cleaved at three sites to produce 40- and 44-kDa fragments. The two cleavage sites were between the NH(2)-terminal pleckstrin homology domain (PH domain) and the kinase domain (TVAD(108 downward arrow)G and EEMD(119 downward arrow)F) and in the COOH-terminal regulatory domain (SETD(434 downward arrow)T). The loss of COOH-terminal domain of the Akt protein reduced its kinase activity and the overexpression of NH(2)-terminal and COOH-terminal-deleted Akt fragment increased the sensitivity to apoptosis-inducing stimuli. These results indicate that caspase-dependent cleavage of anti-apoptotic Akt turns off the survival signals, resulting in the acceleration of apoptotic cell death.
Insights
The anti-apoptotic protein Akt is cleaved by caspases during apoptosis, inactivating its survival signals. This caspase-mediated cleavage accelerates programmed cell death.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The Akt (also known as Protein Kinase B or RAC-PK) is a serine/threonine kinase.
- Akt mediates crucial cell survival signals and protects against apoptosis.
- Kinase activity of Akt is essential for transmitting survival signals.
Purpose of the Study:
- To investigate the role of Akt in apoptosis.
- To determine the mechanism of Akt down-regulation during apoptosis.
- To elucidate the relationship between caspase activity and Akt function in cell death.
Main Methods:
- Western blotting to detect Akt protein levels during apoptosis.
- Treatment with caspase inhibitors to block Akt cleavage.
- In vitro cleavage assays using purified Akt protein and active caspases.
- Site-directed mutagenesis to identify Akt cleavage sites.
- Overexpression of Akt deletion mutants to assess functional consequences.
Main Results:
- Akt protein levels were significantly down-regulated during apoptosis.
- Caspase inhibition prevented Akt down-regulation, indicating caspase-mediated cleavage.
- In vitro assays showed Akt is cleaved by caspases at three specific sites, generating 40- and 44-kDa fragments.
- Cleavage in the COOH-terminal regulatory domain reduced Akt kinase activity.
- Overexpression of truncated Akt fragments lacking the NH(2)-terminal or COOH-terminal domains increased sensitivity to apoptosis.
Conclusions:
- Caspase-dependent cleavage of Akt inactivates its pro-survival function.
- This cleavage event contributes to the acceleration of apoptotic cell death.
- Targeting Akt cleavage could be a strategy to modulate apoptosis in disease contexts.