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Akt phosphorylation site found in human caspase-9 is absent in mouse caspase-9
E Fujita1, A Jinbo, H Matuzaki
1Division of Development and Differentiation, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, 187-8502, Japan.
Abstract:
Caspase-9 is one caspase upstream of caspase-3 and its activation is stimulated by Apaf-1/cytochrome c and inhibited by Akt signals. BAD phosphorylation by Akt is an essential step for growth factor-mediated inhibition of caspase activation. Recently, it was shown that human caspase-9 is phosphorylated by Akt and that its protease activity is reduced. To clarify the molecular mechanism of regulation of caspase-9 activation in neuronal apoptosis, we isolated two alternative splicing products of mouse caspase-9, caspase-9L and caspase-9S, from a P19 embryonal carcinoma cell cDNA library. Curiously, the Akt phosphorylation sites and motifs found in human caspase-9 were absent in both mouse caspase-9L and -9S. Mouse caspase-9 was not phosphorylated by activated Akt in vitro. Reverse transcription polymerase chain reaction analysis showed that the absent Akt motif is not limited to caspase-9 expressed in P19 embryonal carcinoma cells but also occurs in caspase-9 expressed in mouse, rat, and monkey. These results suggest that inhibition of caspase-9 activation by Akt-dependent phosphorylation is not generalized across species.
Insights
Akt-dependent phosphorylation inhibits caspase-9 activation in humans but not in mice, rats, or monkeys. This study identifies species-specific regulation of caspase-9, impacting neuronal apoptosis pathways.
Area of Science:
- Molecular Biology
- Cellular Biology
- Neuroscience
Background:
- Caspase-9 activation is crucial for apoptosis, regulated by Apaf-1/cytochrome c and inhibited by Akt signaling.
- Akt-mediated BAD phosphorylation inhibits growth factor-induced caspase activation.
- Human caspase-9 is phosphorylated by Akt, reducing its protease activity.
Purpose of the Study:
- To investigate the molecular mechanisms regulating caspase-9 activation in neuronal apoptosis.
- To identify and characterize alternative splicing products of mouse caspase-9.
- To determine if Akt-dependent phosphorylation regulates mouse caspase-9 activity.
Main Methods:
- Isolation of alternative splicing products of mouse caspase-9 (caspase-9L and caspase-9S) from P19 embryonal carcinoma cell cDNA library.
- In vitro kinase assays using activated Akt and mouse caspase-9.
- Reverse transcription polymerase chain reaction (RT-PCR) analysis to detect Akt phosphorylation motifs in caspase-9 across species.
Main Results:
- Two alternative splicing products, caspase-9L and caspase-9S, were isolated from mouse caspase-9.
- Mouse caspase-9L and -9S lacked the Akt phosphorylation sites found in human caspase-9.
- Mouse caspase-9 was not phosphorylated by activated Akt in vitro.
- RT-PCR confirmed the absence of the Akt motif in caspase-9 from mouse, rat, and monkey.
Conclusions:
- Akt-dependent phosphorylation does not inhibit caspase-9 activation in mice, rats, or monkeys.
- The regulation of caspase-9 activation by Akt-dependent phosphorylation is not conserved across species.
- This suggests species-specific differences in the apoptotic pathways involving caspase-9.