c-Abl tyrosine kinase regulates caspase-9 autocleavage in the apoptotic response to DNA damage

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Initiator caspase-9 activation is critical for apoptosis induction via developmental signals, oncogenic transformation, and genotoxic stress.
• The c-Abl tyrosine kinase plays a role in the cellular apoptotic response to DNA damage.

Purpose of the Study:

• To investigate the direct interaction between c-Abl and caspase-9.
• To elucidate the role of c-Abl in the regulation of caspase-9 activation during genotoxic stress.

Main Methods:

• In vitro binding assays to demonstrate direct interaction between c-Abl and caspase-9.
• In vitro and cellular phosphorylation assays to identify phosphorylation sites on caspase-9 by c-Abl.
• Inhibition of c-Abl activity using STI571 and assessment of downstream caspase activation and apoptosis.
• Site-directed mutagenesis of caspase-9 (Y153F) to evaluate the functional significance of Tyr-153 phosphorylation.

Main Results:

• Direct binding of c-Abl to caspase-9 was demonstrated.
• c-Abl was shown to phosphorylate caspase-9 on Tyr-153 both in vitro and in cells subjected to DNA damaging agents.
• Inhibition of c-Abl by STI571 blocked DNA damage-induced autoprocessing of caspase-9 and activation of caspase-3.
• A caspase-9 mutant (Y153F) exhibited attenuated DNA damage-induced processing, caspase-3 activation, and apoptosis.

Conclusions:

• Caspase-9 autoprocessing is regulated by c-Abl.
• The phosphorylation of caspase-9 by c-Abl on Tyr-153 is a key mechanism in the apoptotic response to genotoxic stress.