PKC activation contributes to caspase-mediated eIF2alpha phosphorylation and cell death

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• Stress responses in cells involve the phosphorylation of eukaryotic initiation factor 2-alpha (eIF2alpha), impacting cell fate.
• Protein kinase C (PKC) is a signaling enzyme implicated in various cellular processes, including cell death.

Purpose of the Study:

• To investigate the role of PKC activation in stress-induced eIF2alpha phosphorylation, caspase activation, and cell death.
• To elucidate the interplay between PKC, caspases, and eIF2alpha phosphorylation in Spodoptera frugiperda (Sf9) cells.

Main Methods:

• Cell death was quantified using flow cytometry.
• Caspase activation was measured via substrate hydrolysis and protein cleavage assays.
• eIF2alpha phosphorylation and cytochrome c levels were assessed using western blotting.

Main Results:

• PKC activation by PMA enhanced UV or cycloheximide-induced caspase activation, eIF2alpha phosphorylation, and apoptosis in Sf9 cells.
• PMA also boosted cytochrome c-induced caspase activation and eIF2alpha phosphorylation in cell extracts.
• These effects were more potently inhibited by a caspase inhibitor (z-VAD-fmk) than a PKC inhibitor (calphostin).

Conclusions:

• Caspase activation is both a cause and consequence of eIF2alpha phosphorylation.
• PKC activation, downstream of caspase activation, further amplifies caspase activity, eIF2alpha phosphorylation, and ultimately cell death.
• Caspases play a crucial role in orchestrating multiple signaling pathways that promote cell death.