Apoptosis is an evolutionarily conserved mechanism necessary for the homeostasis in multicellular organisms. The cysteine protease caspase-3 has a key role in apoptosis for its central role in the execution of the apoptotic cascade. The molecular mechanisms that regulate caspase-3 activation are not completely understood. Previously, our laboratory demonstrated that caspase-3 is phosphorylated by PKCδ in human monocytes. However, the role of caspase-3 phosphorylation during apoptosis has not been elucidated. In this investigation, it was observed that PKCδ is necessary for caspase-3 phosphorylation. An interaction motif in caspase-3 was identified to be necessary for PKCδ interaction. Five PKCδ phosphorylated sites in caspase-3 were mapped utilizing mass spectroscopy. Phosphorylation of specific sites promoted caspase-3 autocatalytic cleavage and apoptosis, in vitro and in vivo. Caspase-3 phosphorylation acts in a positive feedback mechanism to amplify the apoptotic cascade. These results suggest a novel regulatory mechanism to control caspase-3 apoptotic activity and execution of cell death.
Furthermore, the apoptotic and immunological heterogeneity in CD14+CD16+ and CD14+CD16- monocytes was analyzed. It was observed that CD14+CD16+ cells were more susceptible to undergo spontaneous apoptosis, in part due to upregulation of the activity of the caspases. CD14+CD16+ monocytes release more TNF-α compared to the CD14+CD16- counterparts. Furthermore, the expression of the members of the PKC family was characterized in monocyte subsets. These results suggested that elevated expression of PKCε may play a role in the pro-inflammatory role of CD14+CD16+ monocytes. The current investigation highlights the importance of caspase-3 and the members of the PKC family as important regulators of cell death and survival pathways.