Phosphoproteomics Study on the Activated PKCδ-Induced Cell Death

The proteolytic activation of protein kinase C delta (PKC delta) generates a catalytic fragment called PKC delta-CF, which induces cell death. However, the mechanisms underlying PKC delta-CF-mediated cell death are largely unknown. On the basis of an engineering leukemic cell line with inducible expression of PKC delta-CF, here we employ SILAC-based quantitative phosphoproteomics to systematically and dynamically investigate the overall phosphorylation events during cell death triggered by PKC delta-CF expression. Totally, 3000 phosphorylation sites were analyzed. Considering the fact that early responses to PKC delta-CF expression initiate cell death, we sought to identify pathways possibly related directly with PKC delta by further analyzing the data set of phosphorylation events that occur in the initiation stage of cell death. Interacting analysis of this data set indicates that PKC delta-CF triggers complicated networks to initiate cell death, and motif analysis and biochemistry verification reveal that several kinases in the downstream of PKC delta conduct these networks. By analysis of the specific sequence motif of kinase-substrate, we also find 59 candidate substrates of PKC delta from the up-regulated phosphopeptides, of which 12 were randomly selected for in vitro kinase assay and 9 were consequently verified as substrates of PKC delta. To our greatest understanding, this study provides the most systematic analysis of phosphorylation events initiated by the cleaved activated PKC delta, which would vastly extend the profound understanding of PKC delta-directed signal pathways in cell death. The MS data have been deposited to the ProteomeXchange with identifier PXD000225.