Phosphoproteomics Study on the Activated PKCδ-Induced Cell Death

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