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.

蛋白激酶Cδ（protein kinase Cδ，PKCδ）的蛋白水解激活可产生一个名为PKCδ-CF的催化片段，该片段能够诱导细胞死亡。然而，PKCδ-CF介导的细胞死亡的具体分子机制目前仍未被完全阐明。本研究基于一株可诱导表达PKCδ-CF的工程化白血病细胞系，采用基于SILAC的定量磷酸化蛋白质组学技术，系统且动态地探究了PKCδ-CF表达诱导细胞死亡过程中的整体磷酸化事件。本次研究共分析了3000个磷酸化位点。鉴于PKCδ-CF表达引发的早期应答即可启动细胞死亡，我们通过进一步分析细胞死亡启动阶段的磷酸化事件数据集，旨在筛选出可能与PKCδ直接相关的信号通路。对该数据集的相互作用分析表明，PKCδ-CF可触发复杂的调控网络以启动细胞死亡；基序分析与生化验证结果显示，PKCδ下游的数种激酶介导了该调控网络的构建与运行。通过分析激酶-底物的特异性序列基序，我们从上调的磷酸化肽段中筛选得到59个PKCδ候选底物；其中随机选取12个开展体外激酶实验验证，最终确认其中9个为PKCδ的真实底物。据我们所知，本研究首次对切割激活型PKCδ所启动的磷酸化事件开展了最为系统的分析，这将极大深化我们对PKCδ介导的细胞死亡信号通路的认知。本研究的质谱（Mass Spectrometry, MS）数据已提交至ProteomeXchange数据库，登录号为PXD000225。