Abstract
The focus of the present study was to characterize the phosphoproteome of cytotoxic T cells and to explore the role of the serine threonine kinase PKD2 (Protein Kinase D2) in the phosphorylation networks of this key lymphocyte population. We used Stable Isotope Labeling of Amino acids in Culture (SILAC) combined with phosphopeptide enrichment and quantitative mass-spectrometry to determine the impact of PKD2 loss on the cytotoxic T cells phosphoproteome. We identified 15,871 phosphorylations on 3505 proteins in cytotoxic T cells. 450 phosphosites on 281 proteins were down-regulated and 300 phosphosites on 196 proteins were up-regulated in PKD2 null cytotoxic T cells. These data give valuable new insights about the protein phosphorylation networks operational in effector T cells and reveal that PKD2 regulates directly and indirectly about 5% of the cytotoxic T-cell phosphoproteome. PKD2 candidate substrates identified in this study include proteins involved in two distinct biological functions: regulation of protein sorting and intracellular vesicle trafficking, and control of chromatin structure, transcription, and translation. In other cell types, PKD substrates include class II histone deacetylases such as HDAC7 and actin regulatory proteins such as Slingshot. The current data show these are not PKD substrates in primary T cells revealing that the functional role of PKD isoforms is different in different cell lineages.
Highlights
From the ‡Division of Cell Signalling and Immunology
PKD2 Regulated Phosphoproteome in Cytotoxic T Cells is dependent on its localization and cell context presumably reflecting that the localization of protein kinase D (PKD) plays a key role determining the nature of PKD substrates in different cell populations [15]
PKD1 and PKD2 are highly homologous kinases but it remains to be determined whether the PKD1 substrates identified in nocodazole-treated HEK293 cells are relevant to signaling pathways controlled by endogenous PKD2 in antigen receptor activated primary T cells
Summary
Cell Culture, and SILAC Labeling—P14 T-cell receptor transgenic mice (P14-TCR) PKD2 null mice [4, 5], and wild-type littermates were bred and maintained under specific pathogen-free conditions in the Wellcome Trust Biocenter at the University of Dundee in compliance with U.K. The SILAC labeling was performed in three biological replicates, where the P14 wild-type CTLs cells comprised the “light” condition and PKD2 null CTLs were labeled with “heavy” amino acids in two experiments After TCR stimulation, cells were washed once in cold PBS and were subjected to cytosol and nuclear fractionation as previously described [17]. The wild-type cells comprised the “light” condition, whereas the PKD2 knockout CTLs were labeled with “heavy” amino acids (experiments 1 and 2). Data were acquired on a FACSCalibur (Becton Dickinson) and were analyzed with FlowJo software (Treestar, Ashland, OR)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
