title: Quantitative phosphoproteomics of cytotoxic T cells to reveal protein kinase d 2 regulated networks.
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.
authors.affiliation: From the ‡Division of Cell Signalling and Immunology. College of Life Sciences University of Dundee, Dundee, Scotland, U.K.
authors.forename: María N
authors.initials: MN
authors.lastname: Navarro
authors.affiliation: From the ‡Division of Cell Signalling and Immunology. College of Life Sciences University of Dundee, Dundee, Scotland, U.K.
authors.forename: Juergen
authors.initials: J
authors.lastname: Goebel
authors.affiliation: From the ‡Division of Cell Signalling and Immunology. College of Life Sciences University of Dundee, Dundee, Scotland, U.K.
authors.forename: Jens L
authors.initials: JL
authors.lastname: Hukelmann
authors.affiliation: From the ‡Division of Cell Signalling and Immunology. College of Life Sciences University of Dundee, Dundee, Scotland, U.K. d.a.cantrell@dundee.ac.uk.
authors.forename: Doreen A
authors.initials: DA
authors.lastname: Cantrell
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D017252:CD3 Complex / Q000235:genetics / Q000378:metabolism; D002247:Carbon Isotopes; D005786:Gene Expression Regulation; D053263:Gene Regulatory Networks; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism; D007553:Isotope Labeling; D008213:Lymphocyte Activation / Q000187:drug effects; D051379:Mice; D008822:Mice, Transgenic; D009587:Nitrogen Isotopes; D009928:Organ Specificity; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013154:Spleen / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D050396:TRPP Cation Channels / Q000172:deficiency / Q000235:genetics*
chemical_list: D000906:Antibodies; D017252:CD3 Complex; D002247:Carbon Isotopes; D006655:Histone Deacetylases; D009587:Nitrogen Isotopes; D010749:Phosphoprotein Phosphatases; D010750:Phosphoproteins; D020543:Proteome; D050396:TRPP Cation Channels; C543274:Hdac7 protein, mouse