Quantitative phosphoproteomics of cytotoxic T cells to reveal Protein Kinase D 2 regulated networks

Maria N. Navarro, Juergen Goebel, Jens L. Hukelmann, Doreen A. Cantrell (Lead / Corresponding author)

    Research output: Contribution to journalArticle

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    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 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 3,505 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.
    Original languageEnglish
    Pages (from-to)3544-3557
    Number of pages14
    JournalMolecular & Cellular Proteomics
    Volume13
    Issue number12
    DOIs
    Publication statusPublished - Dec 2014

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    T-cells
    T-Lymphocytes
    Phosphorylation
    Proteins
    Substrates
    Phosphopeptides
    Histone Deacetylases
    Lymphocytes
    Protein-Serine-Threonine Kinases
    Cell Lineage
    Protein Transport
    Transcription
    protein kinase D
    Sorting
    Isotopes
    Chromatin
    Mass spectrometry
    Actins
    Mass Spectrometry
    Protein Isoforms

    Cite this

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    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 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 3,505 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.",
    author = "Navarro, {Maria N.} and Juergen Goebel and Hukelmann, {Jens L.} and Cantrell, {Doreen A.}",
    note = "Copyright {\circledC} 2014, The American Society for Biochemistry and Molecular Biology.",
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    Quantitative phosphoproteomics of cytotoxic T cells to reveal Protein Kinase D 2 regulated networks. / Navarro, Maria N.; Goebel, Juergen; Hukelmann, Jens L.; Cantrell, Doreen A. (Lead / Corresponding author).

    In: Molecular & Cellular Proteomics, Vol. 13, No. 12, 12.2014, p. 3544-3557.

    Research output: Contribution to journalArticle

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    AU - Navarro, Maria N.

    AU - Goebel, Juergen

    AU - Hukelmann, Jens L.

    AU - Cantrell, Doreen A.

    N1 - Copyright © 2014, The American Society for Biochemistry and Molecular Biology.

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    Y1 - 2014/12

    N2 - 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 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 3,505 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.

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