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Novel Ser/Thr protein phosphatase 5 (PP5) regulated targets during DNA damage identified by proteomics analysis.

ABSTRACT: The DNA damage response likely includes a global phosphorylation signaling cascade process for sensing the damaged DNA condition and coordinating responses to cope with and repair the perturbed cellular state. We utilized a label-free liquid chromatography-mass spectrometry approach to evaluate changes in protein phosphorylation associated with PP5 activity during the DNA damage response. Biological replicate analyses of bleomycin-treated HeLa cells expressing either WT-PP5 or mutant inactive PP5 lead to the identification of six potential target proteins of PP5 action. Four of these putative targets have been previously reported to be involved in DNA damage responses. Using phospho-site specific antibodies, we confirmed that phosphorylation of one target, ribosomal protein S6, was selectively decreased in cells overexpressing catalytically inactive PP5. Our findings also suggest that PP5 may play a role in controlling translation and in regulating substrates for proline-directed kinases, such as MAP kinases and cyclin-dependent protein kinases that are involved in response to DNA damage.

SUBMITTER: Ham BM

PROVIDER: S-EPMC2818885 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Novel Ser/Thr protein phosphatase 5 (PP5) regulated targets during DNA damage identified by proteomics analysis.

Ham Bryan M BM Jayachandran Hemalatha H Yang Feng F Jaitly Navdeep N Polpitiya Ashoka D AD Monroe Matthew E ME Wang Ling L Zhao Rui R Purvine Samuel O SO Livesay Eric A EA Camp David G DG Rossie Sandra S Smith Richard D RD

Journal of proteome research 20100201 2

The DNA damage response likely includes a global phosphorylation signaling cascade process for sensing the damaged DNA condition and coordinating responses to cope with and repair the perturbed cellular state. We utilized a label-free liquid chromatography-mass spectrometry approach to evaluate changes in protein phosphorylation associated with PP5 activity during the DNA damage response. Biological replicate analyses of bleomycin-treated HeLa cells expressing either WT-PP5 or mutant inactive PP ...[more]

PMID: 20039704

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Project description:BackgroundProtein Ser/Thr phosphatase 5 (PP5) and its Saccharomyces cerevisiae homolog protein phosphatase T1 (Ppt1p) each contain an N-terminal domain consisting of several tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain that is related to the catalytic subunits of protein phosphatases 1 and 2A, and calcineurin. Analysis of yeast Ppt1p could provide important clues to the function of PP5 and its homologs, however it has not yet been characterized at the biochemical or cellular level.ResultsThe specific activity of recombinant Ppt1p toward the artificial substrates 32P-myelin basic protein (MBP) and 32P-casein was similar to that of PP5. Dephosphorylation of 32P-MBP, but not 32P-casein, was stimulated by unsaturated fatty acids and by arachidoyl coenzyme A. Limited proteolysis of Ppt1p removed the TPR domain and abrogated lipid stimulation. The remaining catalytic fragment exhibited a two-fold increase in activity toward 32P-MBP, but not 32P-casein. Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment. Ppt1p was localized throughout the cell including the nucleus. Levels of PPT1 mRNA and protein peaked in early log phase growth.ConclusionsMany characteristics of Ppt1p are similar to those of PP5, including stimulation of phosphatase activity with some substrates by lipids, and peak expression during periods of rapid cell growth. Unlike PP5, however, proteolytic removal of the TPR domain or C-terminal truncation only modestly increased its activity. In addition, C-terminal truncation did not prevent further activation by lipid. This suggests that these regions play only a minor role in controlling its activity compared to PP5. Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments. The observation that Ppt1p is most highly expressed during early log phase growth suggests that this enzyme is involved in cell growth or its expression is controlled by metabolic or nutritional signals.

Dataset Information

Novel Ser/Thr protein phosphatase 5 (PP5) regulated targets during DNA damage identified by proteomics analysis.

Publications

Novel Ser/Thr protein phosphatase 5 (PP5) regulated targets during DNA damage identified by proteomics analysis.

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