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Phosphoproteomic analysis reveals an intrinsic pathway for the regulation of histone deacetylase 7 that controls the function of cytotoxic T lymphocytes.

Navarro MN, Goebel J, Feijoo-Carnero C, Morrice N, Cantrell DA - Nat. Immunol. (2011)

Bottom Line: A significantly overrepresented group of molecules identified included transcription activators, corepressors and chromatin regulators.A focus on chromatin regulators showed that CTLs had high expression of the histone deacetylase HDAC7 but continually phosphorylated and exported this transcriptional repressor from the nucleus.Screening of the CTL phosphoproteome has thus identified intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators and determine the CTL functional program.

View Article: PubMed Central - PubMed

Affiliation: The College of Life Sciences, Division of Immunology and Cell Biology, The University of Dundee, Dundee, Scotland, UK.

ABSTRACT
Here we report an unbiased analysis of the cytotoxic T lymphocyte (CTL) serine-threonine phosphoproteome by high-resolution mass spectrometry. We identified approximately 2,000 phosphorylations in CTLs, of which approximately 450 were controlled by T cell antigen receptor (TCR) signaling. A significantly overrepresented group of molecules identified included transcription activators, corepressors and chromatin regulators. A focus on chromatin regulators showed that CTLs had high expression of the histone deacetylase HDAC7 but continually phosphorylated and exported this transcriptional repressor from the nucleus. Dephosphorylation of HDAC7 resulted in its accumulation in the nucleus and suppressed expression of genes encoding key cytokines, cytokine receptors and adhesion molecules that determine CTL function. Screening of the CTL phosphoproteome has thus identified intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators and determine the CTL functional program.

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Ingenuity Pathway Analysis of consistent phosphorylations in CTLs. SILAC and HILIC/IMAC purification protocols were independently performed four times, including amino acid labeling switch. The isotope combination (unstimulated/stimulated) was as follows: experiment 1, R10K8/R0K0 (Fig. 1), experiment 2, R0K0/R6K6, experiment 3 and 4, R0K0/R6K6 (not shown). CTLs were stimulated for 1 h with cognate peptide in experiments 1, 2 and 3 and for 10 min in experiment 4. Reproducible phosphorylations in three out of four experiments were considered for analysis. (a) Ingenuity Pathway Analysis of 742 phosphorylations on 473 different proteins found in at least three of the four conducted SILAC experiments. (b) Ingenuity Pathway Analysis of TCR-regulated 94 phosphorylations on 78 different proteins consistently found in our screening using 1.5 fold as threshold for regulation. (c) Representation of the frequency of kinases predicted to be active in CTL using MaxQuant software analysis. MaxQuant uses a sequence window ± 6 amino acids around the identified phosphorylation site to determine the kinase that could phosphorylate this motif.
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Figure 2: Ingenuity Pathway Analysis of consistent phosphorylations in CTLs. SILAC and HILIC/IMAC purification protocols were independently performed four times, including amino acid labeling switch. The isotope combination (unstimulated/stimulated) was as follows: experiment 1, R10K8/R0K0 (Fig. 1), experiment 2, R0K0/R6K6, experiment 3 and 4, R0K0/R6K6 (not shown). CTLs were stimulated for 1 h with cognate peptide in experiments 1, 2 and 3 and for 10 min in experiment 4. Reproducible phosphorylations in three out of four experiments were considered for analysis. (a) Ingenuity Pathway Analysis of 742 phosphorylations on 473 different proteins found in at least three of the four conducted SILAC experiments. (b) Ingenuity Pathway Analysis of TCR-regulated 94 phosphorylations on 78 different proteins consistently found in our screening using 1.5 fold as threshold for regulation. (c) Representation of the frequency of kinases predicted to be active in CTL using MaxQuant software analysis. MaxQuant uses a sequence window ± 6 amino acids around the identified phosphorylation site to determine the kinase that could phosphorylate this motif.

Mentions: To determine the biological reproducibility of the results in Fig. 1 we repeated the experiments four times including amino acid labeling switch. Collective analysis of pooled data from multiple experiments confirmed that CTLs have high basal serine phosphorylation and TCR triggering modifies approximately 20% of these serine phosphorylations. A full list of the basal and TCR regulated serine-threonine phosphorylations identified in at least 3 out of 4 experiments is shown in the supplementary data (see full list in Supplementary Table 2). We also used the Ingenuity program to perform a canonical pathway analysis and molecular and cellular function pathway analysis on the 742 consistent phosphorylations on 473 proteins identified in the CTLs (Fig. 2a). This analysis indicated that the CTL phospho-proteome was significantly overrepresented by proteins that control RNA post-transcriptional modifications, protein synthesis, cell death, gene transcription and polymerization of actin (listed by groups in Supplementary Fig. 3a,b). TCR triggering consistently changed 94 phosphorylations on 81 proteins (including both upregulated and downregulated phosphorylations), representing approximately 17% of all phosphorylated proteins found in our screen (Tables2, 3). Bioinformatics was used to assign the kinases most likely to phosphorylate the CTL phosphoproteins (Fig. 2c and Table 4) and indicated activity of a minimum of at least 18 serine-threonine kinases in CTLs.


Phosphoproteomic analysis reveals an intrinsic pathway for the regulation of histone deacetylase 7 that controls the function of cytotoxic T lymphocytes.

Navarro MN, Goebel J, Feijoo-Carnero C, Morrice N, Cantrell DA - Nat. Immunol. (2011)

Ingenuity Pathway Analysis of consistent phosphorylations in CTLs. SILAC and HILIC/IMAC purification protocols were independently performed four times, including amino acid labeling switch. The isotope combination (unstimulated/stimulated) was as follows: experiment 1, R10K8/R0K0 (Fig. 1), experiment 2, R0K0/R6K6, experiment 3 and 4, R0K0/R6K6 (not shown). CTLs were stimulated for 1 h with cognate peptide in experiments 1, 2 and 3 and for 10 min in experiment 4. Reproducible phosphorylations in three out of four experiments were considered for analysis. (a) Ingenuity Pathway Analysis of 742 phosphorylations on 473 different proteins found in at least three of the four conducted SILAC experiments. (b) Ingenuity Pathway Analysis of TCR-regulated 94 phosphorylations on 78 different proteins consistently found in our screening using 1.5 fold as threshold for regulation. (c) Representation of the frequency of kinases predicted to be active in CTL using MaxQuant software analysis. MaxQuant uses a sequence window ± 6 amino acids around the identified phosphorylation site to determine the kinase that could phosphorylate this motif.
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Related In: Results  -  Collection

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Figure 2: Ingenuity Pathway Analysis of consistent phosphorylations in CTLs. SILAC and HILIC/IMAC purification protocols were independently performed four times, including amino acid labeling switch. The isotope combination (unstimulated/stimulated) was as follows: experiment 1, R10K8/R0K0 (Fig. 1), experiment 2, R0K0/R6K6, experiment 3 and 4, R0K0/R6K6 (not shown). CTLs were stimulated for 1 h with cognate peptide in experiments 1, 2 and 3 and for 10 min in experiment 4. Reproducible phosphorylations in three out of four experiments were considered for analysis. (a) Ingenuity Pathway Analysis of 742 phosphorylations on 473 different proteins found in at least three of the four conducted SILAC experiments. (b) Ingenuity Pathway Analysis of TCR-regulated 94 phosphorylations on 78 different proteins consistently found in our screening using 1.5 fold as threshold for regulation. (c) Representation of the frequency of kinases predicted to be active in CTL using MaxQuant software analysis. MaxQuant uses a sequence window ± 6 amino acids around the identified phosphorylation site to determine the kinase that could phosphorylate this motif.
Mentions: To determine the biological reproducibility of the results in Fig. 1 we repeated the experiments four times including amino acid labeling switch. Collective analysis of pooled data from multiple experiments confirmed that CTLs have high basal serine phosphorylation and TCR triggering modifies approximately 20% of these serine phosphorylations. A full list of the basal and TCR regulated serine-threonine phosphorylations identified in at least 3 out of 4 experiments is shown in the supplementary data (see full list in Supplementary Table 2). We also used the Ingenuity program to perform a canonical pathway analysis and molecular and cellular function pathway analysis on the 742 consistent phosphorylations on 473 proteins identified in the CTLs (Fig. 2a). This analysis indicated that the CTL phospho-proteome was significantly overrepresented by proteins that control RNA post-transcriptional modifications, protein synthesis, cell death, gene transcription and polymerization of actin (listed by groups in Supplementary Fig. 3a,b). TCR triggering consistently changed 94 phosphorylations on 81 proteins (including both upregulated and downregulated phosphorylations), representing approximately 17% of all phosphorylated proteins found in our screen (Tables2, 3). Bioinformatics was used to assign the kinases most likely to phosphorylate the CTL phosphoproteins (Fig. 2c and Table 4) and indicated activity of a minimum of at least 18 serine-threonine kinases in CTLs.

Bottom Line: A significantly overrepresented group of molecules identified included transcription activators, corepressors and chromatin regulators.A focus on chromatin regulators showed that CTLs had high expression of the histone deacetylase HDAC7 but continually phosphorylated and exported this transcriptional repressor from the nucleus.Screening of the CTL phosphoproteome has thus identified intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators and determine the CTL functional program.

View Article: PubMed Central - PubMed

Affiliation: The College of Life Sciences, Division of Immunology and Cell Biology, The University of Dundee, Dundee, Scotland, UK.

ABSTRACT
Here we report an unbiased analysis of the cytotoxic T lymphocyte (CTL) serine-threonine phosphoproteome by high-resolution mass spectrometry. We identified approximately 2,000 phosphorylations in CTLs, of which approximately 450 were controlled by T cell antigen receptor (TCR) signaling. A significantly overrepresented group of molecules identified included transcription activators, corepressors and chromatin regulators. A focus on chromatin regulators showed that CTLs had high expression of the histone deacetylase HDAC7 but continually phosphorylated and exported this transcriptional repressor from the nucleus. Dephosphorylation of HDAC7 resulted in its accumulation in the nucleus and suppressed expression of genes encoding key cytokines, cytokine receptors and adhesion molecules that determine CTL function. Screening of the CTL phosphoproteome has thus identified intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators and determine the CTL functional program.

Show MeSH
Related in: MedlinePlus