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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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Phosphorylated chromatin regulators in CTLs: class II histone deacetylase 7 (HDAC7). (a)De novo synthesis of the acquired pseudo MS3 spectra in one of the four experiments performed for the three peptides KTVpSEPNLK, KEpSAPPSLR and pTRSEPLPPSATASPLLAPLQPR. * in y and b ion series indicates loss of phosphate. (b) Spectral counting of the two detected class IIa HDACs in CTLs, HDAC7 and HDAC4 separately calculated for phosphopeptide enrichment and 14-3-3 affinity purification screens. Graph represents the averaged spectral counts of the four SILAC experiments ± SEM.
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Figure 3: Phosphorylated chromatin regulators in CTLs: class II histone deacetylase 7 (HDAC7). (a)De novo synthesis of the acquired pseudo MS3 spectra in one of the four experiments performed for the three peptides KTVpSEPNLK, KEpSAPPSLR and pTRSEPLPPSATASPLLAPLQPR. * in y and b ion series indicates loss of phosphate. (b) Spectral counting of the two detected class IIa HDACs in CTLs, HDAC7 and HDAC4 separately calculated for phosphopeptide enrichment and 14-3-3 affinity purification screens. Graph represents the averaged spectral counts of the four SILAC experiments ± SEM.

Mentions: The HDAC7 phosphorylations identified in the HILIC-IMAC SILAC analyses of CTLs predict that HDAC7 purified from CTLs would constitutively bind 14-3-3 proteins. To explore this possibility we used 14-3-3 affinity purification and SILAC-mass spectrometry analysis to accurately identify and quantify HDAC7-14-3-3 associations (see Supplementary Methods and37). HDAC7 was consistently identified in the 14-3-3 complexes purified from control and TCR-triggered CTLs; there was no effect of TCR triggering on the 14-3-3 binding of HDAC7 (Table 6). We also did a peptide de novo synthesis of the acquired pseudo MS3 spectra for the three HDAC7 peptides purified on the 14-3-3 complexes; KTVpS(178)EPNLK, KEpS(204)APPSLR and pT(342)RSEPLPPSATASPLLAPLQPR (Fig. 3a). The acquired m/z values of the peptide fragments of the according parental ion after collision induced fragmentation (CID) allowed the identification of the peptide sequence and supported HDAC7 S178 and S204 as correctly assigned phosphorylation sites. The data to support HDAC7 T342 phosphorylation were more equivocal. The b4* and the y18 ion of TRSEPLPPSATASPLLAPLQPR (Fig. 3a) were identified indicating that the phosphorylated site was N-terminal of the glutamic acid residue at position 4 of the peptide. However, there was no fragment ion signal closer to the N-terminus than b4* and y18 making a phosphorylation of S344 and T342 equally likely. However, RS (arginine-serine) sequences are known to be resistant to cleavage by trypsin arguing that the actual site of phosphorylation would be S344 rather than T342. In this respect, previous studies have identified S344 rather than T342 as a phosphorylation site in HDAC7 (refs.25,28).


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)

Phosphorylated chromatin regulators in CTLs: class II histone deacetylase 7 (HDAC7). (a)De novo synthesis of the acquired pseudo MS3 spectra in one of the four experiments performed for the three peptides KTVpSEPNLK, KEpSAPPSLR and pTRSEPLPPSATASPLLAPLQPR. * in y and b ion series indicates loss of phosphate. (b) Spectral counting of the two detected class IIa HDACs in CTLs, HDAC7 and HDAC4 separately calculated for phosphopeptide enrichment and 14-3-3 affinity purification screens. Graph represents the averaged spectral counts of the four SILAC experiments ± SEM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3110993&req=5

Figure 3: Phosphorylated chromatin regulators in CTLs: class II histone deacetylase 7 (HDAC7). (a)De novo synthesis of the acquired pseudo MS3 spectra in one of the four experiments performed for the three peptides KTVpSEPNLK, KEpSAPPSLR and pTRSEPLPPSATASPLLAPLQPR. * in y and b ion series indicates loss of phosphate. (b) Spectral counting of the two detected class IIa HDACs in CTLs, HDAC7 and HDAC4 separately calculated for phosphopeptide enrichment and 14-3-3 affinity purification screens. Graph represents the averaged spectral counts of the four SILAC experiments ± SEM.
Mentions: The HDAC7 phosphorylations identified in the HILIC-IMAC SILAC analyses of CTLs predict that HDAC7 purified from CTLs would constitutively bind 14-3-3 proteins. To explore this possibility we used 14-3-3 affinity purification and SILAC-mass spectrometry analysis to accurately identify and quantify HDAC7-14-3-3 associations (see Supplementary Methods and37). HDAC7 was consistently identified in the 14-3-3 complexes purified from control and TCR-triggered CTLs; there was no effect of TCR triggering on the 14-3-3 binding of HDAC7 (Table 6). We also did a peptide de novo synthesis of the acquired pseudo MS3 spectra for the three HDAC7 peptides purified on the 14-3-3 complexes; KTVpS(178)EPNLK, KEpS(204)APPSLR and pT(342)RSEPLPPSATASPLLAPLQPR (Fig. 3a). The acquired m/z values of the peptide fragments of the according parental ion after collision induced fragmentation (CID) allowed the identification of the peptide sequence and supported HDAC7 S178 and S204 as correctly assigned phosphorylation sites. The data to support HDAC7 T342 phosphorylation were more equivocal. The b4* and the y18 ion of TRSEPLPPSATASPLLAPLQPR (Fig. 3a) were identified indicating that the phosphorylated site was N-terminal of the glutamic acid residue at position 4 of the peptide. However, there was no fragment ion signal closer to the N-terminus than b4* and y18 making a phosphorylation of S344 and T342 equally likely. However, RS (arginine-serine) sequences are known to be resistant to cleavage by trypsin arguing that the actual site of phosphorylation would be S344 rather than T342. In this respect, previous studies have identified S344 rather than T342 as a phosphorylation site in HDAC7 (refs.25,28).

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