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Expression data from P14 TCR cytotoxic T cells overexpressing HDAC7 phosphorylation deficient mutant

ABSTRACT: The present study reports an unbiased analysis of the cytotoxic T cell serine-threonine phosphoproteome using high resolution mass spectrometry. Approximately 2,000 phosphorylations were identified in CTLs of which approximately 450 were controlled by TCR signaling. A significantly overrepresented group of molecules identified in the phosphoproteomic screen were transcription activators, co-repressors and chromatin regulators. A focus on the chromatin regulators revealed that CTLs have high expression of the histone deacetylase HDAC7 but continually phosphorylate and export this transcriptional repressor from the nucleus. HDAC7 dephosphorylation results in its nuclear accumulation and suppressed expression of genes encoding key cytokines, cytokine receptors and adhesion molecules that determine CTL function. The screening of the CTL phosphoproteome thus reveals intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators in CTLs and determine the CTL functional program. We used Affymetrix microarray analysis to explore the molecular basis for the role of HDAC7 in CTLs and the impact of GFP-HDAC7 phosphorylation deficient mutant expression on the CTL transcriptional profile.

ORGANISM(S): Mus musculus

PROVIDER: GSE27092 | GEO | 2011/03/13

SECONDARY ACCESSION(S): PRJNA137455

REPOSITORIES: GEO

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Expression data from P14 TCR cytotoxic T cells overexpressing HDAC7 phosphorylation deficient mutant

Project description:The present study reports an unbiased analysis of the cytotoxic T cell serine-threonine phosphoproteome using high resolution mass spectrometry. Approximately 2,000 phosphorylations were identified in CTLs of which approximately 450 were controlled by TCR signaling. A significantly overrepresented group of molecules identified in the phosphoproteomic screen were transcription activators, co-repressors and chromatin regulators. A focus on the chromatin regulators revealed that CTLs have high expression of the histone deacetylase HDAC7 but continually phosphorylate and export this transcriptional repressor from the nucleus. HDAC7 dephosphorylation results in its nuclear accumulation and suppressed expression of genes encoding key cytokines, cytokine receptors and adhesion molecules that determine CTL function. The screening of the CTL phosphoproteome thus reveals intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators in CTLs and determine the CTL functional program. We used Affymetrix microarray analysis to explore the molecular basis for the role of HDAC7 in CTLs and the impact of GFP-HDAC7 phosphorylation deficient mutant expression on the CTL transcriptional profile. In vitro generated P14 TCR cytotoxic T cells were retrovirally infected with a construct encoding GFP-HDAC7 phosphorylation deficient mutant, sorted in base of GFP expression (GFP positive and GFP negative) and processed for microarray analysis in three biological replicas.

2011-03-13 | E-GEOD-27092 | biostudies-arrayexpress

Phosphoproteomic analyses of IL-2 signaling reveals integrated JAK-dependent and independent phosphorylation networks that drive cytotoxic T cell fate

Project description:High-resolution mass spectrometry analysis of Interleukin 2 (IL-2) and Janus kinase (JAK) controlled protein phosphorylations in cytotoxic T lymphocytes (CTL) revealed JAKs coupled IL-2 receptors to diverse and complex serine/threonine kinase-substrate networks. These involved intricate, co-ordinated phosphorylation of transcription factors, chromatin regulators within the nuclear environment, cytosolic mRNA translational machinery, regulators of GTPases, vesicle trafficking proteins and the actin and microtubule cytoskeleton. We also identified an IL-2-JAK independent SRC family Tyr kinase controlled signaling network that regulates ~10% of the CTL phosphoproteome. One key signaling pathway in CTL is mediated by phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and the serine/threonine kinase AKT. Strikingly, SRC family kinase dependent but JAK independent signaling controlled PIP3 levels and AKT activity in CTL. IL-2-JAK controlled signaling pathways thus coordinate with IL-2 independent networks of protein phosphorylation to program CTL fate.

2016-08-25 | PXD004645 | Pride

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

Project description: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 Labelling 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.

2014-10-03 | PXD001076 | Pride

Effect of rapamycin and KU-0063794 on CTL gene expression

Project description:Comparison of transcriptional profile of CD8 cytotoxic T lymphocytes terated with the mTORC1 inhibitor rapamycin or the mTOR inhibitor KU-0063794 and comparison with proteomic analysis. Abstract: High resolution mass spectrometry maps the cytotoxic T lymphocyte (CTL) proteome and the impact of mammalian target of rapamycin complex 1 (mTORC1) on CTL. We show that the CTL proteome is dominated by metabolic regulators and granzymes and that mTORC1 selectively represses and promotes expression of a protein subset (~10%) including key CTL effector molecules and signaling proteins. mTORC1 also controlled flux through a subset of metabolic pathways rather than acting as an on/off switch for global CTL metabolism. Proteomic data highlighted the potential for mTORC1 negative control of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production in CTL. Further work revealed that mTORC1 represses PIP3 production and determines the mTORC2 requirement for activation of the serine/threonine kinase AKT. Unbiased proteomic analysis thus provides a comprehensive understanding of CTL identity and mTORC1 control of CTL function.

2015-07-15 | GSE70925 | GEO

Spectral library based analysis of arginine phosphorylations in Staphylococcus aureus

Project description:Reversible protein phosphorylation is one of the major mechanisms in the regulation of protein expression and protein activity, controlling physiological functions of the important human pathogen Staphylococcus aureus. Phosphorylations at serine, threonine and tyrosine are known to influence for example protein activity in central metabolic pathways and the more energy-rich phosphorylations at histidine, aspartate or cysteine can be found as part of two component system sensor domains or mediating bacterial virulence. In addition to these well-known phosphorylations, the phosphorylation at arginine residues plays an essential role. Hence, the deletion mutant S. aureus COL ΔptpB (protein tyrosine phosphatase B) was studied since the protein PtpB is assumed to be an arginine phosphatase. A gel-free approach was applied to analyse the changes in the phosphoproteome of the deletion mutant ΔptpB and the wild type in growing cells, thereby focusing on the occurrence of phosphorylation on arginine residues. In order to enhance the reliability of identified phosphorylation sites at arginine residues, a subset of arginine phosphorylated peptides was chemically synthesised. Combined spectral libraries based on phosphoenriched samples, synthetic arginine phosphorylated peptides and classical proteome samples provide a sophisticated tool for the analysis of arginine phosphorylations. This way, 212 proteins phosphorylated on serine, threonine, tyrosine or arginine residues were identified within the mutant ∆ptpB and 102 in wild type samples. Among them, 207 arginine phosphosites were identified exclusively within the mutant ∆ptpB, widely distributed along the whole bacterial metabolism. This identification of putative targets of PtpB allows further investigation of the physiological relevance of arginine phosphorylations and provides the basis for reliable quantification of arginine phosphorylations in bacteria.

2017-12-06 | PXD007167 | Pride

Phosphoproteomic analysis of CD2 signaling reveals an AMPK-dependent targeted granule secretion in human cytotoxic T cells

Project description:The in-depth analysis of co-stimulatory signaling enhancing the activity of cytotoxic T cells represents a major approach towards the development of immunotherapies. Here we describe that CD2 co-stimulation plays a critical role in the functioning of human cytotoxic T cells (CTLs). We found that CD2 promotes the formation of functional immune synapses by orchestrating the polarization of lytic granules towards the synaptic interface. To broadly explore the CD2 signaling network, we undertook an analysis of protein phosphorylation in CD2-stimulated CTLs, which revealed 549 unique CD2-regulated phosphorylation events in 373 proteins. CD2 stimulation elicited an intricated network of signaling events participating in the regulation of T cell metabolism, vesicular trafficking, autophagy, cell polarity and cytoskeleton organization. We further show that a functionally critical node of this network is the AMP-activated protein kinase (AMPK), which regulates granule polarization at the CTL synapse. Polarized trafficking of granules is driven by the concerted action of TAK1/LKB1/CamKK2 kinases, which locally activate AMPK enriched on CTL lysosome membranes, illustrating a novel functional cross-talk between vesicular compartments in CTLs. Our results thus establish CD2 signaling as key for regulating targeted granule secretion in CTLs and reveal an AMPK-dependent mechanism to explain how AMPK-activating drugs boost anti-tumour CTL activity.

2020-04-28 | PXD013840 | Pride

Reprogramming of CTLs into natural killer-like cells in celiac disease

Project description:Celiac disease is an intestinal inflammatory disorder induced by dietary gluten in genetically susceptible individuals. The mechanisms underlying the massive expansion of interferon g–producing intraepithelial cytotoxic T lymphocytes (CTLs) and the destruction of the epithelial cells lining the small intestine of celiac patients have remained elusive. We report massive oligoclonal expansions of intraepithelial CTLs that exhibit a profound genetic reprogramming of natural killer (NK) functions. These CTLs aberrantly expressed cytolytic NK lineage receptors, such as NKG2C, NKp44, and NKp46, which associate with adaptor molecules bearing immunoreceptor tyrosine-based activation motifs and induce ZAP-70 phosphorylation, cytokine secretion, and proliferation independently of T cell receptor signaling. This NK transformation of CTLs may underlie both the self-perpetuating, gluten-independent tissue damage and the uncontrolled CTL expansion leading to malignant lymphomas in severe forms of celiac disease. Because similar changes were detected in a subset of CTLs from cytomegalovirus-seropositive patients, we suggest that a stepwise transformation of CTLs into NK-like cells may underlie immunopathology in various chronic infectious and inflammatory diseases. Keywords: NKG2C; LAK; CTL; NK receptor; IEL; Mucosal Immunity; Celiac Disease

2006-05-15 | GSE4592 | GEO

Analysis of the Candida albicans phosphoproteome

Project description:Candida albicans is an important human fungal pathogen in both immunocompetent and immunocompromised individuals. In response to environmental stimuli, C. albicans regulates complex phenotypic transitions between morphological states, mating competence, and biofilm formation. In addition, other processes related to virulence, stress resistance, and cell wall structure are also highly regulated. Based on the phenotypes of mutants lacking different kinases, it is clear that protein phosphorylation plays an important role in the regulation of these pathways. Here, we present a qualitative analysis of the phosphoproteome of C. albicans hyphae. We identified 19,590 unique peptides that corresponded to 15,906 unique phosphosites on 2,896 proteins. The ratios of serine, threonine and tyrosine phosphosites were 80.01%, 18.11%, and 1.81%, respectively. The majority of proteins (2,111) contained at least two detected phosphorylation sites. Consistent with findings in other fungi, cytoskeletal proteins were among the most highly phosphorylated proteins, and there were differences in GO terms for proteins with serine and threonine versus tyrosine phosphorylation sites. This large-scale analysis identified phosphosites in protein components of Mediator, an important transcriptional co-regulatory protein complex. In vitro studies revealed Cdk8 (Ssn3), a kinase within the Mediator complex, was sufficient to catalyze a subset of these phosphorylations suggesting the potential for autoregulation. These data represent the deepest single analysis of a fungal phosphoproteome, and will lay the groundwork for future analyses of the C. albicans phosphoproteome and specific phosphoproteins.

2015-03-25 | PXD001844 | Pride

IFN-γ Upregulates Survivin and Ifi202 Expression to Induce Survival and Proliferation of Tumor-Specific T Cells

Project description:Transcriptional profiling of mouse tumor-specific cytotoxic T lymphocytes (CTL) comparing activation-induced gene expression profiles induced by tumor-specific antigen (Ag) and CD3 mAb. CTLs were stimulated with either Ag or CD3 mAb for 3 and 24 h, respectively. The stimulated CTLs were compared to un-stimulated CTLs. The objective was to identify differential gene expression profiles induced by the two activation (Ag vs CD3 mAb) conditions. Two-condition experiments, un-stimulated vs stimulated CTLs. Biological replicates: 3 replicates for each comparison. Unstimulated CTLs were compared to stimulated CTLs (Ag and CD3 mAb, respectively).

2010-12-03 | E-GEOD-22890 | biostudies-arrayexpress

A microRNA-directed program of cytotoxic CD8+ T cell differentiation

Project description:Acquisition of effector properties is a key step in the generation of cytotoxic T lymphocytes (CTLs). Here we show that inflammatory signals regulate Dicer expression in CTL, and that deletion or depletion of Dicer in mouse or human activated CD8+ T cells causes upregulation of perforin, granzyme and effector cytokines. Genome-wide analysis of miRNA changes induced by exposure of differentiating CTLs to IL-2 and inflammatory signals identifies miR-139 and miR-150 as components of a miRNA network that controls perforin, eomesodermin (Eomes) and IL-2Ra expression in differentiating CTLs and whose activity is modulated by IL-2, inflammation and antigenic stimulation. Overall our data show that strong IL-2R and inflammatory signals act through Dicer and miRNAs to control the cytolytic program and other aspects of effector CTL differentiation. Comparison of control and Dicer knock-out CTLs differentiated in vitro; Comparison of wild type CTLs differentiated in vitro with or without inflammatory stimuli; Comparison of effector and memory precursor CTLs isolated from mice infected with LCMV-Armstrong

2013-12-02 | E-GEOD-51393 | biostudies-arrayexpress

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