Dataset Information

Nuclear phosphoproteomics analysis of resting and IL-2-stimulated T lymphocytes

ABSTRACT: The aim of the present project was to study in detail the site-specific phosphorylation events occurring both in resting as well as in T lymphocytes stimulated with IL-2 for five minutes. For that purpose we combined SILAC-based quantitative mass spectrometry analysis with phosphopeptide enrichment using TiO2 beads. We performed three biological replicas of the same experiment which resulted in the identification of above 8500 unique phosphosites corresponding to more than 3000 proteins. From the 6145 phosphosites that were consistently quantified in at least 2 out of the 3 replicas performed, we detected that 390 were regulated by IL-2 being the up-regulated phosphosites five times more abundant than the down-regulated ones. Those IL-2-dependent phosphosites corresponded to distinct proteins involved in distinct aspects of gene expression and cell cycle regulation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell

DISEASE(S): Acute Leukemia

SUBMITTER: Nerea Osinalde

LAB HEAD: Irina Kratchmarova

PROVIDER: PXD002839 | Pride | 2016-04-18

REPOSITORIES: Pride

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Publications

Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes.

Osinalde Nerea N Mitxelena Jone J Sánchez-Quiles Virginia V Akimov Vyacheslav V Aloria Kerman K Arizmendi Jesus M JM Zubiaga Ana M AM Blagoev Blagoy B Kratchmarova Irina I

Molecular & cellular proteomics : MCP 20160411 6

Anti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2- dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms that are not clearly understood. To study the role of IL-2 in the regulation of nuclear protein function we have performed an unbiased mass spectrometry-based study of the nuclear phosphoproteome of resting and IL- ...[more]

PMID: 27067055

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Project description:We performed a biological and molecular characterization of the novel human multiple myeloma cell line CMA03/06, an IL-6-independent variant of CMA03 cell line previously established in our Institution. We showed that the CMA03/06 cells grows in the absence of IL-6 with a doubling time comparable to CMA03; the addition of IL-6 to the culture medium or co-culture with multipotent mesenchymal stromal cells does not induce an increase in proliferation rate. Interestingly, we provided evidence that IL-6 independence of CMA03/06 cells is not a consequence of the development of an autocrine IL-6 loop, even though the cells maintain the IL-6 signaling pathway responsiveness as demonstrated by STAT1 and STAT3 induction by IL-6. A slight constitutive activation of STAT3 has been observed in CMA03/06 cell line; however STAT3 silencing in CMA03/06 cells did not affect their viability and proliferation suggesting that this pathway is not the only responsible for the IL-6 independency of CMA03/06 cell line. The new cell line showed a lower susceptibility to camptothecin-induced apoptosis compared to CMA03 cells, whereas an increased induction of apoptosis was observed in CMA03/06 cell line after Bortezomib treatment which may suggest the involvement NF-kB pathway in the IL-6 independent growth and survival of CMA03/06 cells. Finally, global gene expression profiling analysis allowed the identification of a list of 308 modulated genes in CMA03/06 versus CMA03 cells, many of which particularly relevant for MM biology. Overall, the novel CMA03/06 cell line may represent a suitable model for studies investigating molecular mechanisms involved in clonal evolution towards IL-6 and/or stroma-independent growth and survival of myeloma cells. This series of microarray experiments contains the gene expression profiles of 3 independent replicas of CMA03 grown in in normal culture conditions and in absence of IL-6, respectively, and 3 independent replicas of CMA03/06 in normal culture conditions. Cell pellets were resuspended in TRIzol Reagent (Life Technologies, Inc., Rockville, MD, USA), total RNA was then purified using the RNeasy® total RNA Isolation Kit (Qiagen, Valencia, CA).5.5 micrograms of single-stranded DNA target obtained from 100 ng of purified total RNA was fragmented and then labeled using the WT Terminal Labeling Kit according to the standard Affymetrix protocol (GeneChip® Whole Transcript (WT) Sense Target Labeling Assay Manual).The fragmented labeled single-stranded DNA target was hybridized for 16 hr 30 minutes at 45°C on GeneChip® Gene 1.0 ST array according to the standard Affymetrix protocol. The arrays were washed and stained in the Affymetrix Fluidics Station 450.

Dataset Information

Nuclear phosphoproteomics analysis of resting and IL-2-stimulated T lymphocytes

Publications

Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes.

Similar Datasets

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