SILAC-based quantification of changes in protein tyrosine phosphorylation induced by Interleukin-2 (IL-2) and IL-15 in T-lymphocytes

Osinalde, Nerea; Sanchez-Quiles, Virginia; Akimov, Vyacheslav; Blagoev, Blagoy; Kratchmarova, Irina

doi:10.1016/j.dib.2015.08.007

Cited by 12 publications

(8 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Protein lysates were run in two parallel lanes of a precast NuPAGE 4–12% Bis-Tris gel (Thermo Fisher Scientific, Inc.) and visualized with colloidal blue (Thermo Fisher Scientific, Inc.). Both gel lanes were separately cut into slices and subjected to in-gel reduction, alkylation and 5 ng/µl trypsin digestion as previously described (18). Derived peptides were concentrated and desalted using C18 STAGE Tips and further analysed by liquid chromatography (LC)-MS/MS.…”

Section: Methodsmentioning

confidence: 99%

NADH dehydrogenase complex�I is overexpressed in incipient metastatic murine colon cancer cells

et al. 2018

Self Cite

View full text Add to dashboard Cite

Colon cancer is one of the most frequently occurring types of cancers in the world. Primary tumours are treated very efficiently, but the metastatic cases are known to have severe outcomes. Therefore, the aim of the present study was to obtain a greater understanding of the transformation of primary colon cancer cells into metastatic phenotypes. Small changes in protein expression provoke the metastatic phenotype transformation. More sensitive methods to detect small variations are required. A murine colon cancer cell line with metastatic characteristics in a very early phase was created in order to investigate the first steps of transformation using a murine liver metastasis model. The protein expression patterns of metastatic and non-metastatic cells were compared using the stable isotope labelling by amino acids in cell culture method in combination with mass spectrometry. Quantitative proteomics data indicated that nicotinamide adenine dinucleotide hydride (NADH) dehydrogenase complex I was overexpressed in metastatic cells with respect to non-metastatic cells. Since the NADH dehydrogenase complex catalyses the oxidation of NADH to NAD+, the functionality of the complex was studied by measuring the amount of NADH. The results revealed that metastatic cells accumulate more NADH and reactive oxygen species. In addition, the mitochondrial membrane potential of metastatic cells was lower than that of non-metastatic cells, indicating that the activity of NADH dehydrogenase and the mitochondrial oxidative chain were decreased in metastatic cells. During the incipient transformation of primary cancer cells, NADH dehydrogenase complex I was overexpressed but then became inactive due to the Warburg effect, which inhibits mitochondrial activity. In the first step of transformation, the high energy demand required in an adverse environment is fulfilled by overexpressing components of the respiratory chain, a fact that should be considered for future anti-metastatic therapies.

show abstract

Section: Methodsmentioning

confidence: 99%

NADH dehydrogenase complex�I is overexpressed in incipient metastatic murine colon cancer cells

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…Eluted immunecomplexes were run in two parallel lanes of a precast NuPAGE 4–12% Bis-Tris Gel (Invitrogen) and visualized with Colloidal Blue (Invitrogen). Both gel lanes were separately cut into slices and subjected to in-gel trypsinization (Promega) as previously described 53 . Briefly, gel slices were subjected to reduction with 10 mM DTT, alkylation by 55 mM chloroacetamide and protein digestion by incubating with trypsin overnight at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

“…Enrichment of phosphopeptides was performed as previously described 53 54 . 5 mm diameter titansphere beads (GL Sciences) were mixed with the equilibration buffer (50 mg/ml DHB in 80%ACN/1%TFA) 1:1 ratio (w/w) and incubated for 30 min at room temperature whereas vacuum-dried samples were adjusted to 60% ACN/1% TFA.…”

Section: Methodsmentioning

confidence: 99%

Changes in Gab2 phosphorylation and interaction partners in response to interleukin (IL)-2 stimulation in T-lymphocytes

Osinalde

Sanchez-Quiles

Blagoev

et al. 2016

Sci Rep

Self Cite

View full text Add to dashboard Cite

Interleukin-2 (IL-2) stimulation results in T-cell growth as a consequence of activation of highly sophisticated and fine-tuned signaling pathways. Despite lacking intrinsic enzymatic activity, scaffold proteins such as Gab2, play a pivotal role in IL-2-triggered signal transduction integrating, diversifying and amplifying the signal by serving as a platform for the assembly of effectors proteins. Traditionally, Gab2-mediated protein recruitment was believed to solely depend on cytokine-induced phosphotyrosine moieties. At present, phosphorylation on serine/threonine residues is also emerging as a key mediator of Gab2-dependent signal regulation. Despite its relevance, IL-2-triggered regulation on Gab2 phosphorylation is yet poorly understood. Combining antibody- and TiO2-based enrichment of the scaffold protein with SILAC quantitative mass spectrometry we disclose the prominent regulation IL-2 exerts on Gab2 serine/threonine phosphorylation by showing that at least 18 serines and 1 threonine, including previously non-reported ones, become phosphorylated in response to cytokine stimulation. Additionally, we decipher the interactome of the docking protein in resting and cytokine-treated T-lymphocytes and besides well-known Gab2 interactors we discover three novel cytokine-inducible Gab2-binding proteins. Thus, our data provide novel insights and a wealth of candidates for future studies that will shed light into the role of Gab2 in IL-2-initiated signal transduction.

show abstract

“…For that, the gels were cut into the following slices: slice A1 from ~15 kDa to ~25 kDa; slice A2 from ~30 kDa to 50 kDa; slice B from 50 kDa to ~130 kDa and slice C from ~140 kDa to up to the gel. These 4 slices were then subjected to in-gel trypsin digestion as described previously (62). In brief, proteins were first reduced with DTT and then, alkylated with chloroacetamide.…”

Section: In-gel Trypsin Digestion and Peptide Extraction-mentioning

confidence: 99%

The ubiquitin ligase Ariadne-1 regulates NSF for neurotransmitter release

Ramirez

Morales

Osinalde

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Ariadne-1 (Ari-1) is an essential E3 ubiquitinligase whose neuronal substrates are yet to be identified. We have used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics to identify putative Ari-1 substrates in Drosophila heads. Sixteen candidates met the established criteria. Amongst those, we identified Comatose (Comt), the homologue of the Nethylmaleimide sensitive factor (NSF). Using an in vivo GFP pulldown approach, we validate Comt/NSF to be an ubiquitination substrate of Ari-1 in fly neurons. The interaction results in the monoubiquitination of Comt/NSF. We also report that Ari-1 loss of function mutants display a lower rate of spontaneous neurotransmitter release due to failures at the pre-synaptic side. By contrast, evoked release in Ari-1 mutants is enhanced in a Ca 2+ dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. The distinct Ari-1 mutant phenotypes in spontaneous versus evoked release indicate that NSF activity discriminates the two corresponding protein ensembles that mediate each mode of release. Our results, thus, provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination.Neurotransmitter release is mediated by a set of protein-protein interactions that include the Nethylmaleimide sensitive factor (NSF), soluble NSF attachment proteins (SNAPs) and SNAP receptors (SNAREs) (1). These proteins assemble into a tripartite complex in order to elicit synaptic vesicle fusion, which is formed by one synaptic vesicle membrane SNARE protein (v-SNARE), Synaptobrevin, and two plasma membrane SNARE proteins (t-SNAREs), Syntaxin and the 25-kDa synaptosome-associated protein (2). Following vesicle fusion, the tripartite SNARE complex disassembles by the activities of NSF and SNAPs. Free t-SNAREs from the plasma membrane can then participate in new priming reactions, while the v-SNAREs can be incorporated into recycled synaptic vesicles (3). These interactions, also routinely used for intracellular vesicle trafficking in all cell types, are conserved across species (4), including Drosophila (5).Deviations on the rate of neurotransmitter release are at the origin of multiple neural

show abstract

SILAC-based quantification of changes in protein tyrosine phosphorylation induced by Interleukin-2 (IL-2) and IL-15 in T-lymphocytes

Cited by 12 publications

References 8 publications

NADH dehydrogenase complex�I is overexpressed in incipient metastatic murine colon cancer cells

NADH dehydrogenase complex�I is overexpressed in incipient metastatic murine colon cancer cells

Changes in Gab2 phosphorylation and interaction partners in response to interleukin (IL)-2 stimulation in T-lymphocytes

The ubiquitin ligase Ariadne-1 regulates NSF for neurotransmitter release

Contact Info

Product

Resources

About