Quantitative phosphoproteome analysis unveils LAT as a modulator of CD3ζ and ZAP-70 tyrosine phosphorylation.

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The plasma membrane scaffolding protein LAT acts as a central organizer of the TCR signaling machinery to activate many functional pathways. LAT-deficient mice develop an autoimmune syndrome but the mechanism of this pathology is unknown. In this work we have compared global dynamics of TCR signaling by MS-based quantitative phosphoproteomics in LAT-sufficient and LAT-defective Jurkat T cells. Surprisingly, we found that many TCR-induced phosphorylation events persist in the absence of LAT, despite ERK and PLCγ1 phosphorylation being repressed. Most importantly, the absence of LAT resulted in augmented and persistent tyrosine phosphorylation of CD3ζ and ZAP70. This indicates that LAT signaling hub is also implicated in negative feedback signals to modulate upstream phosphorylation events. Phosphorylation kinetics data resulting from this investigation is documented in a database (phosphoTCR) accessible online. The MS data have been deposited to the ProteomeXchange with identifier PXD000341.", "claims": [{"id": "claim-method", "statement": "To determine a threshold above or below which a change in phosphopeptide abundance could be considered statistically significant, we computed a global P-value for fold changes from two data sets obtained from cells independently stimulated in identical conditions (File S1, Figures S4–S6).", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:24204825", "locator": "sections[13] \"LAT-dependent TCR-induced Cellular Phosphorylations\" paragraph 2", "quote": "To determine a threshold above or below which a change in phosphopeptide abundance could be considered statistically significant, we computed a global P-value for fold changes from two data sets obtained from cells independently stimulated in identical conditions (File S1, Figures S4–S6)."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [18] with the dataset identifier PXD000341.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:24204825", "locator": "sections[11] \"MS Raw Data\" paragraph 2", "quote": "The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [18] with the dataset identifier PXD000341."}], "state": "complete"}, {"id": "claim-target-result", "statement": "In LAT-deficient Jurkat T cells, many TCR-induced phosphorylation events persist despite repression of ERK and PLCγ1 phosphorylation, while tyrosine phosphorylation of CD3ζ and ZAP70 becomes augmented and persistent.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:24204825", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Most importantly, the absence of LAT resulted in augmented and persistent tyrosine phosphorylation of CD3ζ and ZAP70."}, {"asset": "source:pmid:24204825", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Phosphorylation kinetics data resulting from this investigation is documented in a database (phosphoTCR) accessible online."}, {"asset": "source:pmid:24204825", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "The MS data have been deposited to the ProteomeXchange with identifier PXD000341."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": "In LAT-deficient Jurkat T cells, many TCR-induced phosphorylation events persist despite repression of ERK and PLCγ1 phosphorylation, while tyrosine phosphorylation of CD3ζ and ZAP70 becomes augmented and persistent.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:24204825"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. 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