Quantitative proteomics analysis of signalosome dynamics in primary T cells identifies the surface receptor CD6 as a Lat adaptor–independent TCR signaling hub

Roncagalli, Romain; Hauri, Simon; Fiore, Frédéric Di; Liang, Yinming; Chen, Zhi; Sansoni, Amandine; Kanduri, Kartiek; R, Joly; Malzac, Aurélie; Lähdesmäki, Harri; Lahesmaa, Riitta; Yamasaki, Sho; Saito, Takashi; Malissen, Marie; Aebersold, Ruedi; Gstaiger, Matthias; Malissen, Bernard

doi:10.1038/ni.2843

Cited by 111 publications

(154 citation statements)

References 53 publications

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“…In contrast, upon TCR stimulation, complex formation of ADAP and ZAP70 is not solely dependent on this single tyrosine because a larger TCR-associated complex is maintained in our experiment that contains both molecules (supplemental Fig. S5) (49). An indirect association of ADAP-Y571F with ZAP70 after TCR triggering is also supported by the finding that the other major tyrosine phosphorylation sites of ADAP are not recognized by ZAP70 in pull-down experiments (18,22,56).…”

Section: Discussionsupporting

confidence: 63%

“…Using either Flag-tagged wild type or Y571F mutant of ADAP the presence of ZAP70, LAT, PLC␥1, and SLP76 was verified as previously described for the ZAP70, LAT or SLP76 interactome (supplemental Fig. S5) (49). In contrast to TCR stimulation it was previously shown that this supramolecular assembly is not formed upon chemokine stimulation (39).…”

Section: The Adap and Zap70 Interaction Is Inducible In A Cellularmentioning

confidence: 99%

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Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration

Kuropka

Witte

Sticht

et al. 2015

Molecular & Cellular Proteomics

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Stimulation of T cells leads to distinct changes of theirT cell migration and the establishment of productive T cell/APC interactions are regulated by the activity of integrins. In resting T cells, integrins are expressed in an inactive state that adopts a conformation with low affinity for their ligands. Members of the intercellular adhesion molecule family (ICAM 1-5) are the physiological ligands of lymphocyte functionassociated antigen 1 (LFA-1, ␣L␤2-integrin) whereas vascular cell adhesion molecule (VCAM) and fibronectin are the ligands for the ␤1-integrin very late antigen 4 (VLA-4) (1, 2). Triggering of the T cell receptor (TCR) by peptide-major histocompati-

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Section: Discussionsupporting

confidence: 63%

Section: The Adap and Zap70 Interaction Is Inducible In A Cellularmentioning

confidence: 99%

Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration

Kuropka

Witte

Sticht

et al. 2015

Molecular & Cellular Proteomics

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“…Using homologous recombination in Escherichia coli (30), a chloramphenicolresistance gene bracketed by BspEI and SalI sites was inserted at the 39 end of the Pag1-coding sequence found in exon 9. The chloramphenicolresistance gene was excised using BspEI and SalI digestion and replaced by a XmaI-SalI fragment corresponding to a one-STrEP tag (OST)-(Stop) 2loxP-tACE-CRE-PGK-gb2-neo-loxP cassette (29). Finally, the targeting construct was abutted to a thymidine kinase expression cassette and linearized with FseI.…”

Section: Pag1mentioning

confidence: 99%

“…Affinity purification coupled with mass spectrometry (AP-MS) allows highly sensitive analysis of the dynamics of noncovalent protein complexes. We developed mice that bear a genetic tag that permits AP-MS analysis of protein complexes isolated from primary CD4 + T cells at different time points following T cell activation (29). Considering that this approach provided a systems-level understanding of the TCR signal-transduction network and identified proteins that have not been observed in the context of TCR signaling, to our knowledge, we used it in the current study to decipher the dynamics of the PAG signalosome in thymocytes and CD4 + T cells.…”

Section: T He Recognition Of Peptide-mhc (Pmhc) Ligands By T Cells Anmentioning

confidence: 99%

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Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells

Reginald

Chaoui

Roncagalli

et al. 2015

The Journal of Immunology

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The protein tyrosine kinase LCK plays a key role in TCR signaling, and its activity is dynamically controlled by the tyrosine kinase C-terminal Src kinase (CSK) and the tyrosine phosphatase CD45. CSK is brought in contiguity to LCK via binding to a transmembrane adaptor known as phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG). The lack of a blatant phenotype in PAG-deficient mice has impeded our understanding of the mechanisms through which PAG exerts its negative-regulatory role in TCR signaling. We used quantitative mass spectrometry and both thymocytes and CD4+ T cells from mice in which a tag for affinity purification was knocked in the gene coding for PAG to determine the composition and dynamics of the multiprotein complexes that are found around PAG over 5 min of activation. Most of the high-confidence interactions that we observed were previously unknown. Using phosphoproteomic analysis, PAG showed low levels of tyrosine phosphorylation in resting primary mouse CD4+ T cells; the levels of tyrosine phosphorylation increased and reached a maximum 2 min after stimulation. Analysis of the dynamics of association of the protein tyrosine phosphatase PTPN22 and lipid phosphatase SHIP-1 with PAG following T cell activation suggests that both cooperate with CSK to terminate T cell activation. Our findings provide a model of the role for PAG in mouse primary CD4+ T cells that is consistent with recent phosphoproteomic studies of the Jurkat T cell line but difficult to reconcile with former biochemical studies indicating that PAG is constitutively phosphorylated in resting T cells and rapidly dephosphorylated once the TCR is engaged.

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Attenuation of Murine Collagen‐Induced Arthritis by Targeting CD6

Ruth

Rasmussen

et al. 2020

Arthritis & Rheumatology

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Objective CD6 is an important regulator of T cell function that interacts with the ligands CD166 and CD318. To further clarify the significance of CD6 in rheumatoid arthritis (RA), we examined the effects of targeting CD6 in the mouse model of collagen‐induced arthritis (CIA), using CD6‐knockout (CD6‐KO) mice and CD6‐humanized mice that express human CD6 in lieu of mouse CD6 on their T cells. Methods We immunized wild‐type (WT) and CD6 gene–KO mice with a collagen emulsion to induce CIA. For treatment studies using CD6‐humanized mice, mice were immunized similarly and a mouse anti‐human CD6 IgG (UMCD6) or control IgG was injected on days 7, 14, and 21. Joint tissues were evaluated for tissue damage, leukocyte infiltration, and local inflammatory cytokine production. Collagen‐specific Th1, Th9, and Th17 responses and serum levels of collagen‐specific IgG subclasses were also evaluated in WT and CD6‐KO mice with CIA. Results The absence of CD6 reduced 1) collagen‐specific Th9 and Th17, but not Th1 responses, 2) the levels of many proinflammatory joint cytokines, and 3) serum levels of collagen‐reactive total IgG and IgG1, but not IgG2a and IgG3. Joint homogenate hemoglobin content was significantly reduced in CD6‐KO mice with CIA compared to WT mice with CIA (P < 0.05) (reduced angiogenesis). Moreover, treating CD6‐humanized mice with mouse anti‐human CD6 monoclonal antibody was similarly effective in reducing joint inflammation in CIA. Conclusion Taken together, these data suggest that interaction of CD6 with its ligands is important for the perpetuation of CIA and other inflammatory arthritides that are T cell driven.

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Quantitative proteomics analysis of signalosome dynamics in primary T cells identifies the surface receptor CD6 as a Lat adaptor–independent TCR signaling hub

Cited by 111 publications

References 53 publications

Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration

Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration

Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells

Attenuation of Murine Collagen‐Induced Arthritis by Targeting CD6

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