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Adhesion and degranulation promoting adapter protein (ADAP) is a central hub for phosphotyrosine-mediated interactions in T cells.

Sylvester M, Kliche S, Lange S, Geithner S, Klemm C, Schlosser A, Grossmann A, Stelzl U, Schraven B, Krause E, Freund C - PLoS ONE (2010)

Bottom Line: While three tyrosine phosphorylation sites have been characterized biochemically, the binding capabilities and associated functions of several other potential phosphotyrosine motifs remain unclear.Furthermore, using a peptide pulldown approach in combination with stable isotope labeling in cell culture (SILAC) we identified SLP-76, PLCgamma, PIK3R1, Nck, CRK, Gads, and RasGAP as phospho-dependent binding partners of a central YDDV motif of ADAP.The phosphorylation-dependent interaction between ADAP and Nck was confirmed by yeast two-hybrid analysis, immunoprecipitation and binary pulldown experiments, indicating that ADAP directly links integrins to modulators of the cytoskeleton independent of SLP-76.

View Article: PubMed Central - PubMed

Affiliation: Protein Engineering Group, Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany.

ABSTRACT
TCR stimulation leads to an increase in cellular adhesion among other outcomes. The adhesion and degranulation promoting adapter protein (ADAP) is known to be rapidly phosphorylated after T cell stimulation and relays the TCR signal to adhesion molecules of the integrin family. While three tyrosine phosphorylation sites have been characterized biochemically, the binding capabilities and associated functions of several other potential phosphotyrosine motifs remain unclear. Here, we utilize in vitro phosphorylation and mass spectrometry to map novel phosphotyrosine sites in the C-terminal part of human ADAP (486-783). Individual tyrosines were then mutated to phenylalanine and their relevance for cellular adhesion and migration was tested experimentally. Functionally important tyrosine residues include two sites within the folded hSH3 domains of ADAP and two at the C-terminus. Furthermore, using a peptide pulldown approach in combination with stable isotope labeling in cell culture (SILAC) we identified SLP-76, PLCgamma, PIK3R1, Nck, CRK, Gads, and RasGAP as phospho-dependent binding partners of a central YDDV motif of ADAP. The phosphorylation-dependent interaction between ADAP and Nck was confirmed by yeast two-hybrid analysis, immunoprecipitation and binary pulldown experiments, indicating that ADAP directly links integrins to modulators of the cytoskeleton independent of SLP-76.

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Nck coprecipitation with ADAP.Formation of a complex containing ADAP and Nck is increased after stimulation via TCR or chemokine receptor. Immunoprecipitation of ADAP from lysates of regular or SLP-76-deficient Jurkat T cells with a polyclonal antibody and detection of Nck after western blotting (upper panel). The antibody does not distinguish between Nck1 and Nck2. Detection of ADAP protein with a monoclonal antibody (center) and tyrosine phosphorylation (lower panel) on the same membrane. Data are representative for two experiments.
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pone-0011708-g004: Nck coprecipitation with ADAP.Formation of a complex containing ADAP and Nck is increased after stimulation via TCR or chemokine receptor. Immunoprecipitation of ADAP from lysates of regular or SLP-76-deficient Jurkat T cells with a polyclonal antibody and detection of Nck after western blotting (upper panel). The antibody does not distinguish between Nck1 and Nck2. Detection of ADAP protein with a monoclonal antibody (center) and tyrosine phosphorylation (lower panel) on the same membrane. Data are representative for two experiments.

Mentions: To test whether a complex between ADAP and Nck could be detected in cellular lysates, we performed immunoprecipitation experiments. Nck was found in a complex with immune precipitated ADAP from Jurkat T cells. The amount of coprecipitated Nck increased strongly after stimulation with OKT3 or SDF-1 (Fig. 4), in agreement with a stimulation-dependent phosphorylation of ADAP and a subsequent interaction with Nck. To further rule out the possibility that the interaction between ADAP and Nck is solely mediated by SLP-76, we performed experiments in SLP-76-deficient Jurkat cells. A small amount of ADAP-bound Nck was found in unstimulated Jurkat cells lacking SLP-76 while TCR-stimulation increased coprecipitation sharply in these cells. SDF-stimulation yielded the same amount of Nck in the absence or presence of SLP-76, indicating that chemokine stimulation renders the ADAP-Nck interaction independent of SLP-76 in both cell lines. Complex formation between Nck and ADAP followed the same tendency as ADAP phosphorylation, while the degree of Nck phosphorylation did not change significantly after stimulation (data not shown).


Adhesion and degranulation promoting adapter protein (ADAP) is a central hub for phosphotyrosine-mediated interactions in T cells.

Sylvester M, Kliche S, Lange S, Geithner S, Klemm C, Schlosser A, Grossmann A, Stelzl U, Schraven B, Krause E, Freund C - PLoS ONE (2010)

Nck coprecipitation with ADAP.Formation of a complex containing ADAP and Nck is increased after stimulation via TCR or chemokine receptor. Immunoprecipitation of ADAP from lysates of regular or SLP-76-deficient Jurkat T cells with a polyclonal antibody and detection of Nck after western blotting (upper panel). The antibody does not distinguish between Nck1 and Nck2. Detection of ADAP protein with a monoclonal antibody (center) and tyrosine phosphorylation (lower panel) on the same membrane. Data are representative for two experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908683&req=5

pone-0011708-g004: Nck coprecipitation with ADAP.Formation of a complex containing ADAP and Nck is increased after stimulation via TCR or chemokine receptor. Immunoprecipitation of ADAP from lysates of regular or SLP-76-deficient Jurkat T cells with a polyclonal antibody and detection of Nck after western blotting (upper panel). The antibody does not distinguish between Nck1 and Nck2. Detection of ADAP protein with a monoclonal antibody (center) and tyrosine phosphorylation (lower panel) on the same membrane. Data are representative for two experiments.
Mentions: To test whether a complex between ADAP and Nck could be detected in cellular lysates, we performed immunoprecipitation experiments. Nck was found in a complex with immune precipitated ADAP from Jurkat T cells. The amount of coprecipitated Nck increased strongly after stimulation with OKT3 or SDF-1 (Fig. 4), in agreement with a stimulation-dependent phosphorylation of ADAP and a subsequent interaction with Nck. To further rule out the possibility that the interaction between ADAP and Nck is solely mediated by SLP-76, we performed experiments in SLP-76-deficient Jurkat cells. A small amount of ADAP-bound Nck was found in unstimulated Jurkat cells lacking SLP-76 while TCR-stimulation increased coprecipitation sharply in these cells. SDF-stimulation yielded the same amount of Nck in the absence or presence of SLP-76, indicating that chemokine stimulation renders the ADAP-Nck interaction independent of SLP-76 in both cell lines. Complex formation between Nck and ADAP followed the same tendency as ADAP phosphorylation, while the degree of Nck phosphorylation did not change significantly after stimulation (data not shown).

Bottom Line: While three tyrosine phosphorylation sites have been characterized biochemically, the binding capabilities and associated functions of several other potential phosphotyrosine motifs remain unclear.Furthermore, using a peptide pulldown approach in combination with stable isotope labeling in cell culture (SILAC) we identified SLP-76, PLCgamma, PIK3R1, Nck, CRK, Gads, and RasGAP as phospho-dependent binding partners of a central YDDV motif of ADAP.The phosphorylation-dependent interaction between ADAP and Nck was confirmed by yeast two-hybrid analysis, immunoprecipitation and binary pulldown experiments, indicating that ADAP directly links integrins to modulators of the cytoskeleton independent of SLP-76.

View Article: PubMed Central - PubMed

Affiliation: Protein Engineering Group, Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany.

ABSTRACT
TCR stimulation leads to an increase in cellular adhesion among other outcomes. The adhesion and degranulation promoting adapter protein (ADAP) is known to be rapidly phosphorylated after T cell stimulation and relays the TCR signal to adhesion molecules of the integrin family. While three tyrosine phosphorylation sites have been characterized biochemically, the binding capabilities and associated functions of several other potential phosphotyrosine motifs remain unclear. Here, we utilize in vitro phosphorylation and mass spectrometry to map novel phosphotyrosine sites in the C-terminal part of human ADAP (486-783). Individual tyrosines were then mutated to phenylalanine and their relevance for cellular adhesion and migration was tested experimentally. Functionally important tyrosine residues include two sites within the folded hSH3 domains of ADAP and two at the C-terminus. Furthermore, using a peptide pulldown approach in combination with stable isotope labeling in cell culture (SILAC) we identified SLP-76, PLCgamma, PIK3R1, Nck, CRK, Gads, and RasGAP as phospho-dependent binding partners of a central YDDV motif of ADAP. The phosphorylation-dependent interaction between ADAP and Nck was confirmed by yeast two-hybrid analysis, immunoprecipitation and binary pulldown experiments, indicating that ADAP directly links integrins to modulators of the cytoskeleton independent of SLP-76.

Show MeSH
Related in: MedlinePlus