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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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Cellular effects of tyrosine mutations.Mutations of ADAP tyrosines alter adhesion and migration of Jurkat T cells. A, B Adhesion assays of Jurkat cells on plates coated with fibronectin (A) or ICAM-1 (B) after overexpression of ADAP (wild type) or tyrosine to phenylalanine mutations at indicated positions. White bars: number of adhering cells without stimulation. Black bars: number of adhering cells after stimulation of cells with OKT3. C Migration of Jurkat cells through a transwell chamber in response to SDF-1. A-C: Average of absolute cell numbers from three experiments ± SD. “*” indicates significant deviation from stimulated ADAP (wild type) determined with Student's t-test.
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pone-0011708-g002: Cellular effects of tyrosine mutations.Mutations of ADAP tyrosines alter adhesion and migration of Jurkat T cells. A, B Adhesion assays of Jurkat cells on plates coated with fibronectin (A) or ICAM-1 (B) after overexpression of ADAP (wild type) or tyrosine to phenylalanine mutations at indicated positions. White bars: number of adhering cells without stimulation. Black bars: number of adhering cells after stimulation of cells with OKT3. C Migration of Jurkat cells through a transwell chamber in response to SDF-1. A-C: Average of absolute cell numbers from three experiments ± SD. “*” indicates significant deviation from stimulated ADAP (wild type) determined with Student's t-test.

Mentions: To assess the functional importance of the identified phosphorylation sites, we created a set of single tyrosine to phenylalanine mutations in the context of full length ADAP, namely at positions 559, 571, 595, 625, 651, 755, 771, and 780. In addition, we prepared the double mutant Y595F/Y651F that was previously utilized by Geng et al. [11] to overcome a possible functional redundancy of the YDDV motifs. The individual constructs were overexpressed in Jurkat cells and tested for their ability to modulate integrin-dependent processes. Overexpression of wild type ADAP increases TCR-triggered LFA-1 adhesion to ICAM-1 about twofold as previously reported [13], [20] (Fig. 2B). This increase was statistically significant (p<0.05 in an independent Student's t-test). Mutating tyrosines 755, 771 or 780 abolished the adhesion promoting effect of ADAP overexpression, while T cells overexpressing ADAP with single mutations at positions 595 or 651 were indistinguishable from cells transfected with wild type ADAP. However, when both sites were mutated, ADAP failed to increase adhesiveness in agreement with previous reports [13]. Adhesion to fibronectin (Fig. 2A) showed comparable effects for all mutations with the exception of Y559F, which showed signal attenuation in this assay, while it displayed wild-type behavior for ICAM-1.


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)

Cellular effects of tyrosine mutations.Mutations of ADAP tyrosines alter adhesion and migration of Jurkat T cells. A, B Adhesion assays of Jurkat cells on plates coated with fibronectin (A) or ICAM-1 (B) after overexpression of ADAP (wild type) or tyrosine to phenylalanine mutations at indicated positions. White bars: number of adhering cells without stimulation. Black bars: number of adhering cells after stimulation of cells with OKT3. C Migration of Jurkat cells through a transwell chamber in response to SDF-1. A-C: Average of absolute cell numbers from three experiments ± SD. “*” indicates significant deviation from stimulated ADAP (wild type) determined with Student's t-test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908683&req=5

pone-0011708-g002: Cellular effects of tyrosine mutations.Mutations of ADAP tyrosines alter adhesion and migration of Jurkat T cells. A, B Adhesion assays of Jurkat cells on plates coated with fibronectin (A) or ICAM-1 (B) after overexpression of ADAP (wild type) or tyrosine to phenylalanine mutations at indicated positions. White bars: number of adhering cells without stimulation. Black bars: number of adhering cells after stimulation of cells with OKT3. C Migration of Jurkat cells through a transwell chamber in response to SDF-1. A-C: Average of absolute cell numbers from three experiments ± SD. “*” indicates significant deviation from stimulated ADAP (wild type) determined with Student's t-test.
Mentions: To assess the functional importance of the identified phosphorylation sites, we created a set of single tyrosine to phenylalanine mutations in the context of full length ADAP, namely at positions 559, 571, 595, 625, 651, 755, 771, and 780. In addition, we prepared the double mutant Y595F/Y651F that was previously utilized by Geng et al. [11] to overcome a possible functional redundancy of the YDDV motifs. The individual constructs were overexpressed in Jurkat cells and tested for their ability to modulate integrin-dependent processes. Overexpression of wild type ADAP increases TCR-triggered LFA-1 adhesion to ICAM-1 about twofold as previously reported [13], [20] (Fig. 2B). This increase was statistically significant (p<0.05 in an independent Student's t-test). Mutating tyrosines 755, 771 or 780 abolished the adhesion promoting effect of ADAP overexpression, while T cells overexpressing ADAP with single mutations at positions 595 or 651 were indistinguishable from cells transfected with wild type ADAP. However, when both sites were mutated, ADAP failed to increase adhesiveness in agreement with previous reports [13]. Adhesion to fibronectin (Fig. 2A) showed comparable effects for all mutations with the exception of Y559F, which showed signal attenuation in this assay, while it displayed wild-type behavior for ICAM-1.

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