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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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Direct binding of Nck to ADAP.Direct phosphorylation dependent interaction of ADAP and Nck SH2 in vitro. A Left: Purified ADAP (full length), ADAP-C, hSH3N or hSH3C were in vitro phosphorylated with Fyn as indicated. Subsequently, proteins were incubated with immobilized GST-Nck1 SH2. The washed GSH matrix was boiled in sample buffer, proteins separated by SDS-PAGE and stained with Coomassie. Band at approx. 24 kDa is GST as an impurity from GST-Nck SH2 preparation. Right: Protein flow through after pre-incubation (without Fyn) and subsequent incubation with GST-Nck1 SH2 to demonstrate protein stability and purity. B Phosphotyrosine detection after western blotting of same protein samples as in A. C Phosphotyrosine detection of protein samples after pre-incubation without or with Fyn as a control of phosphorylation by Fyn. hSH3N phosphorylation was not readily detected by the antibody used.
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pone-0011708-g006: Direct binding of Nck to ADAP.Direct phosphorylation dependent interaction of ADAP and Nck SH2 in vitro. A Left: Purified ADAP (full length), ADAP-C, hSH3N or hSH3C were in vitro phosphorylated with Fyn as indicated. Subsequently, proteins were incubated with immobilized GST-Nck1 SH2. The washed GSH matrix was boiled in sample buffer, proteins separated by SDS-PAGE and stained with Coomassie. Band at approx. 24 kDa is GST as an impurity from GST-Nck SH2 preparation. Right: Protein flow through after pre-incubation (without Fyn) and subsequent incubation with GST-Nck1 SH2 to demonstrate protein stability and purity. B Phosphotyrosine detection after western blotting of same protein samples as in A. C Phosphotyrosine detection of protein samples after pre-incubation without or with Fyn as a control of phosphorylation by Fyn. hSH3N phosphorylation was not readily detected by the antibody used.

Mentions: Phosphorylated SLP-76 is known to bind the SH2 domain of Nck [28], [29]. Nck-SH2 binding predictions (SMALI) [30], [31] indicated that a direct recognition of the YDDV motifs of ADAP is probable. To test this possibility, yeast two-hybrid (Y2H) interaction analysis of ADAP-C with Nck1/2 and other ADAP interaction candidates was performed in the absence and presence of active Fyn kinase (Fig. 5). As expected, the EVH1 domain of VASP interacted independently of Fyn with ADAP-C [32]. Both interactions known to require ADAP phosphorylation, i.e. ADAP-C with SLP-76 and Fyn SH2, were recapitulated with high specificity. Interestingly, in our assay Y2H signals for interaction with full length Nck1 and 2 were strictly dependent on the presence of Fyn kinase, indicating a direct, phosphorylation-dependent interaction of ADAP-C with Nck. Other candidate proteins such as PIK3R1, RasGAP, CRK or Gads were tested but no colony growth was observed (data not shown). The directness and phosphorylation dependency of the ADAP-Nck interaction was further probed by pulldown experiments with recombinantly expressed and purified protein. Purified ADAP (full length) and ADAP-C bound immobilized GST-Nck1/2-SH2 while the isolated hSH3 domains could not (Fig. 6A, lanes 1–8). In addition, binding between ADAP and Nck was strongly enhanced if ADAP had been phosphorylated in vitro by Fyn kinase (Fig. 6B, lanes 3 and 5). Phosphorylation of Fyn itself and ADAP constructs is shown in Fig. 6C.


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)

Direct binding of Nck to ADAP.Direct phosphorylation dependent interaction of ADAP and Nck SH2 in vitro. A Left: Purified ADAP (full length), ADAP-C, hSH3N or hSH3C were in vitro phosphorylated with Fyn as indicated. Subsequently, proteins were incubated with immobilized GST-Nck1 SH2. The washed GSH matrix was boiled in sample buffer, proteins separated by SDS-PAGE and stained with Coomassie. Band at approx. 24 kDa is GST as an impurity from GST-Nck SH2 preparation. Right: Protein flow through after pre-incubation (without Fyn) and subsequent incubation with GST-Nck1 SH2 to demonstrate protein stability and purity. B Phosphotyrosine detection after western blotting of same protein samples as in A. C Phosphotyrosine detection of protein samples after pre-incubation without or with Fyn as a control of phosphorylation by Fyn. hSH3N phosphorylation was not readily detected by the antibody used.
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Related In: Results  -  Collection

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pone-0011708-g006: Direct binding of Nck to ADAP.Direct phosphorylation dependent interaction of ADAP and Nck SH2 in vitro. A Left: Purified ADAP (full length), ADAP-C, hSH3N or hSH3C were in vitro phosphorylated with Fyn as indicated. Subsequently, proteins were incubated with immobilized GST-Nck1 SH2. The washed GSH matrix was boiled in sample buffer, proteins separated by SDS-PAGE and stained with Coomassie. Band at approx. 24 kDa is GST as an impurity from GST-Nck SH2 preparation. Right: Protein flow through after pre-incubation (without Fyn) and subsequent incubation with GST-Nck1 SH2 to demonstrate protein stability and purity. B Phosphotyrosine detection after western blotting of same protein samples as in A. C Phosphotyrosine detection of protein samples after pre-incubation without or with Fyn as a control of phosphorylation by Fyn. hSH3N phosphorylation was not readily detected by the antibody used.
Mentions: Phosphorylated SLP-76 is known to bind the SH2 domain of Nck [28], [29]. Nck-SH2 binding predictions (SMALI) [30], [31] indicated that a direct recognition of the YDDV motifs of ADAP is probable. To test this possibility, yeast two-hybrid (Y2H) interaction analysis of ADAP-C with Nck1/2 and other ADAP interaction candidates was performed in the absence and presence of active Fyn kinase (Fig. 5). As expected, the EVH1 domain of VASP interacted independently of Fyn with ADAP-C [32]. Both interactions known to require ADAP phosphorylation, i.e. ADAP-C with SLP-76 and Fyn SH2, were recapitulated with high specificity. Interestingly, in our assay Y2H signals for interaction with full length Nck1 and 2 were strictly dependent on the presence of Fyn kinase, indicating a direct, phosphorylation-dependent interaction of ADAP-C with Nck. Other candidate proteins such as PIK3R1, RasGAP, CRK or Gads were tested but no colony growth was observed (data not shown). The directness and phosphorylation dependency of the ADAP-Nck interaction was further probed by pulldown experiments with recombinantly expressed and purified protein. Purified ADAP (full length) and ADAP-C bound immobilized GST-Nck1/2-SH2 while the isolated hSH3 domains could not (Fig. 6A, lanes 1–8). In addition, binding between ADAP and Nck was strongly enhanced if ADAP had been phosphorylated in vitro by Fyn kinase (Fig. 6B, lanes 3 and 5). Phosphorylation of Fyn itself and ADAP constructs is shown in Fig. 6C.

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