Figure 1.
Sequence features of ADAP and three dimensional domain structures.
A Schematic overview of ADAP primary structure indicating all tyrosine sequences, structured domains, binding partners, and major sequence features. B Three-dimensional representations of ADAP domains hSH3N (left, reduced variant, PDB ID 2GTJ) and hSH3C (right, PDB ID 1RI9). The surface is shown in transparent grey, secondary structure features are colored in red, green, and yellow. Residues that were found to be phosphorylated are shown with side chains in blue.
Table 1.
Overview of phosphorylation data on all tyrosine and selected serine and threonine residues.
Figure 2.
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.
Figure 3.
Overview of SILAC based peptide binding assay. In our case, the ADAP peptide (586–600) was used either in its non-phosphorylated or tyrosine-phosphorylated form. Labeled or unlabeled Jurkat proteins were incubated with immobilized peptides on agarose beads. Bound proteins were separated by SDS-PAGE and identified by LC-MS/MS.
Table 2.
Enriched proteins after binding to immobilized phosphorylated compared to non-phosphorylated ADAP peptide around Y595 and MS analysis.
Figure 4.
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.
Figure 5.
Kinase dependency of ADAP interactions.
Analysis of phospho-dependent interactions in a modified Y2H system. Diploid yeast colonies (SD2-agar, top) expressing indicated bait, prey, and kinase constructs were assayed for growth on SD4-agar (bottom). Y2H interaction between ADAP-C and VASP is independent of Fyn kinase. In case of the SLP-76 (fl), Fyn SH2 (aa 151-246) and Nck1/2 (fl) preys, growth on SD4 is strictly dependent on the presence of active Fyn, indicating direct, phospho-dependent protein interactions.
Figure 6.
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.