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

Abstract

Stimulation of T cells leads to distinct changes of their adhesive and migratory properties. Signal propagation from activated receptors to integrins depends on scaffolding proteins such as the adhesion and degranulation promoting adaptor protein (ADAP)(1). Here we have comprehensively investigated the phosphotyrosine interactome of ADAP in T cells and define known and novel interaction partners of functional relevance. While most phosphosites reside in unstructured regions of the protein, thereby defining classical SH2 domain interaction sites for master regulators of T cell signaling such as SLP76, Fyn-kinase, and NCK, other binding events depend on structural context. Interaction proteomics using different ADAP constructs comprising most of the known phosphotyrosine motifs as well as the structured domains confirm that a distinct set of proteins is attracted by pY571 of ADAP, including the ζ-chain-associated protein kinase of 70 kDa (ZAP70). The interaction of ADAP and ZAP70 is inducible upon stimulation either of the T cell receptor (TCR) or by chemokine. NMR spectroscopy reveals that the N-terminal SH2 domains within a ZAP70-tandem-SH2 construct is the major site of interaction with phosphorylated ADAP-hSH3(N) and microscale thermophoresis (MST) indicates an intermediate binding affinity (Kd = 2.3 μm). Interestingly, although T cell receptor dependent events such as T cell/antigen presenting cell (APC) conjugate formation and adhesion are not affected by mutation of Y571, migration of T cells along a chemokine gradient is compromised. Thus, although most phospho-sites in ADAP are linked to T cell receptor related functions we have identified a unique phosphotyrosine that is solely required for chemokine induced T cell behavior.

Fig. 1.

Phosphotyrosine sites of ADAP and interaction partners. A, Schematic overview of the ADAP primary structure indicating interaction partners of different phosphotyrosine sites identified by peptide pull-down approaches (22, 23). Black arrows show SH2-pTyr interaction sites. White arrows show interactions dependent on proline rich sequences (PRS). B, Fyn kinase catalyzed in vitro phosphorylation of full-length ADAP. Identification and relative quantification of phosphorylation degrees of individual tyrosine residues was obtained by mass spectrometry. Phosphorylation degrees were estimated by comparing relative MS peak intensities of the corresponding peptide/phosphopeptide pairs as described (33, 34).

Fig. 2.

Proteomics approach for identification of ADAP binding partners. A, Proteins are metabolically labeled in cell culture using “light” or “heavy” lysine and arginine (SILAC). Phosphorylated and nonphosphorylated bait proteins (GST-ADAP^486–783 or GST-hSH3^N) were incubated with the heavy and light labeled Jurkat T cell lysate, respectively. After combining the samples, proteins were separated by SDS-PAGE. After tryptic in-gel-digestion, peptides were analyzed by nanoLC-MS/MS and proteins were identified and quantified using the MaxQuant software. B–E, Scatter plots of heavy/light (forward) and light/heavy (reverse) isotopic ratios of identified proteins from quantitative pull-down experiments using phosphorylated and nonphosphorylated baits. Proteins that showed phosphorylation-dependent enrichment are highlighted. B, GST-ADAP^486–783 incubated with Jurkat T cell lysate. For clarity reasons, only enriched proteins that contain SH2-domains are highlighted. C, GST-hSH3^N incubated with Jurkat T cell lysate. D, GST-hSH3^N incubated with Jurkat T cell lysate under oxidizing conditions. E, GST-hSH3^N incubated with primary human T cell lysate (¹⁸O/¹⁶O labeling). F, Summary of potential phosphorylation-dependent interaction partners identified from pull-down experiments shown in (B–E). Enriched proteins are indicated in red whereas gray color indicates no enrichment/identification. Ox. = oxidizing conditions. Prim. = primary human T cell lysates.

Fig. 3.

Binding studies of the ADAP-ZAP70 interaction. A, Overlay of a region of ¹H-¹⁵N-HSQC spectra reflecting changes in ¹⁵N-labeled hSH3^N upon ZAP70 binding. The overlay of phosphorylated hSH3^N in the absence (dark blue) and presence (red) of equimolar amounts of ZAP70-tSH2 is shown in the left panel whereas the corresponding control experiment with nonphosphorylated hSH3^N is shown on the right (light blue in the absence and red in the presence of ZAP70-tSH2). Resonances of residues displaying significant line broadening are indicated by amino acid type and number. B, Three-dimensional structure representation of the hSH3^N domain (PDB ID: 2GTJ) indicating the residues that are affected strongly by ZAP70-tSH2 binding. Resonances of phosphorylated hSH3^N clearly reduced in intensity upon addition of ZAP70-tSH2 are colored orange (residues L515, A516, S573, L574). Y571 is colored in red. C, Superimposed regions of ¹H-¹⁵N-HSQC spectra reflecting changes in ¹⁵N-labeled ZAP70-tSH2 upon hSH3^N binding. The overlay of ZAP70-tSH2 is shown for spectra taken in the absence (red) and presence of equimolar amounts of phosphorylated hSH3^N (dark blue, left panel) or unphosphorylated hSH3^N (light blue, right panel). D, Three-dimensional structure representation of ZAP70-tSH2 domain (PDB ID: 1M61) indicating the two residues (R17, L40) that are affected strongly by phosphorylated hSH3^N binding. E, Microscale thermophoresis data showing the binding of Y571-phosphorylated Snap-hSH3^N (25 nm) (solid dots) and the corresponding nonphosphorylated domain (25 nm) (triangles) upon addition of increasing concentrations of ZAP70-tSH2 (41 nm to 44 μm). The affinity (K_d = 2.26 ± 0.21 μm) was determined by fitting the data (representing the mean ± S.D. from three independent experiments) according to the law of mass action. The graph on the right exemplarily shows the thermophoretic running behavior of Y571-phosphorylated Snap-hSH3^N upon ZAP70-tSH2 binding at 20% MST power.

Fig. 4.

Co-immunoprecipitation studies of ADAP and ZAP70. A, Jurkat T cells were either left untreated, stimulated with anti-CD3 antibodies (TCR) or CXCL12 for the indicated time points. Lysates were used for immunoprecipitation using anti-ZAP70 antibody. Precipitates were analyzed by Western blotting with the indicated antibodies. WL denotes whole lysates. B, Schematic representation of the suppression/re-expression vectors used in this study. C, Jurkat T cells were transfected with suppression/re-expression constructs which do not suppress endogenous ADAP (shC) or reduce the protein level of ADAP (shADAP) and re-express a Flag-tagged shRNA-resistant wild type form of ADAP (shADAP-WT) or its Y571F mutant (shADAP-Y571F). At 48 h post-transfection, whole-cell extracts were harvested, separated by SDS-PAGE, transferred, and blotted for ADAP, Flag, and β-actin (loading control). The suppression of ADAP, re-expressed Flag-tagged WT, and Y571 mutant were quantified using the ImageQuant software to determine the endogenous ADAP band intensity ratio. D, E, Jurkat T cells were transfected as described in (C) and stimulated with CXCL12 (D) or anti-CD3 antibodies (E) for the indicated time points. Lysates were used for immunoprecipitation using an anti-Flag antibody. Precipitates were analyzed by Western blotting using the indicated antibodies. TCR-stimulated Jurkat T cell lysate served as positive control.

Fig. 5.

The Y571 mutant of ADAP does not affect TCR-mediated adhesion but chemokine-directed T cell migration. A–D, Jurkat T cells were transfected as described in Fig. 4B and 4C. A, Cells were analyzed for their ability to form conjugates with DDAO-S.E. (red)-stained Raji B cells that were loaded with (+) or without (-) superantigen (SA). The percentage of conjugates was assessed by flow cytometry. B, Cells were analyzed for their ability to adhere to ICAM-1-coated wells in a resting state or upon stimulation with anti-CD3 antibody (TCR), phorbol myristate acetate (PMA), or MnCl₂. Adherent cells were counted and calculated as percentage of input (2 × 10⁵ cells). C, Cells were analyzed for their ability to adhere to ICAM-1-coated wells in a resting state or upon stimulation with CXCL12 or MnCl₂. Adherent cells were counted and calculated as a percentage of input (2 × 10⁵ cells). D, Cells were placed into the upper part of transwell chambers coated with ICAM-1. Subsequently, cells were incubated in the absence or presence of CXCL12 in the lower chamber for 2 h. Migrated T cells (lower chamber) were counted and calculated as percentage of input cell numbers. Data represent the mean ± S.D. of three independent experiments (*p ≤ 0.05).