Identification of Phosphorylation-Dependent Interaction Partners of the Adapter Protein ADAP using Quantitative Mass Spectrometry: SILAC vs ¹⁸O-Labeling

Abstract

The immune adapter protein ADAP (adhesion and degranulation promoting adapter protein) plays an important role in integrin-dependent migration and adhesion processes as a consequence of T cell stimulation. ADAP undergoes multiple phosphorylation events during T cell receptor (TCR) or chemokine receptor stimulation. The role of individual phosphotyrosines for protein complex formation and the regulation of cellular adhesion are still under debate. Here, we use peptide pull-down assays and quantitative mass spectrometry to identify interaction partners of site-specifically phosphorylated ADAP sequences. Phosphotyrosine peptide motifs covering Y595, Y625, and Y771 and the corresponding nonphosphorylated sequences were covalently coupled to agarose beads and incubated with Jurkat T cell lysates. For unambiguous differentiation between phosphorylation-specific and nonspecific protein interaction, we employed two different isotope labeling techniques: stable isotope labeling of amino acids in cell culture (SILAC) and enzymatic (18)O-labeling, both in combination with high-resolution mass spectrometry. In addition to previously known SH2 domain-based interactions of ADAP with SLP76, we identified novel ADAP interaction partners - such as the Ras GTPase activating protein - which belong to the larger TCR proximal signaling complex. The results show that both isotope labeling techniques are well suited for distinguishing phosphorylation-specific peptide-protein interactions from the background.

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

“…For example, because the surface residues of protein domains such as SH2 are less well conserved for regions outside the binding pocket, the ADAP hSH3 N domain surface geometry and charge distribution in the vicinity of Y571 might well be incompatible with the defined docking of certain SH2 domain containing proteins. In line with this argument and considering that we use a large excess of bait protein in our domain-based pull-down we do not enrich any of the other SH2 domain containing proteins when compared with previous phosphopeptide enrichments (18,22). Phosphorylated Y571 is predominantly recognized by the N-terminal SH2 (N-SH2) domain of ZAP70 and the residues displaying the largest signal reduction in our NMR studies (R17, L40) are identical to those that are line-broadened when ZAP70-tSH2 binds to the singly phosphorylated ITAM peptides pYNEL or pYDVL (37).…”

“…For pull-downs using 16 O/ 18 O-labeling, gel lanes corresponding to eluates from GST-hSH3 N and Y571-phosphorylated GST-hSH3 N were cut into 20 slices in a parallel fashion. Tryptic in-gel digestion and 16 O/ 18 O-labeling was performed as described (22). Both pull-down experiments (SILAC and 16 O/ 18 Olabeling) were performed at least twice with switched isotopic labeling (forward and reverse experiments).…”

“…Beside these well characterized interactions, several other SH2-domain containing binders were identified by pull-down approaches using phosphorylated peptide baits (Fig. 1A) (22,23). Most of the so far characterized SH2-pTyr interactions in ADAP are mapped to unstructured regions.…”

Multivalent Binding of Formin-binding Protein 21 (FBP21)-Tandem-WW Domains Fosters Protein Recognition in the Pre-spliceosome

“…Tryptic in-gel digestion of proteins and nano-LC-MS/MS experiments were performed as described previously (13). In brief, tryptic peptides were separated by a reversed-phase capillary liquid chromatography system (Eksigent 2D nanoflow LC, Axel Semrau GmbH) connected to an LTQ-Orbitrap XL mass spectrometer (Thermo Scientific).…”

SORLA regulates calpain‐dependent degradation of synapsin

“…LC-MS measurements were performed as described [44]. In brief, excised protein spots were cleavages was allowed for tryptic, chymotryptic, and Asp-N peptides, respectively.…”