Identification of phosphorylation-dependent interaction partners of the adapter protein ADAP using quantitative mass spectrometry: SILAC vs (18)O-labeling.
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.
Lange
Sabine
S
Leibniz-Institut fur Molekulare Pharmakologie, Robert-Rossle-Str. 10, Berlin, Germany.
Sylvester
Marc
M
Schümann
Michael
M
Freund
Christian
C
Krause
Eberhard
E
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000276:immunology; D008958:Models, Molecular; D010103:Oxygen Isotopes / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
D048868:Adaptor Proteins, Signal Transducing; D000596:Amino Acids; C106957:FYB1 protein, human; D010103:Oxygen Isotopes; D019000:Phosphotyrosine