Leibniz-Institut fur Molekulare Pharmakologie, Robert-Rossle-Str. 10, Berlin, Germany.
J Proteome Res. 2010 Aug 6;9(8):4113-22. doi: 10.1021/pr1003054.
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.
免疫衔接蛋白 ADAP（黏附和脱颗粒促进衔接蛋白）在 T 细胞刺激后整合素依赖性迁移和黏附过程中发挥重要作用。ADAP 在 T 细胞受体（TCR）或趋化因子受体刺激期间经历多次磷酸化事件。对于蛋白复合物形成和细胞黏附调节的个别磷酸酪氨酸的作用仍存在争议。在这里，我们使用肽下拉测定和定量质谱来鉴定特异性磷酸化 ADAP 序列的相互作用伙伴。磷酸酪氨酸肽基序覆盖 Y595、Y625 和 Y771 以及相应的非磷酸化序列被共价偶联到琼脂糖珠上，并与 Jurkat T 细胞裂解物孵育。为了在磷酸化特异性和非特异性蛋白相互作用之间进行明确区分，我们采用了两种不同的同位素标记技术：稳定同位素标记的细胞培养物中的氨基酸（SILAC）和酶（18）O 标记，两者均与高分辨率质谱联用。除了先前已知的 ADAP 与 SLP76 基于 SH2 结构域的相互作用外，我们还鉴定了新的 ADAP 相互作用伙伴，如 Ras GTP 酶激活蛋白，它们属于更大的 TCR 近端信号复合物。结果表明，两种同位素标记技术都非常适合区分磷酸化特异性肽-蛋白相互作用与背景。
