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To examine the characteristics of the interaction of the Fc⑀RI␥ ITAM with the SH2 domains of p72 syk , the binding of an 125 I-labeled dual phosphorylated Fc⑀RI␥ ITAM-based peptide to the p72 syk SH2 domains was monitored utilizing a novel scintillation proximity based assay. The K d for this interaction, determined from the saturation binding isotherm, was 1.4 nM. This high affinity binding was reflected in the rapid rate of association for the peptide binding to the SH2 domains. Competition studies utilizing a soluble C-terminal SH2 domain knockout and N-terminal SH2 domain knockouts revealed that both domains contribute cooperatively to the high affinity binding. Unlabeled dual phosphorylated peptide competed with the 125 I-labeled peptide for binding to the dual p72 syk SH2 domains with an IC 50 value of 4.8 nM. Monophosphorylated 24-mer Fc⑀RI␥ ITAM peptides, and phosphotyrosine also competed for binding, but with substantially higher IC 50 values. This, and other data discussed, suggest that high affinity binding requires both tyrosine residues to be phosphorylated and that the preferred binding orientation of the ITAM is such that the N-terminal phosphotyrosine occupies the C-terminal SH2 domain and the C-terminal phosphotyrosine occupies the N-terminal SH2 domain. Src homology 2 (SH2)1 domains are regions of approximately 100 -120 amino acid residues present in a variety of proteins including tyrosine kinases, tyrosine phosphatases, phospholipases, and other signal transducing proteins (1-6). These domains bind with high affinity to tyrosine containing motifs in associating proteins, such as specific cytokine and immunoglobulin receptor subunits, adapter proteins, tyrosine kinases, and other signaling molecules such as STATs, when these motifs are phosphorylated by the action of tyrosine kinases (1-5, 7, 8). This allows recruitment of SH2 domain-containing signaling molecules and phosphotyrosine-containing signaling molecules into receptor-linked signal transduction assemblies (9 -13). The tyrosine-containing motifs are composed of phosphorylated tyrosine residues followed by 3-4 amino acids (e.g. pYXX(L/I)) which carry the sequence-specific information for SH2 recognition (14 -23). These motifs can occur in isolation or in tandem, thus can bind single SH2 domains (e.g. of src related tyrosine kinases) (4, 24 -26) or dual SH2 domains (e.g. of p70 zap and p72 syk ) (27-34). Certain antigen receptor subunits, such as the subunit of the T cell receptor (TCR), the Ig␣ and Ig␤ subunits of the B cell receptor and the ␤ and ␥ subunits of the high affinity IgE receptor (Fc⑀RI), contain tyrosine motifs in tandem and these have been termed immunoglobulin receptor tyrosine activation motifs (ITAMs) (24, 27, 32, 34 -36). In hematopoietic cell signaling, tyrosine-phosphorylated ITAMs have been shown to be critical for signaling interactions via their association with the SH2 domains of tyrosine kinases. For example, p70zap and the src-related kinases p59 fyn and p56 lck , appear to play a role in TCR-mediated T cell a...

This work demonstrates that reversible cyanoenone Michael acceptors, such as the tpAIMs and sAIMs, can be specifically tuned to regulate redox sensitive cysteine residues on key signaling molecules, an approach with significant promise for innovative drug development.

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