Abstract
Specific peptide ligand recognition by modular interaction domains is essential for the fidelity of information flow through the signal transduction networks that control cell behavior in response to extrinsic and intrinsic stimuli. Src homology 2 (SH2) domains recognize distinct phosphotyrosine peptide motifs, but the specific sites that are phosphorylated and the complement of available SH2 domains varies considerably in individual cell types. Such differences are the basis for a wide range of available protein interaction microstates from which signaling can evolve in highly divergent ways. This underlying complexity suggests the need to broadly map the signaling potential of systems as a prerequisite for understanding signaling in specific cell types as well as various pathologies that involve signal transduction such as cancer, developmental defects and metabolic disorders. This report describes interactions between SH2 domains and potential binding partners that comprise initial signaling downstream of activated fibroblast growth factor (FGF), insulin (Ins), and insulin-like growth factor-1 (IGF-1) receptors. A panel of 50 SH2 domains screened against a set of 192 phosphotyrosine peptides defines an extensive potential interactome while demonstrating the selectivity of individual SH2 domains. The interactions described confirm virtually all previously reported associations while describing a large set of potential novel interactions that imply additional complexity in the signaling networks initiated from activated receptors. This study of pTyr ligand binding by SH2 domains provides valuable insight into the selectivity that underpins complex signaling networks that are assembled using modular protein interaction domains.
Highlights
Signaling immediately downstream of receptor tyrosine kinases (RTKs) is accomplished in large part by the recruitment of phosphotyrosine interacting proteins to sites of tyrosine phosphorylation on the activated receptors and their associated scaffold proteins [1,2,3]
Peptide arrays for Src homology 2 (SH2) interactions within the fibroblast growth factor (FGF)/Ins/ Insulin Growth Factor (IGF)-1 signaling pathways The use of addressable peptide arrays is a reproducible and semi-quantitative approach that has been extensively validated for studying protein interactions with peptide ligands [37,38,39]
To investigate connections between SH2 domain proteins and their putative phosphorylated docking sites on cell surface receptors, we developed addressable arrays consisting of 192 phosphotyrosine peptides
Summary
Signaling immediately downstream of receptor tyrosine kinases (RTKs) is accomplished in large part by the recruitment of phosphotyrosine (pTyr) interacting proteins to sites of tyrosine phosphorylation on the activated receptors and their associated scaffold proteins [1,2,3]. SH2 domains interact with phosphorylated tyrosinecontaining peptide sequences [7,8,9,10,11] and in doing so they couple activated protein tyrosine kinases (PTKs) to intracellular pathways that regulate many aspects of cellular communication in metazoans [12,13]. The human genome encodes 111 SH2 domain proteins [14,15] that represent the primary mechanism for cellular signal transduction immediately downstream of PTKs. As one might expect, SH2 domain proteins play an essential role in development and have been linked to a wide array of human malignancies including cancers, diabetes, and immunedeficiencies [14,16]
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