Insulin receptor substrate-1 (IRS-1) is a multi-domain protein that mediates signal transduction from receptors for insulin and other growth factors to a variety of downstream molecules through both tyrosine-phosphorylation-dependent and -independent interactions. While the tyrosine-phosphorylation-dependent interactions mediated by IRS-1 have been well characterized, the molecular basis underlying the tyrosine-phosphorylation-independent IRS-1 interactions is largely unknown. We previously detected, in an in vitro binding assay, interactions of Nck-2 Src homology (SH) 3 domains with IRS-1. We show here that IRS-1 associates with Nck-2 in vivo. Additionally, we have investigated the molecular basis underlying the IRS-1–Nck-2 complex formation. We have found that (i) mutations at the highly conserved tryptophan within the Nck-2 SH3 domains markedly reduced the association with IRS-1, (ii) interactions mediated by multiple SH3 domains enhance the complex formation of Nck-2 with IRS-1, (iii) deletion of either the phosphotyrosine-binding/Shc and IRS-1 NPXY-binding (PTB/SAIN) domains or the Pre-C-terminal domain of IRS-1, but not the pleckstrin homology (PH) domain, reduced the Nck-2 binding, (iv) PTB/SAIN domains or the Pre-C-terminal domain alone is capable of interacting with Nck-2, and (v) the IRS-1–Nck-2 interaction occurs in the absence of other proteins and therefore is direct. These results establish that IRS-1 is a bona fide target of the Nck-2 SH3 domains and reveal that IRS-1 forms a complex with Nck-2 via direct interactions mediated by multiple domains from both binding partners.

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