Src homology 3 domain-dependent interaction of Nck-2 with insulin receptorsubstrate-1

Insulin receptor substrate-1 (IRS-1) is a multi-domain protein that mediate s signal transduction from receptors for insulin and other growth factors t o a variety of downstream molecules through both tyrosine-phosphorylation-d ependent 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 i nteractions is largely unknown. We previously detected, in an in vitro bind ing 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 formati on. We have found that (i) mutations at the highly conserved tryptophan wit hin the Nck-2 SH3 domains markedly reduced the association with IRS-1, (ii) interactions mediated by multiple SH3 domains enhance the complex formatio n 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 b inding, (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 t he absence of other proteins and therefore is direct. These results establi sh that IRS-1 is a bonafide target of the Nck-2 SH3 domains and reveal that IRS-1 forms a complex with Nck-2 via direct interactions mediated by multi ple domains from both binding partners.