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Src homology 3 domain-dependent interaction of Nck-2 with insulin receptor substrate-1.

ABSTRACT: 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.

SUBMITTER: Tu Y

PROVIDER: S-EPMC1221658 | biostudies-other | 2001 Mar

REPOSITORIES: biostudies-other

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Insulin-like growth factor-I-stimulated insulin receptor substrate-1 negatively regulates Src homology 2 domain-containing protein-tyrosine phosphatase substrate-1 function in vascular smooth muscle cells.

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Project description:Adapter protein with a pleckstrin homology (PH) and an Src homology 2 (SH2) domain (APS) and SH2-B are adapter proteins and substrates that interact with the activation loop of the insulin-receptor (IR) kinase. These proteins are homologous and share substantial sequence similarity. We previously showed [Ahmed, Smith and Pillay, FEBS Lett. 475, 31-34], for the first time, that insulin-stimulated phosphorylation of APS led to interaction with c-Cbl in 3T3-L1 adipocytes and in transfected Chinese-hamster ovary (CHO) cells. In the present study, we find that insulin stimulates the membrane translocation and phosphorylation of APS to a much greater extent than SH2-B, despite the structural similarity of these proteins. Expression of APS or SH2-B delays IR tyrosine and IR substrate (IRS) dephosphorylation. This enhancement of signalling is also observed downsteam of the receptor. In control cells that lack APS, following insulin stimulation, extracellular-signal-regulated kinase (ERK) and Akt kinase reach maximal activation and then decline to basal levels by 60 min. In contrast, in APS- and SH2-B-expressing cells, ERK and Akt kinase activation remains at peak levels at 60 min. These effects may occur because these proteins either stabilize the active conformation or prevent dephosphorylation of the IR. We therefore conclude that, despite the ability to couple to c-Cbl, APS functions as a positive regulator of IR signalling and, although SH2-B is a poor substrate for the IR, its association with the IR allows it to regulate pathways downstream of the receptor independently of its phosphorylation.

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A novel domain mediates insulin-induced proteasomal degradation of insulin receptor substrate 1 (IRS-1).

Project description:Insulin receptor substrate-1 (IRS-1) plays a pivotal role in insulin signaling, therefore its degradation is exquisitely regulated. Here, we show that insulin-stimulated degradation of IRS-1 requires the presence of a highly conserved Ser/Thr-rich domain that we named domain involved in degradation of IRS-1 (DIDI). DIDI (amino acids 386-430 of IRS-1) was identified by comparing the intracellular degradation rate of several truncated forms of IRS-1 transfected into CHO cells. The isolated DIDI domain underwent insulin-stimulated Ser/Thr phosphorylation, suggesting that it serves as a target for IRS-1 kinases. The effects of deletion of DIDI were studied in Fao rat hepatoma and in CHO cells expressing Myc-IRS-1(WT) or Myc-IRS-1(Δ386-430). Deletion of DIDI maintained the ability of IRS-1(Δ386-434) to undergo ubiquitination while rendering it insensitive to insulin-induced proteasomal degradation, which affected IRS-1(WT) (80% at 8 h). Consequently, IRS-1(Δ386-434) mediated insulin signaling (activation of Akt and glycogen synthesis) better than IRS-1(WT). IRS-1(Δ386-434) exhibited a significant greater preference for nuclear localization, compared with IRS-1(WT). Higher nuclear localization was also observed when cells expressing IRS-1(WT) were incubated with the proteasome inhibitor MG-132. The sequence of DIDI is conserved more than 93% across species, from fish to mammals, as opposed to approximately 40% homology of the entire IRS-1. These findings implicate DIDI as a novel, highly conserved domain of IRS-1, which mediates its cellular localization, rate of degradation, and biological activity, with a direct impact on insulin signal transduction.

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