Insulin receptor substrate-1 mediates phosphatidylinositol 3'-kinase and p70S6k signaling during insulin, insulin-like growth factor-1, and interleukin-4 stimulation.

Insulin receptor substrate 1 (IRS-1) is the principle cellular substrate for insulin and insulin-like growth factor I (IGF-I) receptor signaling. After phosphorylation of tyrosine residues within the YMXM or YXXM motifs, IRS-1 associates with phosphatidylinositol-3 kinase (PI3K). This signaling pathway and the presence of an IRS-1-like molecule have been demonstrated in IRS-1-transfected and in nontransfected hematopoietic cell lines, respectively. IGF-I has been implicated in lymphocyte development and function, and recently, we showed that functional type-I IGF receptors are present on human thymocytes and peripheral T cells. In this study, we addressed IGF-I signal transduction in nontransformed, freshly isolated, human thymocytes, as well as in blood T cells. Using Western blot analysis, we found that IGF-I induced phosphorylation of a 160-180-kD protein (pp170) in human thymocytes and that phosphorylated pp170 becomes associated with PI3K and is recognized by anti-IRS-1. In blood T cells, this immunoreactive IRS-1 (irIRS-1) is less abundantly expressed than in thymocytes, as assessed with immunoblotting. As a consequence, phosphorylated pp170 was not or hardly detectable after stimulation with IGF-I, and irIRS-1 was not detected in PI3K immunoprecipitates from lysates of IGF-I-stimulated T cells. However, IGF-I induced the tyrosine phosphorylation of other cellular proteins, indicating that differential expression of irIRS-1 contributes to a distinct signaling pathway in T cells.

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Insulin-Like Growth Factor I-Induced Degradation of Insulin Receptor Substrate 1 Is Mediated by the 26S Proteasome and Blocked by Phosphatidylinositol 3′-Kinase Inhibition

Molecular and Cellular Biology ◽

10.1128/mcb.20.5.1489-1496.2000 ◽

2000 ◽

Vol 20 (5) ◽

pp. 1489-1496 ◽

Cited By ~ 87

Author(s):

Adrian V. Lee ◽

Jennifer L. Gooch ◽

Steffi Oesterreich ◽

Rebecca L. Guler ◽

Douglas Yee

Keyword(s):

Growth Factor ◽

Insulin Receptor ◽

Tyrosine Phosphorylation ◽

26S Proteasome ◽

Insulin Receptor Substrate ◽

Phosphatidylinositol 3 Kinase ◽

Insulin Receptor Substrate 1 ◽

Erk Activation ◽

Igf I ◽

Phosphatidylinositol 3

ABSTRACT Insulin receptor substrate 1 (IRS-1) is a critical adapter protein involved in both insulin and insulin-like growth factor (IGF) signaling. Due to the fact that alteration of IRS-1 levels can affect the sensitivity and response to both insulin and IGF-I, we examined the ability of each of these ligands to affect IRS-1 expression. IGF-I (10 nM) stimulation of MCF-7 breast cancer cells caused a transient tyrosine phosphorylation of IRS-1 that was maximal at 15 min and decreased thereafter. The decrease in tyrosine phosphorylation of IRS-1 was paralleled by an apparent decrease in IRS-1 levels. The IGF-mediated decrease in IRS-1 expression was posttranscriptional and due to a decrease in the half-life of the IRS-1 protein. Insulin (10 nM) caused tyrosine phosphorylation of IRS-1 but not degradation, whereas high concentrations of insulin (10 μM) resulted in degradation of IRS-1. IGF-I (10 nM) stimulation resulted in transient IRS-1 phosphorylation and extracellular signal-related kinase (ERK) activation. In contrast, insulin (10 nM) caused sustained IRS-1 phosphorylation and ERK activation. Inhibition of 26S proteasome activity by the use of lactacystin or MG132 completely blocked IGF-mediated degradation of IRS-1. Furthermore, coimmunoprecipitation experiments showed an association between ubiquitin and IRS-1 that was increased by treatment of cells with IGF-I. Finally, IGF-mediated degradation of IRS-1 was blocked by inhibition of phosphatidylinositol 3′-kinase activity but was not affected by inhibition of ERK, suggesting that this may represent a direct negative-feedback mechanism resulting from downstream IRS-1 signaling. We conclude that IGF-I can cause ligand-mediated degradation of IRS-1 via the ubiquitin-mediated 26S proteasome and a phosphatidylinositol 3′-kinase-dependent mechanism and that control of degradation may have profound effects on downstream activation of signaling pathways.

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