Cellular Compartmentalization in Insulin Action: Altered Signaling by a Lipid-Modified IRS-1

Abstract

While most receptor tyrosine kinases signal by recruiting SH2 proteins directly to phosphorylation sites on their plasma membrane receptor, the insulin receptor phosphorylates intermediary IRS proteins that are distributed between the cytoplasm and a state of loose association with intracellular membranes. To determine the importance of this distribution to IRS-1-mediated signaling, we constructed a prenylated, constitutively membrane-bound IRS-1 by adding the COOH-terminal 9 amino acids from p21 ras , including the CAAX motif, to IRS-1 (IRS-CAAX) and analyzed its function in 32D cells expressing the insulin receptor. IRS-CAAX migrated more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than did IRS-1 and demonstrated increased levels of serine/threonine phosphorylation. Insulin-stimulated tyrosyl phosphorylation of IRS-CAAX was slightly decreased, while IRS-CAAX-mediated phosphatidylinositol 3-kinase (PI3-kinase) binding and activation were decreased by approximately 75% compared to those for wild-type IRS-1. Similarly, expression of IRS-CAAX desensitized insulin-stimulated [ 3 H]thymidine incorporation into DNA by about an order of magnitude compared to IRS-1. By contrast, IRS-CAAX-expressing cells demonstrated increased signaling by mitogen-activated protein kinase, Akt, and p70 S6 kinase in response to insulin. Hence, tight association with the membrane increased IRS-1 serine phosphorylation and reduced coupling between the insulin receptor, PI3-kinase, and proliferative signaling while enhancing other signaling pathways. Thus, the correct distribution of IRS-1 between the cytoplasm and membrane compartments is critical to the normal balance in the network of insulin signaling.Many growth factors regulate cellular processes through activation of tyrosine kinases (11,55,58). Ligand binding to the extracellular region of the receptor results in activation of the intracellular tyrosine kinase domain of the receptor and receptor autophosphorylation (35). In most cases, tyrosyl autophosphorylation of the intracellular portion of the receptor directly recruits binding of downstream signaling partners through specialized phosphotyrosine-binding domains, e.g., SH2 domains in signaling proteins (26). The receptor thus nucleates a signaling complex containing numerous SH2 domain-containing signaling proteins (SH2 proteins) on the plasma membrane.The insulin receptor, like most growth factor receptors, contains intrinsic tyrosine kinase activity and undergoes tyrosine autophosphorylation during ligand stimulation (35). However, unlike most other growth factor receptors, the insulin receptor does not directly recruit SH2 proteins. Instead, the insulin receptor phosphorylates intracellular substrate proteins (such as the IRS proteins, IRS-1, IRS-2, IRS-3, and IRS-4) on multiple tyrosine residues, and these IRS proteins in turn recruit SH2 proteins such as phosphatidylinositol 3Ј-kinase (PI3Ј-kinase), GRB-2/mSos, and SHP-2 into a signaling complex (35). Previous studies have shown that the IRS protein...

Membrane Targeting of Grb2-associated Binder-1 (Gab1) Scaffolding Protein through Src Myristoylation Sequence Substitutes for Gab1 Pleckstrin Homology Domain and Switches an Epidermal Growth Factor Response to an Invasive Morphogenic Program

“…Although in contrast to our data, membrane targeting of IRS-1 led to enhanced activation of downstream pathways involving Akt and Erk in response to insulin (Kriauciunas et al, 2000). Although these results apparently differ from ours, they have in common an increase in the local concentration of signaling proteins in the membrane.…”

Oxidative stress induces insulin resistance by activating the nuclear factor-?B pathway and disrupting normal subcellular distribution of phosphatidylinositol 3-kinase

“…This is based on several reports showing IRS-1 phosphorylation and PI 3-kinase activation specifically in the LDM fraction, though not in whole cell lysates, to be important for insulin action [30,31]. We speculate that the insulin-induced increase in IRS-1 phosphorylation in the LDM fraction leads to recruitment of the p85 subunit for PI 3-kinase to that fraction.…”

“…3a, upper panels). Because the insulin signalling in the LDM fraction has been implicated in several insulin actions including insulin-induced glucose uptake [30,31], we carried out subcellular fractionation studies of skeletal muscles from these rats. Subcellular fractionation data showed insulin-induced tyrosine phosphorylation of IRS-1 in the LDM fraction to be significantly decreased in BSO-treated rats as compared with controls, although the IRS-1 protein amount in this fraction was unchanged (Fig.…”

ETV6-NTRK3 transformation requires insulin-like growth factor 1 receptor signaling and is associated with constitutive IRS-1 tyrosine phosphorylation

“…The N-terminal pleckstrin homology (PH) domain of IRS-1 is not only essential for efficient tyrosine phosphorylation of IRS-1 upon IGF stimulation (Yenush et al, 1998), but also appears to be necessary for the translocation of IRS-1 from the cytoplasm to the inner cell membrane during insulin and IGF stimulation (Razzini et al, 2000). Recent studies with a myristoylated form of IRS-1 in insulin-expressing cells demonstrated that constitutive membrane localization of IRS-1 correlated with decreased p85 binding but with increased Ras-MAPK and PI3K-Akt activation in response to insulin (Kriauciunas et al, 2000). It is therefore possible that IGFRI, like the insulin receptor, may play a role in localizing IRS-1 (and associated Grb2 and p85) to the inner cell membrane in EN-expressing cells where it can efficiently activate components of the Ras-MAPK and PI3K-Akt pathways required for EN transformation.…”

“…Third, IGFRI might be necessary to localize the EN/IRS-1 signaling complexes in such a way as to be able to maximally activate pathways required for transformation. There is increasing evidence that the subcellular localization of IRS-1 is important for its ability to effectively activate downstream pathways (Kriauciunas et al, 2000). IRS proteins exist in a dynamic equilibrium between the cytoplasm and inner cell membrane compartments (Myers and White, 1996).…”