Nuclear translocation of insulin receptor substrate-1 by the simian virus 40 T antigen and the activated type 1 insulin-like growth factor receptor

32D cells are a murine hemopoietic cell line that undergoes apoptosis upon withdrawal of interleukin-3 (IL-3) from the medium. 32D cells have low levels of the type 1 insulin-like growth factor (IGF-I) receptor and do not express insulin receptor substrate-1 (IRS-1) or IRS-2. Ectopic expression of IRS-1 delays apoptosis but cannot rescue 32D cells from IL-3 dependence. In 32D/IRS-1 cells, IRS-1 is detectable, as expected, in the cytosol/membrane compartment. The SV40 large T antigen is a nuclear protein that, by itself, also fails to protect 32D cells from apoptosis. Co-expression of IRS-1 with the SV40 T antigen in 32D cells results in nuclear translocation of IRS-1 and survival after IL-3 withdrawal. Expression of a human IGF-I receptor in 32D/IRS-1 cells also results in nuclear translocation of IRS-1 and IL-3 independence. The phosphotyrosine-binding domain, but not the pleckstrin domain, is necessary for IRS-1 nuclear translocation. Nuclear translocation of IRS-1 was confirmed in mouse embryo fibroblasts. These results suggest possible new roles for nuclear IRS-1 in IGF-I-mediated growth and anti-apoptotic signaling.     

Nuclear translocation of insulin receptor substrate-1 by oncogenes and Igf-I. Effect on ribosomal RNA synthesis

The insulin receptor substrate-1 (IRS-1) is one of the major substrates of both the insulin and IGF-I receptors and is generally localized in the cytosol/membrane fraction of the cell. We show here that a substantial fraction of IRS-1 is translocated to the nucleus in mouse embryo fibroblasts (MEF) expressing the simian virus 40 T antigen. Nuclear translocation of IRS-1 occurs also in MEF stimulated with IGF-I or in MEF expressing the oncogene v-src. Nuclear translocation of IRS-1 can be demonstrated by confocal microscopy, immunohistochemistry, or subcellular fractionation. An antibody to IRS-1 immunoprecipitates from nuclear fractions (but not from cytosolic fractions) the upstream binding factor, which is a key regulator of RNA polymerase I activity and ribosomal RNA (rRNA) synthesis. In agreement with this finding, in 32D murine hemopoietic cells, nuclear translocation of IRS-1 correlates with a markedly increased rRNA synthesis. Our experiments suggest that nuclear IRS-1 may play a specialized role in rRNA synthesis and/or processing.     

Oxygen tension regulates the stability of insulin receptor substrate-1 (IRS-1) through caspase-mediated cleavage

The insulin and insulin-like growth factor-1 (IGF-1) receptors mediate signaling for energy uptake and growth through insulin receptor substrates (IRSs), which interact with these receptors as well as with downstream effectors. Oxygen is essential not only for ATP production through oxidative phosphorylation but also for many cellular processes, particularly those involved in energy homeostasis. The oxygen tension in vivo is significantly lower than that in the air and can vary widely depending on the tissue as well as on perfusion and oxygen consumption. How oxygen tension affects IRSs and their functions is poorly understood. Our findings indicate that transient hypoxia (1% oxygen) leads to caspase-mediated cleavage of IRS-1 without inducing cell death. The IRS-1 protein level rebounds rapidly upon return to normoxia. Protein tyrosine phosphatases (PTPs) appear to be important for the IRS-1 cleavage because tyrosine phosphorylation of the insulin receptor was decreased in hypoxia and IRS-1 cleavage could be blocked either with H(2)O(2) or with vanadate, each of which inhibits PTPs. Activity of Akt, a downstream effector of insulin and IGF-1 signaling that is known to suppress caspase activation, was suppressed in hypoxia. Overexpression of dominant-negative Akt led to IRS-1 cleavage even in normoxia, and overexpression of constitutively active Akt partially suppressed IRS-1 cleavage in hypoxia, suggesting that hypoxia-mediated suppression of Akt may induce caspase-mediated IRS-1 cleavage. In conclusion, our study elucidates a mechanism by which insulin and IGF-1 signaling can be matched to the oxygen level that is available to support growth and energy metabolism.     

Regulation of insulin/insulin-like growth factor-1 signaling by proteasome-mediated degradation of insulin receptor substrate-2

Insulin and insulin-like growth factor-1 (IGF-1) regulate metabolism and body growth through homologous receptor tyrosine kinases that phosphorylate the insulin receptor substrate (IRS) proteins. IRS-2 is an important IRS protein, as it mediates peripheral insulin action and beta-cell survival. In this study, we show that insulin, IGF-1, or osmotic stress promoted ubiquitin/proteasome-mediated degradation of IRS-2 in 3T3-L1 cells, Fao hepatoma, cells and mouse embryo fibroblasts; however, insulin/IGF-1 did not promote degradation of IRS-1 in 3T3-L1 preadipocytes or mouse embryo fibroblasts. MG132 or lactacystin, specific inhibitors of 26S proteasome, blocked insulin/IGF-1-induced degradation of IRS-2 and enhanced the detection of ubiquitinated IRS-2. Insulin/IGF1-induced ubiquitination and degradation of IRS-2 was blocked by inhibitors of phosphatidylinositol 3-kinase (wortmannin or LY294002) or mTOR (rapamycin). Chronic insulin or IGF-1 treatment of IRS-1-deficient mouse embryo fibroblasts inhibited IRS-2-mediated activation of Akt and ERK1/2, which was reversed by lactacystin pretreatment. By contrast, IRS-1 activation of Akt and ERK1/2 was not inhibited by chronic insulin/IGF-1 stimulation in IRS-2-deficient mouse embryo fibroblasts. Thus, we identified a novel negative feedback mechanism by which the ubiquitin/proteasome-mediated degradation of IRS-2 limits the magnitude and duration of the response to insulin or IGF-1.     

Receptors for insulin and insulin-like growth factor-1 and insulin receptor substrate-1 mediate pathways that regulate islet function

The insulin/insulin-like growth factor-1 (IGF-1) signalling pathways are present in most mammalian cells and play important roles in the growth and metabolism of tissues. Most proteins in these pathways have also been identified in the beta-cells of the pancreatic islets. Tissue-specific knockout of the insulin receptor (betaIRKO) or IGF-1 receptor (betaIGFRKO) in pancreatic beta-cells leads to altered glucose-sensing and glucose intolerance in adult mice, and betaIRKO mice show an age-dependent decrease in islet size and beta-cell mass. These data indicate that these receptors are important for differentiated function and are unlikely to play a major role in the early growth and/or development of the pancreatic islets. Conventional insulin receptor substrate-1 (IRS-1) knockouts manifest growth retardation and mild insulin resistance. The IRS-1 knockouts also display islet hyperplasia, defects in insulin secretory responses to multiple stimuli both in vivo and in vitro, reduced islet insulin content and an increased number of autophagic vacuoles in the beta-cells. Re-expression of IRS-1 in cultured beta-cells is able to partially restore the insulin content indicating that IRS-1 is involved in the regulation of insulin synthesis. Taken together, these data provide evidence that insulin and IGF-1 receptors and IRS-1, and potentially other proteins in the insulin/IGF-1 signalling pathway, contribute to the regulation of islet hormone secretion and synthesis and therefore in the maintenance of glucose homeostasis.     

Human insulin receptor substrate-1 (IRS-1) polymorphism G972R causes IRS-1 to associate with the insulin receptor and inhibit receptor autophosphorylation

The most commonly detected polymorphism in human insulin receptor substrate-1 (IRS-1), a glycine to arginine change at codon 972 (G972R), is associated with an increased risk of Type 2 diabetes and insulin resistance. To determine the molecular mechanism by which this polymorphism may be linked to insulin resistance, we produced recombinant peptides comprising amino acid residues 925-1008 from IRS-1 that contain either a glycine or arginine at codon 972 and the two nearby tyrosine phosphorylation consensus sites (EY(941)MLM and DY(989)MTM), which are known binding sites for the p85alpha regulatory subunit of phosphatidylinositol 3-kinase. The wild type peptide could be phosphorylated at these sites in vitro by purified insulin receptor. Introduction of the G972R polymorphism into the peptide reduced the amount of tyrosine phosphorylation by >60%. Pull-down experiments indicated that there was an association between the IRS-1-(925-1008) peptide and the insulin receptor that was markedly enhanced by the presence of the G972R polymorphism. The use of additional overlapping fragments localized this interaction to domains between residues 950-986 of IRS-1 and residues 966-1271 of the insulin receptor, containing the tyrosine kinase domain of the receptor. In addition, the IRS-1-(925-1008) G972R peptide acted as a competitive inhibitor of insulin receptor and insulin-like growth factor-1 receptor autophosphorylation. Taken together, these data indicate that the G972R naturally occurring polymorphism of IRS-1 not only reduces phosphorylation of the substrate but allows IRS-1 to act as an inhibitor of the insulin receptor kinase, producing global insulin resistance.     

Differential roles of the insulin and insulin-like growth factor-I (IGF-I) receptors in response to insulin and IGF-I

Insulin and insulin-like growth factor-I (IGF-I) receptors are highly homologous tyrosine kinase receptors that share many common steps in their signaling pathways and have ligands that can bind to either receptor with differing affinities. To define precisely the signaling specific to the insulin receptor (IR) or the IGF-I receptor, we have generated brown preadipocyte cell lines that lack either receptor (insulin receptor knockout (IRKO) or insulin-like growth factor receptor knockout (IGFRKO)). Control preadipocytes expressed fewer insulin receptors than IGF-I receptors (20,000 versus 60,000), but during differentiation, insulin receptor levels increased so that mature adipocytes expressed slightly more insulin receptors than IGF-I receptors (120,000 versus 100,000). In these cells, insulin stimulated IR homodimer phosphorylation, whereas IGF-I activated both IGF-I receptor homodimers and hybrid receptors. Insulin-stimulated IRS-1 phosphorylation was significantly impaired in IRKO cells but was surprisingly elevated in IGFRKO cells. IRS-2 phosphorylation was unchanged in either cell line upon insulin stimulation. IGF-I-dependent phosphorylation of IRS-1 and IRS-2 was ablated in IGFRKO cells but not in IRKO cells. In control cells, both insulin and IGF-I produced a dose-dependent increase in phosphorylated Akt and MAPK, although IGF-I elicited a stronger response at an equivalent dose. In IRKO cells, the insulin-dependent increase in phospho-Akt was completely abolished at the lowest dose and reached only 20% of the control stimulation at 10 nm. Most interestingly, the response to IGF-I was also impaired at low doses, suggesting that IR is required for both insulin- and IGF-I-dependent phosphorylation of Akt. Most surprisingly, insulin- or IGF-I-dependent phosphorylation of MAPK was unaltered in either receptor-deficient cell line. Taken together, these results indicate that the insulin and IGF-I receptors contribute distinct signals to common downstream components in response to both insulin and IGF-I.     

Micro RNA 145 targets the insulin receptor substrate-1 and inhibits the growth of colon cancer cells

The insulin receptor substrate-1 (IRS-1), a docking protein for both the type 1 insulin-like growth factor receptor (IGF-IR) and the insulin receptor, is known to send a mitogenic, anti-apoptotic, and anti-differentiation signal. Several micro RNAs (miRs) are suggested by the data base as possible candidates for targeting IRS-1. We show here that one of the miRs predicted by the data base, miR145, whether transfected as a synthetic oligonucleotide or expressed from a plasmid, causes down-regulation of IRS-1 in human colon cancer cells. IRS-1 mRNA is not decreased by miR145, while it is down-regulated by an siRNA targeting IRS-1. Targeting of the IRS-1 3'-untranslated region (UTR) by miR145 was confirmed using a reporter gene (luciferase) expressing the miR145 binding sites of the IRS-1 3'-UTR. In agreement with the role of IRS-1 in cell proliferation, we show that treatment of human colon cancer cells with miR145 causes growth arrest comparable to the use of an siRNA against IRS-1. Taken together, these results identify miR145 as a micro RNA that down-regulates the IRS-1 protein, and inhibits the growth of human cancer cells.     

Action of insulin receptor substrate-3 (IRS-3) and IRS-4 to stimulate translocation of GLUT4 in rat adipose cells

The insulin receptor initiates insulin action by phosphorylating multiple intracellular substrates. Previously, we have demonstrated that insulin receptor substrates (IRS)-1 and -2 can mediate insulin's action to promote translocation of GLUT4 glucose transporters to the cell surface in rat adipose cells. Although IRS-1, -2, and -4 are similar in overall structure, IRS-3 is approximately 50% shorter and differs with respect to sites of tyrosine phosphorylation. Nevertheless, as demonstrated in this study, both IRS-3 and IRS-4 can also stimulate translocation of GLUT4. Rat adipose cells were cotransfected with expression vectors for hemagglutinin (HA) epitope-tagged GLUT4 (GLUT4-HA) and human IRS-1, murine IRS-3, or human IRS-4. Overexpression of IRS-1 led to a 2-fold increase in cell surface GLUT4-HA in cells incubated in the absence of insulin; overexpression of either IRS-3 or IRS-4 elicited a larger increase in cell surface GLUT4-HA. Indeed, the effect of IRS-3 in the absence of insulin was approximately 40% greater than the effect of a maximally stimulating concentration of insulin in cells not overexpressing IRS proteins. Because phosphatidylinositol (PI) 3-kinase is essential for insulin-stimulated translocation of GLUT4, we also studied a mutant IRS-3 molecule (IRS-3-F4) in which Phe was substituted for Tyr in all four YXXM motifs (the phosphorylation sites predicted to bind to and activate PI 3-kinase). Interestingly, overexpression of IRS-3-F4 did not promote translocation of GLUT4-HA, but actually inhibited the ability of insulin to stimulate translocation of GLUT4-HA to the cell surface. Our data suggest that IRS-3 and IRS-4 are capable of mediating PI 3-kinase-dependent metabolic actions of insulin in adipose cells, and that IRS proteins play a physiological role in mediating translocation of GLUT4.     

Differential subcellular localization of insulin receptor substrates depends on C-terminal regions and importin beta

Insulin receptor substrates (IRSs) play essential roles in signal transduction of insulin and insulin-like growth factors. Previously, we showed that IRS-3 is localized to the nucleus as well as the cytosol, while IRS-1 and 2 are mainly localized to the cytoplasm. In the present study, we found that importin β directly interacts with IRS-3 and is able to mediate nuclear transport of IRS-3. Importin β interacted with the pleckstrin homology domain, the phosphotyrosine binding domain and the C-terminal region of IRS-3; indeed all of these fragments exhibited predominant nuclear localization. By contrast, almost no interaction of importin β with IRS-1 and -2 was observed, and their C-terminal regions displayed discrete spotty images in the cytosol. In addition, using chimeric proteins between IRS-1 and IRS-3, we revealed that the C-terminal regions are the main determinants of the differing subcellular localizations of IRS-1 and IRS-3.     

Differential modulation of the tyrosine phosphorylation state of the insulin receptor by IRS (insulin receptor subunit) proteins.

In response to insulin, tyrosine kinase activity of the insulin receptor is stimulated, leading to autophosphorylation and tyrosine phosphorylation of proteins including insulin receptor subunit (IRS)-1, IRS-2, and Shc. Phosphorylation of these proteins leads to activation of downstream events that mediate insulin action. Insulin receptor kinase activity is requisite for the biological effects of insulin, and understanding regulation of insulin receptor phosphorylation and kinase activity is essential to understanding insulin action. Receptor tyrosine kinase activity may be altered by direct changes in tyrosine kinase activity, itself, or by dephosphorylation of the insulin receptor by protein-tyrosine phosphatases. After 1 min of insulin stimulation, the insulin receptor was tyrosine phosphorylated 8-fold more and Shc was phosphorylated 50% less in 32D cells containing both IRS-1 and insulin receptors (32D/IR+IRS-1) than in 32D cells containing only insulin receptors (32D/IR), insulin receptors and IRS-2 (32D/IR+IRS-2), or insulin receptors and a form of IRS-1 that cannot be phosphorylated on tyrosine residues (32D/IR+IRS-1F18). Therefore, IRS-1 and IRS-2 appeared to have different effects on insulin receptor phosphorylation and downstream signaling. Preincubation of cells with pervanadate greatly decreased protein-tyrosine phosphatase activity in all four cell lines. After pervanadate treatment, tyrosine phosphorylation of insulin receptors in insulin-treated 32D/IR, 32D/ IR+IRS-2, and 32D/IR+IRS-1F18 cells was markedly increased, but pervanadate had no effect on insulin receptor phosphorylation in 32D/IR+IRS-1 cells. The presence of tyrosine-phosphorylated IRS-1 appears to increase insulin receptor tyrosine phosphorylation and potentially tyrosine kinase activity via inhibition of protein-tyrosine phosphatase(s). This effect of IRS-1 on insulin receptor phosphorylation is unique to IRS-1, as IRS-2 had no effect on insulin receptor tyrosine phosphorylation. Therefore, IRS-1 and IRS-2 appear to function differently in their effects on signaling downstream of the insulin receptor. IRS-1 may play a major role in regulating insulin receptor phosphorylation and enhancing downstream signaling after insulin stimulation.     

Subcellular localization of IRS-1 in cell proliferation and differentiation.

The type 1 insulin-like growth factor receptor (IGF-IR) and its docking protein, insulin receptor substrate-1 (IRS-1), play important roles in cell transformation, cell differentiation and aging. IRS-1 and other IRS proteins can, under certain conditions, localize to the nuclei of cells, where they undergo interactions with nuclear and nucleolar proteins. In this study, we confirm and extend these observations, demonstrating that IRS-1 is preferentially nuclear in growing cells. Differentiation and inhibition of ribosomal RNA synthesis cause subcellular redistribution of IRS-1 and other nuclear proteins to the cytoplasm.     

Differential activation of insulin receptor substrates 1 and 2 by insulin-like growth factor-activated insulin receptors

The insulin-like growth factors (insulin-like growth factor I [IGF-I] and IGF-II) exert important effects on growth, development, and differentiation through the IGF-I receptor (IGF-IR) transmembrane tyrosine kinase. The insulin receptor (IR) is structurally related to the IGF-IR, and at high concentrations, the IGFs can also activate the IR, in spite of their generally low affinity for the latter. Two mechanisms that facilitate cross talk between the IGF ligands and the IR at physiological concentrations have been described. The first of these is the existence of an alternatively spliced IR variant that exhibits high affinity for IGF-II as well as for insulin. A second phenomenon is the ability of hybrid receptors comprised of IGF-IR and IR hemireceptors to bind IGFs, but not insulin. To date, however, direct activation of an IR holoreceptor by IGF-I at physiological levels has not been demonstrated. We have now found that IGF-I can function through both splice variants of the IR, in spite of low affinity, to specifically activate IRS-2 to levels similar to those seen with equivalent concentrations of insulin or IGF-II. The specific activation of IRS-2 by IGF-I through the IR does not result in activation of the extracellular signal-regulated kinase pathway but does induce delayed low-level activation of the phosphatidylinositol 3-kinase pathway and biological effects such as enhanced cell viability and protection from apoptosis. These findings suggest that IGF-I can function directly through the IR and that the observed effects of IGF-I on insulin sensitivity may be the result of direct facilitation of insulin action by IGF-I costimulation of the IR in insulin target tissues.     

Regulation of insulin receptor substrate-1 expression levels by caveolin-1

The insulin receptor substrate-1 (IRS-1), a docking protein of the type 1 insulin-like growth factor receptor (IGF-IR) plays a significant role in cell proliferation and differentiation. The expression of IRS-1 is down-regulated in mouse embryo fibroblasts (MEFs) with a deletion of caveolin-1 (cav1) genes (KO cells). Levels of IRS-1 mRNA are not affected. Re-introduction of cav1 into KO cells rescues IRS-1 expression. Stabilization of protein levels is reciprocal and a strict correlation between IRS-1 and cav1 levels was confirmed in five cell lines, and in mouse tissues. IRS-1 binds through its phosphotyrosine binding (PTB) domain to tyrosine 14 (Y14) of cav1, the residue phosphorylated by IGF-1 stimulation and by v-src. The down-regulation of IRS-1 in cav-/- cells occurs via the proteasome pathway. These results indicate a novel mechanism for the regulation of IRS-1 expression levels, an important finding in view of IRS-1 role in cell proliferation and transformation.