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.     

Insulin-like growth factor I receptor signaling in transformation by src oncogenes.

R- cells, a line of mouse embryo fibroblasts with a targeted disruption of the insulin-like growth factor I (IGF-I) receptor genes, are refractory to transformation by several viral and cellular oncogenes. Using colony formation in soft agar as a measure of full transformation, we report here that R- cells can be transformed by v-src, although they still cannot be transformed by the activated c-src527 (mutation at tyrosine 527 to phenylalanine), which readily transforms mouse embryo cells with a wild-type number of IGF-I receptors (W cells). Although v-src is a more potent inducer of tyrosine phosphorylation than c-src527, the extent of phosphorylation of either insulin receptor substrate 1 or Shc, two of the major substrates of the IGF-I receptor, does not seem sufficiently different to explain the qualitative difference in soft agar growth. v-src, however, is considerably more efficient than c-src527 in its ability to tyrosyl phosphorylate, in R- cells, the focal adhesion kinase, Stat1, and p130cas. These results indicate that v-src, but not c-src527, can bypass the requirement for a functional IGF-I receptor in the full transformation of mouse embryo fibroblasts and suggest that qualitative and quantitative differences between the two oncogenes can be used to identify some of the signals relevant to the mechanism(s) of transformation.     

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.     

Inhibition of epidermal growth factor receptor biosynthesis caused by the src oncogene product, pp60v-src.

We have previously shown that an intracellular mechanism down regulates epidermal growth factor (EGF) receptor levels in rodent fibroblasts transformed by the src oncogene (W. J. Wasilenko, L. K. Shawver, and M. J. Weber, J. Cell. Physiol. 131:450-457, 1987). We now report that this down regulation is due to an inhibition of EGF receptor biosynthesis. With Rat-1 (R1) cells infected with a temperature-sensitive src mutant, we found that 125I-labeled EGF binding to cells began to decrease soon after the activation of pp60v-src by shift down to the permissive temperature for transformation. This effect of src on EGF receptors was reversible. Pulse-chase studies with [35S]methionine-labeled cells revealed that the tyrosine protein kinase activity of pp60v-src had little if any effect on EGF receptor degradation rate. By contrast, the expression of pp60v-src caused a large reduction in the apparent rate of EGF receptor biosynthesis. Northern (RNA) blot analysis demonstrated that pp60v-src also caused marked reductions in the steady-state level of EGF receptor mRNA. These data indicate that one way the expression of the src oncogene can affect the machinery of growth control is by affecting the expression of specific genes for growth factor receptors.     

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.     

Phosphorylation and activation of epidermal growth factor receptors in cells transformed by the src oncogene.

Because functionally significant substrates for the tyrosyl protein kinase activity of pp60v-src are likely to include membrane-associated proteins involved in normal growth control, we have tested the hypothesis that pp60v-src could phosphorylate and alter the signaling activity of transmembrane growth factor receptors. We have found that the epidermal growth factor (EGF) receptor becomes constitutively phosphorylated on tyrosine in cells transformed by the src oncogene and in addition displays elevated levels of phosphoserine and phosphothreonine. High-performance liquid chromatography phosphopeptide mapping revealed two predominant sites of tyrosine phosphorylation, both of which differed from the major sites of receptor autophosphorylation; thus, the src-induced phosphorylation is unlikely to occur via an autocrine mechanism. To determine whether pp60v-src altered the signaling activity of the EGF receptor, we analyzed the tyrosine phosphorylation of phospholipase C-gamma, since phosphorylation of this enzyme occurs in response to activation of the EGF receptor but not in response to pp60v-src alone. We found that in cells coexpressing pp60v-src and the EGF receptor, phospholipase C-gamma was constitutively phosphorylated, a result we interpret as indicating that the signaling activity of the EGF receptor was altered in the src-transformed cells. These findings suggest that pp60v-src-induced alterations in phosphorylation and function of growth regulatory receptors could play an important role in generating the phenotypic changes associated with malignant transformation.     

The use of Rous sarcoma virus transformation mutants with differing tyrosine kinase activities to study the relationships between vinculin phosphorylation, pp60v-src location and adhesion plaque integrity

Tyrosine-specific phosphorylation of cellular proteins has been implicated in the neoplastic transformation of cells by Rous sarcoma virus (RSV). One of the putative substrates for the src gene product (pp60v-src) of RSV is the cytoskeletal protein vinculin, giving rise to the hypothesis that tyrosine-specific phosphorylation of vinculin disrupts adhesion plaque integrity, leading to the characteristic rounded morphology of RSV-transformed cells. We have investigated this hypothesis by analysing the properties of fibroblasts transformed by conditional and non-conditional mutants of RSV which confer different morphologies on infected cells, with respect to formation of microfilament bundles, formation of vinculin-containing adhesion plaques, the deposition of a fibronectin-containing extracellular matrix, the localization of pp60v-src and the tyrosine-specific phosphorylation of vinculin. Cells transformed by the temperature-sensitive (ts) RSV mutant LA32 cultured at 41 degrees C were morphologically normal, and contained prominent microfilament bundles and well-developed adhesion plaques. However, these cells had a fully active pp60v-src kinase, had pp60v-src concentrated in their adhesion plaques and contained vinculin which was heavily phosphorylated on tyrosine residues. Cells transformed by a recovered avian sarcoma virus, rASV 2234.3 exhibited a markedly fusiform morphology with pp60v-src concentrated in well-developed adhesion plaques and an elevation of the phosphotyrosine content of vinculin. Cells transformed by LA32 at restrictive temperature comprise morphologically normal cells, indistinguishable from untransformed CEF, yet which contain tyrosine-phosphorylated vinculin and suggest that neither tyrosine-specific phosphorylation of vinculin nor pp60v-src concentration in adhesion plaques is sufficient for the rounded morphology of RSV-transformed cells.     

Connexin 43 in LA-25 cells with active v-src is phosphorylated on Y247, Y265, S262, S279/282, and S368 via multiple signaling pathways

Modulation of gap junction structures and gap junctional communication is important in maintaining tissue homeostasis and can be controlled via phosphorylation of connexin 43 (Cx43) through several different signaling pathways. Transformation of cells by v-src has been shown to down-regulate gap junction communication coincident with an increase in tyrosine phosphorylation on Cx43. Activation of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) also lead to down-regulation via phosphorylation on specific serine residues. Using phosphospecific anti-Cx43 antibodies generated by the authors' laboratory to specific tyrosines (src substrates) and serine residues (MAPK and PKC substrates) to probe LA-25 cells (which express temperature-sensitive v-src), the authors show that distinct tyrosine and serines residues are phosphorylated in response to v-src activity. They show that tyrosine phosphorylation appears to occur predominantly in gap junction plaques when src is active. In addition, src activation led to increased phosphorylation of apparent MAPK and PKC sites in Cx43. These results indicate all three signaling pathways could contribute to gap junction down-regulation during src transformation in LA-25 cells.     

Expression and function of IRS-1 in insulin signal transmission.

IRS-1 is a major insulin receptor substrate which may play an important role in insulin signal transmission. The mRNA for IRS-1 in rat cells and tissues is about 9.5 kilobases (kb). Rat liver IRS-1 was stably expressed in Chinese hamster ovary (CHO) cells (CHO/IRS-1). Although its calculated molecular mass is 131 kDa, IRS-1 from quiescent cells migrated between 165 and 170 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IRS-1 was phosphorylated strongly on serine residues and weakly on threonine residues before insulin stimulation. Insulin immediately stimulated tyrosine phosphorylation of IRS-1, and after 10-30 min with insulin its apparent molecular mass increased to 175-180 kDa. Expression of the human insulin receptor and rat IRS-1 together in CHO/IR/IRS-1 cells increased the basal serine phosphorylation of IRS-1 and strongly increased tyrosine phosphorylation during insulin stimulation. Purified insulin receptors directly phosphorylated baculovirus-produced IRS-1 exclusively on tyrosine residues. By immunofluorescence, IRS-1 was absent from the nucleus, but otherwise distributed uniformly before and after insulin stimulation. Some IRS-1 associated with the insulin receptor during insulin stimulation. In addition, a phosphatidylinositol 3'-kinase associated with IRS-1 during insulin stimulation, and this association was more sensitive to insulin in CHO cells overexpressing the insulin receptor (CHO/IR cells), more responsive to insulin to CHO/IRS-1 cells, and both sensitive and responsive in CHO/IR/IRS-1 cells. Similarly, insulin-stimulated DNA synthesis was more sensitive to insulin in CHO/IR cells, and more responsive in CHO/IRS-1 cells; however, insulin-stimulated DNA synthesis was sensitive but poorly responsive to insulin in CHO/IR/IRS-1 cells. Together, these results suggest that IRS-1 is a direct physiologic substrate of the insulin receptor and may play an important role in insulin signal transmission.     

Overexpression of human insulin receptor substrate 1 induces cellular transformation with activation of mitogen-activated protein kinases.

The receptor insulin substrate 1 protein (IRS-1) is a specific substrate for insulin receptor tyrosine kinase. Expression and tyrosyl phosphorylation of IRS-1 play an important role during normal hepatocyte growth, and the gene is overexpressed in hepatocellular carcinoma tissue. We determined if IRS-1 overexpression directly contributes to cellular transformation. The human IRS-1 gene was subcloned into a mammalian expression vector driven by the cytomegalovirus early promoter. NIH 3T3 cells transiently transfected with this vector subsequently developed transformed foci. Several stably transfected cell lines were established, and they grew efficiently under low-serum conditions and formed colonies when plated in soft agar. Cell lines overexpressing IRS-1 displayed increased tyrosyl phosphorylation of IRS-1 and association with Grb2 but not with the p85 subunit of phosphatidylinositol 3' kinase. Since Grb2 is a component of the son-of-sevenless-Ras pathway and upstream in the mitogen-activated protein kinase (MAPK) cascade, enzymatic activities of the major components of this cascade, such as MAPK kinase and MAPK were evaluated and found to be substantially increased in three independent cell lines with IRS-1 protein overexpression. Such cells, when injected into nude mice, were highly tumorigenic, and there may be a correlation between the degree of MAPK activation and tumor growth rate. This report describes the generation of a transformed phenotype by overexpression of a molecule without a catalytic domain far upstream in the signal transduction cascade and suggests that prolonged activation of MAPKs by this mechanism may be one of the molecular events related to hepatocellular transformation.     

The insulin-like growth factor I receptor is required for Akt activation and suppression of anoikis in cells transformed by the ETV6-NTRK3 chimeric tyrosine kinase

Signaling through the insulin-like growth factor I receptor (IGF-IR) axis is essential for transformation by many dominantly acting oncoproteins. However, the mechanism by which IGF-IR contributes to oncogenesis remains unknown. To examine this, we compared transformation properties of the oncogenic ETV6-NTRK3 (EN) chimeric tyrosine kinase in IGF-IR-null R- mouse embryo fibroblasts with R- cells engineered to reexpress IGF-IR (R+ cells). We previously showed that R- cells expressing EN (R- EN cells) are resistant to transformation but that transformation is restored in R+ cells. We now show that while R- EN cells have intact Ras-extracellular signal-regulated kinase signaling and cell cycle progression, they are defective in phosphatidylinositol-3-kinase (PI3K)-Akt activation and undergo detachment-induced apoptosis (anoikis) under anchorage-independent conditions. In contrast, R+ cells expressing EN (R+ EN cells) suppress anoikis and are fully transformed. The requirement for IGF-IR in R- EN cells is overcome by ectopic expression of either activated Akt or a membrane-targeted form of EN. Moreover, compared to R- EN cells, R+ EN cells show a dramatic increase in membrane localization of insulin receptor substrate 1 (IRS-1) in association with EN. Since EN is known to bind IRS-1 as an adaptor protein, our findings suggest that IGF-IR may function to localize EN/IRS-1 complexes to cell membranes, in turn facilitating PI3K-Akt activation and suppression of anoikis.     

Insulin and IGF-I signaling through the insulin receptor substrate 1

The insulin and insulin-like growth factor-I (IGF-I) receptors are tyrosine kinases. Consequently, an approach to investigating signaling pathways from these receptors is to characterize proteins rapidly phosphorylated on tyrosine in response to insulin and IGF-I. In many cell types the most prominent phosphotyrosine (Ptyr) protein, in addition to the receptors themselves, is a protein of ?160 kD, now known as the insulin receptor substrate 1 (IRS-1). We have purified IRS-1 from mouse 3T3-L1 adipocytes, obtained the sequences of tryptic peptides, and cloned its cDNA based on this information. Mouse IRS-1 is a protein of 1,231 amino acids. It contains 12 tyrosine residues in sequence contexts typical for tyrosine phosphorylation sites. Six of these begin the sequence motif YMXM and two begin the motif YXXM. Recent studies have shown that the enzyme phosphatidylinositol 3-kinase (PI 3-kinase) binds tightly to the activated platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1) receptors, through interaction of the src homology 2 (SH2) domains on the 85 kD subunit of PI 3-kinase with Ptyr in one of these motifs on the receptors. We have found that, upon insulin treatment of 3T3-L1 adipocytes, a portion of the Ptyr form of IRS-1 becomes tightly complexed with PI 3-kinase. Since IRS-1 binds to fusion proteins containing the SH2 domains of PI 3-kinase, association most likely occurs through this domain. The association of IRS-1 with PI 3-kinase activates the enzyme about fivefold. Thus, one signaling pathway from the insulin and IGF-I receptors probably proceeds as follows: tyrosine phosphorylation of IRS-1, tight association of IRS-1 with PI 3-kinase with accompanying activation of the kinase, elevation of the PI 3-phosphates. © 1993 Wiley-Liss, Inc.     

A Swiss 3T3 variant cell line resistant to the effects of tumor promoters cannot be transformed by src.

To study the relationship between oncogenesis by v-src and normal cellular signalling pathways, we determined the effects of v-src on 3T3-TNR9 cells, a Swiss 3T3 variant which does not respond mitogenically to tumor promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We found that src was unable to transform these variant cells, whether the oncogene was introduced by infection with a murine retrovirus vector or by transfection with plasmid DNA. 3T3-TNR9 cells were not inherently resistant to transformation, since infection with similar recombinant retroviruses containing either v-ras or v-abl did induce transformation. Further analysis of Swiss 3T3 and 3T3-TNR9 cell populations infected with the v-src-containing retrovirus revealed that although the amount of v-src DNA in each was approximately the same, the level of the v-src message and protein and the overall level of phosphotyrosine expressed in the infected variants was much less than in infected parental cells. Cotransfection experiments using separate v-src and neo plasmids revealed a decrease in the number of G418-resistant colonies when transfections of TNR9 cells occurred in the presence of the src-containing plasmid, suggesting a growth inhibitory effect of v-src on 3T3-TNR9 cells, as has also been found for TPA itself. Since v-src cannot transform this variant cell line, which does not respond mitogenically to the protein kinase C agonist TPA, we suggest that src makes use of the protein kinase C pathway as part of its signalling activities.     

Transformation and pp60v-src autophosphorylation correlate with SHC-GRB2 complex formation in rat and chicken cells expressing host-range and kinase-active, transformation-defective alleles of v-src.

The biochemical properties of several pp60v-src substrates believed to participate in src-mediated transformation were examined in cells expressing a kinase-active, transformation-defective v-src allele (v-src-F172 delta/Y416F) and its parental allele, v-src-F172 delta, a host-range--dependent allele that transforms chicken cells to a fusiform morphology, but does not transform rat cells. Because pp60v-src-F172 delta is dependent on autophosphorylation for transforming ability, these alleles provide a unique opportunity to examine the role of pp60v-src autophosphorylation in regulating substrate interactions. Increased pp125FAK tyrosine phosphorylation and high levels of pp60v-src-associated phosphotidylinositol-3' kinase activity were detected specifically in chicken cells exhibiting round, refractile transformation but not in cells transformed to a fusiform morphology. Increased pp125FAK kinase activity, but not increased pp125FAK tyrosine-phosphorylation correlated with pp60v-src autophosphorylation and increased anchorage-independent growth. Thus, pp125FAK and PI3'K may participate in morphological transformation by v-src. Furthermore, association of phosphorylated SHC with the adapter GRB2 correlated with increased anchorage-independent growth (and autophosphorylation) in both rat and chicken cells independent of the morphological phenotype induced. Therefore, host-range dependence for transformation may be regulated through association of SHC with GRB2, thus implicating SHC as a crucial substrate for src-dependent transformation.     

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.     

Linker insertion-deletion mutagenesis of the v-src gene: isolation of host- and temperature-dependent mutants.

The host cell regulators and substrates of the Rous sarcoma virus transforming protein pp60v-src remain largely unknown. Viral mutants which induce a host-dependent phenotype may result from mutations which affect the interaction of pp60v-src with host cell components. To isolate such mutants and to examine the role of different regions of src in regulating pp60v-src function, we generated 46 linker insertion and 5 deletion mutations within src. The mutant src genes were expressed in chicken embryo fibroblasts and in rat-2 cells by using retrovirus expression vectors. Most linker insertions within the kinase domain (residues 260 to 512) inactivated kinase activity and transforming capacity, while most insertions in the N-terminal domain and at the extreme C terminus were tolerated. A number of mutations generated a host-dependent phenotype. Insertions after residues 225 and 227, within the N-terminal regulatory domain (SH2), produced a fusiform transformation in chicken embryo fibroblasts and abolished transformation in rat-2 cells; a similar phenotype also resulted from two deletions affecting SH2 (residues 149 to 174 and residues 77 to 225). Insertions immediately C terminal to Lys-295, which is involved in ATP binding, also produced a conditional phenotype. Insertions after residues 299 and 300 produced a temperature-sensitive phenotype, while insertions after residues 304 and 306 produced a host cell-dependent phenotype. An insertion which removed the major tyrosine autophosphorylation site (Tyr-416) greatly reduced transformation of rat-2 cells, a property not previously observed with other mutations at this site. We conclude that mutations at certain sites within src result in conditional phenotypes. These sites may represent regions important in interactions with host cell components.     

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.